.- v! RETURNING MATERIALS: MSU mace 1‘ n book drop to , remove this checkout from ’ w your record. FINES wm ' be charged if book is returned after the date stamped below. til 2'. .. . . ‘ ‘fl N"Ilh"c\:"r -. u '. v [ LIB war” -’ Lislisim 2,3,..gmmmmwn .. -~ " \2'. , THE EFFECTS OF MOUSE MUTANT 3W1 UPON EMBRYONIC DEVELOPMENT: HISTOLOGICAL AND TISSUE CULTURE STUDIES By Linda Christine Chaney A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Zoology and Neuroscience Program 1981 ABSTRACT THE EFFECTS OF MOUSE MUTANT 3V1 UPON EMBRYONIC DEVELOPMENT: HISTOLOGICAL AND TISSUE CULTURE STUDIES By Linda Christine Chaney The t mutants (t_haplotypes), a complex group of alleles identi- fied in mice in association with the gene Brachyury (I), are divided into seven complementation groups. In the homozygous state, alleles from each complementation group affect different stages of early embryonic development. This study examined the effects of one mutant (trl) after it was backcrossed into the C3H.BlO strain of mice. Four different types of experiments were performed in order Ewl to study the effect of mouse mutant upon the embryonic development of the central nervous system. First, the gene was successfully transferred from a balanced lethal (Ii/itvl) “pen bred" stock to an inbred strainof mice, C3H.BlO, in order to reduce the heterogeneous genetic background of the original strain and to permit the study of the action of 3&1 in the absence of mutant genes Iland a}. The gene is presently maintained in two strains: a balanced lethal line, .I:[ityl(generation F12) and a backcross-intercross line (generation N5F4). Second, normal (1/3,.31581) and abnormal (ty1[tvl) embryos at days 9 to 15 of gestation were subjected to gross examination after dissection from amniotic sacs and histological examination at the light microscope level. On the basis of gross morphology, mutant embryos could not be easily distinguished from normal littermates Linda Christine Chaney prior to ll days post-coitus (29). From ll to l5 days pg_mutant embryos could be identified by their relatively smaller size, retarded growth (younger developmental age than normal littermates), enlarged hearts and degenerating neural tissues. At the light microscope level, mutant embryos could be identified from serially sectioned material as early as 9 days pg_by the presence of pycnotic cells within the mantle layer of the rostral portion of the rhombencephalon. At later stages of development, mutant embryos became more easily recognized in sectioned material by the increased number of pycnotic cells in the ventral rhombencephalon and neural tube. As mutant embryos developed from 9 to l3 days 29, a specific pattern of cellular pycnosis appeared within the neural tube. Pycnotic cells were always confined to the mantle layer and first appeared in the ventral region of the rostral portion of the rhombencephalon. The number of pycnotic cells increased with the age of the embryo along the ventral neural tube in rostral and caudal directions, extended into regions of the dorsal neural tube in the area of the caudal rhombencephalon and continued in the dorsal neural tube towards the tail. Related neural ectoderm structures (otic vesicle, cranial and spinal ganglia) did not appear to be affected. In general, non- neural structures continued to develop even in the presence of an abnormally developing neural tube. By 12 to 13 days pg, mutant embryos were in the process of being resorbed, and non-neural as well as neural structures contained pycnotic cells. Linda Christine Chaney Third, embryonic tailbud, forelimb bud, or heart tissue was cultured in 11359 to determine whether the mutant was a general cell lethal and to attempt to identify embryonic genotypes prior to the appearance of gross abnormalities. Due to segregation distortion in males (90% 3‘“ and 10% 3: bearing sperm fertilize ova), 45% of the embryos within a litter are expected to be 381/381. Successful cellular outgrowth appeared in 94% of all cultures (190 cultures representing embryos from days 9 to 15 pg), indicating that the primary action of the gene does not affect all cells of the mutant embryo. Fourth, determinations of embryonic genotypes by assaying fibro- blasts for the presence of two private H-2 antigens (D-2 and 108) were erratic. Results from assays measuring the incorporation of 125I-iododeoxyuridine into the DNA of viable cells following treatment with mouse anti-H-2.2 or mouse anti-H-Zml and complement suggest that very low concentrations of antigens are present on these embryonic fibroblasts. Unlike some t haplotypes acting earlier in embryonic development which appear to be lethal for all cells of the mutant embryo, the action of gene 5’1 is specific for cells within the neural tube. These studies support the hypothesis that death of homozygous mutant embryos results from death of specific cell types within the neural tube and subsequent aberrant cell-cell interactions and disorgani- zation. DEDICATION To my husband, Bill ii ACKNOWLEDGEMENTS I would like to thank my thesis advisor, Dr. James H. Asher, Jr., for his guidance and help during the course of my graduate career. Support and encouragement by members of my graduate committee, Drs. Martin Balaban, Thomas Jenkins, and Charles Tweedle, were greatly appreciated. I would like to thank Dr. Walter J. Esselman for pro- viding access to his tissue culture facilities and supplies. The artistic talents of Ms. Karin Grimnes, who provided the final ink drawings of embryos, are respectfully acknowledged. Financial support from the Department of Zoology and Neuroscience Program was also appreciated and helped towards the completion of this degree. Special thanks goes to my friends and companions who listened to my many research problems and discussed possible solutions. To my husband, Bill, I am especially grateful. His continued support, wit, enthusiasm, and firm shoulder made completion of the work much easier. iii TABLE OF CONTENTS Page LIST OF TABLES ......................... Vi LIST OF FIGURES ........................ Vii LIST OF ABBREVIATIONS USED IN TEXT AND FIGURES ......... ix LITERATURE REVIEW ....................... l The I Locus ........................ l Historical Background ................ l I Mutations ..................... 4 The t_Locus ........................ 7 Genetic Complementation ............... 8 Embryonic Development ................ lO Sperm Maturation and Behavior ............ l4 Genetic Recombination ................ 15 Purpose of Thesis ..................... 19 MATERIALS ........................... 2] METHODS ............................ 23 Animal Breeding ...................... 23 Timed Matings ....................... 25 Preparation of Embryos for Light Microscopy ........ 25 Reconstruction of Mouse Embryos from Transverse Sections . 27 Growth of Primary Explants ................ 27 Subculture of Primary Explants or Cell Lines ....... 28 Mouse Anti-H-ZtWI Production ............... 28 NIH Mouse Alloantisera .................. 29 One Stage Lymphocytotoxicity Assay ............ 3O Igdination of Fibroblasts ................. 3l 1 5I—iododeoxyuridine Fibroblast Microcytotoxicity Assay l ........................ 32 125I-iododeoxyuridine Fibroblast Microcytotoxicity Assay 2 ........................ 33 Photography ........................ 34 RESULTS ............................ 35 Characteristics of Breeding Lines ............. 35 Characteristics of Embryos on the Basis of Gross Morphology ....................... 39 iv TABLE OF CONTENTS (Continued) RESULTS (continued) Characteristics of Embryos on the Basis of Histology Day 9.29 . . . . . . . . . . . . . . . . . . . . . . : Day 9% 3g ...................... Day 10 pg ...................... Day l3 pg ...................... Day 153 ...................... Primary Explants and Subcultures ............. Histocompatibility Typing ................. DISCUSSIONV ........................... Differences in Embryonic Observations . . w1' W1 ...... In Vitro Growth of Primary Explants From t [t Embryos Determination of Embryonic Genotypes Based on H-2 Typing SUMMARY AND PROSPECTIVE RESEARCH ................ LIST OF REFERENCES ....................... 99 107 109 111 112 LIST OF TABLES m 3293. l Origin of Various I_Mutations .............. 5 2 Summary of t_Mutations ................. ll 3 Embedding Protocol for Embryonic Tissue ......... 26 4 Harris' Alum Hematoxylin and Eosin Staining Protocol . . 26 5 Experimental Design for Cytotoxicity Assays ....... 3l 6 125I-iododeoxyuridine Labeling Mix ........... 32 7 Breeding Data for C3H.BlO, C3H.BlO-13V1. and C3H.BlO-3M1 ....................... 37 8 Segregation Distortion in Males and Females of the Backcross-Intercross Line ................ 38 9 Average CR Lengths and Range of CR Lengths of Embryos at Different Stages of Development ........... 40 lo Variation of Age and CR Length of Embryos Hithin Litters ......................... 42 ll Number of Implants, Aborts, and Abnormal Embryos . . . . 43 12 Primary Explants From Embryos Representing Days 9 to 15.EE ....... I ................... 82 13 Specificity of Three Samples of Anti-H-Zml Using y: and HE“ Spleen Cells ............... 90 l4 Specificity of Anti-H-2.2 and Anti-H-ZtWI Using Spleen Cells Isolated From Four Strains of Mice ........ 9O l5 Determination of H-2 Antigens of Normal (1(1) C3H.BlO Fibroblasts Using Two Different Assays ......... 93 l6 Number of Expected and Observed Embryos Homozygous for 3V1 ......................... IOO vi tau. . 3... e“# Figure 10 ll 12 l3 14 LIST OF FIGURES Chromosome 17 of the house mouse ............ Tests for genetic complementation between two different t_mutations in the absence or presence of segregation distortion (SD) .............. Proposed regions of the tail-interacting factor (T-int), se regation distortion factor (A), and lethality Factor (L. in complete t_haplotypes .............. Mating schemes used to maintain mouse mutanttw1 . . . . Normal and abnormal mouse embryos from days 9 to lo; pg_photographed after dissection ............ Transverse sections of normal and abnormal mouse enbryos at day 9 pg .................. Transverse sections of normal and abnormal mouse embryos at day 9} pg .................. Transverse sections of normal and abnormal mouse embryos at day 10.29 .................. Transverse sections of normal and abnormal mouse embryos at day lO E .................. Transverse sections of normal and abnormal mouse embryos at day l0; pg ................. Embryos from days ll and l2 pg photographed after dissection ....................... Transverse sections of normal and abnormal mouse embryos at day ll pg .................. Transverse sections of normal and abnormal mouse embryos at day ll pg .................. Transverse sections of normal and abnormal mouse embryos at day 12 pg_ .................. vii 18 24 46 49 51 54 56 58 61 63 65 68 Figure 15 16 17 18 19 20 21 22 23 24 25 LIST OF FIGURES (continued) Transverse sections of normal and abnormal mouse embryos at day 12 pg .................. Transverse sections of normal and abnormal mouse embryos at day 12 pg .................. Embryos from days 13 and 15 pg photographed after dissection ....................... Transverse sections of normal and abnormal mouse embryos at day 13 pg. .................. Transverse sections of normal and abnormal mouse embryos at day 13 pg .................. Transverse sections of normal and abnormal mouse embryos at day 15 pg .................. Inverted phase contrast photographs of outgrowths from primary embryonic explants (x370) ......... Inverted phase contrast photographs of outgrowths from primary embryonic explants (x370) ........... Incorporation of 125IUdR into actively dividing fibro- blast cells ...................... Determination of antibody titer of anti-H-2.2 ..... Reconstructed composite view of mouse embryos from serially-sectioned material .............. viii Page 70 72 74 75 78 8O 84 LIST OF ABBREVIATIONS USED IN TEXT AND FIGURES b Red blood cell f Nerve fiber L Marginal layer M Mantle layer m Mitotically dividing cell n Notochord p Pycnotic cell S Lumen of spinal cord v Vertebrae CR Crown-rump length cr Lumen in region of caudal rhombencephalon rr Lumen in region of rostral rhombencephalon PBS Calcium-magnesium free phosphate buffered saline IUdR Iododeoxyuridine ix LITERATURE REVIEW The intriguing genetic and developmental features of the T-complex in the house mouse (Mus musculus) have been recently reviewed (Bennett, 1975; McLaren, 1976; Sherman and Hudl, 1977; Klein and Hammerberg, 1977). The complex, located on chromosome 17 near the major histocom— patibility locus (H22), is composed of sets of dominant I_ and reces- sive t_ mutations. Although the functions of the mutations are unknown, two hypotheses have been formulated to explain the action of the genes: 1) The mutations code for specific cell surface anti- gens that govern cell-cell interactions during embryonic development (organizational failure hypothesis; Bennett, 1975); or 2) the muta- tions code for proteins associated with intermediary metabolism of all cells and required for embryonic development (generalized cell lethal hypothesis; Mintz, 1964; Hudl and Sherman, 1976; Nudl, gt gl., 1977). The following review will summarize the current knowledge about dominant .1 and recessive .5 mutations. The T Locus Historical Bagkground: The study of the I_locus region began in 1924 when Dobrovolskaia-Zavadskaia identified a male mouse with altered tail morphology (Dobrovolskaia-Zavadskaia, 1972). The muta- tion had arisen spontaneously in a laboratory stock of mice, was found to be inherited, and was designated as Brachyury (I). In crosses 2 between heterozygous parents (17:), mice with short tails and normal tails were produced but the total number of progeny was reduced. The lethality of 17: embryos was subsequently confirmed by histologi- cal examinations of embryos (Chesley, 1935). In outcrosses of short tail mice to wild mice, progeny without tails were occasionally observed, instead of progeny with short tails (Dobrovolskaia-Zavadskaia and Kobozieff, 1932). In crosses between these tailless mice and mice with normal tails, one-half of the progeny had short tails and the other half had normal tails. On the other hand, when tailless mice were intercrossed, only tailless progeny were obtained and the litter size was drastically reduced. The investi- gators concluded that taillessness resulted from the interaction of Brachyury with another recessive mutation, designated as g. The embryonic lethality of the first g mutation in the homozygous state was later confirmed by Gluecksohn-Schoenheimer (1940). Dunn and coworkers continued to study the Ifcomplex and found the presence of additional g_mutations in laboratory and wild mice (see review by Klein and Hammerberg, 1977). Discovery and identifi- cation of g_mutations was possible only in the presence of the nggpyy .ggy gene. The I_locus is associated with several other gene loci found on chromosome 17 (Figure 1A). The guaking (95) gene affects the synthesis of myelin in the central nervous system; homozygotes have marked rapid tremors which disappear at rest (Green, 1968). The I complex associated protein (Igpzl) gene codes for a protein charac- terized by two dimensional gel electrophoresis and found in high concentrations in testicular cells (Silver, gt 31., 1979). The Lg! H‘— .0 7? _| n 1'3 -i —J —-I O 2 fl -.h :I: l N 7(1— H...— mm... (D a..- Figure 1. Chromosome 17 of the house mouse. A. A partial map of chromosome 17 in the house mouse. 8. An expanded view of the H-2 complex. (lg!) gene is thought to be a g_mutation and causes low segregation distortion (to be discussed later) in males (Lyon and Mason, 1977). The tufted (3:) gene affects hair growth and maintenance (Green, 1968). The tightly-linked group of genes found in the major histo- compatibility complex (H-Z; Figure 18) are identified from studies on allograft rejections, are designated as H-2 haplotypes, and code for specific cell surface proteins (Klein, 1975). These loci, located on the proximal portion of chromosome 17, play a role in mapping various factors found in association with g_mutations. The nomenclature used in discussing I and g_mutations is complex and often confusing. The superscripts x and y (13, 1?, g}. or 3?) are general symbols used to represent two different mutations. With respect to I mutations, the superscript usually refers to the labora- tory in which the mutation was first identified (See Table 1). With respect to g_mutations, mutations extracted from wild populations of mice are designated with a superscript w and number (gyl). Muta- tions discovered in Dunn's laboratory are designated by a serial number in superscript (3?). Mutations discovered outside of Dunn's laboratory and derived from laboratory stocks are designated in super- script by initials of the identifying laboratory and a serial number (ghz, MRC Radiobiological Research Unit, Harwell, England; gAEz, Albert Einstein College of Medicine, New York). ,T Mutations: Several spontaneous, and many radiation-induced, mutations have been described (Bennett, 1975; Bennett, ggngl., 1975). Characteristics common to all 1 mutations studied to date are: 1) 15713 embryos die jp_ggggg_at mid-gestation; 2) 18/1? heterozygotes also die and thus have not been found to complement each other; 3) recombination has not yet been observed between different I_muta- tions (Klein and Hammerberg, 1977); 4) EDI]? embryos may differ pheno- typically from 1¥71¥ embryos (five phenotypic classes have been recog- nized; Bennett, e_t_gl_., 1975); 5) all 1 mutations interact in _t_r_a_n_swith recessive g_mutations to produce tailless progeny; 6) segregation distortion does not occur in males heterozygous for I; and 7) 13/: mice, except Ibp[:, express the wild type protein coded by gene 1gp;13 (Alton, ggug1., 1980). The five different phenotypic classes repre- sented by mutations I, Ihp, Th, 19", and IF are described below. A summary of the origin of various I_mutations is given in Table 1. Embryos homozygous for I die at 10-3/4 days of gestation (Bennett, 1975). The placental connections required for nourishment and main- tenance of the embryo are not established. The primary defect is thought to involve the inability of the primitive streak to undergo normal differentiation, thereby altering the development of the noto- chord and mesoderm. ‘Ipnglggg studies have shown that a structurally abnormal 171 neural tube was an effective inducer of cartilage formation in normal somites; however, I7I_somites could not produce cartilage in Table 1 Origin of Various I Mutations T Mutation Origin Reference 1; Brachyury1 Spontaneous in laboratory Dobrovadskaia- stock Zavaskaia, 1927 ‘Ihp Hairpin allele1 Spontaneous in an AKR Johnson, 1974 mouse ‘Ih Hanwell allele1 Spontaneous in laboratory Lyon, 1959 stock 19F] Orleans allele1 Spontaneous in a Swiss/ Alton, ggngl., Orleans mouse 1980 f Curtailed a1 Ieie1 Radiation-induced Searle, 1966 _His Wisconsin allele Spontaneous in laboratory Alton, ggflgl., stock 1980 19 Jackson allele Spontaneous in a BALB/cHu Bennett, gg gl , mouse 1975 IFJ Jackson allele Spontaneous in a C57BL/6 Bennett, gt_gl_, mouse 1975 .19R4 Oak Ridge 4 allele Radiation-induced Bennett, gt gl_, 1975 Ihg Hertwig's allele Spontaneous in laboratory Bennett, gg a1., stock 1975 1Represents one of the five different phenotypic classes 6 the presence of a normal neural tube (Bennett, 1958). In aggregation studies involving the association of dissociated normal and mutant embryonic cells, aggregates formed by 171 cells were smaller than those formed by normal cells (Yanagisawa and Fujimoto, 1977b, 1978). Results from these experiments imply that cell surface antigens in- volved in cell-cell interactions are aberrant. Cell lines established from 17: embryos are also unable to form normal sized aggregates and are being examined in greater detail to identify the cellular aberra- tion caused by the I_mutation (Yanagisawa and Fujimoto, 1977a; Yanagi- sawa, _e_g_a_l_., 1980). Embryos homozygous forlhp die at 7 days of gestation (Bennett, 1975). The viability of the heterozygote is dependent upon the parental origin of the gene (Johnson, 1974). Heterozygotes, whose Ihp gene is maternally inherited, die ipqggggg; paternally inheritedlhp alleles are not deleterious. In addition to the unusual maternal effects of the gene, the mutation maps as a deletion extending from the I locus distally through the 1gp:l_locus (see Figure 1A; Alton, gt p1,, 1980). Embryos homozygous for 1h die at 8 days of gestation (Lyon, 1959). The embryo sac is half the normal size, and embryonic ectoderm ceases proliferation prematurely. Embryos homozygous for TOrl die at 61 to 7 days of gestation when the embryonic ectoderm prematurely ceases to proliferate (Bennett, gt gl,, 1975). The extraembryonic ectoderm and endoderm, ectoplacen- tal cone, and yolk sac are apparently normal. The mutation maps as a deletion extending from the I_locus through the g§_(guaking) locus (see Figure 1A; Erickson, gt g1,, 1978; Alton, gt g1,, 1980). The 7 deletion does not include the 1gp:l_locus, as was observed in the dele- tion for Ihp. Hence, the wild type allele (1gp;1§) is present at the-1gp:1_locus. Embryos homozygous for IF die at 101 days of gestation and appear to be similar to embryos homozygous for I (Searle, 1966). However, TF7IF embryos are more severely affected. The posterior body is more abbreviated, the neural folds fail to close, and the anterior limb buds are absent. Several other spontaneously occurring or radiation-induced muta- tions have phenotypes similar to one of the mutations (I, Ihp, 1h, IOrl , or IF) previously described (Table 1). Consequently, new I mutations are classified into one of five different classes on the basis of the homozygous lethal phenotypes and the interactions with different I mutations. The included list of mutations is not meant to be comprehensive but only meant to indicate the different origins of.1_mutations. Mutation to dominant lelike alleles occurs spontaneously with high frequency in laboratory stocks and radiation-induced mice (Bennett, 1975; Bennett, ggngl., 1975). These mutations have not, however, been found in wild populations of mice. The t Locus The recessive 3 mutations are far more heterogeneous and variable than the dominant I mutations. The g_mutation in combination with the wild type allele (:jg) has no visible morphological effects; how- ever, the g_mutation in combination with the I mutation produces progeny without tails. Embryos homozygous for g can be either viable (3Y75Y). 8 semi-lethal (pf/pf), or lethal (31/31) (Bennett, 1975; Klein and Hammer- berg, 1977). For purposes of this review, only lethal g mutations (19) and their effects on embryonic development, sperm maturation and behavior, and genetic recombination will be discussed. The lethal g_mutations have several features in common with each other: 1) I/g_mice are viable and usually tailless while jjt_mice are phenotypically normal; 2) embryos heterozygous for non-complementing lethal alleles (pf/g?) die jg ggggg; 3) male mice heterozygous for lethal alleles in two different complementation groups (32/5?) are usually viable and sterile whereas female mice are usually viable and fertile; 4) segregation distortion occurs in male mice heterozy- gous for lethal 3 mutations (1]g_orli/g) but not in female mice; 5) suppression of recombination occurs between the I_1ocus and the .522 locus during meiosis in male and female mice heterozygous for lethal g_mutations; 6) alleles within the same complementation group generally have the same H-2 haplotype. These characteristics will be discussed in greater detail. Genetic Complementation: On the basis of genetic complementation, the lethal g_mutations can be divided into seven complementation groups 12, I_1v73’ tha-l’ 39: identified by one typically acting mutation: .g tw5’ t9 1., 1980). If two different lethal g_mutations complement each , and 371 (Bennett, 1975; Klein and Hammerberg, 1977; Guénet, _e_i_:_ other, then matings between two different balanced lethal (173? and (11;?) mice will produce offspring with normal tails and no tails (Figure 2A). On the other hand, if two different lethal g_mutations do not complement each other, then such crosses will only produce offspring without tails (Figure 28). Cross: d g .125: x .I:££ tx-+ ty-+ (tailless) (tailless) Frequency of ‘Izgi gametes (l-d1) .Izif gametes (l-dz) gametes: x y g '1 gametes (d1) 3; -_i; gametes (d2) Genotypes of T-tf T-tf T-tf tx-+ expected _ x_ _ y_ progeny: Tptf t + ty + t + Phenotypes Of A dies tailless tailless normal tail progeny: , ° 8. dies tailless tailless dies Expected fre- ( )( ) ( ) ( ) quency of l-d 1-d 1-d d l-d d d d progeny: l 2 2 1 1 2 l 2 Expected pro- portion without 50, 25% 25% 25% 25% d1=d2=0.5 Expected pro- portion with SD, d1=0.9 5% 45% 5% 45% and d2=0.5 Figure 2. Tests for genetic complementation between two different t_mutations in the absence or presence of segregation dis- tortion (SD). A. Phenotypes of progeny in the presence of genetic complementation. B. Phenotypes of progeny in the absence of genetic complementation. 10 The effects of mutations within the seven groups of lethal 3 mutations appear at different stages of embryonic development and affect different cellular structures. Although mutations within the same complementation group are defined as alleles on the basis of genetic non-complementation, studies on different g_mutations within the same complementation group have shown detectable differences on the type and time of alteration of cellular morphology (Hillman and Hillman, 1975; Spiegelman, 1978). These differences may, however, result from the interactions of the g_mutation with other genes found in different genetic backgrounds. The alleles at all noneg_loci found in one population of mice may differ drastically from those found in another population of mice. These allelic variations constitute differences in the genetic background of g_mutations and may alter the expression of a single mutation such that it has two very different expressions in the two different populations of mice. A brief synopsis of the developmental defect observed in each complementation group is given below. A summary of.g mutations is given in Table 2. Embryonic Development: Embryos homozygous for alleles within the first, 312 , complementation group represent the earliest acting g_mutations. The effects of the gene are first visible at day l and the embryos die by 2 to 3 days of gestation (Bennett, 1975; Spiegelman, 1975; McLaren, 1976; Klein and Hammerberg, 1977). The embryos reach the morula stage but fail to form the blastocyst. The arrested morulae have large, rounded cells containing fewer polysomes and more lipids lz/g}2 embryos are arrested at the late morula stage; embryos homozygous for 5832, than normal morulae (Spiegelman, 1978). The majority of.g 11 Table 2 Summary ofig Mutations Complementation Predominant T/t Group Member Alleles H-2 Haplotype Phenotype .EIZ .EIZ"£w32 H_2t12 Tailless £173 373 . N-zm11 Tailless gypa'] - gypa‘l ND2 Tailless ._O 1:0,‘56,‘£30,-£h16 H_2w5 Tailless £117, £1113 Normai3 _w5 EwS’ £w6’ twlO’ Ewll, EwIB, H_2tw5 Tailless Ew14’ _w15’ IwIG’ £w17, £w37, Ew38’ Ew39’ £w41’ _w46’ _w47, £w74, £w75, EwBO’ Ew81 E9 $4,.E9":w18’ £w30’ EwSZ ND Tailless Ewl Ewl’ I113 _w12’ IwZO’ Ew21 H_ztwl Tailless tw71’ Ew72 1Exception to classification of g_mutations on the basis of H-2 typing and genetic complementation. 2ND - not yet determined. These g_mutations retain the ggfactor but have lost the Ifjpg_factor. 12 12 a second member of the t_ complementation group, are arrested at the early morula stage (Hillman and Hillman, 1975). Studies involving glz/glz ++ 31/: and _t;"‘32/g“'32 ++ y: chimeras show that the mutation £32 appears to alter the cell surface and prevents the integration of Ew32/Ew32 12 cells with normal cells whereas the g mutation permits the interaction of EAZ/gFZ cells with normal cells (Mintz, 1964; Spie- gelman, 1978). The cell surface differences produced by $12 or gV32 mutations apparently interfere with the establishment of tight junctions between cells (Spiegelman, 1978). 73, comple- Embryos homozygous for the allele within the second, 3? mentation group are recognized at day 3 and die at days 4 to 5 of gestation (Bennett, 1975; McLaren, 1976; Hammerberg and Klein, 1977). Embryos reach a blastocyst stage, induce a decidual reaction in the uterus, and die shortly after implantation (Spiegelman, gg g1., 1976). The trophectoderm and ectoplacental cone fail to form a close associa- tion with the uterine decidua. The mutation appears to alter the function of the trophectoderm. tha-l Embryos homozygous for the allele within the third, __ , com- plementation group die after implantation (Guénetpggmgl.. 1980). The gypa'] is unknown. The mutation is the specific tissue affected by most recently discovered g_mutation in wild mice and appears to comple- ment all other 3 mutations. Embryos homozygous for alleles within the fourth, g9, complemen- tation group are first detected at 5} days and die at 6 to 7 days of gestation (Bennett, 1975; McLaren, 1976; Klein and Hammerberg, 1977). The egg cylinder fails to lengthen and the ectoderm does not differentiate into the embryonic and extraembryonic components. 13 Attempts to culture trophectoderm and/or inner cell mass of 39/39 and 55/55 embryos i_ vitro have failed (Nudl and Sherman, 1978; Hogan, ‘ggng1., 1980). Examination of B-glucuronidase activity in gé/g§ cells cultured 1p vitro shows that macromolecular synthesis stops at the egg cylinder stage (Hudl and Sherman, 1978). The primary effect of the t6 mutation has been hypothesized to be a metabolic lesion which kills inner cell mass cells quickly but has a more gradual effect upon trophoblast cells. 5 , complemen- Embryos homozygous for alleles within the fifth, EU tation group are recognized at 61 days and die by 8 to 10 days of gestation (Bennett, 1975; McLaren, 1976; Klein and Hammerberg, 1977). Embryonic and extraembryonic ectoderm forms, but the growth and main- tenance of the egg cylinder stops. Attempts to culture inner cell masses of gUS/trs embryos ipngiggg have failed, and the lethal period jp_vitro corresponds to that observed 1p utero (Hudl and Sherman, 1976; Hogan, gt g1., 1980). However, jp_vitro culture of grslgrs trophectoderm in the absence of the inner cell mass has been successful (Sherman, 1975). The differential results in the ability to culture inner cell mass (embryonic derivative) or trophectoderm (extraembryonic derivative) imply that various cell types are not equally sensitive to the effects of the g mutation or that death of trophectodermal cells is due to secondary degenerative effects resulting from the primary lesion in the inner cell mass (Hudl and Sherman, 1976). Embryos homozygous for alleles within the sixth, g9, complementa- tion group are first visible at 6 days and die at 8 to 10 days of gestation (Bennett, 1975; McLaren, 1976; Klein and Hammerberg, 1977). Formation and differentiation of the primitive streak begins, but 14 mesodermal differentiation is aberrant. Mesodermal cells are abnormal in shape, fail to establish intercellular junctions, and are deficient in microfilaments (Spiegelman, 1975; Bennett, 1978; Snow and Bennett, w18/fw18 1978). Ip_vitro cultures of.g embryos, ectopic implants and mutant ++ normal chimera studies show that some cells are capable of surviving beyond the usual period of lethality exhibited by mutant embryos jp_pgggg (Hudl, pp 11., 1977). An explanation for the ability of some embryonic cells to survive in an environment outside of the uterus is not readily apparent. 1, complemen- Embryos homozygous for alleles within the seventh, gr tation group have been reported to be visible as early as day 9 and die between 11 to 21 days of gestation. The neural tube forms but cells in the ventral half of the neural tube become pycnotic and die (Bennett, gt 31., 1959a, b). Sperm Maturation and Behavior: The effects of lethal g_mutations in the heterozygous state on sperm maturation and behavior fall into two categories: 1) the effects causing segregation distortion in I/g or :/g males mated with females; and 2) the effects causing sterility in 33/3? males (Bennett, 1975; Klein and Hammerberg, 1977). With respect to the cause of segregation distortion in males, in which fertilization of ova by sperm from 1/3 or I/g_males does not usually result in typical Mendelian ratios of expected phenotypic offspring, recent studies have shown that sperm bearing lethal g_mutations are not morphologically defective and have the same life span as sperm bearing the wild type allele (McGrath and Hillman, 1980a). The distor- tion does not occur in metaphase I of meiosis since equal numbers of t: and 1¢bearing chromosomes are present (Hammerberg and Klein, 15 12 1975a). Transmission frequency of.g -bearing sperm 1p vitro, for example, is Mendelian (McGrath and Hillman, 1980b) and is identical to the transmission frequency jp_pjyg when matings are delayed until the time of ovulation (Braden, 1958). Braden (1958) has hypothesized that sperm bearing lethal g_mutations are physiologically superior to sperm bearing the wild type allele and that this physiological advantage is time dependent. The superiority of gybearing sperm is expressed in normal matings but not in delayed matings, implying that the sperm are responding to extrinsic factors present in the uterine and oviducal environments. The sterility of 33/3? male mice may be due to lack of spermato- zoan maturation (McGrath and Hillman, 1980b). Histological and ultra- structural studies indicate that no unique spermatid or spermatozoan defects are found in sperm produced by pf/g? males (Hillman and Nadijcka, 1978a,b, 1980; Nadijcka and Hillman, 1980). However, fewer spermatozoa are found in the female reproductive tract (Tucker, 1980). Results of 1p.!iggg fertilization of ova with sperm from 53/5? males in which all female-derived physical and physiological barriers are removed show that fertilization is still not possible (McGrath and Hillman, 1980b). The sperm from 55/3? males resemble immature normal sperm in their ability to fertilize ova. Thus, the decreased number of sperm is not the primary cause of sterility. The lack of spermatozoan maturation may result from their inability to mature en route through the epididymis, to undergo capacitation, or to arrive at the site of fertilization. Genetic Recombination: The distance between the I locus and 5:2 locus is. approximately 15 cM (Bennett, 1975; Klein and Hammerberg, 1977). 16 In the presence of lethal g_mutations, however, only 1%-2% recombination occurs during meiosis between the I locus and the 3;; locus. The recombination suppression effect observed in the presence of lethal g_mutations is not absolute since recombinants can be identified. Various hypotheses proposed to explain the suppression effect include the presence of deletions or structural rearrangements in moderately repetitive DNA (iDNA) intercalated between structural genes (Geyer- Duszynska, 1964; Lyon and Mason, 1977). Lyon and coworkers have investigated the recombinant g_mutants in order to determine the cause of recombination suppression and to attempt to map the lethal g mutations (Lyon and Meredith, 1964a,b,c; Lyon and Bechtol, 1977; Lyon and Mason, 1977; Lyon, gt 31., 1979a). Their results suggest that suppression is due to either an intrinsic failure of the chromatin material to participate in crossing-over during meiotic pairing or a mismatching of normal or abnormal hetero- chromatin, thus preventing chiasma formation (Lyon, ggugl., 1979a). Recently, experiments involving recombination between two chromosomes carrying extensive overlapping segments ofig chromatin suggest that recombination suppression is not due to an intrinsic inability of g chromatin to undergo crossing-over but due to mismatching of normal and variant chromatins (Silver and Artzt, 1981). Regions of DNA in two over-lapping 3 mutations, gylz, an(111.1117 , are homologous and will permit normal crossing-over whereas regions of :_DNA and g_DNA are non-homologous and will inhibit crossing-over. The evidence described above does not eliminate the possibility of deletions occurring within similar regions and causing the mis- matching of chromatin. However, the looped out chromosomal material 17 found on cytological examinations of chromosomes in some g_mutant mice (Geyer-Duszynska, 1964) and thought to represent deleted regions of DNA could also result from regions of non-homologous pairing in an otherwise normal chromosome. More studies are needed to distin- guish between the two hypotheses. Three properties usually associated with g_mutations include: 1) the interaction with [_mutations to produce tailless progeny; 2) the presence of a segregation distortion factor in males; and 3) the lethality of developing embryos homozygous for g, The various phenotypes of mice produced following exceptional recombination events indicate that g_mutations may have at least three altered regions of the chromosome, each coding for one of the three properties usually found in g_mutations (Lyon and Mason, 1977). Since the mutation covered an extended region of the chromosome and includes more than one locus, the 3 mutations with all three properties are now referred to as com- pletenghaplotypes. The region interacting with Brachyury (I) to produce taillessness is referred to as I}int (Figure 3) and maps close to I (Lyon and Mason, 1977). The A_region causes low segregation distortion in the presence of the wild type allele and interacts with .Irint and L_to give a high segregation distortion ratio (Lyon and Mason, 1977; Hammerberg, 1981). The L region is responsible for lethal- ity observed in homozygous g/p_embryos and maps in the pp region (Lyon and Mason, 1977). 18 T qk Tcp-l low tf H-2 1 Jill/[L 1 1 41111411 1 G— T (Ill!!! 1 | 1141/ HIT 1 T-int A L Figure 3. Proposed regions of the tail-interacting factor (Irint), segregation distortion factor (A), and lethality factor (p) in complete g haplotypes. Another gene locus recently identified and mapped within the I - H32 interval is _Tgp;l_ (Silver, gggl., 1979; Silver, fig” 1980; Danska and Silver, 1980). The Igp;l_gene codes for a protein with a molecular weight of 63,000 daltons that is found on all cellular surfaces, but appears in greatest quantities on testicular cells (0.4% of total protein; Silver, ggflgl., 1979). Three alleles have been identified: .Igp;lP is found in wild type and I mutant mice; 1gp;1§ is associated with complete 5 haplotypes; and 192:1? (null allele) is associated with the IF p mutation. Specific H-2 haplotypes are also associated with specific p_muta- tions (Hammerberg and Klein, 1975b,c; Hammerberg, ggwgl., 1976; Haupt- field, ggwgl., 1976). Since recombination suppression exists and includes the 5;; locus, g and H;g_appear to be inherited as a unit, a "super gene" (Snell, 1968). A strong correlation exists between the .2 mutation and H-2 haplotype. Alleles within a complementation group carry the same H-2 haplotype; alleles within different complementation groups are usually associated with different H-2 haplotypes (see Table 2). In summary the suppression of recombination betweenliandifl;g are thought to arise from mismatching of chromatin. As a result, genes 19 found within the interval are inherited as a unit and the term p_haplo- type is used instead of g_mutation or allele to define this set of genes. Purpose of Thesis The alleles found in the complementation group of g_mutations acting late in embryonic development, 531, have not been studied exten- sively. The/g?"1 mutations appear to act after organogenesis has begun whereas all other p mutations act prior to the initiation of organo- Jiwl genesis. In the one study that has reported the effects of muta- tions on embryonic development, data obtained from embryonic studies Iwl’ £w3, Ew12 thO involving , and __ were summarized and emphasis was placed on the effects observed after 13 days of gestation (Bennett, 23 21.. 1959a,b). Complementation studies of gvl, £33, 3312 IwZO , and with mutations from other complementation groups indicate that the grl haplotypes differ drastically with respect to their complemen- tation ability (Lyon, ggngl., 1979b). Since little information was 1 mutation and since non- available on the early effects of the t? complementing gr] haplotypes probably differ in their effects on em- bryonic development (as noted for other non-complementary.g_haplotypes), theg"1 mutation was selected for the study of the gene action at early time periods of development. Research on 5’1 was divided into four phases. First, the gene was transferred from the original "pen bred" stock to an inbred strain of mice, C3H.BlO, in order to establish more vigorous breeding lines, to eliminate the heterogeneous genetic background, and to examine the effects off"1 in the absence of genes I and 3?; Second, normal 20 and abnormal embryos at days 9 to 15 of gestation were subjected to gross examination after dissection from amniotic sacs and histological examination by light microscopy in order to determine the effects oft!”1 on embryonic development. Third, primary explants from mouse embryos derived from C3H.BlO-g?"1 matings were cultured jp_vitro in order to determine if: explants would attach to the substrate and cellular outgrowth would continue longer jp_vitro than 1p.vivo; any specific cell types from primary explant outgrowth would become estab- lished ip_vitro; and cell lines could be subcultured from primary explants. Fourth, the H-2 types of cells grown jp_vitro were assayed in an attempt to identify mutant from normal embryos without sacri- ficing tissue for histological examination. MATERIALS Paraplast, melting point 56-57°C, microcentrifuge tubes (250ml capacity), 3" x 1" glass slides, and Permountwere purchased from Fisher Scientific, Fair Lawn, New Jersey. Glass coverslips and 35 mm sterile tissue culture dishes were purchased from Corning, Corning, New York. Falcon MicroTest II plates and lids were purchased from Becton, Dickinson & Co., Cockeysville, Maryland. H-2 typing reagents 0-2, 0-23, 0-28, and 0-33, prepared by Drs. G. Snell and M. Cherry, were obtained from the Transplantation and Immuno- logy Branch NIAID of the National Institute of Health, Bethesda, Mary- land. Fetal calf serum, CMRL 1066 (10x), mycostatin, rabbit complement, and trypan blue were purchased from Grand Island Biological Co., Grand Island, New York. Penicillin G and streptomycin sulfate were purchased from Calbiochem-Behring Corp., La Jolla, California. Hematoxylin and eosin were obtained from MC/B, Norwood, Ohio. Albumin fixative came from Harleco, Gibbstown, New Jersey. Bovine pancreas type III trypsin, fluorodeoxyuridine, glutamine, and asparagine were purchased from Sigma, St. Louis, Missouri. 125I-iododeoxyuridine (NEX 072) was obtained from New England Nuclear, Boston, Massachusetts. MEM/Hanks balanced salt solution, MEM, and non-essential amino acids (lOOmM) were purchased from International Scientific Institute, Inc., Cary, Illinois. Flow Laboratories, Rockville, Maryland, was the source of 100 _nfl sodium pyruvate. All other chemicals were analytical reagent grade. 21 22 C3H mice were obtained from the Jackson Laboratories, Bar Harbor, Maine. Breeding pairs of C3H.BlO mice were kindly provided by Dr. D. Shreffler, University of Michigan, Ann Arbor, Michigan. Breed- ing pairs of Izgf/gylai_mice were kindly provided by Dr. H. O. McDevitt, Stanford University, Stanford, California. The C57BL/105cSn strain was already established in the laboratory by Dr. James H. Asher, Jr. METHODS Animal Breeding Normal (i/i) mating pairs of C3H.BlO mice having agouti (A/A) coat color were kindly provided by Dr. D. Shreffler, Department of Genetics, University of Michigan. The C3H.BlO strain, carrying the H-2 histocompatibility haplotype of the C57BL/10 strain backcrossed on the C3H background, is maintained by full sib mating and is pre- sently at generation N10F27+17. Three breeding pairs of mice having black and tan coat colors 3V1 as balanced lethals (gF/gF) and carrying the mutations I and (1}gj/gylgj) were obtained from Dr. H. O. McDevitt, Department of Medicine, Stanford University. In order to overcome the poor repro- ductive capability of the original stock of Izgf/gvlai_mice and to reduce the heterogeneous genetic background of the original strain, genes land 5"” were successfully transferred from the balanced lethal "pen bred" stock to the inbred strain of mice, C3H.BlO. (Similar crosses with C57BL/1OScSn were not successful; see Results.) Mutant twl is presently maintained in two ways: as a balanced lethal line, designated as C3H.BlOe11V1 (generation F13; Figure 4A) and as a back- cross-intercross line, designated as C3H.BlOegU1 (generation N5F4; Figure 4B). Animals were kept on an artificial light-dark cycle (13 hours light, 11 hours dark). Food and water were given gg_libitum. 23 24 T-tf X T-tf tW1_+ l TAMI-‘1’ T-tf T-tf tW1-+ T-tf t"1-+ €”1-+ (dies) (tailless) (dies) A. Balanced Lethal Line C3H.BlO C3H.BIO-tf”1 .izi x T-tf +-+ tw.1_+ BACKGROSS T-tf x t"1-+ +.+ +-+ INTERCROSS i) 1;: Iii-:2 l-_t_f' T-tf +-+ +-+ +-+ tV1-+ (normal tail) (normal tail) (short tail) (tailless) B. Backcross-Intercross Line Figure 4. Mating schemes used to maintain mouse mutant 331. 25 Timed Matings Males and females of appropriate genotypes (C3H.BlO :1: x C3H.BlO 35/: or C3H.BlO-t)”1 fig“ x C3H.BlO-gm 3;“) were mated, and females were examined twice daily for vaginal plugs. At 9, 91, 10, 105, 11, 12, 13, and 15 days post-coitus (day of observation of a vaginal plug = day 0), pregnant females were killed by cervical dislocation. Embryos were sterilely removed, placed in sterile calcium- magnesium free phosphate buffered saline (PBS), dissected free from amniotic sacs under a Wild M7A dissection microscope, and transferred to 35 mm sterile culture dishes containing PBS. Embryos were staged according to Grfineberg (1943) and crown-rump (CR) lengths were measured with an ocular micrometer. Tailbud, forelimb bud, or heart tissues were excised and transferred to sterile vials for tissue culture. Prior to fixation in Bouin's solution (Heesner, 1960), embryos were photographed with a Canon F-l camera using an Olympus 28 mm macrolense. Preparation of Embryos for Light Microscopy Embryos fixed in Bouin's solution were dehydrated in graded etha- nols, cleared in xylenes, and embedded in paraplast (Table 3; Neesner, 1960). Embryos were serially sectioned at 8 um on an A0 rotary micro- tome. Sections were mounted on glass slides with albumin, stained with hematoxylin and eosin (Table 4; Luna, 1968), and examined on a Zeiss Universal II microscope with camera attachment at magnifi- cations to x250. 26 Table 3 Embedding Protocol for Embryonic Tissue Solutions Time, min Solutions Time, min 30% ethanol 20 100% ethanol 20 50% ethanol 20 100% ethanol 20 70% ethanol 20 100% xylenes 3O 80% ethanol 20 100% xylenes 30 95% ethanol 20 Paraplast1 3O 95% ethanol 20 Paraplast 30 1Infiltration of paraplast performed under 15 lbs. of vacuum at 60°C. Table 4 Harris' Alum Hematoxylin and Eosin Staining Protocol Solutions Time, min Xylenes 4 Xylenes 4 100% ethanol 2 100% ethanol 2 95% ethanol 2 70% ethanol 2 Tap water 1 3 Harris' alum hematoxylin 10 Tap water 1/3 Tap water 1/3 0.9% hydrochloric acid 3 dips Tap water 1 dip Tap water 2 5 Alcoholic eosin Y 1/2 95% ethanol 3 dips 95% ethanol 3 dips 100% ethanol 2 100% ethanol 2 Xylenes 3 Xylenes 3 Xylenes 3 Coverslip with Permount; dry overnight on warming tray. THarris' alum hematoxylin prepared as described in Luna, 1968. 2 Alcoholic eosin Y prepared as described in Luna, 1968. 27 Reconstruction of Mouse Embryos from Transverse Sections In order to reconstruct a composite view of a neural tube from a serially sectioned embryo, every fifth section was projected onto a sheet of paper with a Bausch and Lomb microscope slide projector, and the neural tube, heart, and esophagus were traced. Every fifth section was then examined with a Zeiss Universal II microscope for placement of pycnotic cells within the embryonic tissue on the traced sections. Traced sections were sequentially aligned at every two mm on graph paper, and the extent or width of the neural tube, heart, and esophagus and pycnotic cells were marked with dots. The general outline of a saggital view was constructed by joining the appropriate dots for the neural tube, heart, and esophagus. The width of the embryO‘was magnified approximately 43 times and the length was slightly distortedby a factor of 1.18. Growth of Primary Explants Tailbud, forelimb bud, or heart tissues excised from embryos were transferred under sterile conditions from vials to 35 mm culture dishes containing 0.5 ml of CMRL 1066 medium supplemented with 10% heat inactivated (56°C for 30 minutes) fetal calf serum, 2.0 pM_g1ut- amine, 50 units/m1 of penicillin, 50 units/m1 of streptomycin, and 100 units/ml of mycostatin (CMRL 1066; Oldham and Herberman, 1976; Murrell, 1979) Primary explants were placed in a humidified incubator at 37°C in an atmosphere of 5% C02, 95% air. During the first week, explants were observed daily on an Olympus CK inverted stage microscope magnified to x200 for attachment and cellular outgrowth. Subsequent observations were made every three to four days prior to feeding cultures 28 by replacing old medium with one ml of fresh CMRL 1066. Cultures contaminated with bacteria, yeast, or fungi were discarded. Subculture of Primary Explants or Cell Lines CMRL 1066 medium from primary explants or cell lines to be subcul- tured was removed and the culture was rinsed twice with two mls of sterile PBS. Each culture was incubated with 0.5 mls of 0.05% trypsin (bovine pancrease type III) in PBS containing 50 units/ml each of peni- cillin and streptomycin at 37°C in an atmosphere of 5% C02, 95% air for three minutes. Excess trypsin was removed and the culture was incu- bated an additional two minutes. Approximately 1 to 3 mls of CMRL 1066 was added to stop the enzymatic disaggregation of cells by trypsin. Cells were dispersed by trituration with a Pasteur pipet. Cell density was determined by counting cells in a drop of medium in a hemacytometer on an American Optical Series 20 microscope magnified to x200. Cells were diluted to the appropriate cell density (2-4 x 105 cells/ml) and plated in either 35mm tissue culture dishes at 2-4 x 105 cells/dish or wellsof MicroTest II plates at 2-4 x 104 cells/well. Subcultures were grown in a humidified incubator at 37°C in an atmosphere of 5% C02, 95% air and fed every three to four days by replacing old medium with fresh CMRL 1066. Subsequent subculturing was generally performed before cells reached confluency. Mouse Anti-H-ztw‘ Production In order to produce mouse alloantisera to antigen 108 (H-ZtWI), heterozygous C3H.BlOegV1 (fiftyl) and normal C3H.BlO (1/:) male mice were used as donors and recipients, respectively. A spleen was sterilely removed from a iytYl male mouse, rinsed in sterile PBS, 29 and placed in a 35 mm sterile culture dish containing two mls of PBS. The spleen sac was punctured with a 25 gauge needle, and cells were squeezed through holes in the spleen sac with forceps. The empty spleen sac was discarded. Cells were dispersed by passing through 25 and 27 gauge needles sequentially, and counted in a hemacytometer on an American Optical Series 20 microscope. The splenic cell concen- tration was adjusted to 5-6 x 107 cells/m1. Normal C3H.BlO (i/i) male mice five to six weeks old were injected weekly for seven weeks in the intraperitoneal cavity with 0.2 mls of freshly prepared spleen cell suspension containing 2-4 x 107 cells (Snell, pg gl., 1976). One‘week after the last injection, recipient mice were anesthetized with chloroform and bled via heart puncture with a 25 gauge needle and one ml syringe. Blood was pooled into three conical test tubes and allowed to clot at room temperature for three hours. The clots were freed from the sides of the tubes and allowed to contract overnight at 4°C. Antiserum was removed with a Pasteur pipet from each tube to a clean conical tube, spun at 700 x g for 10 minutes at 4°C in an IEC CRU-SOOO centrifuge, aliquoted into 0.5 m1 samples, and stored at -70°C. Prior to use, an aliquot was thawed and heat inactivated at 56°C for 30 minutes. Antiserum was subsequently stored at -20°C. NIH Mouse Alloantisera Four H-2 typing reagents (D-Z, D-23, 0-28, and D-33) were received gggggg from NIH. Each was reconstituted with one ml of sterile dis- tilled water, aliquoted with a Hamilton syringe into microcentrifuge tubes as lOO-ZOO ul samples, and stored at -70°C. For use, sera was 3O thawed to room temperature; subsequent storage was at -20°C. The activity of the antisera was not affected by refreezing. One Stage Lymphocytotoxicity Assay Lymphocytes were isolated from mouse spleens of appropriate geno- types as described above (Mouse Anti-H-2tW1 Production), centrifuged in an IEC CRU-SOOO centrifuge at 300 x g for 10 minutes at 4°C, and resuspended in four mls of MEM/Hanks medium. Cells were counted in a hemacytometer and the cell density was adjusted to give a final concentration of 3-5 x 107 cells/m1. Appropriate dilutions of anti- sera were made in HEM/Hanks. Rabbit complement, previously absorbed with agarose, was diluted 1:3, complement:Mem/Hanks medium. A modification of the Amos assay (Amos, gngl,, 1969) was used to test for the H-2 antigens found on lymphocytes with mouse alloanti- sera H-ZtWI, H-2.2, and H-2.33. To a clean test tube were added 20 ul of cells, 20 ul of diluted antisera and 20 ul of diluted rabbit comple- ment (C'). The mixture was vortexed and incubated for 60 minutes at 37°C in an atmosphere of 9% C02, 91% air. The reaction was stopped by placing the test tube on ice and adding 50 ul of cold MEM/Hanks. Prior to counting, 20 ul of 0.4% trypan blue was added. The number of viable cells, which exclude trypan blue, and the number of dead cells, which take up trypan blue, were counted in a hemacytometer on an American Optical Series 10 Microstar microscope. An experimental design, shown in Table 5, indicates the controls needed for cytotoxi- city assays. The percent lysis was calculated as: 100% L # dead cells in complete assay _ # dead cells in C' control ] toth’? of calls in complete assay total # of cells in C' control 31 Table 5 Experimental Design for Cytotoxicity Assays Anti- Rabbit Cells to Purpose Test tube serum complement be tested Cell control 1 - - + C' control 2 - + + Antiserum control 3 + - + Complete assay 4 + + + Iodination of Fibroblasts Mouse embryonic fibroblasts from primary explants or cell lines were subcultured as described above (Subculture of Primary Explants or Cell Lines) and dispersed into MicroTest II wells at a concentration of 2 x 104 cells/well to 1 x 102 cells/well. Cultures were incubated 24 hours at 37°C in an atmosphere of 5% C02, 95% air. CMRL 1066 medium was removed, cultures were washed once with PBS, and 0.1 m1 of 125I-iododeoxyuridine labeling mix (IZSIUdR; Table 6; LeMevel and Hells, 1973; O'Toole and Clark, 1976) was added to each well with a Clay Adams pipeter. Cells were placed in a well-humidified incubator at 37°C in an atmosphere of 5% C02, 95% air for 18 hours. The label was removed by aspiration, cells were washed twice with 0.2 m1 of PBS, and cells were detached from the wells by incubation in the presence of 0.25% trypsin in PBS for 30 minutes at 37°C. Released cells from each well were removed to a gamma vial, each wellwas washed twice with distilled water, and the wash was added 32 Table 6 125I-iododeoxyuridine Labeling Mix Stock solutions: Labeling MEM MEM 85 mls Heat inactivated fetal calf serum 10 mls 100x Non-essential amino acids 1 m1 Glutamine (3 mg/ml) 1 m1 Asparagine (2 mg/ml) 1 ml 100 mM sodium pyruvate 1 ml PeniETllin and streptomycin, 50 units/m1 1 ml 7.5% sodium bicarbonate 3 mls TITS—ml? Fluorodeoxyuridine (FUdR) 10'5 M 125I-iododeoxyuridine 50 uCi/ml Labeling Mix (lzsIUdR) Labeling MEM stock 4.5 mls FUdR stock 0.5 mls 125I-iododeoxyuridine stock 0.05 mls 5. m s to the gamma vial containing freed cells. Vials were counted in a Beckman Biogamma II counter. 125I-iododeoxyuridine Fibroblast Microcytotoxicity Assay 1 Cultured embryonic cells were subcultured in MicroTest II wells at 1-2 x 104 cells/well and incubated for 24 hours at 37°C in an atmosphere of 5% C02, 95% air. CMRL 1066 medium was removed, cells were washed twice with 0.2 m1 of PBS, and 0.1 ml of 125IUdeas added to each well. Cells were cultured in a well-humidified incubator 33 for 18 hours at 37°C in an atmosphere of 5% C02, 95% air. The label was removed and cells were washed twice with 0.2 m1 of PBS. 30 ul of diluted antisera and 30 ul of rabbit complement diluted 1:9 with labeling MEM supplemented with 5% heat inactivated fetal calf serum were added to cultures and mixed on a vortex. Cells were incubated for six hours at 37°C in an atmosphere of 5% C02, 95% air. Antisera and complement were removed, and cells were detached from MicroTest II wells by incubating with 0.25% trypsin. Detached cells and subsequent lwashes with PBS were placed in gamma vials (as described in Iodina- tion g: Fibroblasts) and counted in a Beckman Biogamma II counter. The percent lysis was calculated as: CPM in viable ex erimental cells E 1 ’ 1(CPM in C' control + CPM in Antisera control)] 100% 125I-iododeoxyuridine Fibroblast Microcytotoxicity Assay 2 Cultured embryonic cells were subcultured into MicroTest II wells at 1-2 x 104 cells/well and incubated 24 hours at 37°C in an atmosphere of 5% C02, 95% air. CMRL 1066 medium was removed and cells were washed three times with 0.2 m1 of PBS. 30 ul of antisera, appropriately diluted in labeling MEM supplemented with 5% heat inacti- vated fetal calf serum, and 30 ul of rabbit complement diluted 1:9 with labeling MEM supplemented with 5% heat inactivated fetal calf serum*were added. Cells were incubated for 30 minutes at 37°C in an atmosphere of 5% C02, 95% air. The supernatant was removed, each 125IUdR was well was washed once with 0.2 ml of PBS, and 0.1 m1 of added to each well. Cultures were incubated for 18 hours at 37°C in an atmosphere of 5% C02, 95% air. The label was removed, cells 34 were washed once with 0.2 ml of PBS, and cells were removed to gamma counting vials after the 0.25% trypsin treatment described above (Iodination pf Fibroblasts). Vials were counted in the Beckman Bio- gamma II counter. The percent lysis was calculated as: L CPM in viable experimental cells ] 100% i1C CPM in C' contrOl + CPM in Antisera control) Photpgraphy Embryos were photographed after dissection with a Canon F-l camera and a 28 mm Olympus macrolense attached to bellows. Cultures were photographed with a camera mounted on a Leitz Diavert inverted phase contrast microscope. Tissue sections were photographed with a camera mounted on a Zeiss Universal II microscope. Kodak Panatomic X (ASA 32) filnlwas purchased in bulk, rolled in film canisters in appropriate lengths, and used in all photographic work. Panatomic X filnlwas developed at room temperature as follows: 8; minutes in Kodak Microdol X diluted 1:3, Microdol X: water, with agitation every 30 seconds; 30 seconds in 1.5% acetic acid with con- stant agitation; 3 minutes in Kodak Acid Fixer with constant agita- tion; 20 minutes in running tap water; and a rinse in Kodak photo- flo solution. Prints were made on Polycontrast Rapid RC II paper with a Leitz enlarger and developed as follows: 11 minutes in Kodak Dektol diluted 1:2, Dektol:water; 15 seconds in 1.5% acetic acid; 4 minutes in Kodak Acid Fixer; and 4 minutes in running tap water. RESULTS Characteristics of BreedinggLines The original Iggf/gylei strain obtained from Dr. H. O. McDevitt was characterized by black and tan coat color, poor reproduction (only 33% of all breeders reproduced), small litter size (3 pups per litter), and male segregation distortion factor of 0.8 (H. 0. McDevitt, personal communication). Since mice with black and tan coat colors are notoriously poor breeders (J. H. Asher, Jr., personal communication), black and tan mice carrying Ifgf/tWI -: were mated with either C57BL/lOScSn (black coat color, g/g) or C3H.BlO (agouti coat color, A/A) normal (3/3) mice to try to establish more vigorous breeding lines and to eliminate the heterogeneous background of the “pen bred" stock. The resultant progeny'were characterized by either short tails (Iii/ti) or normal tails (pm-flit). Short tail and progeny characterized by taillessness and black (matings involving CS7BL/lOScSn mice) or agouti (matings involving C3H.BlO mice) coat were selected as future breeders. Efforts to establish breeding lines ofgf”1 backcrossed onto C57BL/1OScSn background were unsuccessful. Tailless females with black coat colors were not capable of breeding. An anatomical ab- normality in the pelvic region appearing after two generations of 35 36 backcrossing prevented copulation. Consequently, attempts to establish 3Y1 on C57BL/TOScSn were discontinued. Efforts to establish breeding lines oflgf"1 on C3H.BlO background were successful. Animals were always selected for agouti coat color in order to eliminate the breeding problems associated with the black t and tan gene, p_. Two breeding schemes used to maintain the mutant 5Y1 are shown in Figure 4. The balanced lethal line (C3H.Blofilgyl; Figure 4A) will only produce viable tailless (Ikiflivlfi) progeny in the absence of recombination since I:E:[I:£: and gyleijtwl -1 animals die jg ggggg. The backcross-intercross line (C3H.Blofpvl; Figure 4B) is dependent upon maintaining a breeding nucleus of normal C3H.BlO mice to be used in the backcross portion of the breeding scheme. ' Tailless progeny are backcrossed with normal C3H.BlO mice to produce short tail (I:gf/:::) and normal tail (trlg:/:::) offspring. Short tail and normal tail animals are intercrossed; the tailless progeny are subsequently backcrossed to continue the breeding line. The breeding problems described by McDevitt were not as prevalent in animals with a C3H.BlO background. Data are summarized in Table 7 for each mating system with respect to the percent of successfully reproductive females, average litter size at birth, and death rate at weaning. In the balanced lethal line, the percent of females that reproduce has increased and the average litter size has doubled compared to values for McDevitt's stock. The decrease in litter size from F1 through F10 was due to inbreeding depression (Green, 1968, 1981); litter size has subsequently increased. The high death rate detected at weaning in all three strains results from the in- ability of breeding females to nourish pups. Females stressed by 37 Table 7 Breeding Data for C3H.BlO, 03H.BloeItW1, and C3H.BlO-3Y1 % Reproductive Avg. Litter Death Rate 2 Generation Females Size at Birth at Meaning, % C3H.BlO N10F27+1 100 6.1 18.2 N10F27+2 86 6.2 26.5 N10F27+9 100 5.0 25.3 N10F27+10 78 5.1 9.3 N10F27+16 80 7.4 10.8 3 N10F27+17 "- 6.0 11.9 c.ili.l.ilO-Tt"’1 F1 67 6.8 5.9 F2 88 5.5 9.3 F9 50 4.2 15.8 F10 33 3.3 15.4 3 F12 "" 6.8 14.7 C3H.BlO-t"1 N1 100 7.9 4.2 NZF1 100 7.4 11.9 N3F2 100 6.0 12.3 N4F3 100 6.4 15.6 1Females giving birth to one or more litters were considered to be fertile. 2Based on the number of pups born and the number of pups weaned at 3three weeks of age. Data not available since these females are currently being mated. 38 fluctuating environmental conditions form mammary tumors, thus inhibi- ting lactation. The male and female segregation distortion factors were deter- mined in the backcross-intercross line. Phenotypic examinations of progeny from the backcross matings revealed the number of I_and £31 gametes fertilizing ova. Mice carrying 371 had normal tails whereas mice carrying I had short tails. In matings of I/gyl males x ‘1/:_fema1es, the percent of segregation distortion was high and in- creased from generation N1 to N4F3. The current male segregation distortion is 0.9. In matings of I/pyl females x j/:_males, a slight but insignificant distortion occurred (Table 8). The male to female ratio did not differ from normal Mendelian expectations in either case. Table 8 Segregation Distortion in Males and Females of the Backcross- Intercross Line Segregation Distortion T/t“"1 Males TytVl Females Generation of Progeny :73“ 17: z t“ x2 :It“ .17: % 3.“ x2 N1 49 13 79.0 20.91 6 4 60.0 0.4 N2F1 56 10 84.8 32.11 39 36 52.0 0.1 N3F2 35 4 89.7 24.61 61 40 60.4 4.42 N4F3 49 5 90.7 35.91 61 49 55.5 1.3 1 2 x0.005 [1] ‘ 7°88 2 2 - x0.05 [1] ’ 3°84 39 Characteristics of Embryos on the Basis of Gross Morphology Matings in normal C3H.BlO mice (3]: x 1/19 and heterozygous 0311.1310-3‘"1 mice (_+_/_t_“'1 x i/g'”) were performed. Embryos for histo- logical examinations were collected from pregnant females at days 9, 9}, 10, 10;, 11, 12, 13, and 15 days post-coitus (pg). Information obtained at the time of dissection included the number of implants and embryos in each litter. For each embryo, the crown-rump (CR) length was measured, age based on Grfineberg's description of external embryonic features was determined, presence or absence of a heart beat was noted, and presence of any abnormal morphology was identified at the gross level. The gross morphological data were used to examine the effects of gfl on embryonic development in a new genetic background. In the following discussion, embryos from j/gvl x :ngl matings could have three possible genotypes. Homozygous normal (1/1) and hetero- zygous (i/grl) embryos were indistinguishable and will be considered as normal (1/3); homozygous mutant (tPI/3V1) embryos will be considered as abnormal. Data on average CR lengths of embryos and range of CR lengths for a given embryonic age are summarized in Table 9. Embryos were staged according to Grfineberg's criteria; the embryonic age as based on external morphology, not the expected age as determined from the day of plug (day 0), was used in summarizing the information. CR lengths for abnormal embryos were excluded if the age of the embryo was found to be retarded as compared to normal litter mates. Compari- sons of average sizes and range of sizes in embryos from 03H.BlO or C3H.BlOepyl matings at different embryonic ages showed few discre- pancies. The sizes and ranges were comparable in embryos collected .cmas mgu mo cowpop>ou usaucmum .maoumogups cowuummmpv <~z up; a co smug—0L3... .332. cm .3...» bus-ammo:— mOthEm mo .3452” oo.~_-oo.e_ mm.. a Any mm.m_ m~.¢_--.m~ Ne.o w A_~V mm.m_ m. e~.cp-~e.m mm.o u Amv Ne.m ma.o_-e~.m 66.o w AGNV 6m.m m. “5.x -ep.6 am.o u Aa_v -.~ mm.m -mm.e m6.o h Am_v mp.w NP mm.a -am.m om.c w ANFV P..c No.6 -mm.e mm.o a Amwv mp.o PP mm.m -mm.m _m.o w Aemv Na.e pm.m -mo.e ¢~.o a Aeev m~.¢ mo. No.4 -Pm.m Pe.o a Aomv Km.m No.4 -mm.m m~.o n Amwv ep.e o_ mo.e -em.~ em.o w Ammv m~.m. mm.m -mm.~ em.o a Am_v m~.m «a mo.m -_6.P Nom.o aHAemv mm.~ mo.m -mm._ ~w~.o wfiammv m~.N a as as as as man .om< .m~_m :_ mmcmm .mem ommgm>< .mNPm cm magma .m~¢m muncm>< uwcoagnsm mme.oaz ~3w-opm.=mu mmcwumz o—m.:mu acmsqum>mo Co magnum acmcomm_c an moxgasm ea msuozmg mo mo mwcmm can mgamcma mu wmmcm>< m m—amh 41 from either mating, indicating that the presence of mutant 381 did not affect the development of normal embryos within litters. The values listed are similar to previously published data for other mouse strains (Altman and Katz, 1979). The expected litter age was used to compare ranges in age and CR lengths within litters from C3H.BlO and C3H.BlO-t)"1 matings (Table 10). Prior to 10; days pg, the range in age and size of embryos 1 embryos could from either mating was very similar. Homozygous 5? not be identified. However, at 10% days pg, discrepancy in range of age appeared in litters derived from C3H.BlO and C3H.BlOggV1 matings. In litters derived from C3H.BlO matings at 11 to 15 days pg, the range of age decreased, indicating that embryos within a litter were of equivalent ages. Embryos identified as developmentally younger than their littermates at earlier stages of development had grown and attained the same age and developmental features characteristic of its littermates at later stages of development. Conversely, the increased range in age and size of litters derived from C3H.BlOegU1 matings was indicative of the presence of abnormally developing embryos. Subsequent histological studies showed these "retarded" embryos to Ewl. Ewl be homozygous for Contrary to a previous report on mutant (Bennett, ggugl., 1959b), the decreased size of an embryo could be used only after day 11 pg to identify the embryo as a mutant homozy- gous for 581. The number of implants and aborts in litters derived from C3H.BlO and C3H6810f£r1 matings are summarized in Table 11. The number of implants per litter was 8.5 1 0.84 and 8.7 i 1.24 for C3H.BlO and C3H.BlOegyl, respectively. The mutant 5Y1 did not appear to affect 42 mpimm.¢ mpipp m~.¢—i-.mp mp m. e~.opiem.m mpimp m~.o~im~.m mpim~_ mp ~e.m i—m.m Npim mm.m imm.o N_ N. mm.m inv.¢ Fpiop mm.e imm.e _p _F mm.m imm.m «ofinwm Fm.m -mo.m mopim mop ~m.e imm.~ opimm mm.¢ im¢.m caiam op n¢.m -o_.N «mum -.m imm.p «mum am mo.m upm.p mi«m ~a.~ ioo.~ mimm m as when as when Ame .om< .o~wm =_ magma .mm< :w manna .m~¢m cw magma .mmm,:~ magma vmuumaxm mmewbaz 32m-c.m.:mo mmcvuuz opm.=mu 5268624 ea;o_z usages“ co gonzo; «8 sea oa< Lo eopoa_ca> op mpamh 43 .coms mga mo cowumw>mu ugmucmum .mcoppm:_smxm _muwmopoumws Lmamm cmpepacmu? monomznosoz m .mmmmgpcmgmq :_ cm>_m mgwuuwp mo gonsazw mea.o a me._ me~._ a ma.@ mmm.o a a.o mam.o a mm.m m oo.~ N oo.o_ A_V op co._ m oo.m Amv em m_ m om.~ m om.a ANS m, om._ 8 ca.“ any mm m, o. oo.F m mm.m Amy aw mm.p a ~6.~ Amy «N N. m oo._ m 56.8 Amy om oo.~ m mm.m Amv mu P_ m oo.~ a ~8.a Amv mm Km.o a cm.¢ ARV me he. a ma.o m oo.op Aev ca om.o _ oo.m ANV a, o_ o oo.P N oo.m “NV op oo.o o om.m Amy K, am «N no._ m 56.x Hflmv cw oo.P a om.m PAav Fm m 233%... hwy...“ 2%”? an“? amfifi mug“ Emu? Sagas amfifi mm“ mme_oaz ~36-o_m.=mu ameaoaz o_m.=mu aooooaxm mozgnsm pastocn< was .mucoa< .mucmpaEH we conga: pp mpnmh 44 the ability of embryos to implant in the uterine wall after fertili- zation. The .average abort rate per litter for C3H.BlO and C3H.BlO-fl1 matings was 0.9 i 0.55 and 1.49 i 0.64, respectively. The abort 1 ETall/Evil rate for CBH.BlOegU excludes embryos identified histologically but includes those homozygotes that could not be identified. After the embryos derived from C3H.BlOepyl matings were examined histologically, the number of homozygous 331 embryos for each time period were determined. The percent of abnormal embryos (aborts plus abnormal embryos) was found to increase from 12.5% to 70% with increasing age. The ability to identify homozygous 3V1 embryos and its correlation with the percent of abnormal embryos expected and observed will be discussed later. Characteristics of Embryos on the Basis of Histology Histological examinations by light microscopy of serially sectioned embryos derived from C3H.BlO-tW1 matings at different stages of develop- ment were performed. Emphasis was placed upon the ability to identify 1/Ew1 homozygous gr embryos as early as possible and the determination of the effects of the mutant gene, especially in homozygous EwI/Ewl embryos, at different stages of embryonic development. The gross morphological data together with the histological data wOuld provide a description of the effect of mutant gyl. Day 9 pc: Homozygous abnormal embryos were impossible to dis- tinguish from normal littermates at day 9 pg on the basis of CR length or gross morphology (Figure 5A,B). No edema, reduction in size, or other gross abnormalities such as an enlarged heart were present. Only 2 of 21 embryos were identified as tflxtVl after tissue 45 .343 9:95 Wm m: 5 36:2 .u_ .233 9:5 a 3. E. 3562 .m .363 335 fl 2 an .8222 .a .A~.wva excasm mm.op xmu _mscoz .u .Am.omxv ozgaew.mm.m aou peacocn< .m .Am.omxv ozgasm.wm a hen posse: .< .m:_c:upzu oeuw> mH.com ummpuxm 8cm: meanpwmh .covuummmwo cmueo umsamcmopoga mm.mo_ on a want sage moxgnsm wmzos Poscocna uco _mscoz .m mgsmwd 46 at " “are .53 I 1 -. . 45,414“. 4. 47 sections stained with hematoxylin and eosin were examined by light microscopy. Degenerating and pycnotic cells were characterized by the presence of darkly staining chromatin material in the mantle layer of the ventral (basal) neural tube at the rostral border of the rhombencephalon (Figure 6A-C). Nucleated red blood cells, whose nuclei stained with hematoxylin and were centrally located within the cells, were easily distinguished from pycnotic cells. Actively dividing cells of the neural tube were prevalent in all embryos and were confined to the ventricular zone (Boulder Committee, 1970). Pycnoses were absent in the regions rostral and caudal to the degener- ating rhombencephalon (Figure 6D-F). The width of the neural tube (measured from the ependymal to marginal layers), neural ectoderm derivatives, cranial and spinal ganglia, somites, notochord, and mesenchyme were normal in appearance. Day 9; pc: The identification of normal and abnormal embryos at day 9&ng,on the basis of size differences or abnormalities in gross structures was not possible. Although detection of a 91 day ‘pg abnormal embryo from an expected 9; day pg litter was not made histologically, one abnormal 9; day embryo from a 10 day pg litter was found. The embryo was characterized histologically by the presence of pycnotic cells in the mantle layer of rostral and caudal portions of the ventral rhombencephalon (Figure 7A-B, D-E). Pycnotic cells were absent from other regions of the neural tube (Figure 7C-F). The width of the neural tube was decreased only in regions containing pycnoses. Actively dividing cells were present in the ventricular zone, but the mantle layer of affected neural tube regions was not as wide as the mantle layer in comparable normal neural tube regions. Figure 6. 48 Transverse sections of normal and abnormal mouse embryos at day 9 pg, A. Rostral portion of rhombencephalon in the region of the eye from a normal mouse embryo (x73). Magnification of region outlined in A. Note the presence of mitotically dividing cells in the ventricular zone and the cellular arrangement found in the mantle layer of the neural tube. Nucleoli appear as darkly staining granules within cells of the neural tube (x290). Rostral portion of rhombencephalon in the region of the eye from an abnormal mouse embryo. Note the presence of mitotically dividing cells con- fined to the ventricular zone and of a few pyc- notic cells in the mantle layer of the neural tube (x290). Caudal portion of rhombencephalon in the region pf the otic vesicle from a normal mouse embryo x73 . Magnification of region outlined in 0. Note the presence of mitotically dividing cells con- fined to the ventricular zone of the neural tube (x290). Caudal portion of rhombencephalon in the region of the otic vesicle from an abnormal mouse embryo. Note the presence of mitotically dividing cells confined to the ventricular zone and the absence of pycnotic cells in the mantle layer of the neural tube (x290). 49 50 .Aoomxv Laxap opucae oga =_ mp—mu o_uo:oxa 5o oucmmna ten 63:» _agam: mo zany: muoz .ozgaso wanes pusgocaa an ace» taco pa:_qm ea :oFmos Fauamu .u .AooMxv gmxap «pages on» =_:u_3 Amzogsav mp—mu upaocoaa mo muzmmmga use was» panama ea swap: ummmmcumu apugmppm ouoz .oxgaso «macs Peacocao ca scam m_u.mm> u.»o age we =o_mog as“ :p :o_e=amu:oaeosc we copugca panama .m .AooMxv some. m—ucee may cpsu_3 Am:ogsav m—pmu u_uo:uxq ea oucommsa ace maau _egamc mza mo sacwz vomeoguov x—ugm_—m oaoz .oagaeo omaos peacocae an acne mam age we ccwmog use =_ co—ezqmugoasozg mo :owugoa —ugumo¢ .o .Acomxv egosoouo: mg» we accommca ace maze _eczm: use we =uu.: muoz .oagaso «mace pesto: 8 56cm taco panama ea chmog _ouamo .u .Acomxv goxap a—ucas mg» case’s m—poo uPHocuaa yo oucomaa wee mesa pmgzo: mo guu_3 ouoz .oagnso «mace _asgo: a scam o_o_mo> upuo as» no :ovmoc may :. copmgaoucmneogg ea copueoa panama .m .Accmxv coho. m—pcae use :_:a_3 m—pmu uwuoguxq mo oucmmaa new man» —oL=m: mo guc.z muoz .ozgaso mmsos —msgo: a scam mam ogu ea :opmoc mg“ a. :o—egnoocmangg we :o_ugoa pneumom .< .lm.*m xou an mozgasm mmaos paseocae ace passe: mo mcovuuom ongo>mzmsh .n meampu 51 52 Other neural ectoderm derivatives, cranial and spinal ganglia, somites, notochord, and mesenchyme were normal in appearance. Day 10 pc: The identification of abnormal embryos from normal littermates was still not possible on the basis of size reduction or abnormal gross morphology (Figure 5C,D). Several homozygous embryos identified histologically were characterized by the presence of pyc- notic cells in the mantle layer of the ventral (basal) portion of the mesencephalon and rhombencephalon and absence of pycnotic cells in the dorsal and ventral regions of the neural tube rostral to the mesencephalon and caudal to the rhombencephalon (Figures 8 and 9). The largest number of pycnotic cells appeared to be in the mantle layer of the ventral portion of the rostral rhombencephalon, where pycnoses first appeared at day 9 pg. The number of pycnoses decreased in affected regions of the neural tube rostral and caudal to the rostral rhombencephalon; few pycnoses were observed at the level of the otic vesicle. The width of the neural tube was smaller than normal in affected regions. Dividing cells were occasionally observed in the ventricular zone. The marginal layer was either absent or reduced in size. Other neural ectodermal derivatives as well as non-neural structures appeared to be normal. Day 10} pc: Abnormal embryos could not be distinguished from normal littermates unless the age and CR length were obviously re- tarded (Figure 5E,F). Pycnotic cells were confined to the mantle layer of the ventral portion of the rhombencephalon and, occasionally, the mesencephalon (Figure 10). The degree of degeneration did not appear to be as severe as that observed at day 10 pg, In the affected regions, width of the neural tube was slightly smaller and the 53 e.e.m.< ’ .Aoomxv meeN _e:_mges e me meemmee mgu eee .meeN mpaees men we save: emmemgmme mg» .gmxep m—ueee mg» ea emcpmeeu mppmo e_ue:eze me amass: mg» muez .u :— emeppuae =e.mmg ee ce'mme.w_cmez .Ammxv m=e~ cm—ae'cu:m> mgu :. uemmmce mam mP—me mcpep>pe a—peewueu_z .Azegeev mp—me o_ae:e>e mepcpmmm »_xgee me geese: mgu eee maze pegeme mga we seep: emmemgeme mgu muez .exgasm mazes peacecem :e Sega mam mg» we :epmme mg» g, eepecemeemesege we =e_ugee pegmmez .Aocmxv memewm pecegem: mcpe_mu:ee meeN pmewmces nee "namepaecem: map:_eu:eo m=e~ m—uces mm—pmm m=_u_>_e xp—eu_uemws oepcveaeee meeN Lepauvmu=m> "mean _egemc mg» g. mmce~ mmcgu we muemmmge mg» mmez .< :. em=__u=e :e_ams me eepueuF$F=mmx .Amuxv menu _egemc mga ee seep: ms» wee meeN cepzepgucm> mg» ea emepuceu mppmu acwep>we a_peu_ueu_s we meemmmce mg» maez .eagasm mazes Pesee: e seem mam mgu ye :evmme mg» :. ce—msemeemesegg 3e eepugee —egume¢ .< .lm.o_ see we mexgasm mmeee pesgeeem ece pesge: ee mcewaomu mmcm>m=ech .m enema; 54 55 mi .Aoomxv eageEm pesto: m cw em>cmmee mega em m—eegeesee mw emxep mpacee mg» a. xuwmeme —_me mgm use meem Le_:u_gu=m> mg» em emewweeu mam mpwmu mewew>we appmewueuwz .exgesm mmees passecee :e eegw eeeu pmewem me» we sewagee peezeo .Aoomxv meeN gepemwcm=m> mgu ew m__me aewew>we »_—euwaeaws we mucmmmge may mmez .exgesm mmees —esce= e secw egee .mewem ms» we sewagee peeseu .Acomxv < cw em>cmmee mesa e» emceeseo x_m;mw—m emmemceme m. mesa wegeme mg» we anew: mgw .eagasm mazes —e5gecee :e Eecw m—uwmm> owue men we eewmmm mzu cw ce—egemecmesegg we eewugee peeeeu 48me e555 mazes .952. m Eecw m—uwmm> ewue mg» we :ewmmg msa cw :epegemecmeeegs we sewagee weeeeo .< .lm.op see me meagasm mmees —msge:ne use peace: we meewmomm mmgm>mcmcw .m msemww 56 Figure 10. 57 Transverse sections of normal and abnormal mouse embryos at day 10; pg. A. E. Rostral portion of rhombencephalon in the region of the eye from a normal mouse embryo. Note the presence of the three distinct zones of the neural tube (x75). Rostral portion of rhombencephalon in the region of the eye from an abnormal mouse embryo. Note the presence of the ventricular and mantle zones and the absence of the marginal zone (x75). Caudal portion of the rhombencephalon in the region of the otic vesicle of a normal mouse embryo. Note the width of the neural tube and the presence of three zones (x75). Caudal portion of the rhombencephalon in the region of the otic vesicle from an abnormal mouse embryo. Note the decreased width of the neural tube and the absence of the marginal zone (x75). Caudal portion of the spinal cord from a normal mouse embryo. Note the presence of mitotically dividing cells in the ventricular zones and neuro- blasts in the mantle zone (x75). Caudal portion of the spinal cord from an abnormal mouse embryo. Note the presence of mitotically dividing cells in the ventricular zone. The width of the neural tube appears to be smaller than that observed in E (x75). 58 59 marginal layer was not as prevalent. Other neural and non-neural structures appeared to be normal. Day 11 pc: Abnormal embryos were usually distinguished from normal embryos at day 11 pg by the reduction in CR length, retarded developmental age, and appearance of opaque degenerating tissues (Figure 11 A-D). An enlarged heart or wavy neural tube was rarely observed and could not be used to identify abnormal embryos. Pyc- notic cells were found in the mantle layer of the: mesencephalon, ventral regions of the diencephalon and telencephalon, ventral and dorsal regions of the rhombencephalon, and ventral and dorsal regions of the spinal cord (except for the spinal cord just rostral to the tailbud) (Figures 12 and 13). Pycnoses_were pgygg_observed in the most ventral portion of the neural tube above the notochord. The width of the neural tube was decreased and the size of the marginal layer was reduced, while the overall shape of the neural tube was maintained. The apparent number of mesenchymal cells appeared to fluctuate in all embryos. Neural ectoderm derivatives, cranial and spinal ganglia, and somites appeared to be normal. Eye and ear develop- ment continued and ossification centers began to appear, especially around the notochord. Since day 11 embryos were retarded in develop- mental age, the development Of other neural and non-neural structures was compared to younger, normal embryos of an equivalent developmental age. Day 12 pc: By day 12 pg abnormal embryos were easily distinguished from normal littermates by the presence of reduced CR length, retarded developmental age, and degenerating tissues (Figure llE-G). In the 6O .AF.¢wxv m cw excesm ea mueELmumw. .exgeEm westeeem mm.~— .Am.wwxv m cw ezeesm op maeEgmuuww .exgeEm _esgecnm mm.~— .Am.e_xv exceem Peacec.mm.~_ .Ao.__xv u cw excesm em muesgmumww .engEm weasecee.mm pp .Am.wva excesm peacecem mm.pp .Ae.mwxv < cw exgeEm e» maeEgmuuww .exgesm Peacecee mm.pp mam: meeewwew .Am.F—xv eagesm _msce=.mm ,— .mcwcempee egmw>.mfl Lew .cewummmmwe cmuwe emgeegmeuece.mm Np use pp whee seew xmo . xea . am: . e m m E. .e an .e an .m see .< emmwoxm mexgesu .2 8:3... 62 .Amwxv usesmeaes msu m>eee z—pewgwu sewmms ms» sw usmmee use sexew m_»ses ms» we wwes pegusm> msa sw useew mse m_—me uwaesexe mesa muez .ezsesm pessesee se eesw mpuwmm> uwue ms» we sewmms ms» sw se—essmmsmeeesg we sewages Peuzeu .Amwxv usesueues ms» m>eee xwmomswu mesa —eg=ms msu we sewmmc msu s. usmmee use mes» pegsms ms» we w_es _egusm> ms» ea umswwsem mge mp—me owuesoxs Hes» mmez .ezgasm pesgesee se seew mpowmm> uwue msu we sewmms msu sw se—essmusmesess we sewages —eu:eu .Aocmxv emxep mpmses ms» em umswwseu m—pme uwuesexe we mesmmmse msu mmez .ezgaem muses weasesee se sesw mam ms» we sewmms ms» sw se_essmosmeeesg we sewusee pesames .Amwxv heme. peswmsee e we mosmmee ms» use gmxe. mwuses ms» sw usaew mp—mm uwaesuzs we senses ummemeusw ms» maez .ewsasm Fessesee se segw mxm msu we sewmms msu sw sepesemosmesess we sewusee pesumes .Amwxv aeswmmwu mewss mw cmxe— peswmses msw .smxe— m—uses ms» we suuwz.use auwmsmu _Pmu msu mmez .ewgesm weases e seew mwewmm> ewme ms» we sewmmg msu sw sewesemusmesesg we sewuses peuseu .< .mm.p_ Aeu me meagesm mazes —esgesee use _eEses we msewuomm mesm>msesw .N. oc=e_m 64 .Accmxv Amzessev useseeues ms» m>eee sewmms ms» sw aemuxm amzep m_usee ms» sw m—pme uwuesexs we mosmmmge ms» maez .a sw szesm mesa pegsms msu we wpes pesusm> ms» we sewaeowwwsme: .u .Amaxv one» _acsoe age we sewases pegusm> msa we sexep m—uses msa sw m—pmu uwaesezs ms» we asmsmswwseu ms» maez .ezgasm mazes pesgesee se sesw useo peswsm ms» we sewmms peuseu .s .Amwxv m__me owaesuws we musmmee ms» use usee peswem ms» we meesm _eLmsma ms» maez .exgesm mesee _esgesee se sesw useo peswem msu we sewmms peuseo .u .Amwxv smae— mpuses ms» sw m_—me owuesuxs :mw e we mesmmmss ms» use mes» pessms may we smuwz ummemsemu msu muez .ewsesm mmses pessesee se secw «gems msu we sewmms msu sw ugee .eswem .m .Amwxv gmAe— weswases muswumwu use sm»e_ m—uses msa we xuwmsmu _pmu msu mmez .exgeem mmses _eEees e secw ugems ms» we sewmmg msa sw useu weswsm .< .lm.—— aeu ue measesm mmeee pessesee use pesges we msewuemm mmsm>msesw .m. mesmww 65 66 most extreme cases, the neural tube was completely pycnotic and the overall shape was generally discernible but disorganized (Figures 14C, 15C-D, and 16C). Other neural and non-neural structures displayed pycnotic cells. In less extreme cases, pycnoses were still confined to the neural tube (Figures 148, 158, 168). The mantle and marginal layers of the neural tube were decreased in size. In either case, eye, ear, and cartilage development had progressed to the same stage found in a comparable littermate even though other neural or non- neural structures were obviously degenerating. Day 13 pc: By day 13 pg, the easily identified abnormal embryos were in the process of being resorbed (Figure 17A-C). The tissues were fragile and easily torn during dissection. Histological exami- nations Showed that neural as well as non-neural structures were pycnotic (Figures 18 and 19). The general outlines of structures were observed but cells within were disorganized and pycnotic. The development of various organ systems appeared to be arrested at dif- ferent stages of development. Day 15 pc: Abnormal embryos were carefully dissected free from amniotic sacs and fixed (Figure 17D-E). Obviously, all structures were pycnotic upon histological examination by light microscopy (Figure 20). Primary Explants and Subcultures Tailbud, forelimb bud, or heart tissues were excised from 207 embryos representing days 9 to 15 pg and cultured jg yipgg as des- cribed in Methods. Primary explants were transferred to untreated 35 mm tissue culture dishes and usually attached to the plastic Figure 14. Transverse sections of normal and abnormal mouse 67 embryos at day 12 pg, A. A portion of the basal neural tube in the region of the isthmus from a normal mouse embryo. Mitotically dividing cells are present in the ventricular zone. A large number of cells are present in the mantle zone (x300). A portion of the basal neural tube in the region of the isthmus from an abnormal mouse embryo. Note the large number of pycnotic cells and decreased cell density in the mantle zone (x75). A portion of the basal neural tube in the region of the isthmus from an abnormal mouse embryo. Note the loss of structural integrity, cellular disorganization and cellular pycnosis in the neural tube (x75). 69 .Amwxv mp—mu aswussessem use mesa _eeems ms» we sewueewsemsemwu se_:_pme use xuwgmmmsw pessuussum we xee_ ms» muez .ewgeEm ~e§sesee se segw sem\mzm ms» we sewmms ms» sw se_essmesmesess mse we sewusee _euseu .Amwxv mesa —es=ms msu we sewmee -wsemgemwu sepappmm use auwsmmusw peseuueeem we seep ms» muez .exgesm muses pesgesee se sesw mzm ms» we sewmms msu sw sewesemmsmams ms» we sewages < .mexv mse~ —eswmgee e we see_ use mse~ mpuses ms» ea ums—wseo mP—me ewuesua we senses ms» muez .exgesm —esgesee se sesw semxmzm msu we sewmms msu sw se—esemusmeEess msa we sewuses peueeu .Amwxv mse~ peswases meswumwu men use mse~ mpuses msu we zuwmsmu __eu msa muez .exsesm mazes pesLes e sesw sem\mxm ms» we sewmmg msu sw sewesemusmneess ms» we sewages _eueeu .< elm Np aeu «e mexgesm mmees _essesee use _eEses we msewuomm mesm>msegw .e. oc=e_s 7O Figure 16. 71 Transverse sections of normal and abnormal mouse embryos at day 12 p_, A. Caudal portion of spinal cord from a normal mouse embryo (x300). Caudal portion of spinal cord from an abnormal mouse embryo. Note the presence of pycnotic cells in the alar and basal regions of the mantle layer and absence of pycnotic cells above the notochord (x75). Caudal portion of spinal cord from an abnormal mouse embryo. Note the lack of structural integrity and general cellular disorganization of the neural tube (x75). .A¢.wwxv exgesm wesgesee.mm m— wen . .Ae.esxv asceEo wascoeae.mm m. sea . u o .A~.mwxv exessm pessesee mm.m— see .u .Am.m—xv exssem wesLesee mm.m— zen .m < .Ao.u_xv exgsEm —esses.mm mp xeo . 73 .mswseawem esew> mw.sew ummwexm mam: mueepwew .sewuemmmwu smewe umseesmeuess mm.m~ use mp exeu sesw mezgesm .~_ ocsewa 75 .Amwxv msmmwu aswusuegssm me ppm: we mean pegsms ms» sw sewueewsemsemwu se—sp—mo use xuwsamusw we xue— msa muez .eagesm meses —esgesae se Eesw se—essmusmmms msa we sewusee < .Acmxv sewueewsemse Lepappmu umswwmuip—mz.»se we mesmmee use mes» pegsms msm sw mwpme owuesews we memesss mmsep ms» maez .ewgesm meses _eeLesee se sesw se—essmosmmes ms» we sewases < .Amwxv mesa pessms ms» msw>emp smeww m>sms we use mes» _essms msu sw mw_me uwuesuxs we musmmese msu muez .m s. szesm mesa pessms ms» we sewuses pesasm> we sewueewwwsme: .Aeexv ease _acsoe use we smxe— mwuses msu sw m—pmu uwaesews we musmmmss msm maez .exsesm mmses peacesee se eesw m—uwmm> ewue ms» we sewmms msu sw sepessmusmesess ms» we sewage: peuseu .Acmxv mmseN weswmgee use m—uses peELes we mmsmmmgs msu muez .exgasm mmses wesges e sesw sem\m>m ms» we sewmms ms» s. se—esemosmums ms» we sewage: < .< .lm.m_ xeu we measesm meses —essesee use peace: we usewuumm mnem>msesh .e_ ocsews 77 .Amwxv sewueewsemsemwu sepappme use xuwgmmmsw pegsuessmm we seep ms» muez .ezgeEm meses weasesee se sesw useu _eswsm msa we sewusee _euseo .u .Amwxv usmmmss mme mp—mo ewuesmws 3mw < .smwep mwusee ms» we zuwmsmu ——me use suuw: ummemumu ms» muez .exgesm meses _esgesee se eesw useu peswsm msu we sewuses _euseo .m .Aomxv easesm mmsee _eeLes e Eesw usee _eswsm msa we sewesee —eu=eu .< .lm.m_ aeu He mexsssm mmsee _eseesee use peaces we mseweemm mmLm>msesw .e. measwa 78 79 .Amwxv uwaesmzs mwe m—kmu —_e aesu muez .ewgesm muses _eEeesee se Eesw sewesemusmums ms» we sewage < .Aomxv ewuesoxs use m—pmu p—e gases» sm>m m—eeswmem—u mse mmwsauusam hue msw_u=e msu muez .ewgesm mmsee weasesse se sesw useu —eswsm msu we sewages < .Acmxv m—pmo uwuesuxs we emu—s msu sw mewemmgm> we seweesgew use usesoemes msu we musmmmse msa maez .ewgesm muses —esgesee se seew usee weswsm msu we sewases peuueu .Acmxv mmsmmwe wpe sw m—pmu uwaesexs we mesmmmse use me upeesm se—esemmsmwu ms» mums: we msw—ase pesmsmm msm muez .ewuesm mmses —eEgesee se Eecw mam msm we sewmms msu sw sepesemesmwu msa we sewuwes < .Aomxv sewueewsemse sewsp—me we ewe. msu use mwwmo ._e we mmmesezs ms» muez .ewsesm meses pesgesee se sesw eeems msu we sewmmw ms» sw uweu weswem .< .lm.mw Aeu we newnesm mmses weswesee use wesges we msewummm mmsm>msesw .o~ msemww 81 substratum within 24 hours. Within 48 hours, cellular outgrowth appeared from the cut edge of the primary explant. Cells were either fibroblast-like or epithelial-like and surrounded the primary explant (Penso and Balducci, 1963). After at least a week in culture, cells preparing to undergo mitotic division detached from the substrate and appeared as rounded cells on the substrate surface. Continued growth was noted by the increase in circumference of cells surrounding the explant. Cellular outgrowth was usually limited to a circular focus of 10 to 15 mm in diameter. By this stage, cell confluency was reached and the culture could be used for subculturing and estab- lishing cell lines. For an explant culture to be considered successful, three criteria had to be met: 1) the explant had to attach to the substratum; 2) cellular outgrowth had to be observed; and 3) cells had to be viable for a minimum of three weeks. Of the 207 explants cultured jphyippg, 86% met these criteria and were considered successful cul- tures (Table 12). However, this value is an underestimate of the rate of success since explants were cultured from living as well as dying embryos. Obviously, tissue excised for jp_ng g culture from a dying embryo may not be expected to attach to a substratum and display cellular outgrowth. 0f the 207 explants, l7 (8%) were known to be derived from embryos dying jp_gpgpg_(lable 12). Therefore, 176 of 190 explants from viable embryos (94%) were successfully cul- tured. Tissue excised from either normal or abnormal embryos produced four different types of primary cultures when observed by light micro- scopy. Most cultures from tailbud or forelimb bud tissues were composed 82 Table 12 Primary Explants From Embryos Representing Days 9 to 15 pg Expected Embryo Number 1 No. Successful] Age, Days of Explants No. Failed 9 29 (4)2 28/1 (1 13 9; 29 (4) 27/2 10 36 (4) 33/3 101 42 (5) 37/5 (1) 11 28 (4) 25/3 12 25 (3) 18/7 (7) 13 10 (2) 7/3 (3) 15 8 (1) 3/5 (5) 1 One explant per embryo. Number in parentheses refers to the total number of litters. Number in parentheses refers to culture of tissue from resorbing embryos. 2 3 of fibroblast-like cells (Figure 21A). These Type I cultures were composed of very narrow, elongated and spindly cells arranged in parallel fashion. In these cultures, a state of confluency was reached very quickly. Cell boundaries were barely discernible. Monolayers of cells were only observed at the peripheral edges of the culture. Rounded, birefringent cells, probably representing cells undergoing mitotic division, were prominent. At the periphery, cells having more than two long processes were occasionally observed. These stel- late we mesmmmee ms» use mameweeseww ms» we meesm xwuswsm .msew ms» maez .mwsuwau __ meww .m .uee awe—eeseww mmsmu e se Amzessev mwwmo msmmswswmgwe .umusees we Lenses msu meez .mssmwse H meww .< .Aeamxs meeepsxo ewsozsesm aseewse Eeuw msezesmuse we mssegmeeess emeceseu mmese umusm>s_ ._~ mesmww 84 85 of covering a flat surface, the cells tended to form circular mounds with little or no contact between mounds. In regions where fibroblast cells were absent, large amorphous cells with transparent cytoplasm and "fringed" edges were present. Confluency of cells was not reached as quickly as in cultures of Type I. In Type III cultures, forelimb bud or heart explants were the source of epithelial-like cells (Figure 21C,D). Long, spindly shaped fibroblasts were absent. The flattened cells were characterized by their uniform size and polygonal cell boundaries. Cells in Type III cultures were slower to grow than cells in Type I or Type II cultures. Cells prevalent in Type IV cultures were neither fibroblast- like nor epithelial-like (Figure 22A,8). Cells of different shapes and sizes were present and were extremely difficult to observe because of their transparency. Vacuolated and granulated cells accumulated with increasing age of the culture. In addition, large clear cells with highly refractive membranes appeared. Extra-cellular "debris", presumably forméd by cultured cells, was suspended in culture media and increased in concentration between media changes. In contrast to fibroblasts found in Type I cultures, fibroblasts in Type IV cul- tures appeared at the peripheral edges of the cultures and never obliterated the unusual cells. Trypsinization of Type IV cultures was extremely difficult. Large areas of cells were often left attached to the substratum, and extra-cellular debris continued to be produced by cells subsequent to the trypsinization treatment. Cells prevalent in cultures derived from heart explants of I/I embryos usually had two or more processes and were highly refractive 86 1.1..m_wmu msoEe mmeusee sews—wee we mmsmmee msp maez .mexsesm w\w sesw muse—so .u .mseppso >H meww .m .mwpme umuewesesm use ummewesee> use mmeesm usmemwwwu we wwwme we mosmmmse esp meez .mssuwee >H mexw .< .Aoaexv megawaxo mwsexseEm aseswse Eesw mspzesmuue we essesmepese mmesmsem mmess ummsm>sH .NN ec=e_a 88 (Figure 22C). The cells were scattered around the primary explant and rarely contacted each other. The general features of fibroblast- like or epithelial-like cells observed in culture types I-IV described above rarely appeared in the few (7) I/I_cultures examined. Several primary cultures were subcultured for the production of cell lines and to keep a viable representative example of the original cell outgrowth from the primary explant. The cells were passed either before reaching confluency, or shortly thereafter. Usually the cell type found as the predominant form in the primary culture was established in the subculture. Once a cell line appeared to be established, subculturing could be performed every four to five days. Histocompatibility Typing Homozygous lethal 381 embryos cannot be distinguished from normal littermates prior to day 11 pg of development since there is no reduction in CR length and/or abnormalities in gross structures. If mutant embryos can only be determined after histological examina- tions, then biochemical and other histochemical studies cannot be performed simultaneously. In an attempt to distinguish normal and abnormal embryos without sacrificing all of the embryonic tissues, fibroblast-like cells grown from jpflng g explant cultures described above were assayed for the presence of the H-2 antigens. The H-2 haplotype of fibroblasts is representative of the genotype of an embryo. Production of mouse alloantisera to H-ZtWI, determination of the specificity of mouse alloantisera to H-Z antigens, and 89 examination of different assays for detection of H-2 antigens using mouse alloantisera were performed. twl Anti-H-2 was produced by injection of normal (i/i) 03H.810 male mice with spleen cells isolated from i/EUI mice (:,H-2b/gyl, twl H-ZtWI). An antibody response to H-2 as well as other antigens not shared by the two strains was elicited. Blood samples from twelve mice were pooled to form three samples of H-ZtWI: H-ZtWI-l, H-ZtWI-Z, and H-th1-3. Specificity and titer of each antiserumiwere determined in a complement-mediated lymphocytotoxicity assay using spleen cells isolated from C3H.BlO y: and 0311.810-1;W1 mice (Amos, e_t in 1969). The results, shown in Table 13, indicated that all three antisera were specific for the H-thl haplotype, even though the antibody titer (reciprocal titer of 10) was relatively low. Antiserum to H-2.33, one of the private antigens for the H-2b haplotype, was also tested. As expected, :/:_cells were lysed only by the anti-H-2.33 antiserum andgltW1 H-ZtWI. The three antisera (H-ZtWI-l, H-ZtWI-Z, and H-2t"1-3) were cells were lysed by both anti-H-2.33 and anti- considered to be equivalent and used interchangeably. The H-Z haplotypes of four strains of mice, C3H.BlO (:11), C3H.BlO- t‘“ (i/g‘“), 0311310113“ (_T/g“), and C3H (i/:) were tested with mouse alloanti-H-2.2, a private H-2b antigen that had been previously tested, and mouse alloanti-H-ZtWI in a complement-mediated lymphocyto- toxicity assay. The known H-2 haplotypes for each donor strain and results of the assay are summarized in Table 14. The results indicated that C3H.BlO (1/:) cells were lysed only by anti-H-2.2. The j/gyl cells were lysed by both anti-H-2.2 and anti-H-ZtWI. Thai/g?"1 cells were lysed by anti-H-ZtW1 but not by 9O Tab1e 13 Specificity of Three Samples of Anti-H-ZtWI Using i/: and :ngI Spleen Cells % Lysis Anti serum 1/_+_ :/_t_""1 Anti-H-thI-l 1/10 dilution 6 46 1/20 dilution 6 32 Anti-H-ZtNI-Z 1/10 dilution 6 31 1/20 dilution 6 27 Anti-H-ZtWI-3 1/10 dilution 6 44 1/20 dilution 6 32 Anti-H-2.33 1/80 dilution 76 53 Table 14 twl Specificity of Anti-H-2.2 and Anti-H-2 Using Spleen Cells Isolated From Four Strains of Mice Donor Known H-2 Type % Lysis with g; Spleen Cells H-2b H-ZtWI Anti-H-2.21 Anti-H-zt"1‘ C3H.BlO (35/3) + - 20.3 0 C3H.BlO (:13Y1)3 + + 21.1 31.9 C3H.BlO (1717114 25 + o 46.2 C3H (1/1) - - o 12.2 1 2 3 4 Antisera diluted 1/50. Antisera diluted 1/10. Cells from mice of the C3H.BlOegvl strain. Cells from mice of the.C3H.810-T_t"'1 strain. 5H-Z haplotype associated with the I_mutation is unknown. 91 anti-H-2.2, indicating that the H-2.2 antigens associated with I_did not include antigen H-2.2. The C3H :/:_cells were not lysed by anti- tWI. Since the antisera were H-2.2 and cross-reacted with anti-H-Z specific for a given H-2 haplotype, a complement-mediated cytotoxi- city assay was used to determine the H-2 types (and genotypes) of fibroblast-like cells grown from embryonic explants jp_vitro. Prior to the determination of H-2 types on fibroblast-like cells in a complement-mediated fibroblast cytotoxicity assay, the ability of fibroblasts to incorporate 125I-iododeoxyuridine (IZSIUdR) and the cell concentration at which maximum incorporation would occur were examined. 125IUdR, a thymidine analog, is inserted into DNA during DNA biosynthesis prior to mitotic cell division. A constant amount of 125IUdeas added to varying concentrations of normal (11:) cells (1 x 102 cells/well to 2 x 104 cells/well). The results, shown in Figure 23, indicated that: 1) cells were actively dividing and 125IUdR; 2) incorporation of label was dependent 125 would incorporate upon cell concentration; and 3) maximum IUdR incorporation occurred at a cell concentration of l x 104 cells/well. To determine the sensitivity of two different assay protocols and to test for the presence of H-2 antigens of fibroblast-like cells from a primary explant derived from a 10 day pg i/i embryo subcultured twice jg nggg, twodifferent complement-mediated fibroblast cyto- toxicity assays utilizing 125IUdR were examined. In assay 1, 125IUdR was incorporated into DNA of actively dividing cells prior to antibody and complement exposure. In assay 2, complement and antisera were added prior to incorporation of 125IUdR into DNA of the remaining viable cells. 92 .mwwmm umeweegeww mswuw>wu xwm>wpoe emsw suammH we sewuegeeeeesw .mm mesmww 20.._.<¢._.zm0200 Aamo _ 11 L a nu d W . 1v H A o .o... 10 1m 93 The antisera used in assay 1 were specific for two private anti- gens of the H-2b haplotype, H-2.2 and H-2.33. The data, shown in Table 15, indicated that both antigens were present on fibroblast- like cells. More cells were lysed with anti-H-2.2 than with anti- H-2.33. This difference in the degree of cytotoxicity may result from differential expression of H-2.2 and H-2.33 on fibroblast-like cells, differential exposure of each antigen on the cell surface, and/or differential antibody titers to the antigens. Temporal differ- ences of H-2 expression have been noted on embryonic tissues and may explain the variation of expression of the antigens (Ostrand- Rosenberg, gpngl,, 1977; Kirkwood and Billington, 1981). Preferential loss of antigen expression has also been observed on cells grown jpuyigpg and subcultured several times (Ostrand-Rosenberg, gt gl., 1977). Regardless of the reasons for differences using the two anti- sera, both were expressed on the cell surface. Only anti-H-2.2 was used in assay 2 to determine if the method was more sensitive than assay 1 to detect the presence of H-2 antigens on the cell surface. The degree of cytotoxicity was approximately twice that observed in assay 1. The apparent enhanced sensitivity Table 15 Determination of H-2 Antigens of Normal (1/1) C3H.BlO Fibroblasts Using TWO Different Assays % Lysis in Anti-Serum ASsay 1 Assay 2 Anti-H-2.2, diluted 1/10 28.7 54.3 Anti-H-2.33, diluted 1/10 9.3 NTl 1NT - not tested. 94 of assay 2 may result from exposure of cells to complement and antisera 125IUdR incorporation into DNA of dividing cells. If cells prior to react to complement and antisera, few viable cells remain that will become labeled prior to mitotic division. Since label is incorporated into viable cells prior to antibody and complement treatment in assay 1, cell overgrowth may inhibit complement and antibody complex formation on most of the cell surfaces and yield a low cytotoxic value. 0n the basis of this information, assay 2 was used in subsequent experi- ments. The antibody titer of anti-H-2.2 used in assay 2 was determined with normal (1/1) fibroblast-like cells subcultured three times 1g ‘nggg. The data, shown in Figure 24, indicated that maximum cell lysis occurred at an antibody reciprocal titer of 80. The degree of cell lysis decreased in either apparent antibody or antigen excess. At all antibody dilutions the degree of lysis exceeded 20%. The results described above for one normal (1/1) cell line were encouraging because the cell line did express H-2 antigens that could be detected via complement-mediated cytotoxicity measuring 125IUdR incorporation. Subsequently two cultures whose haplotypes were un- known as well as the cell line previously tested were assayed for H-2 antigens. Unfortunately, the detection of H-2 antigens on normal (:11) cells became erratic; the results described above could not be reproduced. Results from assays on the two unknown lines were ambiguous; consequently, determination of H-2 types and genotypes could not be made. N.N-=-_eee we eeeee seeeweee we eeweeewseeeee .eN ecee_e 20:34.0 >o0m_._.z< 0..) ON}. 0*}. on}. 625. can} ave}. 95 — d d - u d - Joe 1 GOP SISA'I % DISCUSSION In the original papers describing the effects of four p_mutations w20 "1 complementation group (twl, 3Y3, gylz, and g from the g_ ) on embryonic development, the mutations were considered to be allelic and identical with respect to the time and expression of gene action (Bennett, gt 31., 1959a,b). Consequently, crosses were made at random to produce embryos of homozygous (38/58) or compound heterozygous (ti/3?. where x and y represent two non-complementing alleles within the 3P1 group) genotypes. Information obtained from histological examinations of embryos at days 9, 10 to 11, 12, 13, 14, and 15 to 20 of gestation was thought to represent the morphological effects 581 complementation group as a whole, but of the g_mutations in the the pooled data obliterated any subtle differences that might have been observed for each homozygous class, i.e., 531/371. LPBKEUB. Ew12/Evin w20/tw20. , and 5_ Other studies on.g mutations within the same complementation group have demonstrated that g "alleles" do not have the same effects upon embryonic development (Hillman and Hillman, 1975; Sherman and Wudl, 1977). Thus, the value of information obtained from pooled observations on homozygous and compound hetero- zygous embryos is questionable for several reasons. First, the mutations were derived from wild populations of mice Ewl tw3 _ located in different geographical locales ( - New York: __ w12 Connecticut; _t_ - California) and an exception inabalanced laboratory 96 97 5 complementation stock carrying the mutation tNIS (an allele of the 3? group; Bennett, ggngl., 1959a). The mutants were crossed to an inbred Brachyury laboratory stock for 2 to 3 generations and subsequently inbred for 3 to 5 generations. Although each mutant had been crossed to a common laboratory stock and inbred several generations, the genetic background of each mutant was still heterogeneous, especially for linked genes, and not comparable to one another. For example, the genetic background of Iggf/gylji would have been different from w20_ the genetic background of 1:3:/ .1, The presence of different modifier genes contributed by the diverse genetic background in each mutant could alter the gene action of the mutation so that phenotypic expression of each might gppggp to be similar, but the cellular pro- cesses affected would be different. Second, in matings between mice heterozygous for g_(I:gf/p§e:_x lfgjjgfej), the number of dead embryos (presumably representing the homozygous mutant genotypes) observed from days 10 to 12 pg were drastically different for each non-complementing 3V1 mutation. In thegw1 complementation group, the percent moles observed ranged from 47% for 571/331 to 96% for 5820/5820 (Bennett, gpngl., 1959a). The variation in numbers of dead embryos observed for each of the four homozygous genotypes may have resulted from differences in content of genetic modifiers associated with each mutation or may have been a reliable index for expressing differences in gene action for each mutation. For example, studies of two alleles in the 332 12 complemen- W32/tW32 tation group, g and 3732, have shown that g}2/p}2 and g embryos differ morphologically from each other (Hillman and Hillman, 1975). Although both mutations arrest development at the morula 98 tw32 12 stage, __ alters cells in the early morula stage and g alters cells at the late morula stage. Since the mutations were not on isogenic backgrounds, it is difficult to ascertain if the variation of expression is due to differences in genetic background or to intrin- sic differences inherent to each of the two g_mutations. Third, results from genetic complementation tests involving alleles of the $31 complementation group with the 39 mutation (g9 complementation group) show that the number of viable offspring varies 1 with each allele in the 5V complementation group (Bennett and Dunn, 1964). The relative viability of offspring heterozygous for £9 and 1 the 3? mutations were: 5Y1. 85%, £83, 90% (strongly complements); £w12’ 59%; and gyzo, 1.2% (weakly complements). Similar observations have been made in genetic complementation tests involving other 5 mutations (Lyon, gg.gl., 1979b). The differences in magnitude of degree of viability is not thought to result from variations in genetic background(8ennett and Dunn, 1964). Chromosomal structural differ- wl ences among the g_ mutations have been proposed to explain the varia- tion of viability observed in complementation tests involving the t0 Ewl mutation and g mutations in the complementation group (Bennett and Dunn, 1964). Existence of such chromosomal structural differences wl would imply that members of the g complementation group are not truly allelic. The variation of degrees in complementation of 3&1 with t0 mutations provides a strong argument for differences of gene action 1 mutation. Each mutation may alter different cellular in each 3? processes and interactions, even though the end result of the gene action encoded by these mutations is manifested as effects on embryonic 99 central nervous system development acting at similar time periods. Until the genes are placed on the same genetic background and homo- zygous mutant embryos are re-examined to determine the mutant effect, the mutations and morphological anomalies caused by the mutations should be considered as separate entities altering similar stages of embryonic development. Since little information was readily available on the gene action w1 of one mutation, g , as expressed in homozygous embryos at 9 to 15 days of gestation, the current study was undertaken.‘ Significant differences were noted in descriptions of embryos homozygous for tw1 given in this thesis and those made by Bennett, ggugl. (1959b). Whether the observed differences result from the various genetic twl backgrounds in which the mutation resides, the methods from*which data was collected (pooled homozygous and compound heterozygous wlxiwl embryos versus single g_ embryo samples), or the influence of 1 the Brach ur (T) mutation upon the t“ mutation (homozygous embryos _____JL_IL _. ._ collected from I:gf/pvlei_x 13gf/gyle:_matings versus fiftyl x :jgyl matings) cannot presently be evaluated. (In the ensuing discussion, references made to Bennett refer to the work performed by Bennett, $31., 1959b.) Differences in Embryonic Observations A summary of the number of embryos examined at different stages tw1 of development and the number of homozygous embryos identified after histological examination by light microscopy is given in Table 16. wl The number of homozygous 5. embryos expected were calculated by using a male segregation distortion factor of 0.9. Since 90%;"1 100 H ee.m m H me. ex .m.o we Leauew sewuseumwu sewuemmgmmm se ummem .mmuemwueses H3m.we amass: use «geese we amass: ms» we saw .mmmmsasmsee sw sm>wm «mew smeEss mmmsewesw we senses weuem mse eegw ueuuwse msm: mseumeese aswuumesm msu sw mme— message we smessz exu m N H mm.N m.e e N m N e_ m. Ne.e N.e N e e m e. m. .N.e e.N_ m_ m_ e_ m eN N_ ee.e e.e e P_ e m eN __ Nm.e e.N_ N_ __ m e Awe eN es. Np.ep N.ew on N e m Ame Ne e, Ne.e N.N_ e. N e N e. «e ee.N ee.e_ N_ NN N e “ANV eN e eNx ~LM\HLI mmmmuesma mewgesu —esgese< ~3M\% mmsee< musewesw . ewes umuemexu :5 we wmessz pemew we smessz we sense: we geese: m2 umuumexm we smessz “um L_ew msemxeese: mewsesm um>emmeo use umpemexm we gmesez mp mpneh 101 bearing sperm and 10% irbearing sperm will fertilize ova, 45% of the embryos in a litter are expected to be homozygous for From t“. days 9 to 10; pg, the number of observed homozygous embryos were deficient. From days 11 to 15 pg, however, the number of observed Ewl and expected homozygous embryos coincided, indicating that embryos homozygous for tW1 were identified at later embryonic stages of develop- ment. In contrast, Bennett was able to identify 74% of the expected number of abnormal embryos at day 9‘pg and 100% of the affected embryos subsequent to day 10 pg. At day 9 pg, the characteristics observed by Bennett in abnormal embryos included: 1) reduction in Size to 1/2 - 3/4 that of normal littermates; 2) presence of microcephaly; and 3) pycnotic cells located in the mantle layer of the ventral portion of the hindbrain, midbrain, and anterior regions of the spinal cord. In the two embryos identified after histological examination in this thesis, the charac- teristics observed for each were that: l) the size of the embryo was similar to a "normal“ littermate; 2) microcephaly was not evident; and 3) pycnotic cells were confined to the mantle layer of the ventral region of the rostral rhombencephalon. The embryos described by Bennett may possibly represent the embryos homozygous for 382° or compound heterozygous for 3720 and gyl’3’ or 12. 20 Approximately 95% of the embryos homozygous for 3? died early and were represented by moles (Bennett, gt 31., 1959a); only 47% of the embryos homozygous for!1 died at the same embryonic age. At days 10 to 11 pg, the characteristics observed in abnormal embryos by Bennett were: 1) retarded (younger developmental age than normal littermates) growth and reduced size; 2) presence of 102 pycnotic cells extending from the hindbrain rostrally in the brain and caudally in the spinal cord; and 3) presence of pycnoses with widespread necrosis and loss of structural integrity in the ventral half of the neural tube. The features described by Bennett were not observed in abnormal embryos described in this thesis until day 11 p_, Few homozygous 3Y1 embryos were identified at days 10 or 10% pg, Of those observed at day 10 pg, pycnotic cells were confined to the mantle layer of the ventral portion of the mesencephalon and rhombencephalon. The focus of degeneration corresponded to that observed at day 9 pg. The decreased width of the neural tube in affected regions may correspond to a decrease in the number of neuro- epithelial cells undergoing mitotic division. Of those observed at day 10} pg, the degree of degeneration did not seem as severe, but the width of the neural tube was decreased and the marginal layer was not prevalent. At day 11 pg, however, abnormal embryos were recognized and the characteristics observed by Bennett, except number 3, were also noted. The loss of structural integrity of the ventral half of the neural tube was rarely present at this stage of develop- ment. At day 12 pg Bennett observed that the degree of necrosis and structural degeneration was never as severe as that observed at day 11 pg; in fact, considerable areas were almost free of pycnosis but deficient in the number of typical neural cells. In embryos described herein, the degree of pycnosis and degeneration in the neural tube was severe and the neural tube was beginning to lose its structural integrity. "Recovery" of the neural tube in abnormal embryos was not present. 103 At day 13 pg, general external features described by Bennett included: edema, microcephaly, and enlarged hearts. Histologically, few pycnotic cells were observed in the brain and spinal cord; spinal ganglia were normal, and vertebral cartilage formation had begun. In abnormal embryos described in this thesis, external features of abnormal embryos included retarded size and occasionally, microcephaly and enlarged hearts. Histologically, the neural tube was disorganized and had lost structural integrity. Although spinal ganglia and ossi- fication centers were present, non-neural as well as neural structures were degenerating. Structures in older embryos (day 15 pg) were almost impossible to identify since tissue was being resorbed. In summary, from observations given herein, the mutation 5V1 acts prior to day 11 pg, but its effects are not overtly noticeable until day 11 pg, The degree of pycnosis and degeneration have been sumnarized for various embryonic stages of development in Figure 25. The initial action of the gene appears to be on neuroepithelial cells or neuroblasts found in the mantle layer of the neural tube. The focus is in the ventral portion of the neural tube at the rostral border of the rhombencephalon (Figure 25A). The presence of pycnotic cells follows a specific pattern of development, spreading rostrally and caudally in the ventral portion of the neural tube and caudally in the dorsal portion of the neural tube (Figure 25A-E). Pycnotic cells were pgygg observed in the most basal portion of the neural tube in the region above the notochord. The biochemical basis of this defect is presently unknown but may correlate with the inability of neuroepithelial cellsto divide and/or neuroblast cells to migrate within the neural tube. Both Figure 25. 104 Reconstructed composite view of mouse embryos from serially sectioned material. The neural tube is depicted by a heavy black line. Dotted lines repre- sent the developing eye and otic vesicle. Pycnotic cells found in sections of the developing neural tube are shown by dots (x12). A. Abnormal day 9 embryo showing restriction of pycnotic cells to the rostral portion of the rhombencephalon. Abnormal day 94 embryo showing the increased number of pycnotic cells in the ventral portion of the neural tube. Abnormal day 10 embryo showing the continued in- crease in numbers of pycnotic cells and confine- ment of pycnoses to the ventral neural tube. Abnormal day 10% embryo showing pycnotic cells in dorsal and ventral regions of the neural tube. Abnormal day 10 embryo from litter dissected at 11 days. Non-neural as well as neural structures are degenerating (only pycnotic cells in the nueral tube are shown). 105 O 106 mitotic and migratory processes may rely upon the synthesis of spe- cific classes of glycosaminoglycans, a group of highly branched carbo— hydrates composed of repeating dissaccharide units and usually linked to a protein core (proteoglycans; Margolis and Margolis, 1979; Lennarz, 1980). Glycosaminoglycans (GAG) are important components of the extracellular matrix (ECM) and may play a role in cellular migration (Toole, 1972, 1973; Pratt, ggng1., 1975), cellular differentiation (Shur and Roth, 1973), and cellular morphology (Cohn, pp 31., 1977; Solursh and Morriss, 1977). A correlation exists in synthesis of GAG by the neural tube and notochord, accumulation of GAG into the basal laminae and ECM, and changes in neural tube and mesenchymal cell morphology (Hay and Meier, 1974; Morriss and Solursh, 1978). tw1 If abnormal synthesis of GAG occurs in embryos homozygous for , then neural tube cell morphology may be altered and cause the secon- dary defects observed in abnormal embryos. One class of enzymes, glycosyltransferases, are required for GAG synthesis (Margolis and Margolis, 1979) and may be involved in cell recognition and migration (Shur and Roth, 1973). One model has suggested that cells migrate over carbohydrate chains (Shur, 1977a). Studies supporting this hypothesis have been performed on different aged chick embryos with various glycosyltransferases (Shur, 1977a,b; Roth, 1979). Of particular importance is the presence of galactosyltransferases in neural tube regions of developing chick embryos (Shur, 1977a), and the teratogenic effects of excess UDP- galactose and UDP-N-acetylglucosamine during gastrulation (Roth, 1979). Since abnormalities in glycosyltransferase activities have been reported in the g_mutants (Shur and Bennett, 1979; Shur, pg gl., 107 1979), it is possible that proteins involved in synthesis of carbo- hydrate containing macromolecules may represent a primary target of the mutant gene 381 during embryonic development. In Vitro Growth of Primary Explants from tWI/tWI Embryos The 3 mutations can be divided into two groups on the basis of time of gene expression: those mutants acting early during embry- onic development and causing death prior to organogenesis and those mutants acting shortly after the beginning of organogenesis and causing death prior to birth (Fujimoto and Yanagisawa, 1979). Studies have shown that primary explants of embryonic tissue from early acting 3 mutations _t_6, gws , and-_t_12 do not grow in culture (Wudl and Sherman, 1976; Wudl, ggngl,, 1977), while primary explants from [/1 embryos, a "late" acting I_mutation, do grow (Ephrussi, 1935; Yanagisawa and Fujimoto, 1977; Yanagisawa, pg_gl,, 1980). 3Y1 is classified as a late acting mutation, and primary explants from mouse embryos of all three genotypes (1/1, i/LWI. and g" llgrl) were cultured jp_ vitro. The data given in this thesis represents the first report on jp_vitro culture of a "late" acting recessive g_mutation. Since the explants originated from embryos in matings between heterozygous (j/gyl) males and females, the number of explants from Lwl embryos homozygous for was expected to be greater than 25%. Segre- gation distortion, a phenomenon that occurs in heterozygous males, alters the expected genotypic ratios from 25% LVI/tfll : 50% 3/5N1 : 25% y: to 45% gwl/t“ : 50% fig“ : 5% :71 because 90% of the sperm 1 will fertilize ova (see Figure 2).‘ Of the 190 primary explants studied, 85 were expected to be 531/331. Since homozygous 5U carrying 33 1 108 embryos die by day 12 pg_jp_utero, tissue excised from these embryos wI/Ewl might also die 1p vitro. If explants from 3 embryos do die jp_vitro, then only 55% of the cultures would be expected to succeed. The number of successful cultures (94%) far exceeded the number pre- explants have been shown to dicted (55%). Hence, homozygous 371 survive jp_vitro long past the time that they would normally die 1 vivo. The length of time that cells survived jp_vitro was important. "lltfil embryos did If explants and cells derived from explants of.g not live past their jpugggpg_equivalent gestational age of death, usually 12 days pg, then the results would support the conclusion that all cells of gUI/gyl embryos were "programmed" for cell death, an observation that has been made to 59/38 and th5/3Y5 embryos (Wudl and Sherman, 1978; Hogan, gphg1,, 1980). However, 94% of the explants survived for at least three weeks. Many lasted for three to four months, when the study ended. Primary explants from embryos which had been identified as 381/331 on the basis of gross and histological examinations of embryos were as viable as primary explants from normal (i/i,‘:/pvl) littermates. Provided that the embryo from which the explants were taken was not dead (as defined by the presence of a beating embryonic heart), the mutant explants had similar cell morphologies, survived ipnyiggg for extended periods of time, and were successfully subcultured. The significance of these studies is that the gene does not appear to affect all cells of the mutant embryo; otherwise in vitro cell growth would not be possible. The gene appears to affect specific cells 109 within the developing neural tube and would, therefore, appear to be an intrinsic cell specific lethal. Determination of Embryonic Genotypes Based on H-2 Typing One property of the g_mutations that can be advantageous is cross-over suppression. Suppression of recombination extends from the I_locus to the distal end of the H12 locus and includes the gfl mutation (Klein and Hammerberg, 1977). As a result, the £31 mutation is tightly linked to the H;g_locus (1% recombination), and the two loci are inherited as a unit. Since the H-2 haplotype, H-thl, asso- ciated with the t“ mutation is distinctly different from the H- 2 haplotype, H-2b, associated with the normal allele (Klein, e_t 31.. 1978; Klein, 1975), the different H-2 haplotypes expressed in cell lines would correlate with the genotypes of the embryos from which the cultured tissue was taken. Three possible embryonic genotypes derived fromg/tW1 xii/tW1 :’ lib/i: I.|_2b; i: flb/LWIs flit“; and E“, flth/iwl’ fltwl. matings and their H-2 haplotypes are: Thus experiments were performed in an attempt to identify haplotypes of fibroblasts growing in tissue culture and derived from primary embryonic explants. Determination of haplotypes would be represen- tative of the genotypes of the embryos. Initial results on the determination of the H-Zb haplotype asso- ciated with :[:_fibroblasts implied that the genotypes of embryos could be ascertained from H-2 types of fibroblasts derived from embry- onic explants. Results from subsequent studies were, however, erratic and might be explained by altered expression of H-2 antigens during 110 culturing and/or low concentrations of the H-2 antigens on the cell surface. Several reports have indicated that the detection of H-2 antigen expression is dependent upon the age of the culture, the number of times that cells were subcultured, the specific haplotype under examina- tion, the tissue source of cultured cells, and the antiserum batch employed (Klein, 1965; Edidin, 1976; Holtkamp, ggwgl., 1979; Ostrand- Rosenberg, ggugl., 1980; Kirkwood and Billington, 1981). Utilization of a more sensitive method, such as combined absorption and cytotoxic assays, may alleviate problems of sensitivity and reproducability (Klein, 1975). In addition, the effects of culturing cells derived from primary embryonic explants on H-2 expression should be investigated to determine if and how expression of H-2 antigens on cultured cells grown_i_pvitro change with time and subculturing. SUMMARY AND PROSPECTIVE RESEARCH Experiments and observations described herein on mouse mutant le indicate that the action of the gene affects only some cell types within the neural tube at specific stages of development. Embryonic tissue derived from homozygous embryos can be grown in vitro and will survive beyond the lethal period observed for homozygous embryos jp_utero. Although the primary action of the gene is unknown, the twl growth and maintenance of the neural tube in homozygous embryos 531 mutation. One possible explanation appears to be inhibited by the is that the 3V1 mutation affects the activity of a protein required for the synthesis of carbohydrate containing macromolecules, and thereby alters the synthesis, composition, and distribution of glycos- aminoglycans in the neural tube and surrounding regions. The hypothesis that the neural tube growth and maintenance are inhibited by the 3V1 mutation will be examined in future studies. Mutant embryos will be analyzed with respect to: l) developmental interrelationships between the processes of proliferation and degenera- tion in the developing CNS, 2) the nature of prenatal necrosis and its cellular consequences at the fine structural level, and 3) the status of GAG within the basal laminae of the neural tube and extra- cellular matrix Of nearby mesenchymal cells. 111 LIST OF REFERENCES LIST OF REFERENCES Altman, P.L. and Katz, 0.0., eds.. 1979. Inbred and Genetically Defined Strains of Labpratory Animals, Part 1. Federation of American Societies for Experimental Biology, Bethesda, Md. Alton, A.K., Silver, L.M., Artz, K. and Bennett, 0. 1980. Molecular analysis of the genetic relationship of trans interacting factors at the I/p_comp1ex. Nature 288, 368-370. Amos, 0.8., Bashir, H., Boyle, W., MacQueen, M. and Tiilikainen, A. 1969. A simple microcytoxicity test. Transplantation 7, 220-223. Bennett,[L 1958. In vitro study of cartilage induction in [/1 mice. Nature 181, 1286. Bennett, 0. 1975. The T-locus of the mouse. Cell 6, 441-454. Bennett, 0. 1978. Genetically programmmed abnormalities of cell inter- actions. Birth Defects: Original Article Series, Vol. XIV, No. 2, 285-303. Bennett, 0. and Dunn, L.C. 1964. Repeated occurrences in the mouse of lethal alleles of the same complementation group. Genetics 49, 947-958. Bennett, 0., Dunn, L.C. and Badenhausen, S. 1959a. A second group of similar lethals in populations of wild house mice. Genetics 44, 795-802. Bennett, 0., Badenhausen, S. and Dunn, L.C. 1959b. The embryological effects of four late-lethal g-alleles in the mouse, which affect the neural tube and skeleton. J. Morph. 105, 105-143. Bennett, 0., Dunn, L.C., Spiegelman, M., Artz, K., Cookingham, J. and Schermerhorn, E. 1975. Observations on a set of radiation-induced dominant I-like mutations in the mouse. Genet. Res., Camb. 26, 95-108. Boulder Committee. 1970. Embryonic vertebrate central nervous system: Revised terminology. Anat. Rec. 166, 257-262. 112 113 Braden, A.W.H. 1958. Influence of time of mating on the segregation ratio of alleles at the I-locus in the house mouse. Nature 181, 786-787. Chesley, P. 1935. Development of the short-tailed mutant in the house mouse. J. exp. Zool. 70, 429-455. Cohn, R.H., Banerjee, 5.0. and Bernfield, M.R. 1977. Basal lamina of embryonic salivary epithelia. J. Cell. Biol. 73, 464-478. Danska, J.S. and Silver, L.M. 1980. Cell-free translation of a I/t complex cell surface-associated gene product. Cell 22, 901-904. DobrovolskaIa-Zavadskaia, N. 1927. Sur la mortification spontanée de la queue chez la souris nouveau-née et sur 1'existence d'un caragtéur (facteur) héréditaire "non viable". Comptes Rendus Societe de Biologie 97, 114-119. Dobrovolskaia-Zayadskaia, N. and Kobozieff, N. 1932. Les souris anoures et a queue filiforme qui se’reproduisent entre elles sans disjonction. Comptes Rendus Societe de Biologie 110, 782-784. Edidin, M. 1976. The appearance of cell-surface antigens in the develop- ment of the mouse embryo: a study of cell-surface differentiation. Ip; Embryogenesis in Mammals, Ciba Foundation Symposium 40, Else- vier, New York, pp.177-197. Ephrussi, B. 1935. The behavior 1p vitro of tissues from lethal embryos. J. exp. Zool. 70, 197-204. Erickson, R.P., Lewis, S.E. and Slusser, K.S. 1978. Deletion mapping of the tycomplex of chromosome 17 of the mouse. Nature 274, 163- 164. Fujimoto, H. and Yanagisawa, K.0. 1979. Effect of the Ifmutation on histogenesis of the mouse embryo under the testis capsule. J. Embryol. exp. Morph. 50, 21-30. Geyer-Duszynska, I. 1964. Cytological investigations on the I-locus in Mus musculus L. Chromosoma 15, 478-502. Gluecksohn-Schonheimer, S. 1940. The effect of an early lethal (59) in the house mouse. Genetics 25, 391-400. Green, E.L., ed. 1968. Biology of the Laboratory Mouse. New York, N.Y., Dover Publications, Inc. Green, E.L. 1981. Genetics and Probability in Animal BreedinggExperi- ments. Oxford University Press, New York. GrUneberg, H. 1943. The development of some external features in mouse embryos. J. Hered. 34, 89-92. 114 Guénet, J- L wBongaane,H ,Gaillard, J. and Jacob, F. 1980. gVPa'I, tw “P '2, tw three new t- haplotypes in the mouse. Genet. 'Res. , Camb. 36, 211- 217. Hammerberg, C. 1981. The influence of TOr r1 upon male fertility in .g-bearing mice. Genet. Res. , Camb. 37, 71- 77. Hammerberg, C. and Klein, J. 1975a. Evidence for postmeiotic effect of t factors causing segregation distortion in mouse. Nature 253, 137- 138. Hammerberg, C. and Klein, J. 1975b. Linkage disequilibrium between H- 2 and t complexes in chromosome 17 of the mouse. Nature 258, 296- 299. Hammerberg, C. and Klein, J. 1975c. Linkage relationships of markers on chromosome 17 of the house mouse. Genet. Res., Camb. 26, 203-211. Hammerberg, C., Klein, J., Artzt, K. and Bennett, 0. 1976. Histocom- patibility-2 system in wild mice. 11. H-2 haplotypes of g-bearing mice. Transplantation 21, 199-213. Hauptfeld, V., Hammerberg, C. and Klein, J. 1976. Histocompatibility- 2 system in wild mice. III. Mixed lymphocyte reaction and cell- mediated lymphocytotoxicity with g-bearing mice. Immunogenetics 3, 489-497. Hay, E.0. and Meier, S. 1974. Glycosaminoglycan synthesis by embryonic inductors: neural tube, notochord, and lens. J. Cell Biol. 62, 889-898. Hillman, N. and Hillman, R. 1975. UltrastrUCtural studies of tW32/t "32 mouse embryos. J. Embryol. exp. Morph. 33, 685- 695. Hillman, N. and Nadijcka, M. 1978. A comparative study of spermiogene- sis in wild-type and Trip-bearing mice. J. Embryol . exp. Morph. 44, 243-261. Hillman, N. and Nadijcka, M. 1978. A study of spermatozoan defects in wild-type and TEE-bearing mice. J. Embryol. exp. Morph. 44, 263-280. Hillman, N. and Nadijcka, M. 1980. Sterility in mutant (th/tLy) male mice. 1. A morphological study of spermiogenesis. 3'. Embryol. exp. Morph. 59, 27- 37. Hogan, 8., Spiegelman, M. and Bennett, 0. 19 O. In vitro development of inner cell masses isolated from to/g and tW57tW5 mouse embryos. J. Embryol. exp. Morph. 60, 419- 428. 115 Holtkamp, 8., Lindahl, K.F., Segall, M. and Rajewsky, K. 1979. Spon- taneous loss and subsequent stimulation of H-2 expression in clones of a heterozygous lymphoma cell line. Immunogenetics 9, 405-421. Johnson, 0.R. 1974. Hairpin tail: A case of post-reductional gene action in the mouse egg? Genetics 76, 795-805. Kirkwood, K.J. and Billington, W.0. 1981. Expression of serologically detectable H-2 antigens on mid-gestation mouse embryonic tissues. J. Embrol. exp. Morph. 61, 207-219. Klein, J. 1965. The ontogenetic development of H-2 antigens in vivo and jp_vitro. In: Blood Groups of Animals, J. MatouSek_TEd.), Dr. W. Junk, Publishers, The Hague, The Netherlands. PP. 405-414. Klein, J. 1975. Biology of the Mouse Histocompatability-Z Complex. New York, Springer-Verlag. Klein, J. and Hammerberg, C. 1977. The control of differentiation by the I-complex. Immunological Reviews 33, 70-104. Klein, J., Flaherty, L., Vanderberg, J.L. and Shreffler, 0.C. 1978. H-2 haplotypes, genes, regions, and antigens: First listing. Immunogenetics 6, 489-512. LeMevel, B.P. and Wells, S.A. 1973. A microassay for the quantitation of cytotoxic anti-tumor antibody: Use of 1 51-iododeoxyuridine as a tumor cell label. J. Natl. Cancer Inst. 50, 803-806. Lennarz, W.J., ed. 1980. The Biochemistry of Glycoproteins and Proteo- glycans. Plenum Press, New York. Luna, L.G., ed. 1968. Manual of Histologic Staining Methods of the Armed Forces Institute of PathOlogy. McGraw Hill Book Co., New York. Lyon, M.F. 1959. A new dominant I-allele in the house mouse. J. Hered. 50, 140-142. Lyon, M.F. and Bechtol, K.B. 1977. Derivation of mutant g-haplotypes of the mouse by presumed duplication or deletion. Genet. Res. Camb. 30, 63-76. Lyon, M.F. and Mason, I. 1977. Information on the nature of g-haplo- types from the interaction of mutant haplotypes in male fertility and segregation ratio. Genet. Res., Camb. 29, 255-266. Lyon, M.F. and Meredith, R. 19648. Investigations of the nature of t-alleles in the mouse: Genetic analysis of a series of mutants Derived from a lethal allele. Heredity 19, 301-312. 116 Lyon, M.F. and Meredith, R. 1964b. Investigations of the nature of t-alleles in the mouse: II. Genetic analysis of an unusual mutant allele and its deriviative. Heredity 19, 313- 325. Lyon, M.F. and Meredity, R. 1964c. Investigations of the nature of Ip-alleles in the mouse: III. Short tests of some further mutant alleles. Heredity 19, 327-330. Lyon, M.F., Evans, E.P., Jarvis, S.E. and Sayers, I. 19798. .g-Haplo- types of the mouse may involve a change in intercalary DNA. Nature 279, 38-42. Lyon, M. F. Jarvis, S. E, Sayers, I. and Johnson, 0. R. 1979b. Comple- mentation reactions of a lethal mouse t- haplotype believed to include a deletion. Genet. Res. , Camb. 33, 153- 161. Margolis, R.U. and Margolis, R.K., eds. 1979. Complex Carbohydrates of Nervous Tissue. Plenum Press, New York. McGrath, J. and Hillman, N. 19808. Sterility in mutant (tL x/tLy) male mice. III. 1p_vitro fertilization. J. Embrol. exp. Morph. 59, 49-58. McGrath, J. and Hillman, N. 1980b. Thel _p_v wtro transmission frequency ofjtheIglz mutation in the mouse. J. Em Bro 1. exp. Morph. 60, 14 -151. McLaren, A. 1976. Genetics of the early mouse embryo. Ann. Rev. Genet. 10, 361-388. Mintz, 8.1964. Formation of genetiga ily mosaic mouse embryos and early development of lethal (t1)-normal mosaics. J. exp. 2001. 157, 273-292. Morriss, G.M. and Solursh, M. 1978. Regional differences in mesen- chymal cell morphology and glycosaminoglycans in early neural-fold stage rate embryos. J. Embryol. exp. Morph. 46, 37-52. Murrell, L. R. 1979. Vertebrate cell culture: An overview. In: Practical Tissue Culture Applications, K. Maramorosch and H. Hiruml, (Eis. 1'.'ACademic Press, New York 9 PP. 9-25. Nadijcka, M. and Hillman, N. 1980. Sterility in mutant (th/tLy) male mice. 11. A morphological study of spermatozoa. J. Embryol. exp. Morph. 59, 39-47. Oldham, R. K. and Herberman R. B. 1976. Determination of cell-mediated cytotoxicity by the1 125 51- labeled iododeoxyuridine microcytotoxicity assay. In: In Vitro Methods in Cell-Mediated and Tumor Immunity, B. R. Bloom and J. R. David, ’(Eds. ), Academic Press, New York, pp. 461 -470. 117 Ostrand-Rosenberg, 5., Hammerberg, C., Edidin, M. and Sherman, M.I. 1977. Expression of histocompatibility-2 antigens on cultured cell lines derived from mouse blastocysts. Immunogenetics 4, 127-136. O'Toole, C. and Clark, E.A. 1976. Guidelines for microcytoxicity assay. In: Methods in_§ell-Mediated and Tumor Immunity, B.R. Bloom and J.R. David, (Edsil, Academic Press, New York, pp. 437- 449. Penso, G. and Balducci, D. 1963. Tissue Cultures in Biological Research. Elsevier Publishing Co., New York. Pratt, R.M., Larsen, M.A. and Johnston, M.C. 1975. Migration of cranial neural crest cells in a cell-free hyaluronate-rich matrix. Devel. Biol. 44, 298-305. Roth, S. 1979. Plasma membrane glycosyltransferases and morphogenesis. In: Mechanisms of Cell Change, J.D. Ebert and 1.5. Okada, (Eds.), John Wiley and Sons, New York, pp. 215-223. Searle, A.G. 1966. Curtailed, a new dominant I-allele in the house mouse. Genet. Res., Camb. 7, 86-95. Sherman, M.I. 1975. The role of cell-cell interaction during early mouse embryogenesis. In: The Early Development of Mammals, M. Balls and A.E. Nild, (Eds.), Cambridge University Press, New York, pp. 145-165. Sherman, M.I. and Nudl, L.R. 1977. .I-complex mutations and their effects. In: Concepts in Mammalian Embryogenesis, M.I. Sherman, ed., The MIT Press, Cambridge, MA., pp. 136-234. Shur, 8.0. 1977. Cell-surface glycosyltransferases in gastrulating chick embryos. I. Temporally and spatially specific patterns of four endogenous glycosyltransferase activities. Devel. Biol. 58, 23-39. Shur, 8.0. 1977. Cell-surface glycosyltransferases on gastrulating chick embryos. II. Biochemical evidence for a surface localization of endogenous glycosyltransferase activities. Devel. Biol. 58,40-55. Shur, 8.0. and Bennett, D. 1979. A specific defect in galactosyl- transferase regulation on sperm binding mutant alleles of the Ijt_locus. Devel. Biol. 71, 243-259. Shur, 8.0. and Roth, S. 1973. The localization and potential function of glycosyltransferases in chick embryos. Am. 2001. 13, 1129-1135. Shur, B.D., Oettgen, P. and Bennett, D. 1979. UDP-galactose inhibits blastocyst formation in the mouse: Implications for the mode of action of I7t-complex mutations. Devel. Biol. 73, 178-181. 118 Silver, L.M. and Artzt, K. 1981. Recombination suppression of mouse t-haplotypes due to chromatin mismatching. Nature 290, 68-70. Silver, L.M., Artzt, K. and Bennett, 0. 1979. A major testicular cell protein specified by a mouse 17; complex gene. Cell 17, 275-284. Silver, L.M., White, M. and Artzt, K. 1980. Evidence for unequal crossing-over within the mouse 17; complex. Proc. Natl. Acad. Sci. USA 77, 6077-6080. Snell, 6.0. 1968. The H-2 locus of the mouse: Observations and speculations concerning its comparative genetics and its poly- morphism. Folia Biol. 14, 335-358. Snell, G.D., Dausset, J. and Nathenson, S., Eds. 1976. Histocompati- bility. Academic Press, New York, pp. 1-66 and 221-227. Snow, M.H.L. and Bennett, D. 1978. Gastrulation in he mouse: Assess- ment of cell populations in the epiblast of 3M1 [3M13 embryos. J. Embryol. exp. Morph. 47, 39-52. Solursh, M. and Morriss, G.M. 1977. Glycosaminoglycan synthesis in rat embryos during the formation of the primary mesenchyme and neural folds. Devel. Biol. 57: 75-86. Spiegelman, M. 1975. Electron microscopy of cell associations in 1} locus mutants. In: Symposium on Embryogenesis in Mammals, K. Elliot and M. O'Connor, (Eds.l:’Elsevier, Amsterdam, The Nether- lands. pp. 199-220. Spiegelman, M. 1978. Fine structure of cells in embryos chimeric for mutant genes at the T73 locus. In: Genetic Mosaics and Chimeras in Mammals. Plenum Press, New York, pp. 59-80. Spiegelman, M., Artzt, K. and Bennett, 0. 1976. Embryological study of a T7; locus mutation (tM73) affecting trophectoderm development. J. Embryol. exp. Morphol. 36, 373-381. Toole, B.P. 1972. Hyaluronate turnover during chondrogenesis in the developing chick limb and axial skeleton. Devel. Biol. 29, 321-329. Toole, B.P. 1973. Hyaluronate and hyaluronidase in morphogenesis and differentiation. Am. 2001. 13, 1061-1065. Tucker, M.J. 1980. Explanation of sterility in t§[§¥ male mice. Nature 288, 367-368. Neesner, F.M. 1960. General Zoological Microtechniques. The Williams and Wilkins Co., Baltimore. 119 Nudl, L.R. and Sherman, M.I. 1976. In vitro studies of mouse embryos bearing mutations at the I}1ocus: EP5 and t12. Cell 9, 523- 531. Nudl, L. R. and Sherman, M. I. 1978. nvitr studies of mouse embryos bearing mutations in the T-complex: J. Embryol. exp. Morph. 48, 127- 151. Nudl, L.R., Sherman, M.J. and Hillman, N. 1977. Nature of lethality of 3 mutations in embryos. Nature 270, 137-140. Yanagisawa, K.0. and Fujimoto, H. 1977a. Viability and metabolic activity of homozygous Brachyury (1) embryos. J. Embryol. exp. Morph. 40, 271-276. Yanagisawa, K. 0. and Fujimoto, H. 1977b. Differences in rotation- mediated aggregation between wild- -type and homozygous Brachyury (T) cells. J. Embryol. exp. Morph. 40, 277- 283. Yanagisawa, K. 0. and Fujimoto,H 1978. Aggregation of homozygous Brachyury (T) cells in the culture supernatant of wild- -type or mutant embryos. Exp. Cell Res. 115, 431 -435. Yanagisawa, K.0., Urushihara, H., Fujimoto, H., Shiroishi, T. and Moriwaki, K. 1980. Establishment and characterization of cell lines from homozygous Brachyury (1]1) embryos of the mouse. Differentiation 16, 185-188.