ALT EREB PATTERN 0F GENE ACTIVITY IN ABNORMAL SEA URCBEN BEORPIEOGENESIS A Dissertahon Ecr his Degree c-E DE}. D. EEECEEESAEE SEEEEE EJflEVERSi'EY Wiliiam Robe-2t Eckberg 1975 1 .b 1‘ hFSfi “" This is to certify that the thesis entitled ,1 _ . Altered Pattern of Gene Activity in Abnormal Sea Urchin Morphogenesis presented by William Robert Eckberg has been accepted towards fulfillment of the requirements for Pho D; degree in 20010“ Major pr fess Dat " / 75“ 0-7639 . n.“ --_v- afarnnfld , ‘ ' ~‘ - " L" n xfl‘ .1' I q ’ ‘ ‘ u 3| U’bgll‘. 5 E“ a j" 4 «\n' 11“] ' ‘- 5‘ 7‘ — an *- « f. — ~ :‘_n r15: . 3-; m' Jim i‘.‘ : . w ‘. R'C'. 07 ' to -; ‘fi 1 was 3.».3 "u : 2r. - Hg.- ; . c . preaq. 'J‘i. it .* J. Lezjation la. Shiny». n e .,L:-L --:-; -, mam-sat” j ...u ~. am. who mm» it " i“ 01 ”‘4", . .fifip, ,3,__.U.3,1,‘..,_...; -:~va_ F’yrw‘afi W .V #5 law j ‘. 1.: L. -‘ ' thficL. : ‘f'v ' “-' “' °”' ’ ‘ in a” m tote“. .«m ‘ ifiktmme J' a? ’saém: "mauve; “W5“! ' '5 , \H" x -. vi". ‘i ‘1 fl _ ‘iuiy-Jaieiuu. saw was exam '~ *: ; . r3» EW'J' L radian-h W‘ ‘2" and by an rm warm new -:- . w» “Le-a. “,me nus. {an-“him“ type; of m mien :, be hetr’rogew my awaiting a. {fatness ir‘ :L‘ H ~A_ a 37 ‘ L - ' M I 'U i ; .l ‘y , ‘ _‘. 4. ”~_ . ' \ i. - ,v/L us. ABSTRACT ALTERED PATTERN OF GENE ACTIVITY IN ABNORMAL SEA URCHIN MORPHOGENESIS BY William Robert Eckberg Early sea urchin embryos can be experimentally manipulated, by addition of excess Zn++ to the sea water, such that abnormal embryos develop with exaggeration of their ectodermal character— istics and suppression of their mesentodermal characteristics. In order to test whether this alteration in development involves changes in the pattern of embryonic gene activity, the rate of transcription and the variety of transcription products in normal and animalized embryos were examined. As an estimate of the rate of transcription, the kinetics of 3H-uridine incorporation into RNA of the embryos was examined. Cleavage stage embryos of both groups incorporate uridine at a low rate which increases after hatching in the normals and begins to level off at the prism stage. This acceleration is delayed by about 6 hours in the animalized embryos but the same rate is achieved by these in later development. This difference is not due to any differential permeability to precursor, nor can it be correlated with a difference in cell number between normal and animalized embryos. Rapidly-labelled RNA was examined by sucrose gradient sedi- mentation and by RNA/DNA hybridization in DNA excess. Rapidly- labelled RNA from both types of embryos consists of heterogeneous, high molecular weight material, although some differences in size “'1I"" classes were observed. This RNA hybridized to non-repetitive DNA sequences readily, but to repetitive DNA sequences to a much lower extent. In order to determine the complexity of the non—repetitive DNA sequences represented in RNA of normal and animalized embryos, hybrid formation between purified radioactive non-repetitive gastrula DNA and excess RNA was determined. RNA was isolated from unfertilized eggs and from normal blastulae and prism larvae and from animalized embryos of comparable ages. The results demonstrate that the complexity of transcription in- creases during the development of both normal and animalized embryos. Experiments in which RNAs isolated from two stages were combined indicated that extensive homology exists between the populations although some differences were detected between embryos of different ages as well as between normal and animal- ized embryos of the same age. The evidence presented above indicates that animalization involves alterations in the pattern of embryonic gene activity and therefore that alterations in embryonic gene activity may be induced in embryos merely by altering the embryos' ionic environment. It is suggested that this effect on gene activity may be a secondary reflection of primary effects of Zn++ on the cell surface. The alterations in gene activity may be causal in the alteration of development. ALTERED PATTERN OF GENE ACTIVITY IN ABNORMAL SEA URCHIN MORPHOGENESIS BY “William Rebert Eckberg A DISSERTATION Submitted to Michigan State University 9in artial fulfillment of the requirements NOS 1. , 'fijn for the degree of . m are I'. _. DOCTOR OF PHILOSOPHY . “21:1. ' 5 - and a K ’ Department of Zoology ion“ .hnp; ~ "'r'. 3 4 p. . 2 ~ ’7‘. f‘ . ' . I ,;' 2 L a . “it . r." ‘ ‘5: 7". a“ §~ ‘1 {i ‘vv . ‘.' ‘ 7' 1 Y / 2‘ I V E; ’ - . , - . - I . .A' . »» .2” _ , LT; y“ .. ‘ iik :‘ 7 a? 14misa ‘ ' $3.133”. . ..’"“i". 7 '7; ,1, 139’“ : ' ‘ a? ”a‘ ACKNOWLEDGMENTS ‘I would like to express special thanks to Dr. Hironobu V‘;Ozaki, my thesis advisor, for his advice, encouragement, .and support during the research for and writing of this dis- L-fiertation. Thanks also to Drs. Neal Band, John Shaver, liritz Rottmann, John Boezi, and the late Charles Thornton ‘;_£or their help in serving on my Committee and for their pro- fifi}t§asional criticism of my ideas and work. ~ Jfltx Thanks also to the Zoology staff, especially to Drs. [Qhornton and Shaver and to Vicki Conklin for their assistance 'H, 6 support whenever I needed anything. \ g . Most importantly, thanks to all my friends here, some of ' *5 on are mentioned above. Without their moral support and ‘ 'e2; se of humor in good and bad times, none of this could have - r o 4 1" yiegn done. '3. TABLE OF CONTENTS Page LIST OF TABLES. . . . . . . . . . . . . . . . . . . . . v LIST OF FIGURES . . . . . . . . . . . . . . . . . . . . vi INTRODUCTION. . . . . . . . . . . . . . . . . . . . . . 1 General Introduction . . . . . . . . . . . . . . . 1 Control of Development by Variable Gene Activity . 2 Development of the Gradient Theory of Sea Urchin Morphogenesis . . 4 Non- genetic Theories of Animalization and. Vegetal- ization . . . . . . . . . . . . . 7 Gene Activity and the Gradients. . . . . . . . . . 9 Gene Activity and Gastrulation . . . . . . . . . . 12 Control of Development by Ions . . . . . . . . . . 13 STATEMENT OF THE PROBLEM. . . . . . . . . . . . . . . . 15 MATERIALS AND METHODS . . . . . . . . . . . . . . . . . 16 Culture of Embryos . . . . . . . . . . . . . . . . 16 Chemical Animalization . . . . . . . . . . . . . 16 Determination of the Number of Cells per Embryo. . l6 Uptake and Incorporation of Radioactive Uridine. . 17 Preparation of Radioactive RNA . . 18 Sucrose Gradient Centrifugation Analysis of Radio— active RNA. . . . . . . . . . . . . 18 Extraction of Sea Urchin Sperm DNA . . . . . . . l9 Shearing and Alkaline Sedimentation Analysis of DNA . . . . . . . . . . . . . 19 Reassociation of Sea Urchin sperm DNA. . . . . . . 20 RNA/DNA Hybridization in DNA Excess. . . . . . . . 21 Preparation of Radioactive Non-repetitive DNA. . . 22 RNA/DNA Hybridization in RNA Excess. . . . . . . . 23 Thermal Stability of Hybrids . . . . . . . . . . . 25 RESULTS . . . . . . . . . . . . . . . . . . . . . . . . 27 Animalization of Sea Urchin Embryos by Treatment with the Zinc Ion . . . . . . . . . . . . . . 27 Rate of Cleavage . . . . . . . . . . . . . . . 30 Rates of Uridine Uptake and Incorporation. . . . . 31 Molecular Size of Rapidly- Labelled RNA . . . . . . 35 Molecular Size of Sheared DNA Fragments. . . . . . 35 Genomic Structure of the Sea Urchin. . . . . . . . 35 Templates for Rapidly—Labelled Heterogeneous RNA . 40 Genomic Representation in RNA of Normal and Animal- ized Embryos. . . . . . . . . . 44 Thermal Stability of RNA/DNA Hybrids . . . . . . . 50 iii ,EAnimalization by Treatment with Excess Zinc ion. . ”‘gflptake and Incorporation of Radioactive Uridine. . .?.£- '“Characteristics of Rapidly-labelled RNA Molecules. “. gStructure of the Sea Urchin Genome . . . ‘ RNA Transcripts of Normal and Animalized Embryos . Alteration of Sea Urchin Development by zinc . . . .-?EConclusions. . . . . . . . . . . . . . . . . . . . ’ 4-") IX. 0 O o a I a o I o o n o I o o o I I o 0 o o 0 Effect of zinc on the Uptake and Incorporation of ' Valine. . . . . . . . . . . . . . . . . . . . - C . {HST 0F REFERENCES. O o o a I o o n o t o I I o D o 0 o ’ . b 70 74 LIST OF TABLES Page Incubation conditions for saturation of radioactive non repetitive DNA with RNA. . . . . 24 Sea urchin sperm DNA reassociation kinetics. . . . 38 Percent of non-repetitive DNA hybridized to RNA from various developmental stages of normal and animalized embryos . . . . . . . . . . . . . 48 Incorporation of 3H—uridine/nuc1eus and incorpor- ation/uptake/nucleus during a 30 min pulse during the development of normal and animali- zed sea urchin embryos . . . . . . . . . . . . . 56 Percent of purified non-repetitive DNA recover- ed from HAP as apparent DNA/DNA duplex at low Cot Values: 0 O O I O O O C I I I O C O I C 0 62 Percent incorporation into TCA-insoluble material of C valine by normal and animalized sea urchin embryos during a 30 min pulse label . . . 73 “Viv" LIST OF FIGURES FIGURE PAGE 1. a. Normal prism larva of Strongylocentrotus purpuratus. Regional differentiation of the gut and the presence of the skeletal spicules are apparent. (x 250) b. Animalized embryo of the same age and from the same batch as the embryo shown in (a) but raised in the presence of 5 x 10-4M ZnSO4 added to the sea water. The outer diameter of the animali— zed embryo is greater due to the fact that the presumptive endodermal and mesodermal cells are a part of the ectoderm. Aggregated descendents of the primary mesenchyme cells are in the blastocoel and skeletal spicules are lacking. (x 250). . . . . . . . . . . . . . . . . . . . . 28 2. Number of cells during the development of normal (0) and animalized (0) sea urchin embryos and major ‘ morphological events occurring in normal develop— ment. h, hatching blastula; B1My-l, early mesen- chyme blastula; Ga-l, first evidence of invagina- tion; -J4/5, gut 1nvaginated 4/5 of body length; Er, prism larva (Whiteley and Baltzer, 1958) . . 31 3. a. Uptake of 3H-uridine during a 60 min pulse by normal (0) and animalized (0) embryos. b. Incorporation of H-uridine into TCA-insoluble material during a 60 min pulse by normal (I) and animalized (0) embryos . . . . . . . . . . . . . 4. Uptake of 3H—uridine during a 30 min pulse by normal (0) and animalized (0) sea urchin embryos during development. . . . . . . . . . . . . . . . . . . 5. Incorporation of 3H-uridine into TCA-insoluble mat- erial by normal (0) and animalized (0) embryos during development . . . . . . . . . . . . . . . 34 6. Sucrose gradient centrifugation analysis of newly— synthesized RNA from normal (a) and animalized (b) sea urchin embryos labelled for 30 min with 3H-uridine beginning 48 hr post—fertilization. . 36 vi 10. 11. 12. 13. 14. 15. Alkaline sucrose gradient sedimentation analysis of H-thymidine-labelled gastrula DNA. The peak (fraction 8) corresponds to an 82 w equal to 6.3 (Abelson and Thomas, 1966) ang'a single- stranded nucleotide length of 450 (Wetmur and Davidson, 1968). . . . . . . . . . . . . . . . . . 37 Reassociation kinetics of 900 and 450 nucleotide long fragments of sea urchin sperm DNA assayed by hydroxyapatite (HAP) chromatography. The open and filled circles represent data obtained in different experiments using independent prepara— tions of 450 nucleotide long DNA fragments; the filled squares represent data from the 900 nucleotide long fragments. . . . . . . . . . . . . 39 Thermal stability of reassociated sperm DNA/DNA hybrids (O) and 3H-gastru1a DNA/sperm DNA hybrids (O). The Tm's are 82.50 and 80.00, respectively . . . . 41 Reassociation kinetics of sea urchin sperm DNA assayed by 81 nuclease (0). Reassociation kinetics of radioactive non-repetitive gastrula DNA in the presence of a loo-fold excess of total sperm DNA assayed by $1 nuclease (I) . . . . . . . . . . . . 42 Kinetics of hybridization of 30 min pulse-labelled RNA from normal prism larvae (0) and from animal-L ized embryos of the same age (I) incubated in the presence of a loo-fold excess of total sperm DNA . 43 Hybridization of radioactive non—repetitive gastrula DNA with RNA from normal prism larvae. Conditions are as given in Table I and in Materials and Methods. . . . . . . . . . . . . . . . . . . . . . 45 Hybridization of radioactive non-repetitive gastrula DNA with RNA from animalized embryos of the same age as the prism larvae shown in Figure 12. Con- ditions are as given in Table I and in Materials and Methods. . . . . . . . . . . . . . . . . . . . 46 Summary of the hybridization data obtained at all stages tested after incubation for 24 x 104 ug/ml x hr as given in Table I. For the combination experiments, the concentration x time factor for each RNA reactant was 24 x 10 ug/ml x hr. . . 49 Thermal denaturation profile of radioactive non- repetitive gastrula DNA/total sperm DNA hybrids (O) and radioactive non- repetitive gastrula DNA/ RNA hybrids (0) in 0. 1M Na assayed by Slnuclease. The Tm of the DNA/DNA hybrids is 78°; the Tm of the RNA/DNA hybrids is 71° . . . . . . . . . . . 51 a? grporation of 3H—uridine per nucleus by normal ' (0) and animalized (0) sea urchin embryos ‘during development. The units on the ordinate represent cpm/nucleus/embryo multiplied by a f constant in order to give the rate of incorpor- ation per nucleus an arbitrarily assigned value of 1.0 at the prism larva stage. . . . . . . . . . 57 a _1&.. Uptake of 14C-va-line during a 30 min Pulse by a; normal (0) and animalized (0) embryos. ".v;b. Incorporation of‘ C-valine into TCA-insoluble ” material during a 30 min pulse by normal (0) and animalized (0) embryos. . . . . . . . . . . . 71 \ O ‘ A n... _" $114211 .' . . ‘ r..-;-435‘ -. \ ... ~" 7 ‘ .' pdi'i? '. 1-1ifiz'i’: ‘ , ~. Iviii .‘ ' I ‘l ii F2117), General Introduction Developmental processes are believed to be controlled by the interaction of the individual's genome with its environment. Differentiation, the selective restriction of a cell's developmental potential, can thus be viewed as the result of such interactions. With such a frame of reference, it may be predicted that (l) alterations in genetic activity may be produced in embryos by altering their environment, and (2) that such alterations would cause alterations in the differentiative patterns of the embryonic cells. Early development of the sea urchin is believed to be controlled by a coordination of two opposing gradients of morphogenetic influence, one maximal at the animal pole and the other maximal at the vegetal pole (reviewed by Runnstrom, 1933; Needham, 1942; Lallier, 1964). Disruption of this co- ordination through chemical or microsurgical manipulation can produce abnormal embryos with exaggeration of their ectodermal characteristics at the expense of their mesentodermal character— istics (called animalized embryos) or with exaggeration of their mesentodermal characteristics at the expense of their ectodermal characteristics (called vegetalized embryos). Strongly animalized embryos develop as a hollow ectodermal sphere with hyperextension of the acronal stereocilia and complete suppression of gut formation; strongly vegetalized embryos develop as a large gut possessing a few small ectoderm- like "blebs" (Horstadius, 1939; Runnstrbm and Immers, 1970). Such abnormal embryos may be produced by altering the embryos' ionic environment (Needham, 1942; Lallier, 1964). Runnstrom (1967) hypothesized that the coordination of the animal and vegetal gradients is mediated through the activity of the embryonic genome. The present study was undertaken to attempt to correlate experimentally-induced alterations in embryonic development with alterations in embryonic gene activity as a test of the first of the above predictions. Specifically, this study tests directly whether animalization (the production of animalized embryos) involves alterations in the activity of the embryonic genome. Control 9: Development Ex Variable 9222 Activity ' The theory of variable gene activity has evolved partially to explain how the fertilized egg is able to differentiate several different types of cells quickly after rapid cleavage. This theory suggests that differentiation is a result of the activity of certain genes or groups of genes and that different groups of genes are active in cells of different states of differentiation. This theory and its relevance to early develop— ment have been extensively reviewed by Davidson (1968). Several important lines of evidence suggest that all cells of an organism contain the same genetic information. Driesch (1892), by exerting pressure on the embryos, was able to distri- ; bute early cleavage nuclei of sea urchin embryos to improper regions of the embryo; such embryos developed normally. Spemann (1938) constricted newt eggs so that the two nuclei of the first cleavage remained in one half of the embryo; after a number of cleavages, a daughter nucleus of the cleaving half escaped into } a. * A the non-nucleated half, thereby inducing it to cleave. If the constriction was then completed such that the embryo was divided in half, two normal twins resulted with the development of one delayed by the length of time required for the original non-nucleated half to acquire a nucleus. Finally, nuclei from various cells of embryonic and larval tissues of varying differentiated states have been transplanted into enucleated unfertilized amphibian eggs and these have been found capable in some cases of supporting normal differentiation (King and Briggs, 1956; DiBerardino and King, 1967; Gurdon, 1968). McCarthy and Hoyer (1964) directly tested the equivalence of the genetic materials (DNA molecules) of cells of different differentiated states by molecular hybridization experiments. In their experiments no differences were detected between the DNAs of various adult mouse tissues. Gene products (RNA molecules) of cells of different differen- tiated states are not all similar, however, as demonstrated by the same authors and by many others for adult tissues and for normally developing tissues. In sea urchin embryos, for example, differences are detected between RNAs of blastulae and prism larvae (Whiteley gt gt., 1966, 1970), of unfertilized eggs and blastulae (Glisin gt_gt., 1966), of unfertilized eggs and cleaving embryos (Hynes and Gross, 1970; Mizuno gt gt., 1974) and between different cells of cleaving embryos (Mizuno gt gt., 1974). Similarly, the amount of genetic activity varies in cells of different differentiated states. Based on the rate of RNA transcription t2 ytttg by isolated chromatin (Marushige and Ozaki, 1967; Ozaki, 1970) and by molecular hybridization to DNA 4 of the ta ytttg transcription products (Chetsanga gt gt., 1970), the activity of the genome in synthesizing RNA molecules increases during the development of the sea urchin embryo. Thus, while the genetic information is c0nstant in the cells of a given individual, the expression of this information is different between different cells. Development Qt Egg Gradient Theory 9; Sgg Urchin Morphogenesis With the discovery by Driesch (1891) that a normal, but small sea urchin embryo can develop from any blastomere isolated from a cleavage stage embryo up through the 4—cell stage, the idea was advanced by Driesch that the sea urchin embryo is an "harmonious equipotential system", in which any part can give rise to a complete larva. Other embryos, on the other hand, were considered to be "mosaics" in that isolated blastomeres could give rise only to partial embryos (see Wilson, 1925). However, after the third cleavage, isolated blastomeres or groups of blastomeres form normal or abnormal embryos, depending upon the regions of the egg contained in the fragments (H3rstadius, 1939; Berg and Cheng, 1962). Other experiments (Boveri, 1901; Horstadius, 1939) demonstrated that regional differentiation was present in the unfertilized sea urchin egg along the animal— vegetal axis. By the sixteen-cell stage this regional differen- tiation is apparent in that three distinct types of blastomeres may be distinguished; tiny "micromeres" at the vegetal pole, a tier of large "macromeres" above them, and another tier of inter- mediate-sized "mesomeres" at the animal pole. Deletion experi- ments have shown that each type of blastomere has its own 5 prospectiVe fate at this stage. Isolated animal half embryos (mesomeres) would develop into a hollow ciliated blastula-like structure with hyperdevelopment of the apical tuft (animalized» embryo), whereas isolated vegetal half embryos (micromeres plus macromeres) typically developed into an embryo with an abnormally large and often evaginated gut (vegetalized embryo). These deletion experiments are consistent with the idea that the sea urchin egg, too, is a mosaic, but Horstadius' (1939) recombination experiments demonstrated that the blastomeres have a broader developmental potential and that the cells of these regions were capable, under the proper influences, of giving rise to other parts of the normal embryo. Comparable alterations in development have been obtained by adding certain chemicals to the embryos' medium. Herbst (1892) added Li+ to the sea water in which sea urchin embryos were developing and observed hyperdevelopment of the gut structures at the expense of the ectodermal structures, or vegetalization. Backstrom and Gustafson (1953) observed that the period of maximum lithium—sensitivity is during early cleavage, the period of embryonic determination. This evidence suggests that lithium— induced vegetalization is a phenomenon of significance to the embryo. MacArthur (1924) suggested that this action was due to a differential suceptability of the animal region to Li+. Other chemical agents promote animalization. These include thiocyanate and iodide (Lindahl, 1936), sulfated organic molecules such as Evans Blue (Lallier, 1955b), Zn++ ions (Lallier, 1955a), and proteolytic enzymes such as trypsin (Harstadius, 1949; Moore, 1952; Runnstrgm and Immers, 1966) and pronase —-‘—-—- —. vnw .‘m, 6 (Lallier, 1969). The sensitivity towards all these agents has been shown to be greatest during the period of embryonic determination (Lallier, 1959, 1964). Experimental results such as these have been cited in support of the theory that sea urchin development is controlled by the interaction of two opposing gradients of morphogenetic influence, One maximal at the animal pole and the other maximal at the vegetal pole (Runnstrgm, 1933). Normal interaction of the two gradients would produce a normal larva, while suppression of one and/or activation of the other would produce animalization or vegetalization, depending upon which gradient is suppressed or activated. Suppression of the vegetal gradient or activation of the animal gradient would give rise to an animalized embryo, and suppression of the animal gradient or activation of the _vegetal gradient would give rise to a vegetalized embryo. The recent discovery that chemicals with morphogenetic activities may be extracted from unfertilized eggs (Horstadius gt gt., 1967; Josefsson and Horstadius, 1969) and from cleaving embryos (Horstadius and Josefsson, 1972; Fujiwara and Yasumasu, 1974b) has given support to the theory that the gradients are chemical in nature, although most of the agents have not yet been pruified highly enough to allow the determination of their molecular structures. An antivegetalizing activity has been characterized, however, as 5-methy1 cytosine (Fujiwara and Yasumasu, 1974b). This molecule is able to reduce vegetalization of normal embryos without producing typical animalization (cf. Horstadius, 1972). An analogue of this moleCule, 2-thio, S-methyl cytosine, has been reported to possess an antivegetalizing and possibly an animalizing activity (Gustafson and Horstadius, 1956). Non-Genetic Theories gt Animalization And Vegetalization The mechanism of the vegetalizing actiOn of Li+ remains unknown. Lindahl (1936) demonstrated that lithium inhibits some reactions of glycolysis, but Backstrdm (1959) has shown that the embryonic hexose monophosphate shunt activity is not affected by Li+ treatment during the period of embryonic determination, suggesting that the effect on glycolysis is independent of the morphogenetic effects. Lindahl and Kiessling (1951) observed that Li+ causes an accumulation of inorganic pyrophosphate in eggs treated during the period of determination and suggested that the formation of ATP is inhibited by Li+. Runnstrdm and Immers (1971) proposed that the colloidal state of the animal region of the embryo was affected by Li+, pre- venting the diffusion of the hypothetical "animalizing substance." Ranzi (1957) reported that vegetalizing agents stabilize proteins in solution against denaturation and suggested that vegetalization results from the stabilization of embryonic proteins. The mechanism of action of the animalizing agents is also unknown. Ranzi (1957) demonstrated that in contrast to the effect of vegetalizing agents, animalizing agents denature proteins in solution and suggested that animalization occurs under c0nditions of protein denaturation. Horowitz (1940) showed that the rate of respiration in thiocyanate—treated embryos does not increase above the level found in normal blastulae, however Backstrdm (1955) demonstrated that the inhibition of respiration by iodosobenzoic acid is minimal during the period of embryonic determination. Backstr5m (1959) also showed that the hexose-monophosphate shunt activity is the same between normal and animalized embryos during the period of embryonic determination. Recent research has suggested that animalizing agents exert their effects at the cell surface (Lallier, 1968, 1972). It has been shown that Zn++ ions increase and Li+ ions decrease the animalizing actions of proteases (Lallier, 1969) and that the action of Li+ in decreasing the animalizing activity of pronase cannot be accounted for by a direct effect of Li+ on the properties of the enzyme. Proteolytic enzymes may act at the cell surface. It is not known whether they are able to enter the cell or whether this would be necessary for them to produce their effect. Recently Lallier (1972) has shown that concanavalin A, a phytohemaglutinin known to interact with the cell surface specifically, is also an animalizing agent and that its effect is enhanced by Zn++. Lallier con— cluded that Zn++, proteolytic enzymes, and concanavalin A all interfere with the same type of cell surface structures. Timourian (1968) demonstrated that the uptake of 652n++ by sea urchin embryos is low during the period of embryonic determination, when they produce their effects on morphogenesis, further indicating that a primary effect may be at the cell surface level. None of these possibilities are mutually exclusive, however. It is not unlikely that different animalizing and vegetalizing agents act at different levels to influence complicated control 9 mechanisms and any one agent may produce effects at more than one level of cellular activity. gggg Activity Agg Egg Gradients Autoradiographic studies have demonstrated regional differ— ences in the incorporation of 14C-adenine into sea urchin embryos (Markman, 1961a). Markman found that at the early blastula stage, adenine is more strongly incorporated into RNA in the animal region of the embryo, while after the mesenchyme blastula stage incorporation is stronger in the vegetal region. Further studies on isolated animal half embryos (Markman, 1967) showed an activation of incorporation at the blastula stage and a decrease at the gastrula stage of the controls. The incorporation of precursor into ribosomal RNA (rRNA) is activated at the mesenchyme blastula stage (Giudice and Mutolo, 1967; Sconzo gt gt., 1970a), the stage at which vegetal differentiation begins to be expreSSed, but this same activation occurs in disaggregated cells of the embryos, even if the cells are not allowed to reaggregate (Sconzo gt gt., 1970b; Hynes gt gt., 1972). Thus, normal cell-to-cell interactions are unnecessary for the activation of rRNA accumulation. 0n the other hand, rRNA accumulation is inhibited in animalized embryos (Pirrone gt gt., 1970; O'Melia and Villee, 1972). Such results may be reconciled by two possible interpretations. The first is that rRNA synthesis, stability, and/or processing is specifically inhibited in animalization; the second is that the levels of the animalizing agents used by the investigators were too toxic and the lack of incorporation observed was really a non-specific effect of toxicity. .a.s¥v 10 The transcription inhibitor, actinomycin D, has been used in the study of sea urchin morphogenesis. Gross and Cousineau (1963, 1964) demonstrated that concentrations of actinomycin D sufficient to block nearly all nuclear RNA synthesis would still permit development to occur through cleavage. Giudice gt gt., (1968) demonstrated that the process of gastrulation is sensitive to actinomycin D treatment at around the time of hatching. Lallier (1963) treated sea urchin embryos during the period of embryonic determination with actinomycin D and found that development was arrested. If the embryos were returned to sea water after the blastula stage had been reached, a great deal of recovery occurred if the concentration of actinomycin D had been low enough. Embryos treated simultaneously with Li+ and actinomycin D became more strongly vegetalized than those raised in Li+ alone, while Zn++ or Evans Blue-treated embryos were less strongly animalized in actinomycin D, suggesting that animalization and not vegetalization was dependent upOn trans- cription. Markman and Runnstram (1963) observed that the animal- ization of animal half embryos is reduced by treatment with actinomycin D, and also (Markman and Runnstrom, 1970) that the animalizing effect of trypsin and the "endogenous" animalizing substance of Harstadius gt gt. (1967) are reduced by treatment with actinomycin D. However, in contrast to the results of Lallier (1963), the same authors reported that treatment of embryos with actinomycin D reduced the level of vegetalization by Li+ (Runnstrgm and Markman, 1966), suggesting that Li+-induced vegalization, too, is gene dependent. v ,..vw,y— 11 That protein synthesis is altered in animalized and vegetalized embryos may also indicate that gene-level control mechanisms are important in animalization and vegetalization. Berg (1968) concluded that the effect of Li+ in reducing the rate of protein synthesis in advanced embryos was a secondary reflection of a primary effect on transcription. Carroll gt gt. (1974) demonstrated that the electrophoretic pattern of newly—synthesized proteins was different between normal and animalized embryos. O'Melia (1972) reported that esterase isozyme activities which appear in normal pluteus larvae do not appear in animalized embryos of the same age. These results, as well, are consistent with an interpretation based on an altered pattern of transcription in the abnormal embryos. The protein synthesis inhibitor, chloramphenicol, has been found to be a vegetalizing agent (Lallier, 1962; Harstadius, 1963; Fujiwara and Yasumasu, 1974a). This finding was originally interpreted to indicate that animal differentiation requires protein synthesis. However, another protein synthesis inhibitor, puromycin, did not act as a vegetalizing agent (Fujiwara and Yasumasu, 1974b), suggesting that this activity may not derive from the inhibitory effect on protein synthesis. Inconsistent effects have been obtained when attempts have been made to animalize or vegetalize embryos or isolated animal and vegetal half embryos by amino acids and amino acid analogues (Gustafson and H5rstadius, 1955, 1957; Fudge, 1959; Bosco and Monroy, 1960). Experiments using inhibitors of nucleic acid and protein synthesis to test for possible gene dependence of animalization and vegetalization have been inconsistent. Experiments using A 12 actinomycin D to test for gene-dependence of animalization have been consistent with such an interpretation, but the results of similar experiments to test the gene-dependence of vegetal- ization have been inconclusive. It has already been shown that the vegetalizing effect of chloramphenicol probably does not derive from its effect on translation; such results clearly emphasize the major limitation of experiments using metabolic inhibitors: that the experimental results obtained using them do not always reflect specific effects of the inhibitors. Thus these data cannot be said to demonstrate conclusively that embryonic gene activity is involved in determination in the sea urchin embryo. Eggg Activity Agg Gastrulation The cell movements involved with gastrulation in the sea urchin embryo have been extensively discussed by Gustafson and Wolpert (1967). Gastrulation begins when the presumptive primary mesenchyme cells, derivatives of the micromeres, detach from the blastula wall, round up, begin characteristic "pulsatory" movements, and migrate into the blastocoel. There they assume a characteristic ring-shapped pattern, fuse into a "cable", and induce an invagination of the presumptive endoderm at the vegetal pole. Cells at the tip of the invaginating archenteron which are destined to become the embryonic mesoderm or "secondary mesenchyme? then send out pseudopodial processes which attach to the blastocoel wall and pull the archenteron in further. Gut formation is completed with the invagination of the ectoderm at the point of contact with the archenteron to form the larval A K 13 mouth. At the same time, the cable of primary mesenchyme is differentiating spicules which elongate to give the larva its typical pluteus or "easel" shape. Microtubules have been suggested to be the primary determinants of cell shape in the cable and pseudopods (Gibbins gt gt., 1969). The ability of the presumptive primary mesenchyme cells to induce gut formation has been suggested to be related to their surface coat of acid mucopolysaccharides (Karp and Solursh, 1974). In addition, it has been shown that serotonin antagonists inhibit the release of primary mesenchyme cells and the onset of invagination and that this inhibition may be reversed by the addition of serotonin (Gustafson and Toneby, 1970, 1971). Treatment with actinomycin D beginning before hatching and extending to the mesenchyme blastula stage also suppresses gastrulation, indicating that gastrulation is gene-dependent (Guidice gt gt., 1968). At this same time in development, novel gene groups are known to be activated (Whiteley gt gt., 1966, 1970). These results suggest that gastrulation is under the c0ntrol of the embryonic genome and that this genomic control may be expressed through the production of serotonin and acid mucopolysaccharides by the presumptive mesenchyme cells. Control Qt Developmggt ty IQEE Inorganic ions have been shown to be able to control patterns of differentiation in other systems as well as sea urchin embryos. Barth and Barth (1969, 1972, 1974) have shown that epidermal explants of amphibian gastrulae can be induced to differentiate into nerve and pigment cells under the __ 1". .‘. l4 influence of Ca++, given the proper ionic environment. Phytohemagglutinin-induced transformation of lymphocytes has been shown to have an absolute requirement for divalent cations (Alford, 1970) and to be accompanied by a temporary uptake of Ca++ from the medium (Allwood gt gt., 1971; Whitney and Sutherland, 1972). Zn++ ions were very effective in supporting transformation even in the absence of Ca++ (Alford, 1970), and Zn++ alone can actually induce trans- formation (Kirchner and Rfihl, 1970). Direct effects of the ionic environment have been demon- strated on the pattern of puffing of salivary chromosomes (Kroeger and Lezzi, 1966). Such ionic alterations can mimic hormonal effects on the puffing pattern. In addition, ions have been shown to modify the intracellular metabolism of cyclic nucleotides (Rasmussen, 1970). It has been hypothesized that an interaction of intracellular inorganic ions with cyclic nucleotides is responsible for differentiation in many systems, including neural induction, slime mold aggregation, mitosis, and gastrulation in embryos, and chemical teratogeneisis (McMahon, 1974). 15 STATEMENT 92 TEE PROBLEM If variable gene activity is an essential component in the control of developmental processes, then alterations in developmental processes should also involve alterations in the activity of embryonic genes. Animalization involves specific morphological alterations in development, ytt. suppression of mesentodermal differentiation and enhancement of ectodermal differentiation. The transcription inhibitor, actinomycin D, interferes with the process of animalization (Lallier, 1963; Markman and Runnstrdm, 1963, 1970), and such experiments led Runnstr5m (1967) to hypothesize that the co- ordination of the animal-vegetal gradients is mediated through the activity of the embryonic genome. In addition, it has been reported that the accumulation of one gene product, rRNA, is inhibited in animalization (Pirrone gt gt., 1970; O'Melia and Villee, 1972). A clear demonstration that animalization is gene-dependent must show that specific alterations in gene activity occur in animalization and that these alterations are responsible for the abnormal morphogenesis. The objective of the present research was to determine, experimentally, whether or not animalization actually involves alterations in the pattern of embryonic gene activity. Two experimental approaches have been taken; one to determine the extent of gene activity (RNA synthesis) quanti- tatively, and the other to examine the diversity of gene products present in normal and animalized embryos. MATERIALS AND METHODS Culture 9: Embryos Gametes of the sea urchin, Strongylocentrotus purpuratus (Pacific Bio—Marine, Venice, California, or Controlled Environ- ments, Bellvue, Washington) were obtained by the injection of isotonic KCl and fertilized (Tyler and Tyler, 1966), and the embryos were cultured at a concentration of 1% v/v in artificial sea water (Instant Ocean, Aquarium Systems, Eastlake, Ohio) at 15°C either in monolayer cultures in petri dishes or in con— tinuous agitation by a stirrer rotated at 30 rev/min. In addition, cultures contained 250 ug/ml streptomycin sulfate to inhibit bacterial growth. Chemical Animalization For animalization, zinc sulfate was added to the sea water to the appropriate concentration. A stock solution of 0.1M ZnSO4 was diluted directly into the sea water. Under these conditions, the ZnSO4 had no effect on the pH of the sea water at the concentrations used. The zinc ion used has been shown to be an effective animalizing agent (Lallier, 1955a, 1959). In any one experiment eggs from the same batch were used for both control and experimental culture. During early cleavage stages, whole cells were counted in living embryos. For later stages, the embryos were fixed in 16 A 17 neutral 1% formalin made in artificial sea water, and squash preparations were made with the aceto-orcein fast green stain (Kurnick and Ris, 1948). The nuclei were then counted using an ocular grid (American Optical 1409A) for reference. The counts were taken to represent the number of cells per embryo. Standard deviations of the nuclear counts from different embryos from the same population were always less than 10% of the mean number of cells for individuals in the population. Uptake and Incorporation of Radioactive Uridine Equal aliquots containing approximately 104 embryos were obtained from the cultures at the times specified, washed, suspended to 0.25 ml in sea water containing 250 ug/ml streptomycin sulfate, and incubated with 2 uCi/ml 3H-S-uridine (specific activity 20 Ci/mmole) Schwartz—Mann, Orangeburg, N.J.) for the length of time specified for each experiment. Incubation was stopped by the addition of ice-cold sea water containing a 104—fold excess of non-radioactive uridine. The embryos were collected on Whatman 3MM paper discs by filtration, washed and dried. For the determination of uptake, the discs were placed directly in a toluene based scintillation fluid containing 4 gm/l PPO and 0.25 gm/l dimethyl POPOP without further treat- ment and their radioactivity was measured by a Packard Tri—Carb 3320 Spectrometer. For the determination of incorporation into RNA, the dried discs were washed for 15 min successively in each of two changes of ice cold 5% trichloroacetic acid (TCA), one of 95% ethanol and one of ethyl ether. The discs were then dried and their radioactivity was measured as above. Filter discs of control and animalized embryos were always processed 18 together in any given experiment. The radioactivity was expressed as the net counts after subtraction of background. The relative net standard error of the counts was always less than a 5% of the total counts. Preparation pf Radioactive RNA Prism stage embryos (48 hr) and animalized embryos of the same age were labelled at a concentration of 10% v/v in arti- ficial sea water containing 250 ug/ml streptomycin sulfate and 3.3 uCi/ml uridine 5-3H for 30 min. RNA was extracted by a modification of the hot phenol-SDS method of Girard (1967). Embryos were homogenized in 5 vol acetate—EDTA (0.01M Na acetate, 0.01M Na EDTA, pH 5.1) containing 0.1% SDS and 0.1% bentonite, and RNA was extracted at 60° by shaking With an equal volume of phenol. The aqueous phase was reextracted twice with phenol at 0° and the RNA precipitated by ethanol. Sucrose Gradient Centrifugation Analysis pf Radioactive RNA Radioactive RNA was layered over a 28 ml linear 2.5-15% sucrose gradient made up in acetate-EDTA and centrifuged in a Spinco SW25.1 rotor at 25,000 rev/min for 18 hr at 4°. Fractions were collected dropwise after bottom puncture, and radioactive RNA coprecipitated by 10% TCA with 100 g/ml yeast RNA. Pre— cipitates were collected on glass fiber filters (Reeve-Angel # 984H), rinsed with 5% TCA, dried at 60°, and their radioactivity determined by liquid scintillation counting. 19 DNA was extracted from sperm by the method of Whiteley SE 21. (1970). One gm of washed sperm was suspended in 125 ml EDTA-Tris (0.1M EDTA, 0.04M Tris-HCl, pH 8.2) and an equal volume of EDTA—Tris containing 2% SDS was added and the lysate heated to 60° for 10 min. The lysate was then treated at 37° -with 50 g/ml pronase for 8 hr. Pronase had been preincubated at 1 mg/ml for 2 hr to destroy nuclease activity. DNA was extracted with an equal volume of phenol and reextracted with an equal volume of chloroform—iso-amyl alcohol (24:1) and precipitated by ethanol. Shearing 229 Alkaline Sedimentation Analysis pf QNA DNA was dissolved at a concentration of 1-2 mg/ml in 0.1 x SSC (0.15M NaCl, 0.015M Na citrate, pH 7.0), sheared in an omnimixer at maximum speed for 5 min, further sheared by soni- cation (20 sec at maximum noise in 2 ml or less volume), dialyzed against distilled water, and lyophilized. DNA was 3 than dissolved in 0.12M PB (equimolar mixture of mono- and disodium phosphate) to the desired concentration (4-8 mg/ml). 1 Sometimes shearing by sonication was performed after lyophilization. Neither the sedimentation nor the reassociation properties of the DNA was affected by delaying sonication. Alkaline sedimentation was performed according to the pro— ‘ cedure of Abelson and Thomas (1966). 50-100 9 DNA was alkalai— denatured (0.33N NaOH, 10 min at room temperature) and layered °‘her a 4.8 ml linear 5—20% sucrose gradient made up in 0.9M Nam, 0.1M NaOH. Gradients were centrifuged at 20°C and 50,000 rev/min for 6 hr in an SWSOL rotor. Fractions were collected 20 dropwise after bottom puncture and the A260 of each fraction was determined. Radioactive samples were coprecipitated with 100 ug/ml sea urchin sperm DNA by 10% TCA and processed as above for RNA. The 520,w was calculated for the peak according to the equation of Abelson and Thomas (1966) and the single- stranded nucleotide length was determined according to Wetmur and Davidson (1968). Samples longer than 400—500 nucleotides were resonicated to that length, except in One experiment in which the reassociation kinetics of 900 nucleotide—long fragments were determined. Reassociation pf Sea Urchin Spgpm 2E5 Sheared DNA (100 ug) was heat denatured (10 min at 100°) and allowed to reassociate at 600 to various gppg (moles nucleo- tide x sec x liter-1, Britten and Kohne, 1968) in either sealed capillary tubes or in MicroFlex tubes (Kontes Glass Company). Reassociation kinetics were measured in two ways: 1. Single and double-stranded components were separated by chromatography on a 1.2 x 1 cm hydroxy—apatite (HAP) column at 60°. Single—stranded DNA was eluted with 10 vol 0.12M PB; double-stranded DNA was eluted with 10 vol 0.5M PB. The percent reassociated was calculated from the absorbance readings (at 260 nm) of the material recovered from the column. The 0.5M PB fraction absorbance was multiplied by a factor of 1.28 to compensate for its hypochromicity (Melli and Bishop, 1969). 2. Single-stranded DNA was degraded by S1 nuclease. Sl nuclease was prepared from Aspergillus oryzae a-amylase (Sigma) through step 4 of the method of Vogt (1973), and concentrated by A W————.——‘ 21 chromatography on a small column of DEAE-cellulose. Reassociated DNA was diluted to 150 ug/ml with 0.1M NaCl, 0.001M ZnSO4, 5% glycerol, 0.03M Na acetate, pH 4.6 and treated with 20-50 units/ml Sl nuclease at 45° for 30 min. For non—radioactive DNA, samples were chilled, precipitated by 5% perchloric acid, filtered through a Millipore filter, and the absorbance (at 260 nm) of the filtrate (single—stranded material) was read. Radioactive samples were precipitated by TCA with 100 ug/ml bovine serum albumin (BSA) and the radioactivity in double-stranded material was determined by liquid scintillation counting as described above for RNA. RNA/DNA Hybridization i2 DNA Excess Before hybridization, radioactive RNA was digested at 37° for 1 hr with 50 ug/ml DNase (RNase-free, Worthington). After additional deproteinization with pronase (50 ug/ml for 1 hr at 37°) and phenol, the RNA was precipitated by ethanol. The RNA was then dissolved in 0.12M PB and sheared to approximately Gs by sonication. Final preparations had specific activities of approximately 1000 dpm/pg and the radioactivity was greater’ than 99.5% alkalai-labile. RNA specific activities from normal and animalized embryos were identical. Sheared radioactive RNA (1 Hg) was added to a loo-fold excess of sperm DNA in 0.12M PB containing 0.1% SDS. After heat denaturation (5 min at 100°) the mixtures were incubated at 60° to the desired DNA 995, diluted to 5 ml in 0.24M PB and divided in half. One half was incubated with 20 Lg/ml RNase (bovine pancreas, Worthington) for 20 min at 37°. RNase had A ' “v" -.- 22 been heated previously to 80° for 10 min in 0.24M PB to destroy DNase activity. The other half was treated similarly but without RNase. Mixtures were precipitated by TCA, and the radioactivity was determined as above. Preparation pf Radioactive Non-repetitive DNA A 1% suspension of embryos was labelled from fertilization to the early gastrula stage (36 hr) with 2 uCi/ml of thymidine methyl—3H (16.7 Ci/mmole, Schwartz-Mann). DNA was purified by the method of Marmur (1961) from nuclei prepared by the procedure of Loeb (1969). Embryos were washed twice in 0.53M NaCl, 0.53M KCl (19:1), once in 1M dextrose, and once in SSC. The final pellet was suspended in 30 vol SSC and homogenized by two passages through a #20 gauge hypodermic needle. The homogenate was mixed with an equal volume of 2M sucrose and centrifuged at 15,000 x g for 30 min. The nuclear pellet was suspended in 5 m1 EDTA-Tris, heated to 60° for 10 min, made 1% in SDS and 1M in NaClO4, shaken with an equal volume of chloroform—isoamyl alcohol and precipitated by ethanol. DNA was dissolved in 5 ml 0.1 x SSC and incubated with 50 ug/ml heat-treated RNase at 37° for 30 min. DNA was made to 0.1M Tris, pH 9.0, 0.1M NaCl, and 1% SDS, shaken with an equal volume of phenol, and precipitated by ethanol. Final preparations had specific activities of about 120,000 dpm/ug. Purified DNA was then dissolved in 0.12M PB and sheared. An aliquot was denatured and allowed to reassociate in a large excess of non-radioactive sperm DNA. The kinetics of reassocia- tion of radioactive and non—radioactive DNA were the same. A 1-4 *vo 23 Radioactive DNA was then heat-denatured and incubated to 99E 30. Single-stranded material was purified from HAP, heat— denatured, reincubated to 92E 60, and repurified from HAP. The purified non-repetitive DNA (single-stranded after both incubations) was dialyzed against distilled water, lyophilized, and dissolved in a small volume of 0.12M PB. That this purified non-repetitive DNA is essentially free from repetitive DNA sequences was shown by the reassociation kinetics of the radioactively labelled non-repetitive DNA in the presence of a large excess of non-radioactive total sperm DNA (fig. 11). RNA/DNA Hybridization i3 RNA Excess Non-radioactive RNA was extracted from unfertilized eggs and from normal blastulae (24 hr) and prism larvae (48 hr) and from animalized embryos of comparable ages as described above. The isolated RNA was further pruified by DNase digestion and cetyltrimethyl ammonium bromide precipitation (Bellamy and Ralph, 1968), sheared, dialyzed against distilled water, lyophilized, and dissolved in 0.5M NaCl, 0.001M EDTA, 0.02M Tris-HCl, pH 7.4, containing 0.1% SDS (after Leong gp al., 1972). Heat-denatured radioactive non-repetitive DNA was incubated with an excess of RNA in the above buffer at 60°. After incubation the mixtures were diluted to 150 ug/ml RNA in the $1 nuclease buffer and divided in half. Half was treated with $1 nuclease as described above, and the other half was treated similarly but without 81 nuclease. Table I summarizes the conditions of incubation and gives the RNA/DNA ratios used. All incubations were to the same equivalent Cot with respect A 24 amgwm H Hoocomfiwos nosdeHOSm mon mmncfimnwoo om Hmmwomonw Macon <0Hcamr Hsoagmnwon wean wz>\oz> mnsw