THE EFFECTS OF ADULT? T§SSUE fRAGHONS 2335:) OF #:4388013 AMERST THESE FRACTEQE‘JS 855 THE EARLY BEVELOF‘MENT 0F RAM PEHMS EMBRWS Thesis for the Degree of M. S. MECHJGAN STAYE? UNEVERSITY PAMELA KAY MCALLBSTER 1,970 “HES‘S L Y BINSING BY , [3 mm: & suus' -,_ ~ ~‘ .. B; 1 mm nu: ”1’9“???“ a 3i; ABSTRACT THE EFFECTS OF ADULT TISSUE FRACTIONS AND OF ANTIBODIES AGAINST THESE FRACTIONS ON THE EARLY DEVELOPMENT OF RANA PIPIENS EMBRYOS By Pamela Kay Mo Allister Three experiments were conducted in an attempt to clarify the role of both antigens and antisera in the processes of early growth and differentiation in'fiéga’pipiens embryos. Hearts and kidneys from adult frogs were fractionated and the fractions and antibodies against them were injected into embryos in the blastula stage. Tissue fractions from both heart and kidney caused inhibition of deve10pment in a significant number of embryos. The effects were non- specific in that the development of the entire embryo was retarded. The injection of absorbed and unabsorbed antisera against heart fractions inhibited the development of the heart in a small number of embryos. The results were specific in that other organs deve10ped nor- mally. Those embryos injected with anti-kidney or with control sera exibited no defects of the heart. THE EFFECTS OF ADULT TISSUE FRACTIONS AND OF ANTIBODIES AGAINST THESE FRACTIONS ON THE EARLY DEVELOPMENT OF RANA PIPIENS EMBRYOS Byzz Pamela Kay Mc Allister A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Zoology 1970 ACKNOWLEDGMENTS I wish to express my thanks to Dr. John R. Shaver, my major professor, for his guidance throughout my graduate studies and for his direction and criticism in the preparation of this manuscript. I would also like to acknowledge Dr. Stephanie H. Barch for her guidance and assistance in the laboratory. I also thank the members of my guidance committee, Drs. Charles S. Thornton and Virginia Mallman for their helpful suggestions and assistance in the preparation of this thesis. ii TABLE OF CONTENTS Page IIIST 0F TABLEOOOOOOOOO...OOIOOOOOOOOOOOO0.0.0.0....OOOOOOOOOOOOOOOOOOiV LIST OF FIGUREOOOOOOOOOIOOOOO0.......OOCOOOOOOOOOOOOOOOOOOOCOOOOOOOOO v IMRWNMHMLH.H.H.H.H.H.u.u.n.u.n.u.u.u.u.u.u.u.u.1 METHOE AND MATERIALS................................................. 6 PREPARATION OF ANTIGENS.......................................... 6. PREPARATION OF ANTISERA.......................................... 6 INJECTION OF EMBRIOS............................................. 8 HISTOLOGICAL PREPARATION......................................... 9 RESULTS...............................................................1O ANALYSIS OF ANTIGENS AND ANTISERA................................1O EFFECTS OF ANTIGEN INJECTION.....................................15 WECTS OF ANTISERA INJECTIONeeeeeeoeoo'eooooeoeeoeeeeoeooooeooeeezl DISCUSSIONOOOOOOOOCOOOO0.0...O...OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO.0.0.028 SWeeoeeeoeoeeoeeeeeeoeooeosoeanoeooeeoesoeeeoeoeeeee000.000.000.031" RWERENCE CITEOOOOOOOQOOO0.00000000COOOIOOOOOOOO00.0.00000000000000035 iii LIST OF TABLES Table Page 1. mums OF TISSUE FRACTION INJmTIONOOOOCCOOO0......00000000000.0.020 2. RBULTS 0F UNABSORBED ANTISERUM INJmTIONOOOOOOOOOOOOOOOOOOOOOOOOOOZu 3. RESULTS OF ABSORBED.ANTISERUM INJECTION............................27 iv LIST OF FIGURES Figure Page 1. 2. 3. 1+. 5. 6. 7. CENTRIFUGATION PROCEDURE FOR TISSUE FRACTIONATION................. 7 ANALYSIS OF HEART FRACTIONS.......................................11 ANALYSIS OF KIDNEY FRACTIONS......................................13 ANALYSIS OF ANTISERA..............................................16 EMBRYOS INJECTED WITH TISSUE FRACTIONS............................18 EMBRYOS INJECTED WITH UNABSORBED ANTISERA.........................22‘ mm INJETED wITH ABSORBED ANTISWOOOOOOOOOOOOOOOOOOOOO0.0.0.25 INTRODUCTION It has been claimed in recent years that the application to an embryo of organ or tissue specific antigens or of antibodies directed against those antigens could produce either a stimulatory or inhibitory effect on the differentiation of the homologous organ or tissue depend- ing on the experimental conditions. Growth, cell interactions, cell migration, and differentiation may all be affected by both antigen and antibody and the net effect may be either positive or negative. -The effects of the antigens or antisera may be species-specific or tissue- Specific or both, or may be totally nonpspecific. Specificity of the stage of develOpment at which the antigens and antibodies exert their effect has also been noted. Numerous experiments have been done to determine the effect of adult tissues both in developing and in regenerating systems. 'weiss (1952) prOposed that adult tissues have an inhibitory effect on the homologous organ in a developing embryo. He cultured chick embryos in extracts with or without the homologous organ. Fewer of those embryos cultured in whole embryo extract containing the homologous organ developed hearts or kidneys than those cultured in incomplete extracts lacking either heart or kidney. Rose (1955) demonstrated that in some cases the presence of adult tissues could Specifically inhibit the development of the homologous organ in gang pipiens embryos. In most cases the embryos were retarded at a specific stage of growth. Specific effects were limited to h of 1 2 26 experiments. Brain-treated animals in these 4 experiments exibited poor morphology of the nervous system, incomplete or missing neural canal, and random arrangement of cells in the neural tube. Heart- treated embryos had small hearts, and those treated with blood failed to develop blood cells. Clark and McCallion (1959) demonstrated that the effects of organ homogenates were Specific since heart homogenates cause reduction in heart size but have no effect on the brain. More recently, McCallion (196h) working with chick embryos has demonstrated that the inhibitory effect of brain on the development of the brain is related to organ specific substances. Brain Specific proteins when injected between 32 and 96 hours result in abnormal growth of the nervous system. Injection of brain proteins and lipoproteins which are also present in other tissues have no such effect on the nervous system. Braverman (1961) demonstrated that the effect of the injection of brain is not only specifically inhibitory to the brain but is Specific for that region of the brain from which the extracts are derived. Extracts from the anterior portion of the brain preferentially inhibit the differentiation of the forebrain, whereas extracts from the hindbrain inhibit the hindbrain and all areas more anterior. Thus there may be a gradient effect such as that demonstrated in regenerating systems. That adult organs have a stimulatory effect on the homologous organ has also been suggested and is supported principally by experiments employing chorioallantoic grafts in the chick. Ebert (1951) observed spleen hypertrophy following adult Spleen grafts. Ebert (195“) further found that the nitrogen content of the host spleen increased indicating an increase of protein content of the organ. He also demonstrated that the increased nitrogen content of the host Spleen resulted from 3 mobilization of 835- methionine labeled proteins from the graft. Tumanishvili and Salmatina (1968) found that embryonic chick liver increases in size upon the injection into chick embryos of adult liver cytoplasm. The increase in liver size followed a burst of mitotic activity. They further demonstrated that injection of nuclear material results in inhibition of mitotic activity and a decrease in liver size. Liver size in these SXperiments was determined by two factors within the cytoplasm and nucleus which stimulate and inhibit mitosis. That the effect of some adult tissues is non-Specific was demonstrated by Van Alten and Fennell (1959). Embryonic chick Spleen was enlarged following grafts of many organs. Both liver and heart were enlarged following Spleen, liver, and duodenal grafts. Andres (1955) measured mitotic indices of liver and mesonephros following the injection of a suspension of liver or mesonephros into chick embryos. The mitotic index of the homologous organ increased while that of the heterologous organ remained the same, was slightly decreased, or was increased but to a lesser extent than the homologous organ. There are several factors which must be taken into account when considering the effect of adult tissues on develOping systems which may account for the contradictory results which have been obtained in the 'works previously described. The Spleen is an immunologically competent organ and thus the increase in size of the host Spleen demonstrated by Ebert, and of other organs demonstrated by Van Alten and Fennell, may be a result of a graft-versus-host reaction. Such Spleen enlargement has been demonstrated by Simonsen (1962) who used genetic markers to deter- mine that Spleen enlargement in chick embryos was due to the invasion of immunocompetent cells into the host Spleen. - a Further, the mode of reSponse to adult tissues may be dependent on time. Undifferentiated tissues appear to be subject to inhibitory con- trol whereas differentiated tissues which have not yet attained adult Size may not. Although the effects of tissue antigens on development may be unpredictable, the effects of antisera do not appear to be so. Antisera may be lethal if applied in high doses or may produce many non-specific defects. Antisera prepared against body tissues may also produce specific defects if applied directly to the embryo in the proper concentration. Lens antiserum, applied in a suitable concentration, has been effective in interfering with lens and eye development (Fowler and Clarke, 1960; Clarke and Fowler, 1960; Langman, 1960, 1963). The reaction of the. embryos was limited to ectodermal tissues and was effective only if the antisera were applied before the 16 somite stage (Langman, 1960). Langman (1963) also demonstrated that the antibody responsible was directed against the a-crystalline. Ebert (1950) studied the effect of tissue antisera on the developing chick. The antisera were lethal in high concentrations and inhibited growth in lower concentrations. At very low concentrations the antisera produced Specific defects in the developing embryos. Those embryos grown in anti-heart sera frequently failed to develop normal somites and lateral plate mesoderm and occasionally failed to develop functional hearts. Neural development in these embryos was normal. Those embryos cultured in anti-brain sera had defects in the anterior portion of the brain. Owens (1960) injected early frog blastulae with anti-heart sera. Heart deveIOpment was selectively inhibited in a Significant number of embryos. The inhibition could not be coorelated with the presence or 5 absence of heart-Specific antibodies in the sera. The effects of antisera in mammalian development have also been extensively explored. A review of this work can be found in Brent (1968). If antisera have an inhibitory effect on developing organs then it could be predicted that antigens, being complementary, would have the opposite effect and would thus stimulate the development of the homologous organ. This study was begun in an attempt to clarify the role of both antigens and antisera in the processes of early growth and differentiation inifigg§_pipiens. It was hoped that the use of substances of greater tissue Specificity than employed in past experiments would result in more predictable results, and that the effects of both the antigens and antisera could be related to the Specificity of the substances used. METHODS AND.MATERIALS A. Preparation of Antigens: Antigens were prepared by a modification of the method of D'Amelio and Perlman (1960). Thirty to thirty-six large frogs are pithed and the heads removed. The hearts and kidneys are removed, washed, blotted on filter paper, and weighed. The tissues are then ground in a glass grinder using 1 gram of tissue to 10 ml. of medium. The kidneys are ground in 0.24 M sucrose in 0.02 M phOSphate buffer at a pH of 7.2. The hearts are ground in 0.24 M urea in phosphate buffer. The homogenate is then centrifuged according to the scheme in Figure 1. The fractions produced are used for injection into rabbits to produce antibodies and also for injection into embryos. B. Preparation of antisera: All antigens were injected into adult rabbits (German checker strain) in the subscapular musculature using 1.5 m1. of antigen solution and 1.5 ml. of complete Freund's adjuvant. The rabbits were given booster injections at one month intervals following the initial injection. Incomplete Freund's adjuvant was used for the booster injections. The rabbits were bled through the marginal ear vein one week after each booster injection. The antisera were tested in Ouchterlony double diffusion plates using 1% ion agar number 2 (Colab) in rabbit saline. The antisera were tested against the homologous antigen solution and against various heterologous antigen fractions. The antisera were then 6 homogenate lSOOXg 5 Mine pellet supernate (discarded) 20,000Xg 15 min. pellet supernate (resuSpended in 0.65% NaCl) MITOCHONDRIAL FRACTION 105,000Xg 90 min. P°11°t SUPERNATE (resuSpended in 0.65% NaCl) FRACTION or W3339d1;95’000x3 pellet resuspended m e in 0.65% N3C1+Oeu¢ y// DOC PELLET ERACTION (microsomes) 105,000Xg 90 Mine /\\\ pellet DOC SUPERNATE (resuSpended in DOC) (80% protein) washed 90 min. DOC RESIDUE (80% RNA) Centrifugation Procedure for Tissue Fractionation Figure 1 8 pooled and fractionated with 33% ammonium sulfate to separate the glo- bulin and albumen fractions. The globulins were dialized against 0.65% NaCl, frozen, and stored until use. C. Injection of Embryos: Adult female frogs were stimulated to ovulate according to the procedure of wright and Flathers (1961). Twenty-four to forty-eight hours later the eggs were squeezed onto glass slides and fertilized. Sperm suSpension was obtained by macerating the testes from two adult males in 0.1 strength Holtfreter's solution. Seven to ten ml. of Holt- freter's was used for each testis. After fertilization the eggs were placed in aerated tap water in finger bowls and kept at 18 degrees until they reached the mid-blastula stage at which time they were injected. Injections were performed with glass micrOpipettes prepared from capil- lary tubing (1.0 mm.0. D.) with a Livingston pipette puller (Otto K. Hebel,, Scientific Instruments, Rutledge, Pa.). A 10 mm. hypodermic syringe was connected to the injecting needle by a length of Intramedic polyethylene tubing (I.D. 0.030" & O.D. 0.048", Clay Adams, Inc., N{Y.) and the system was then filled with water. The needle was filled by withdrawing the plunger of the syringe leaving an air bubble between the water and the injecting fluid. Injections were performed under a binocular dissecting microscope by inserting the needle into the blastocoel cavity and expell— ing the fluid by Squeezing the plunger. Embryos from each female were injected with both control and experimental solutions. Holtfreter's solution was used as a control in all experiments. In those experiments in which antibodies were used, nonpimmune serum was also used as a control. Nonpimmune serum absorbed with liver was used as an additional control 9 in the experiment utilizing absorbed antisera. D. Histological Preparation: After injection, the embryos were maintained at 18 degrees until the controls reached Shumway stage 22 at which time they were fixed in Smith's modification of Bouin's fixative. Randomly selected embryos from each group were then embedded in paraffin, sectioned at 10 microns, stained in hematoxylin and eosin, and examined histologically. RESULTS A. Analysis of antigens and antisera: The analysis of the heart tissue fractions showed that each frac- tion had antigenic determinants in common with the other fractions and in common with kidney (Figure 2). The mitochondrial fraction had fewer antigenically active components than the supernate or the pellet. The mitochondrial antigens were not unique to the mitochondrial fraction since they were also present in the supernate and in the pellet. Heart supernate and whole heart apparently contain the same number and kinds of antigenic determinants when tested with unabsorbed antisera. The uncertainty is due to the large number of precipitin lines formed. The heart pellet produced fewer precipitin lines, none of which were unique 'to the pellet. Using antisera absorbed with liver, it was readily apparent that the supernate either with sucrose or with urea buffer contained at least one antigenic determinant not found in any other fraction. Absorption with other body tissues also failed to remove this component. After absorption with liver, 3 precipitin lines remained. Two of these could be removed by absorption with frog serum, kidney, or skeletal muscle. The third line remained after absorption with these tissues. The analysis of the kidney tissue fractions showed that all the fractions had many determinants in common with heart (Figure 3, a-e). The mitochondrial fraction, supernate, and pellet appeared to be immuno- logically alike and were indistinguishable from the whole kidney 10 11 Figure 2--Analysis of heart fractions. Key to Abbreviations AH - Anti-whole heart serum AK - Anti-whole kidney serum BS - heart supernate HUS - heart urea supernate H - whole heart HM - heart mitochondria HP - heart pellet H DOC S - heart DOC supernate H DOC R - heart DOC residue K - whole kidney Figure 2 13 Figure 3--Analysis of kidney fractions. Key to Abbreviations AK - Anti-whole kidney AH - Anti-whole heart K - whole kidney KM - kidney mitochondria KS - kidney supernate KP - kidney pellet K DOC R - kidney DOC residue K DOC S - kidney DOC supernate H - whole heart AKL - Anti-kidney absorbed with liver 15 homogenate. The DOC residue was only weakly antigenic and produced few precipitin lines when tested against anti-whole kidney. The DOC supernate did not differ from the pellet. When antisera were absorbed with liver, all fractions except the DOC residue formed one or more precipitin lines with the antiawhole kidney sera. The unabsorbed antisera which were used for injection into embryos contained many antibodies which were not specific for the tissue against which they were directed. That is, all antisera contained antibodies against antigenic determinants found in other body tissues as well. In all cases except the anti-heart pellet the antisera also contained organp Specific antibodies. Antisera after absorption in most cases no longer reacted with heterologous tissues. Anti-heart supernate and anti-heart A urea supernate formed two weak precipitin lines with kidney after absorp- tion (Figure 4, c-e). Anti-heart pellet antisera after absorption no longer formed precipitin lines with either heart or kidney (Figure 4, f). Anti-kidney sera after absorption no longer reacted with heart but would react with heterologous kidney fractions (Figure 3, f; Figure 4, aEb). B. Effect of antigen injection: The embryos injected with tissue fractions frequently failed to develop beyond the tailbud stage (Figure 5). All of the heart fractions injected produced this effect in a significant number of cases (Table 1). Those embryos injected with undiluted heart supernate died Shortly after gastrulation. Those embryos injected with whole kidney or kidney supernate also failed to develop beyond the early tailbud stage. The kidney supernate was lethal to 90? of the embryos injected with it. The supernate diluted 1:10 and the kidney pellet did not produce Signi- ficant numbers of retarded embryos. No embryos were found in which the 16 Figure 4-Analysis of antisera Key to Abbreviations AKSL - Anti-kidney supernate absorbed with liver AKPL - Anti-kidney pellet absorbed with liver AHL - Anti-whole heart absorbed with liver AHSL - Anti-heart supernate absorbed with liver AHUSL - Anti-heart urea supernate absorbed with liver AHPL - Anti-heart pellet absorbed with liver K - whole kidney KM - kidney mitochondria KS - kidney supernate KP - kidney pellet H - whole heart HM - heart mitochondria HS - heart supernate HUS - heart urea supernate HP - heart pellet H DOC S - heart DOC supernate 17 Figure 4 18 Figure 5--Embryos injected with tissue fractions. OU‘ 'SZIBJU Control embryo injected with Holtfreter's solution. Section through the heart. - Same embryo as above. Section through the pronephros. Embryo injected with heart supernate diluted 1:10. Section through the heart. Same embryo as above. Section through the pronephros. Embryo injected with kidney supernate. Section through the heart. Same embryo as above. Section through the pronephros. 19 Figure 5 20 Table 1--Results of Tissue Fraction Injection INJECTED WITH $332?“ NORMAL P2253333“ Control . Holtfreter's 5 25 -- wholea _ 3___ 3 0.06 ___ supefmat;E (lethal) _ - _ -- — —— Heart supernate 1:10a_ ~_ 4 2 0.008 ureS—supernate‘ _ 3 _ _3—— 0.06—— — _ {3.1135 __ —3— — 3 0.06 wholea‘ 2 g _ >o.25 Kidney supanatea ——_(90%—lethal)_4 _——2 _ 0.008 _ ___ supernate 1:10f__ 1_ 5_ >0.§0#___ pelleta — — _1 5 >0.55 a - tissue-Specific antigen demonstrated b - tissue-Specific antigen not demonstrated 21 heart or kidney was Specifically affected. C. Effects of antisera injection. The injection of unabsorbed antisera did not cause cessation of development as was observed in the antigen injected embryos. The in- jection of anti-heart sera produced embryos with Specifically reduced hearts in a few instances (Figure 6, Table 2). These embryos were other- ‘wise normal. No control embryos were found with Specifically reduced hearts. No specific inhibition of kidney development were observed. Antisera absorbed with liver produced embryos whose deve10pment was retarded at the tailbud stage in many instances. This was seen in those embryos injected with absorbed control serum as well as those injected with absorbed tissue antisera. A In addition, specific.inhibition of heart development resulted from the injection of antisera against heart fractions (Figure 7, Table 3). The injection of kidney antisera had no Specific effect on development which could be determined. Specific inhibition was found both in anti- sera in which tissue-Specific antibodies were present and in sera in which no Specificity could be demonstrated. Further, antisera in which a specific component was demonstrated failed in some cases to produce inhibition of the homologous organ. In no case was inhibition of dev- elopment of a heterologous organ observed. 22 Figure 6--Embryos injected with unabsorbed antisera. Control embryo injected with non-immune serum. Section through the heart. Same embryo as above. Section through the pronephros. Embryo injected with anti-heart pellet. Section through the heart. ' Same embryo as above. Section through the pronephros. Embryo injected with anti-kidney supernate. Section through the pronephros. Same embryo as above. Section through the pronephros. ’31 MO 0U) > ll I| I 23 Figure 6 24 ooveavmcosoo no: weaponfipce oamwoodmucewao I n oouenpmcosop weaponfipcs OfimdoOlecmwno I e o o n o especaoQSm Idpq< o N a o wpoaaom o m I I 3 I cuiflmmmmmmmSmummmm endow m- -m - m - o -- amwmmmmmmm -nsne o 0 ma N sesammmmo: muq III:ununnmunuunuuummcuunuunaI: m n.smpmammaom Honesoo awesome madame endgame manna seam anz E§§o EH3 SBBBH soapooncH Edmomfiuc< oonnomnscp Ho endomomalm canoe .IS ‘1" m C) IN I "J I A) \I\ ;re 7-oizhryos injected with absorbed antisera. .ztryo injected wi h non-imzune serum absorb with liver. Section through the heart. Same embryo as above. Sec icn through the pronephros. Embryo injected with anti-heart pellet absorbed with liver. Section through the heart. Same embryo as above. Section through the pronephros. Embryo injected with anti-kidney pellet absorbed with liver. Section through the heart. Same embryo as above. Section through the pronephros. 8, 26 Figure 7 27 vopanamcoson no: moaconflaca owmaooamncamno .. n covuupmcosoc anaconda"; oamwoogmucewuo I a o o o w aooaaoa ecceau I ll III III! I0" I :4. o - io n : m 3.13m Eh: o o o m defies: o H o m mmodnoa o 111: :0 w I o oopocnomzm «0.3 endow - - - 2.--}..- Ag: 0 e N l m tire” 3.3 o m H n «3o? q a w a .fimomommwfiu o o o o oqagmwmm c Hogocoo lilo»: to . s H I m I: Miomonhmmmm omoonum amoenmm amnm