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t-complex(tw18) mouse embryos
Cell Differentiation, 13 (1983) 159-170 Elsevier ScientificPublishers Ireland, Ltd.
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Immunoscanning electron microscopy of antigenic determinants of T/t-complex ( t w18) m o u s e embryos Y e h u d a Ben-Shaul 2, K a r e n A r t z t 1 a n d D o r o t h e a B e n n e t t 1 1 Sloan - Kettering Institute for Cancer Research, New York, U.S.A. and 2 Tel Aviv University, Tel A viv, Israel
(Accepted 23 June 1983)
twls antigen(s) have been localized by immunoscanning electron microscopy in 7.5-day mutant embryos using a primary antiserum to twls made and defined on male germ cells and a secondary rabbit anti-mouse Ig antibody coupled to hemocyanin. Homozygotes ( t w t s / t wls) diagnosed on morphological grounds are more densely labeled primarily on those cell types affected by genetically caused dysfunction. Eight-celled t ~ls embryos are not labeled. This finding of embryonic cell type specificity, together with the available evidence for stage specificity, implicate a function for t-antigens in embryonic development. scanning electron microscopy (SEM); immunolabeling; mouse embryo; T/t-complex Introduction The T/t-complex on chromosome 17 of the mouse is an extensive genetic region with multiple effects on embryonic development, sperm structure and function, and chromosomal recombination (Bennett, 1980). Mutant t-haplotypes are defined as alternate forms of a chromosome region composed of more than one locus. A number of recessive lethal t-haplotypes have been identified by their interaction with the dominant mutation T to produce taillessness in T / t mice. By genetic test, lethal t-haplotypes fall into eight different complementation groups; embryos homozygous for each of the complementation groups have distinctive abnormalities of differentiation which occur at specific points during the first ten days of gestation (Bennett, 1981). Serological studies with antisera prepared against sperm carrying various haplotypes have suggested that T/t-complex antigens are expressed only on the cell surface of sperm, testicular cells, early embryos (Yanagisawa et al., 1974; Kemler et al., 1976; Marticorena et al., 1978) and embryonal carcinoma cells (Artzt et
al., 1974). It has been assumed that these antigens on the surface of embryonic cells may control cell-cell interactions, and that the defects in the mutants are due to malfunction in recognition and morphogenetic movements (Gluecksohn-Waelsch and Erickson, 1970; Bennett et al., 1972), although no direct proof of specific antigen localization was available. We have now examined the question of celland stage-specific distribution of t-associated antigens in embryos carrying t w18, which is a recessive lethal mutation belonging to the t 9 complementation group. Homozygotes for t w~8 develop a primitive streak at the usual time, about 7 days of gestation; however, mesoderm cells, derivatives of the primitive streak, are morphologically abnormal and fail to migrate. The embryos subsequently die at 8 or 9 days because of their retarded and defective development of mesodermal structures (Bennett and Dunn, 1960; Moser and Gluecksohn-Waelsch, 1967). Thin section electron microscopy of t w l S / t w18 mesoderm cells has shown that they are abnormal in shape and do not assemble normal intercellular junctions; these defects have
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been attributed to membrane-related failures of cell-cell r e c ~ t i o n and interaction (Spiegelman and Bennett, 1974). In an attempt to correlate morphological abnormalities of t w~8 homozygotes with the presence of specific surface antigens we have employed indirect immunolabeling and scanning electron microscopy (SEM) using hemocyanin molecules as visual markers. We chose to use scanning electron microscopy and immunolabeling with hemocyanin as a visual marker for several reasons. By SEM, relatively large areas of cell surfaces are exposed for examination at high resolution rather than the low resolution obtained by immunofluorescence or only the profiles of membranes observed by transmission electron microscopy. Also the density, distribution or aggregation of the visual marker molecules, if such exist, can readily be seen using SEM. Used at appropriate concentrations of primary antibody, this method provides an effective semi-quantitative way of measuring relative degrees of primary binding without the nonspecific sticking that is characteristic of larger markers such as latex spheres or viral markers. However, the advantage of good quantitation leads to some sacrifice of definition on certain cell types with heavily blebbed or microvillous surfaces, because of the small size and relatively uniform structure of hemocyanin molecules. This approach has yielded interesting results for other embryonic systems (Ben-Shaul et al., 1979, 1980) as well as for t-mutant sperm (Ben-Shaul et al., 1981). Here, it has enabled us to analyze the cell surfaces of different cell types in embryos, to correlate, qualitatively, the presence of the specific antigen(s) with the morphology of homozygotes (twls/twl s) versus littermate controls consisting of heterozygotes ( + / t wls) and normal embryos ( + / + ), and to suggest that the expression of the antigen(s) is cell type and stage specific.
B T B R T F / N e v strain background. Tailles mice were backcrossed to the random-bred CF 1 stock (Carworth Farms) to obtain normal tailed obligate t w18 heterozygotes, which were then crossed together to produce litters containing tw~8/t w18, + / t w18, and + / + embryos. For normal controls ( + / + ) CF 1 mice were used. Litters containing t 12 morulae were obtained from similar crosses.
Collection of embryos Mating was confirmed by the presence of a vaginal plug the following morning, which is designated as day 0 of gestation, t w18 homozygotes die at about the eighth or ninth day of embryonic development. Therefore, to avoid analysis of dead or necrotic cells the study was carried out on embryos during the seventh day, prior to the lethal stage, on the assumption that since morphological abnormalities are often evident then, the relevant t-antigen(s) would also be expressed. Females at the seventh day of pregnancy were killed by cervical dislocation and their uteri removed into Whitten's medium. The uterine muscles were stripped away and the embryos dissected free from the uterine decidua with fine forceps. After washing in medium, the embryos were split longitudinally with a very fine needle into two halves, to expose the surfaces of the three germ layers, and fixed in 2% glutaraldehyde in 0.1 M phosphate buffer. It is only rarely possible to achieve perfect symmetry or lack of distortion in this procedure. Morula stage embryos were collected at the second day of gestation by flushing the oviducts with Whitten's medium. The zona pellucida was removed by gentle digestion with 0.5% pronase (in PBS) for a few minutes under observation with a dissecting microscope. These embryos were washed three times in medium and then incubated for at least 2 h before fixation in glutaraldehyde.
Materials and Methods
Immunolabeling
Mice
Antisera. The procedures used to raise anti-t sera, and to absorb and test for specific anti-t activity were as described previously (Yanagisawa et al., 1974; Artzt and Bennett, 1977). The antiserum used in these experiments was [129/Sv X
Mice were obtained from tailless balanced lethal stocks maintained as closed colonies at the S l o a n - K e t t e r i n g Institute on the i n b r e d
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B A L B / c ] F 1 anti-t w18. This antiserum diluted 1 / 8 was absorbed with 40 × 106 + / + testicular cells to 0.1 ml serum. In cytotoxicity tests after absorption it was negative on + / + cells and retained an average cytotoxic index of 0.30 on + / t w18 testicular cells with a titer of > 1 : 128.
Preparation of marker conjugates Hemocyanin, the visual marker, was extracted from the hemolymph of the 'Jerusalem mountain' snail Levantina hierosolima. The shell was softened by HC1 and punctured at the region of the heart. Drops of hemolymph were collected into a test tube on ice. Shell debris were removed by centrifugation at 300 × g, 20 rain and hemocyanin molecules were collected at 50,000 rpm for 90 rain (Beckman L3-50 ultracentrifuge, Rotor SW 50.1). Concentration of hemocyanin was determined spectrophotometrically (Shaklai and Daniel, 1970) and dilution to the required concentration was in PBS. Conjugation of hemocyanin to rabbit antimouse IgG (Miles) was carried out by two-step procedure: hemocyanin was activated by glutaraldehyde; 3 ml of 20% glutaraldehyde were swirled on ice, 1 ml of 20 m g / m l hemocyanin was added and the mixture was kept for 1 h, before removing excess glutaraldehyde by dialysis against PBS. The activated hemocyanin was added to an IgG solution at a molecular ratio of 5 : 1 at neutral pH. Reaction time was 3 - 4 h. To terminate the reaction 0.2 ml of 2 M glycine was added and the mixture kept for 30 min. The conjugate was dialyzed against PBS overnight and then passed through a Sepharose 4B column to separate excess unconjugated protein and hemocyanin aggregates. (Hemocyanin aggregates are eluted before the clean conjugated hemocyanin preparation.) Confirmation of absence of hemocyanin-conjugate aggregrates was done by negative staining electron microscopy. The working concentration was about 1 mg hemocyanin/ml. An identical procedure was used to prepare goat anti-rabbit IgG-hemocyanin conjugate.
lmmunolabeling Split 7 day embryos, or embryos at the 8-cell stage were first incubated in the appropriate anti-t
serum for 15 min at 37°C followed by 30 min at room temperature. Mouse anti-t w18 was used at a 1 : 1 0 dilution and rabbit anti-t 12 at 1:25. The labeling was carried out in miniwells (Terasaki type) and care was taken that the embryos were totally covered by the antiserum. Following the incubation and 3 washes in PBS the embryos were transferred to IgG-hemocyanin conjugate (rabbit anti-mouse IgG-hemocyanin for labeling of t w18 embryos and goat anti-rabbit hemocyanin for labeling of t 1:) for 30 min at room temperature, washed 3 times in PBS and fixed for 30 min in 2% glutaraldehyde in PBS.
Scanning electron microscopy Fixed embryos were first dehydrated in a series of graded ethyl alcohols and then critical-point dried from liquid CO 2 (Anderson, 1966). Before dehydration, morulae were attached to 13 mm plastic coverslips precoated with 0.25% poly-Llysine (Sigma), and were dried while attached. Dried 7.5-day t ~ s embryos were attached to metal stubs with double Scotch tape. This procedure often results in some distortion of gross morphology because of uneven attachments and twisting (see for example Figs. 1, 3 and 4), but generally leaves cell association intact (see Figs. 5-8). After coating by rotation with gold-paladium (80:20) the specimens were observed and photographed in a Jeol-S35 scanning microscope at 15-30 kV.
Results
Figs. 1 - 4 show low magnification scanning electron micrographs of the inner aspect of morphologically normal embryos (either + / t w18 or + / + ) (Figs. 1, 2) and embryos diagnosed on gross morphological criteria as homozygous tw18/ twm (Figs. 3, 4), just later than 7.5 days of gestation. In morphologically normal embryos either + / + or + / t ~18, the three germ layers are clearly observed as continuous layers throughout the embryo, since the embryonic mesoderm has already migrated from the primitive streak region and penetrated the space between the outer embryonic
i
163 TABLE I Immunolabeling of embryos with mouse anti-t wls antiserum and rabbit anti-mouse Ig-G coupled with hemocyanin Embryos 7.5-7.75 day embryos Preselected homozygotes (tw'8/t w18) Morphologically normal littermates (twls/+ or +/+) 7.5 day embryos pooled from several t wls litters 7.5 day normal C F 1 embryos
t wls embryos 8-celled stage
Number of embryos a 5 4 4
12 13 11 1 10 4 23
Mesoderm
Ectoderm
+ + +
+ +
( + + )-( + + + )
( + )-( + + )
-
-
+ + + + + + -
(+ + ) - ( + + +) ( + )-( + + ) -
Endoderm
+ (-)-( + ) -
+ (-)-( + ) -
+ -
+ + + ~ dense labeling, + + = positive labeling above background, + = weak labeling slightly above background, - = no labeling (see Figs. for examples) a Number of embryos with a specific pattern of labeling as scored under mesoderm, ectoderm and endoderm.
endoderm and the columnar embryonic ectoderm l i n i n g the a m n i o t i c cavity. T h e m e s o d e r m cells a r e s t e l l a t e a n d d i s p l a y t h i n f i l o p o d i a (Figs. 5, 6). I n c o n t r a s t , t * ~ 8 / t w~8 h o m o z y g o t e s s h o w m e s o d e r m cells o n l y in t h e r e g i o n o f t h e p r i m i t i v e streak, where they accumulate between the endoderm and e c t o d e r m , p u s h i n g e c t o d e r m cells to f o r m a ' b u l g e ' i n t h e a m n i o t i c c a v i t y (see Fig. 3). T h e s e ' b u l g e s ' are readily observed under the dissecting microscope, therefore permitting the identification and separation of homozygous embryos from normals o r h e t e r o z y g o t e s . T h e m e s o d e r m cells a r e r o u n d r a t h e r t h a n stellate a n d u s u a l l y d i s p l a y r e l a t i v e l y s m a l l n u m b e r s o f f i l o p o d i a (Fig. 8).
Immunolabeling
Table I summarizes the immunolabeling obs e r v a t i o n s d e s c r i b e d in t h e f o l l o w i n g sections. W e s t u d i e d in d e t a i l o n e l a r g e litter t h a t c o n t a i n e d 5 e m b r y o s d e f i n e d as t w18 h o m o z y g o t e s b y t h e criterion of bulging primitive streak mesoderm, and 8 m o r p h o l o g i c a l l y n o r m a l l i t t e r m a t e s . A l l the h o m o z y g o t e s s h o w t h e p r e s e n c e o f t ~18 a n t i g e n ( s ) o n t h e i r a b n o r m a l m e s o d e r m cells, w h i c h w e r e d e n s e l y l a b e l e d (Figs. 9, 10). I n a f e w cases, a b n o r m a l l y g r o w i n g m e s o d e r m cells h a v e p r o t r u d e d o u t s i d e t h r o u g h the e n d o d e r m layer. S u c h p r o t r u s i o n s w e r e a l s o l a b e l e d (Figs. 11, 12). T h e l a t e r a l s u r f a c e s o f
Fig. 1, 2. Low magnification SEMs of morphologically normal 7.5-day t wl8 segregant embryos. Ec=embryonic ectoderm; En = embryonic endoderm; Arrows = mesoderm cells; E = extra-embryonic region. Scale bars = 100/tm. Figs. 3, 4. Low magnification of morphologically abnormal 7.5-day t W18 homozygous embryos. M = mesoderm cells in a single restricted region. The abnormal embryos are smaller than the normal ones. Scale bars ffi100 pm. Figs. 5, 6. Higher magnification of the three embryonic germ layers in morphologically normal embryos (details from Figs. 2 and 1, respectively). M = mesoderm cells, located between ectoderm and endoderm, stellate and displaying filopodia. En = endoderm cells with short microvilli facing the outer side of the embryo. Ec = columnar ectoderm cells. Scale bars = 10/zm. Figs. 7, 8. Higher magnification of embryonic layers in morphologically abnormal homozygous embryos. Fig. 7 shows that mesoderm cells have not migrated (there are no mesoderm cells in this region). Ec = ectoderm; En = endoderm; detail from the upper left side of Fig. 4 (box). Fig. 8 shows abnormal, round, mesoderm cells (M) with only a few filopodia; detail from the lower right side of Fig. 3 (box). Scale bars =10/~m.
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Figs. 9, 10. Densely labeled mesoderm cells of homozygous 7.5-day embryo. The l a b d is randomly distributed (details from Figs. 3 and 4) (some hemocyanin molecules are indicated by arrows). Bars = 1 #m. Inset is a detail from Fig. 9 (box) showing in 0.5/~m 2 of cell surface area more than 20 hemocyanin molecules. Scale bar = 0.5/zm. Figs. 11, 12. Protrusion of a mesoderm cell through the endoderm of a homozygous embryo. The normal endodermal microvilli are not labeled (see also Fig. 16 for high magnification); some abnormal endodermal blebs (Fig. 11, arrows) arepossibly labeled. En = the outer surfaces of endoderm cells. Fig. 11. Scale bar = 2 # m . Fig. 12. Scale bar = 1 #m.
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Figs. 13, 14. Ectoderm cells of t wls homozygotes showing variability in the density of the label from positive labeling just above background (Fig. 13) to dense labeling (Fig. 14). Some hemocyanin molecules are indicated by arrows. Scale bars = 1 /~m. Inset is detail from Fig. 13 (dark arrow) (magnification × 3) showing very few hemocyanin particles. Fig. 15. A mesoderm cell of a morphologically normal putative heterozygnte. The cell is positively labeled above background. Fig. 16. Endoderm cell from a morphologically normal embryo. The membrane facing a neighboring cell is weakly labeled. The microvilli are devoid of any label. Scale bar = 1 ~m.
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Fig. 17. Ectoderm cells of morphologically normal putative heterozygote embryo, positively labeled. Scale bar = 1 / t m . Fig. 18. Membranes of ectoderm cells from morphologically normal embryo, facing the amniotic cavity, are not labeled. Scale bar = 1 p.m. Figs. 19, 20. Mesoderm and ectoderm (respectively) cells of putative normal embryo showing only background labeling (arrows). Respective cells from CF 1 normal embryos look exactly the same. Scale bars = 1 /~m.
167 ectoderm cells in these embryos were always labeled, although the density of the label was generally lower than that on the mesoderm ceils, and was also more variable. Variability in density was found not only between cells of different embryos, but also between ectodermal cells of the same embryo (Figs. 13, 14). Ectodermal cell surfaces that face the amniotic cavity were generally labeled more weakly than the lateral surfaces. The label was observed on their lateral surfaces but never on their apical borders facing the outside. Embryonic endoderm cells were usually weakly labeled or not labeled at all. Among 8 littermate embryos which were morphologically normal, 4 were not labeled, or were labeled at the background level (i.e. an occasional hemocyanin particle) and are probably normal segregants ( + / / + ). The other four were labeled above background, ranging from 'weak' labeling to 'dense' labeling, and are probably + / / t wm heterozygotes (Figs. 15-20). The numbers of embryos studied here, and in the series reported below are obviously too small to provide statistical assurance that our genotypic assignments are correct, although the proportions of densely labeled, moderately labeled and essentially negative embryos are compatible with the 1 : 2 : 1 genotypic ratios of t w18, and + / / + embryos predicted on the basis of the 0.5 transmission ratio of t wls. Furthermore, it has to be emphasized at this point that the analysis performed was a qualitative one and the term 'weak' or 'dense' does not imply a count of hemocyanin marker molecules per unit of surface area, but rather the density of the marker in relation to labeled mesoderm cells of homozygous embryos. In all the embryos analyzed, both homozygotes and heterozygotes, cells in the extraembryonic region were virtually devoid of label. This observation served as an internal control. In another set of observations, 36 embryos from five litters of + / / t wls parents were analyzed at slightly earlier stages, which precluded preselection of homozygous embryos on morphological criteria. Mesoderm cells of 12 embryos were densely labeled; their embryonic ectoderm was also positively labeled but varied in density. Thirteen other embryos had unequivocal label on their mesoderm cells but the label was not as dense as in the first
group, and their ectoderm was usually weakly labeled. Eleven embryos showed no label on either mesoderm or ectoderm. Control
Beside the usual control where normal mouse serum was used instead of specific antiserum, and which yielded only background labeling, the most important one was the analysis with anti-t wl8 antiserum of normal embryos from litters not segregating for t wls (-k-f-k-X+//+). These mice were mated and dissected at the same time as the experimental ones from + / / t w18 parents, and the embryos were incubated with the same anti-t w18 serum under the same conditions, and processed for scanning microscopy at the same time. Of 11 embryos analyzed in detail only one showed label on mesoderm cells, which was weak but above background. The pictures obtained were identical to unlabeled, morphologically normal embryos from twm litters (see Figs. 19, 20). Stage specificity
To determine whether the expression of t wa8 antigens(s) on the cell surfaces of embryos carrying the t wls mutation is stage specific, an immunolabeling experiment of embryos from + / t wl8 parents at the 8-cell stage was performed. Of 27 embryos examined, only 4 showed very weak labeling, marginally above background (Figs. 21, 22). Since homozygotes could of course not be identified prior to immunolabeling, an additional control experiment was performed to determine whether these early stage embryos could indeed be positively and densely labeled. When morulae of another t-mutant, t ~2, whose homozygous phenocritical period occurs between cleavage and blastocyst stages, were incubated with an appropriate antiserum (rabbit anti-t ~2) and labeled with conjugated hemocyanin markers (goat-anti rabbit IgG-hemocyanin), some of the embryos were heavily labeled (data not shown). Technical problems associated with the small size of post-implantation embryos between 5 and 7 days have so far prevented us from addressing the question of stage specificity more precisely.
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Figs. 21, 22. Eight-celledembryos (Fig. 21) from litters segregating t w18 homozygotes.The labeling is at the background level (Fig. 22, arrows). Fig. 21. Scale bar = 10 #m. Fig. 22. Scale bar = 1 #m.
Discussion This study addressed pivotal questions, namely, whether t-antigens, so well characterized with male germ cells, are detectable in t-mutant embryos and, even more importantly, whether they are present specifically on those tissues displaying genetically induced dysfunction. If true, this would implicate a function for these antigens, and support previously proposed conjectures (Artzt and Bennett, 1977) that these structures play a role in defective cell interaction in the embryo. We chose to study the t ~ 8 mutation because it acts at a time when the embryo is becoming complex, and is accessible to definitive experimental investigation because homozygotes can be diagnosed on morphological grounds. At late 7.5 days there is a clearly definable set of tissues affected in t w j 8 homozygotes, as well as unaffected tissues which continue to develop normally for several days more (Bennett and Dunn, 1960; Moser and GluecksohnWaelsch, 1967). In normal embryos, the em-
bryonic ectoderm feeds cells into the proliferative center of the primitive streak, from where they emerge as stellate and migratory mesoderm. In t w18 homozygotes, however, the embryonic ectoderm and mesoderm fail to achieve this critical step, and it is precisely these cells that are most heavily labeled with anti-t w18 serum. On the other hand, the embryonic endoderm, and the extraembryonic ectoderm and endoderm, which continue to develop normally in the mutants are virtually devoid of label. This offers strong evidence that at 7.5 days the t w18 antigen(s) are exposed primarily on cells affected by the mutation. Our data also provide evidence that the t w18 antigen is not expressed on eight-celled embryos, and thus suggest in a very broad sense that its expression is stage specific. Obviously though, this needs to be confirmed, and the temporal limits of antigen expression defined, by further experiments with embryos at stages intermediate between the 2-day and 7.5-day stages we studied here, as well as with older embryos up to the time of death. We
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must also point out that others (Kemler et al., 1976) have evidence arguing against stage specificity. Using indirect immunofluorescent techniques this group reported results suggesting that morulae homozygous for t ° t wS, t w l s and t w32 all expressed their relevant t-antigen, although only t ~3e embryos show defects at the morula stage. We later assayed t w5 a n d t ~32 morulae by complement dependent cytotoxicity methods, and reported antigen expression on t w32 but not t ~5 embryos (Marticorena et al., 1978). Thus all our data are compatible with stage-specific expression of t-associated antigens, although we have no explanation for the discrepant results of Kemler et al. In any case, the present report is the first time t-antigens have been localized in time and place to the affected tissues in known homozygous embryos. It is worth emphasizing that whatever the biological or molecular defect in t wls, it is a critical one for cell commitment and survival. It was observed by Artzt and Bennett (1972) that t w l s homozygotes transplanted to ectopic sites such as the testis continued to grow although only half survived. However, those growths resulted in tumors composed primarily of ectodermal tissues but not of mesoderm or of any structures dependent on epithelial-mesenchymal interactions. Furthermore, these tumors were frequently histologically malignant and resembled neuroepitheliomas, which suggested that normal mesodermal interactions are necessary for the controlled differentiation of ectoderm. It is an interesting, although unexplained, point that other investigators found that transforming the mutant cells in tissue culture with SV40 produced primarily cell lines of mesodermal origin (myoblasts and adipocytes) (Kelly et al., 1979), which suggested that the defective mutant mesoderm cells may be particularly sensitive targets for viral transformation.
Acknowledgements This work was supported by NSF Grant PCM 77-17835, NIH Grant CA-21651, and by NCI Core Grant CA-08748.
References Anderson, T.F.: Electron microscopy of micro-organisms. In: Physical Techniques in Biological Research, ed. A.W. Pollister, Vol. 3A (Academic Press, N.Y.) pp. 369-387 (1966). Artzt, K. and D. Bennett: Serological analysis of sperm of antigenically cross-reacting T/t-haplotypes and their recombinants. Immunogenetics 5, 97-107 (1977). Artzt, K., D. Bennett and F. Jacob: Primitive teratocarcinoma cells express a differentiation antigen specified by a gene at the T-locus in the mouse. Proc. Natl. Acad. Sci. U.S.A. 71, 811-814 (1974). Bennett, D.: The T-complex in the mouse: an assessment after 50 years of study. Harvey Lect. Ser. 74, 1-24 (1980). Bennett, D.: T / t locus, its role in embryogenesis and its relation to classical histocompatibility systems. In: Ontogeny and Phylogeny of Lymphocyte Functions, Vol. 29: Progress in Allergy (Karger, Basel) pp. 35-53 (1981). Bennett, D. and L.C. Dunn: A lethal mutant (t wls) in the house mouse showing partial duplication. J. Exp. Zool. 143, 203-219 (1960). Bennett, D., E. Goldberg, L.C. Dunn, and E.A. Boyse: Serological detection of a cell surface antigen specified by the T (Braehyury) mutant gene in the house mouse. Proc. Natl. Acad. Sci. U.S.A. 69, 2076-2080 (1972). Ben-Shaul, Y., R.E. Hausman and A.A. Moscona: Visualization of a cell surface glycoprotein, the retina cognin, on embryonic cells by immunolatex labeling and scanning electron microscopy. Dev. Biol. 72, 89-101 (1979). B¢n-Shaul, Y., R.E. Hausman and A.A. Moscona: Age-dependent differences in cognin recognition on embryonic retina cells: immunolabeling and SEM studies. Dev. Neurosci. 3, 66-74 (1980). Ben-Shaul, Y., B. Babiarz and D. Bennett: Visualization of the F9 antigen on sperm from normal and T/t-complex mutants by immunoscanning electron microscopy. Dev. Genet. 2, 171-183 (1981). Glueeksohn-Waelsch, S. and R.P. Erickson: Cellular membranes: a possible link between H-2 and T-locus effects. In: Proc. Syrup. Immunogenetics of the H-2 System, eds. A. Lengerova and M. Vojtiskova (Karger, Basel) pp. 120-122 (1970). Kelly, F., J.-L. Guenet and H. Condamine: Karyologically identified homozygous t w~8 embryos: extrauterine growth properties and transformation by SV40. Cell 16, 919-927 (1979). Kemler, R., C. Babinet, H. Condamine, G. Gachelin, J.-L. Guenet and F. Jacob: Embryonal carcinoma antigen and the T / t locus of the mouse. Proc. NatL Acad. Sci. U.S.A. 73, 4080-4084 (1976). Marticorena, P., K. Artzt and D. Bennett: Relationship of F9 antigen and genes of the T / t complex. Immunogenetics 7, 337-347 (1978). Moser, G. and S. Gluecksohn-Waelseh: Developmental genetics of a recessive allele at the complex T-locus in the mouse. Dev. Biol. 16, 564-576 (1967).
170 Shaklai, N. and E. Daniel: Fluorescence properties of hemocyanin from Leoantina hierosolima. Biochemistry 9, 564-568 (1970). Spiegelman, M. and D. Bennett: Fine structural study of cell migration in the early mesoderm of normal and mutant mouse embryos (T-locus: tg/tg). J. Embryol. Exp. Morphol. 32, 723-738 (1974).
Yanagisawa, K., D. Bennett, E.A. Boyse, L.C. Dunn and A. DiMeo: Serological identification of sperm antigens specified by lethal t-alleles in the mouse. Immunogenetics 1, 57-67 (1974).
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Immunoscanning electron microscopy of antigenic determinants of T/t-complex ( t w18) m o u s e embryos Y e h u d a Ben-Shaul 2, K a r e n A r t z t 1 a n d D o r o t h e a B e n n e t t 1 1 Sloan - Kettering Institute for Cancer Research, New York, U.S.A. and 2 Tel Aviv University, Tel A viv, Israel
(Accepted 23 June 1983)
twls antigen(s) have been localized by immunoscanning electron microscopy in 7.5-day mutant embryos using a primary antiserum to twls made and defined on male germ cells and a secondary rabbit anti-mouse Ig antibody coupled to hemocyanin. Homozygotes ( t w t s / t wls) diagnosed on morphological grounds are more densely labeled primarily on those cell types affected by genetically caused dysfunction. Eight-celled t ~ls embryos are not labeled. This finding of embryonic cell type specificity, together with the available evidence for stage specificity, implicate a function for t-antigens in embryonic development. scanning electron microscopy (SEM); immunolabeling; mouse embryo; T/t-complex Introduction The T/t-complex on chromosome 17 of the mouse is an extensive genetic region with multiple effects on embryonic development, sperm structure and function, and chromosomal recombination (Bennett, 1980). Mutant t-haplotypes are defined as alternate forms of a chromosome region composed of more than one locus. A number of recessive lethal t-haplotypes have been identified by their interaction with the dominant mutation T to produce taillessness in T / t mice. By genetic test, lethal t-haplotypes fall into eight different complementation groups; embryos homozygous for each of the complementation groups have distinctive abnormalities of differentiation which occur at specific points during the first ten days of gestation (Bennett, 1981). Serological studies with antisera prepared against sperm carrying various haplotypes have suggested that T/t-complex antigens are expressed only on the cell surface of sperm, testicular cells, early embryos (Yanagisawa et al., 1974; Kemler et al., 1976; Marticorena et al., 1978) and embryonal carcinoma cells (Artzt et
al., 1974). It has been assumed that these antigens on the surface of embryonic cells may control cell-cell interactions, and that the defects in the mutants are due to malfunction in recognition and morphogenetic movements (Gluecksohn-Waelsch and Erickson, 1970; Bennett et al., 1972), although no direct proof of specific antigen localization was available. We have now examined the question of celland stage-specific distribution of t-associated antigens in embryos carrying t w18, which is a recessive lethal mutation belonging to the t 9 complementation group. Homozygotes for t w~8 develop a primitive streak at the usual time, about 7 days of gestation; however, mesoderm cells, derivatives of the primitive streak, are morphologically abnormal and fail to migrate. The embryos subsequently die at 8 or 9 days because of their retarded and defective development of mesodermal structures (Bennett and Dunn, 1960; Moser and Gluecksohn-Waelsch, 1967). Thin section electron microscopy of t w l S / t w18 mesoderm cells has shown that they are abnormal in shape and do not assemble normal intercellular junctions; these defects have
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been attributed to membrane-related failures of cell-cell r e c ~ t i o n and interaction (Spiegelman and Bennett, 1974). In an attempt to correlate morphological abnormalities of t w~8 homozygotes with the presence of specific surface antigens we have employed indirect immunolabeling and scanning electron microscopy (SEM) using hemocyanin molecules as visual markers. We chose to use scanning electron microscopy and immunolabeling with hemocyanin as a visual marker for several reasons. By SEM, relatively large areas of cell surfaces are exposed for examination at high resolution rather than the low resolution obtained by immunofluorescence or only the profiles of membranes observed by transmission electron microscopy. Also the density, distribution or aggregation of the visual marker molecules, if such exist, can readily be seen using SEM. Used at appropriate concentrations of primary antibody, this method provides an effective semi-quantitative way of measuring relative degrees of primary binding without the nonspecific sticking that is characteristic of larger markers such as latex spheres or viral markers. However, the advantage of good quantitation leads to some sacrifice of definition on certain cell types with heavily blebbed or microvillous surfaces, because of the small size and relatively uniform structure of hemocyanin molecules. This approach has yielded interesting results for other embryonic systems (Ben-Shaul et al., 1979, 1980) as well as for t-mutant sperm (Ben-Shaul et al., 1981). Here, it has enabled us to analyze the cell surfaces of different cell types in embryos, to correlate, qualitatively, the presence of the specific antigen(s) with the morphology of homozygotes (twls/twl s) versus littermate controls consisting of heterozygotes ( + / t wls) and normal embryos ( + / + ), and to suggest that the expression of the antigen(s) is cell type and stage specific.
B T B R T F / N e v strain background. Tailles mice were backcrossed to the random-bred CF 1 stock (Carworth Farms) to obtain normal tailed obligate t w18 heterozygotes, which were then crossed together to produce litters containing tw~8/t w18, + / t w18, and + / + embryos. For normal controls ( + / + ) CF 1 mice were used. Litters containing t 12 morulae were obtained from similar crosses.
Collection of embryos Mating was confirmed by the presence of a vaginal plug the following morning, which is designated as day 0 of gestation, t w18 homozygotes die at about the eighth or ninth day of embryonic development. Therefore, to avoid analysis of dead or necrotic cells the study was carried out on embryos during the seventh day, prior to the lethal stage, on the assumption that since morphological abnormalities are often evident then, the relevant t-antigen(s) would also be expressed. Females at the seventh day of pregnancy were killed by cervical dislocation and their uteri removed into Whitten's medium. The uterine muscles were stripped away and the embryos dissected free from the uterine decidua with fine forceps. After washing in medium, the embryos were split longitudinally with a very fine needle into two halves, to expose the surfaces of the three germ layers, and fixed in 2% glutaraldehyde in 0.1 M phosphate buffer. It is only rarely possible to achieve perfect symmetry or lack of distortion in this procedure. Morula stage embryos were collected at the second day of gestation by flushing the oviducts with Whitten's medium. The zona pellucida was removed by gentle digestion with 0.5% pronase (in PBS) for a few minutes under observation with a dissecting microscope. These embryos were washed three times in medium and then incubated for at least 2 h before fixation in glutaraldehyde.
Materials and Methods
Immunolabeling
Mice
Antisera. The procedures used to raise anti-t sera, and to absorb and test for specific anti-t activity were as described previously (Yanagisawa et al., 1974; Artzt and Bennett, 1977). The antiserum used in these experiments was [129/Sv X
Mice were obtained from tailless balanced lethal stocks maintained as closed colonies at the S l o a n - K e t t e r i n g Institute on the i n b r e d
161
B A L B / c ] F 1 anti-t w18. This antiserum diluted 1 / 8 was absorbed with 40 × 106 + / + testicular cells to 0.1 ml serum. In cytotoxicity tests after absorption it was negative on + / + cells and retained an average cytotoxic index of 0.30 on + / t w18 testicular cells with a titer of > 1 : 128.
Preparation of marker conjugates Hemocyanin, the visual marker, was extracted from the hemolymph of the 'Jerusalem mountain' snail Levantina hierosolima. The shell was softened by HC1 and punctured at the region of the heart. Drops of hemolymph were collected into a test tube on ice. Shell debris were removed by centrifugation at 300 × g, 20 rain and hemocyanin molecules were collected at 50,000 rpm for 90 rain (Beckman L3-50 ultracentrifuge, Rotor SW 50.1). Concentration of hemocyanin was determined spectrophotometrically (Shaklai and Daniel, 1970) and dilution to the required concentration was in PBS. Conjugation of hemocyanin to rabbit antimouse IgG (Miles) was carried out by two-step procedure: hemocyanin was activated by glutaraldehyde; 3 ml of 20% glutaraldehyde were swirled on ice, 1 ml of 20 m g / m l hemocyanin was added and the mixture was kept for 1 h, before removing excess glutaraldehyde by dialysis against PBS. The activated hemocyanin was added to an IgG solution at a molecular ratio of 5 : 1 at neutral pH. Reaction time was 3 - 4 h. To terminate the reaction 0.2 ml of 2 M glycine was added and the mixture kept for 30 min. The conjugate was dialyzed against PBS overnight and then passed through a Sepharose 4B column to separate excess unconjugated protein and hemocyanin aggregates. (Hemocyanin aggregates are eluted before the clean conjugated hemocyanin preparation.) Confirmation of absence of hemocyanin-conjugate aggregrates was done by negative staining electron microscopy. The working concentration was about 1 mg hemocyanin/ml. An identical procedure was used to prepare goat anti-rabbit IgG-hemocyanin conjugate.
lmmunolabeling Split 7 day embryos, or embryos at the 8-cell stage were first incubated in the appropriate anti-t
serum for 15 min at 37°C followed by 30 min at room temperature. Mouse anti-t w18 was used at a 1 : 1 0 dilution and rabbit anti-t 12 at 1:25. The labeling was carried out in miniwells (Terasaki type) and care was taken that the embryos were totally covered by the antiserum. Following the incubation and 3 washes in PBS the embryos were transferred to IgG-hemocyanin conjugate (rabbit anti-mouse IgG-hemocyanin for labeling of t w18 embryos and goat anti-rabbit hemocyanin for labeling of t 1:) for 30 min at room temperature, washed 3 times in PBS and fixed for 30 min in 2% glutaraldehyde in PBS.
Scanning electron microscopy Fixed embryos were first dehydrated in a series of graded ethyl alcohols and then critical-point dried from liquid CO 2 (Anderson, 1966). Before dehydration, morulae were attached to 13 mm plastic coverslips precoated with 0.25% poly-Llysine (Sigma), and were dried while attached. Dried 7.5-day t ~ s embryos were attached to metal stubs with double Scotch tape. This procedure often results in some distortion of gross morphology because of uneven attachments and twisting (see for example Figs. 1, 3 and 4), but generally leaves cell association intact (see Figs. 5-8). After coating by rotation with gold-paladium (80:20) the specimens were observed and photographed in a Jeol-S35 scanning microscope at 15-30 kV.
Results
Figs. 1 - 4 show low magnification scanning electron micrographs of the inner aspect of morphologically normal embryos (either + / t w18 or + / + ) (Figs. 1, 2) and embryos diagnosed on gross morphological criteria as homozygous tw18/ twm (Figs. 3, 4), just later than 7.5 days of gestation. In morphologically normal embryos either + / + or + / t ~18, the three germ layers are clearly observed as continuous layers throughout the embryo, since the embryonic mesoderm has already migrated from the primitive streak region and penetrated the space between the outer embryonic
i
163 TABLE I Immunolabeling of embryos with mouse anti-t wls antiserum and rabbit anti-mouse Ig-G coupled with hemocyanin Embryos 7.5-7.75 day embryos Preselected homozygotes (tw'8/t w18) Morphologically normal littermates (twls/+ or +/+) 7.5 day embryos pooled from several t wls litters 7.5 day normal C F 1 embryos
t wls embryos 8-celled stage
Number of embryos a 5 4 4
12 13 11 1 10 4 23
Mesoderm
Ectoderm
+ + +
+ +
( + + )-( + + + )
( + )-( + + )
-
-
+ + + + + + -
(+ + ) - ( + + +) ( + )-( + + ) -
Endoderm
+ (-)-( + ) -
+ (-)-( + ) -
+ -
+ + + ~ dense labeling, + + = positive labeling above background, + = weak labeling slightly above background, - = no labeling (see Figs. for examples) a Number of embryos with a specific pattern of labeling as scored under mesoderm, ectoderm and endoderm.
endoderm and the columnar embryonic ectoderm l i n i n g the a m n i o t i c cavity. T h e m e s o d e r m cells a r e s t e l l a t e a n d d i s p l a y t h i n f i l o p o d i a (Figs. 5, 6). I n c o n t r a s t , t * ~ 8 / t w~8 h o m o z y g o t e s s h o w m e s o d e r m cells o n l y in t h e r e g i o n o f t h e p r i m i t i v e streak, where they accumulate between the endoderm and e c t o d e r m , p u s h i n g e c t o d e r m cells to f o r m a ' b u l g e ' i n t h e a m n i o t i c c a v i t y (see Fig. 3). T h e s e ' b u l g e s ' are readily observed under the dissecting microscope, therefore permitting the identification and separation of homozygous embryos from normals o r h e t e r o z y g o t e s . T h e m e s o d e r m cells a r e r o u n d r a t h e r t h a n stellate a n d u s u a l l y d i s p l a y r e l a t i v e l y s m a l l n u m b e r s o f f i l o p o d i a (Fig. 8).
Immunolabeling
Table I summarizes the immunolabeling obs e r v a t i o n s d e s c r i b e d in t h e f o l l o w i n g sections. W e s t u d i e d in d e t a i l o n e l a r g e litter t h a t c o n t a i n e d 5 e m b r y o s d e f i n e d as t w18 h o m o z y g o t e s b y t h e criterion of bulging primitive streak mesoderm, and 8 m o r p h o l o g i c a l l y n o r m a l l i t t e r m a t e s . A l l the h o m o z y g o t e s s h o w t h e p r e s e n c e o f t ~18 a n t i g e n ( s ) o n t h e i r a b n o r m a l m e s o d e r m cells, w h i c h w e r e d e n s e l y l a b e l e d (Figs. 9, 10). I n a f e w cases, a b n o r m a l l y g r o w i n g m e s o d e r m cells h a v e p r o t r u d e d o u t s i d e t h r o u g h the e n d o d e r m layer. S u c h p r o t r u s i o n s w e r e a l s o l a b e l e d (Figs. 11, 12). T h e l a t e r a l s u r f a c e s o f
Fig. 1, 2. Low magnification SEMs of morphologically normal 7.5-day t wl8 segregant embryos. Ec=embryonic ectoderm; En = embryonic endoderm; Arrows = mesoderm cells; E = extra-embryonic region. Scale bars = 100/tm. Figs. 3, 4. Low magnification of morphologically abnormal 7.5-day t W18 homozygous embryos. M = mesoderm cells in a single restricted region. The abnormal embryos are smaller than the normal ones. Scale bars ffi100 pm. Figs. 5, 6. Higher magnification of the three embryonic germ layers in morphologically normal embryos (details from Figs. 2 and 1, respectively). M = mesoderm cells, located between ectoderm and endoderm, stellate and displaying filopodia. En = endoderm cells with short microvilli facing the outer side of the embryo. Ec = columnar ectoderm cells. Scale bars = 10/zm. Figs. 7, 8. Higher magnification of embryonic layers in morphologically abnormal homozygous embryos. Fig. 7 shows that mesoderm cells have not migrated (there are no mesoderm cells in this region). Ec = ectoderm; En = endoderm; detail from the upper left side of Fig. 4 (box). Fig. 8 shows abnormal, round, mesoderm cells (M) with only a few filopodia; detail from the lower right side of Fig. 3 (box). Scale bars =10/~m.
164
Figs. 9, 10. Densely labeled mesoderm cells of homozygous 7.5-day embryo. The l a b d is randomly distributed (details from Figs. 3 and 4) (some hemocyanin molecules are indicated by arrows). Bars = 1 #m. Inset is a detail from Fig. 9 (box) showing in 0.5/~m 2 of cell surface area more than 20 hemocyanin molecules. Scale bar = 0.5/zm. Figs. 11, 12. Protrusion of a mesoderm cell through the endoderm of a homozygous embryo. The normal endodermal microvilli are not labeled (see also Fig. 16 for high magnification); some abnormal endodermal blebs (Fig. 11, arrows) arepossibly labeled. En = the outer surfaces of endoderm cells. Fig. 11. Scale bar = 2 # m . Fig. 12. Scale bar = 1 #m.
165
Figs. 13, 14. Ectoderm cells of t wls homozygotes showing variability in the density of the label from positive labeling just above background (Fig. 13) to dense labeling (Fig. 14). Some hemocyanin molecules are indicated by arrows. Scale bars = 1 /~m. Inset is detail from Fig. 13 (dark arrow) (magnification × 3) showing very few hemocyanin particles. Fig. 15. A mesoderm cell of a morphologically normal putative heterozygnte. The cell is positively labeled above background. Fig. 16. Endoderm cell from a morphologically normal embryo. The membrane facing a neighboring cell is weakly labeled. The microvilli are devoid of any label. Scale bar = 1 ~m.
166
Fig. 17. Ectoderm cells of morphologically normal putative heterozygote embryo, positively labeled. Scale bar = 1 / t m . Fig. 18. Membranes of ectoderm cells from morphologically normal embryo, facing the amniotic cavity, are not labeled. Scale bar = 1 p.m. Figs. 19, 20. Mesoderm and ectoderm (respectively) cells of putative normal embryo showing only background labeling (arrows). Respective cells from CF 1 normal embryos look exactly the same. Scale bars = 1 /~m.
167 ectoderm cells in these embryos were always labeled, although the density of the label was generally lower than that on the mesoderm ceils, and was also more variable. Variability in density was found not only between cells of different embryos, but also between ectodermal cells of the same embryo (Figs. 13, 14). Ectodermal cell surfaces that face the amniotic cavity were generally labeled more weakly than the lateral surfaces. The label was observed on their lateral surfaces but never on their apical borders facing the outside. Embryonic endoderm cells were usually weakly labeled or not labeled at all. Among 8 littermate embryos which were morphologically normal, 4 were not labeled, or were labeled at the background level (i.e. an occasional hemocyanin particle) and are probably normal segregants ( + / / + ). The other four were labeled above background, ranging from 'weak' labeling to 'dense' labeling, and are probably + / / t wm heterozygotes (Figs. 15-20). The numbers of embryos studied here, and in the series reported below are obviously too small to provide statistical assurance that our genotypic assignments are correct, although the proportions of densely labeled, moderately labeled and essentially negative embryos are compatible with the 1 : 2 : 1 genotypic ratios of t w18, and + / / + embryos predicted on the basis of the 0.5 transmission ratio of t wls. Furthermore, it has to be emphasized at this point that the analysis performed was a qualitative one and the term 'weak' or 'dense' does not imply a count of hemocyanin marker molecules per unit of surface area, but rather the density of the marker in relation to labeled mesoderm cells of homozygous embryos. In all the embryos analyzed, both homozygotes and heterozygotes, cells in the extraembryonic region were virtually devoid of label. This observation served as an internal control. In another set of observations, 36 embryos from five litters of + / / t wls parents were analyzed at slightly earlier stages, which precluded preselection of homozygous embryos on morphological criteria. Mesoderm cells of 12 embryos were densely labeled; their embryonic ectoderm was also positively labeled but varied in density. Thirteen other embryos had unequivocal label on their mesoderm cells but the label was not as dense as in the first
group, and their ectoderm was usually weakly labeled. Eleven embryos showed no label on either mesoderm or ectoderm. Control
Beside the usual control where normal mouse serum was used instead of specific antiserum, and which yielded only background labeling, the most important one was the analysis with anti-t wl8 antiserum of normal embryos from litters not segregating for t wls (-k-f-k-X+//+). These mice were mated and dissected at the same time as the experimental ones from + / / t w18 parents, and the embryos were incubated with the same anti-t w18 serum under the same conditions, and processed for scanning microscopy at the same time. Of 11 embryos analyzed in detail only one showed label on mesoderm cells, which was weak but above background. The pictures obtained were identical to unlabeled, morphologically normal embryos from twm litters (see Figs. 19, 20). Stage specificity
To determine whether the expression of t wa8 antigens(s) on the cell surfaces of embryos carrying the t wls mutation is stage specific, an immunolabeling experiment of embryos from + / t wl8 parents at the 8-cell stage was performed. Of 27 embryos examined, only 4 showed very weak labeling, marginally above background (Figs. 21, 22). Since homozygotes could of course not be identified prior to immunolabeling, an additional control experiment was performed to determine whether these early stage embryos could indeed be positively and densely labeled. When morulae of another t-mutant, t ~2, whose homozygous phenocritical period occurs between cleavage and blastocyst stages, were incubated with an appropriate antiserum (rabbit anti-t ~2) and labeled with conjugated hemocyanin markers (goat-anti rabbit IgG-hemocyanin), some of the embryos were heavily labeled (data not shown). Technical problems associated with the small size of post-implantation embryos between 5 and 7 days have so far prevented us from addressing the question of stage specificity more precisely.
168
Figs. 21, 22. Eight-celledembryos (Fig. 21) from litters segregating t w18 homozygotes.The labeling is at the background level (Fig. 22, arrows). Fig. 21. Scale bar = 10 #m. Fig. 22. Scale bar = 1 #m.
Discussion This study addressed pivotal questions, namely, whether t-antigens, so well characterized with male germ cells, are detectable in t-mutant embryos and, even more importantly, whether they are present specifically on those tissues displaying genetically induced dysfunction. If true, this would implicate a function for these antigens, and support previously proposed conjectures (Artzt and Bennett, 1977) that these structures play a role in defective cell interaction in the embryo. We chose to study the t ~ 8 mutation because it acts at a time when the embryo is becoming complex, and is accessible to definitive experimental investigation because homozygotes can be diagnosed on morphological grounds. At late 7.5 days there is a clearly definable set of tissues affected in t w j 8 homozygotes, as well as unaffected tissues which continue to develop normally for several days more (Bennett and Dunn, 1960; Moser and GluecksohnWaelsch, 1967). In normal embryos, the em-
bryonic ectoderm feeds cells into the proliferative center of the primitive streak, from where they emerge as stellate and migratory mesoderm. In t w18 homozygotes, however, the embryonic ectoderm and mesoderm fail to achieve this critical step, and it is precisely these cells that are most heavily labeled with anti-t w18 serum. On the other hand, the embryonic endoderm, and the extraembryonic ectoderm and endoderm, which continue to develop normally in the mutants are virtually devoid of label. This offers strong evidence that at 7.5 days the t w18 antigen(s) are exposed primarily on cells affected by the mutation. Our data also provide evidence that the t w18 antigen is not expressed on eight-celled embryos, and thus suggest in a very broad sense that its expression is stage specific. Obviously though, this needs to be confirmed, and the temporal limits of antigen expression defined, by further experiments with embryos at stages intermediate between the 2-day and 7.5-day stages we studied here, as well as with older embryos up to the time of death. We
169
must also point out that others (Kemler et al., 1976) have evidence arguing against stage specificity. Using indirect immunofluorescent techniques this group reported results suggesting that morulae homozygous for t ° t wS, t w l s and t w32 all expressed their relevant t-antigen, although only t ~3e embryos show defects at the morula stage. We later assayed t w5 a n d t ~32 morulae by complement dependent cytotoxicity methods, and reported antigen expression on t w32 but not t ~5 embryos (Marticorena et al., 1978). Thus all our data are compatible with stage-specific expression of t-associated antigens, although we have no explanation for the discrepant results of Kemler et al. In any case, the present report is the first time t-antigens have been localized in time and place to the affected tissues in known homozygous embryos. It is worth emphasizing that whatever the biological or molecular defect in t wls, it is a critical one for cell commitment and survival. It was observed by Artzt and Bennett (1972) that t w l s homozygotes transplanted to ectopic sites such as the testis continued to grow although only half survived. However, those growths resulted in tumors composed primarily of ectodermal tissues but not of mesoderm or of any structures dependent on epithelial-mesenchymal interactions. Furthermore, these tumors were frequently histologically malignant and resembled neuroepitheliomas, which suggested that normal mesodermal interactions are necessary for the controlled differentiation of ectoderm. It is an interesting, although unexplained, point that other investigators found that transforming the mutant cells in tissue culture with SV40 produced primarily cell lines of mesodermal origin (myoblasts and adipocytes) (Kelly et al., 1979), which suggested that the defective mutant mesoderm cells may be particularly sensitive targets for viral transformation.
Acknowledgements This work was supported by NSF Grant PCM 77-17835, NIH Grant CA-21651, and by NCI Core Grant CA-08748.
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170 Shaklai, N. and E. Daniel: Fluorescence properties of hemocyanin from Leoantina hierosolima. Biochemistry 9, 564-568 (1970). Spiegelman, M. and D. Bennett: Fine structural study of cell migration in the early mesoderm of normal and mutant mouse embryos (T-locus: tg/tg). J. Embryol. Exp. Morphol. 32, 723-738 (1974).
Yanagisawa, K., D. Bennett, E.A. Boyse, L.C. Dunn and A. DiMeo: Serological identification of sperm antigens specified by lethal t-alleles in the mouse. Immunogenetics 1, 57-67 (1974).