Effect of the T-mutation on histogenesis of the mouse embryo under the testis capsule

Transcription

1 /. Embryol. exp. Morph. Vol. 5, pp. -3, 79 Printed in Great Britain Company of Biologists Limited 79 Effect of the T-mutation on histogenesis of the mouse embryo under the testis capsule By H. FUJIMOTO 1 AND K. O. YANAGISAWA 1 From the Laboratory of Cell Biology, Mitsubishi-Kasei Institute of Life Sciences, Tokyo SUMMARY Mouse embryos homozygous for the T-mutation show abnormalities, severer at the posterior embryonic regions, by day 9 of gestation and die before day 11 in utero. To analyse developmental potentiality of the T/T embryos, fragments of their anterior and posterior portions were grafted into the testes of adult T/ + mice, and examined histologically for the tissues formed after 1 month. The grafted tissues of the T/T embryos grew beyond the destined lethal stage and gave rise to benign teratomas composed of mature tissues. Although there were some different features of the tissues formed in the teratomas derived from different portions and stages of the embryos, their types were essentially identical between wild-type and the mutant teratomas. Statistical analysis showed that frequency of the cartilage and/or bone formation was significantly lower in the posterior mutant teratomas. It cannot be concluded, however, that this difference is essentially caused by T-mutation. The main conclusion of present experiments is that grafted portions of T/T embryos have the potentiality to develop intoteratomas containing derivatives of all three germ layers. INTRODUCTION In day 8-9 of gestation, mutants homozygous for the!t-gene are distinguishable from normal litter-mates by morphological abnormalities, which show a cephalo-caudal gradient, in the axial organ system. The posterior end is more affected than is the anterior (Chesley, 35). Spiegelman (76) has revealed with the electronmicroscope that by day 9, chordamesodermal cells of the T/T embryo form extensive tubular epithelial structures resembling the neural tube in the posterior region, normally occupied by the notochord and the somites. She speculated that these morphological abnormalities of the T/T embryo might be caused by the incomplete specification of the primitive streak, derivatives. The T/T embryos die by day 11 because of the failure to establish placenta! circulation as a result of abnormalities of the allantois (Gluecksohn- Schoenheimer, 44). Therefore, it is impossible to observe histogenesis of the T/T embryos in utero. If they were able to survive beyond the lethal stage,. 1 Authors' address: Laboratory of Cell Biology, Mitsubishi-Kasei Institute of Life Sciences,. 11 Minamiooya, Machida-shi, Tokyo 4, Japan.

2 22 H. FUJIMOTO AND K. O. YANAGISAWA could their tissues showing morphological abnormalities differentiate into any normal adult tissues? One method of investigating differentiation in early postimplantation embryos of the rodents is by transferring them to extra-uterine sites, e.g. into the anterior chamber of the eye (Grobstein, 51), under the testis capsule (Diwan & Stevens, 76) or under the kidney capsule (Levak-Svajger & Svajger, 74; Svajger & Levak-Svajger, 74). In these sites, the grafts survived long enough to observe their final histogenesis. Present experiments were designed to analyse the effect of the /"-mutation on histogenesis by transferring tissue fragments of the T/T embryos to adult testes. MATERIALS AND METHODS The Brachyury mutant mice were originally supplied by Dr D. Bennett. Adult mice for the present experiments (T/+ and +/ + ) were obtained by heterozygous matings (77+ xt/ + ). Pregnant female mice were sacrificed on days 8 and 9 of gestation (day of vaginal plug = day zero). Embryos were removed from their decidua, and their yolk sac and amnion were removed in Hanks' balanced salt solution under a dissecting microscope. The T/T embryos obtained by heterozygous matings (T/+ xt/+) were distinguished from their normal litter-mates by their typical abnormal morphology. Their development was staged by the number of somites of their normal litter-mates. The embryos from homozygous matings (+ / + x + / + ) served as controls. For morphological observation of the embryos, the removed embryos were usually fixed in Bouin's fluid and embedded in paraplast. They were serially sectioned at 7 /on and stained with haematoxylin and eosin. To observe finer details of younger embryonic tissues, the embryos were fixed in 2-5 % glutaraldehyde followed by 1 % osmium tetroxide and embedded in Epon (Luft, 61). Sections, 1 /im thick, were stained with Ito's solution (Ito & Winchester, 63). To investigate histogenesis of the embryonic tissues, anterior and posterior portions were dissected from +/+ or T/T embryos using fine needles in Eagle's minimum essential medium and grafted into the testes of adult T/+ male mice according to Stevens' method of surgery (Stevens, 68). One graft was dissected from each embryo. The recipient animals were killed one month after grafting. Visible growths were dissected out, while testes without visible growths were removed intact. They were fixed in Bouin's fluid and embedded in paraplast. Sections, 1 pan. thick, were stained with Alcian blue at ph 2-5, haematoxylin and eosin. For the examination of nervous tissue, the silver impregnation method was carried out (Otsuka, 62). All sections were examined under a light microscope for the presence of differentiated tissues. Tissue types were compared with those described in a textbook of histology (Bloom & Fawcett, 75), and identified according to the descriptions of Stevens & Hummel (57).

3 Effect oft-mutation on histogenesis 23 Fig. 1. Sagittal 1 /tm sections of the primitive streak region of the T/T'embryo at the 5 to 6-somite stage, x 16. Inset shows the rosette-forming cells, x 6. RESULTS In order to identify suitable stages for grafting into testes, development of the T/T embryos was morphologically examined with special reference to the unusual tubular structures at the posterior regions. At the early somite stage, the'primitive streak area of the T/T embryo was conspicuously enlarged. From histological observation, it was assumed that mesodermal cells at the caudal extremity of the primitive streak area were arrested (as found by Spiegelman (76)). At the stage when normal Utter-mates have five to six somites, small rosette structures consisting of several cells are found in the posterior region of the T/T embryo (Fig. 1). At more advanced stages, they develop into tubular structures (Figs 2 A, B). By about the 24- somite stage, most chordamesodermal cells arrange themselves into tubular structures, but somites are not formed. Necrosis is severer at the posterior end of the embryo. Although the T/T embryo grows until about the 3-somite stage, the portion posterior to the forelimb buds degenerates. Finally the T/T embryo dies at about the 36-somite stage. On the basis of such observation, grafting experiments of the T/T and wildtype embryos were undertaken at the following two stages. One was the, 4- to 1-somite stage (day 8) when the unusual tubular structures began to be formed. The other was the 15- to -somite stage (day 9) at which this structure had almost been established, yet necrosis in the posterior region of the T/T embryo had not become too marked. The anterior and posterior portions of the embryos at these stages were dissected as shown in Fig. 3, and grafted into the adult testes. All successful grafts resulted in tumorous growths. Size of the growths from the posterior embryonic portions was generally larger than that from the anterior in each genotype. Size of the growths derived from the T/T embryos was slightly smaller than that from wild-type embryos at each stage.

4 24 H. FUJIMOTO AND K. O. YANAGISAWA Fig. 2. Seven /*m sections of the posterior region of the T/Tembryo at the 14-somite stage. The chordamesodermal cells form tubular epithelial structure. (A) Sagittal section. The abnormal tube connects with the neural plate at its front region, x 11. (B) Cross-section. x41. CA, caudal artery; H, hind gut; NP, neural plate; T, tubular epithelium. Histological observation showed that these growths were benign teratomas composed of various mature tissues, but not embryonal carcinoma cells. The features of histological organization were essentially identical in both genotypes. There were, however, differences between the features of tissues formed in the teratomas derived from the anterior embryonic regions and those from the posterior. Skin associated with hair follicles and sebaceous glands was conspicuous in the posterior teratomas (Fig. 4A). It occupied a small area in the anterior teratomas. Melanocytes were present in the dermis and the basal layer of the epidermis. Melanocytes in the posterior teratomas were more abundant than in the anterior. Adult nervous tissues were main components of the anterior teratomas at day 9 (Fig. 4B). They occupied peripheral area of the teratomas from other stages or portions of embryos. In some cases, neurons appeared to have formed end plates on striated muscle (Fig. 4C). Ganglionic cells were found among developing tissues (Fig. 4D). The number of ganglionic cells in anterior teratomas was less than that of the posterior ones. Adipose tissue and

5 Effect oft-mutation on histogenesis 25 Fig. 3. Diagram to illustrate dissected regions of the embryos. They were cut with fine needles along broken lines. Arrows indicate anterior and posterior grafted portions of the embryos. (A) Wild-type embryo at day 8. (B) The T/T embryo at day 8. (C) Wild-type embryo at day 9. (D) The T/T embryo at day 9. connective tissue were observed surrounding each organoid. Striated muscle cells, cartilage and bone with marrow are found in the adipose tissue (Fig. 4E). They were prominent structures in the anterior teratomas. Blood cells in capillaries were found in many sections, though it was not possible to determine whether these tissues belonged to the teratoma or to the host. Smooth muscle layers are observed encircling cavities lined with epithelium (Fig. 4F). These epithelia in the posterior teratomas contained mainly goblet cells secreting mucus, while they were mainly keratinized in the anterior. Usually, the epithelia of these two types were adjacent to each other. They were referred to as alimentary tubes. Respiratory tubes, comprising ciliated columnar epithelium with glands encircled with cartilage, were characteristic structures in the anterior teratomas (Figs 4G, H). From these histological observations, types of tissues identified in each teratomas are scored in Table 1. The grafts of both genotypes gave rise to mature tissues derived from all three germ layers. In many cases, single teratomas contained all the tissues listed in Table 1, but in some teratomas certain of the tissues were missing. At day 9, 55 % of the posterior mutant teratomas D

6 26 H. FUJIMOTO AND K. O. YANAGISAWA

7 Age of the embryo... Effect oft-mutation on histogenesis 27 Table 1. Incidence of mature tissues found in teratomas Grafted portion of the embryo... Genotype of the embryo... No. of successful grafts Ectodermal derivatives Epidermis Hair follicles Sebaceous glands Nervous tissue Ganglionic cells Melanocytes in dermis Mesodermal derivatives Cartilage and/or bone Striated muscle Smooth muscle Adipose tissue Connective tissue Endodermal derivatives Epithelium of alimentary tube Epithelium of respiratory tube Anterior 16 a T/T 12 a a,x" = l-22,p>-2;b, x 2 = 1-44,P>-2; c, x e, x 2 = 3, P>1; f, x* = 1*93, P>-l; g, x 2 X" test; after Yates (43) for small numbers. Day 8 > A r Posterior A + / + 2P c d T/T b 14c 1 7d Day 9 A Anterior Posterior A + / + T/T + / + 1 e 8 9 f 4 12* e 4 4 f 6 98 h T/T 1 h = 3-86, P<5; d, x 2 = 2-49, P>5; = -46, P>-3; h, x 2 = 18, P<-1. FIGURE 4 Tissues in teratomas produced by fragments of the TIT embryos in the testis. (A) Skin from a posterior portion of the embryo at day 9. Keratinized epithelium with hair follicles, sebaceous glands, dermal connective tissue and adipose tissue. x9. (B) Nervous tissue from an anterior portion of the embryo at day 9. Stained with silver impregnation method, x. (C) Nerve and striated muscle from an anterior portion of the embryo at day 9. Stained with silver impregnation method, x 36. (D) A ganglion embedded in adipose tissue from a posterior portion of the embryo at day 9. x 36. (E) Endochondral ossification and red bone marrow in a teratoma derived from an anterior portion of the embryo at day 8. x 72. (F) Intestine from a posterior portion of the embryo at day 9. Epithelium contains goblet cells, x 36. (G) Respiratory tube from an anterior portion of the embryo at day 9. x 57. (H) Ciliated columnar epithelium. Enlargement of a part of (G). x 36. A, adipose tissue; B, bone; C, cartilage; CO, connective tissue; DC, degenerating cartilage; G, gland; N, nerve; S, seminiferous tubule of the host; SM, smooth muscle; ST, striated muscle.

8 28 H. FUJIMOTO AND K. O. YANAGISAWA (11/) were defective in some of the tissues examined, compared with 15 % in the wild-type (3/). In the mutant teratomas, missing tissue was cartilage and/or bone in 99 % of the cases (1/11). The frequency of cartilage and/or bone formation is significantly lower (P< 1, Table 1) in the mutant compared to wild-type. Similarly, differentiation of these tissues is lower in the posterior mutant teratomas at day 8 (i><5, Table 1). Of the posterior teratomas 67 % (14/) were deficient in some tissues examined, compared with 1 % in the wild-type (2/). In the mutant teratomas, 5 out of 14 defective teratomas failed to form only cartilage and/or bone. Two were deficient in cartilage and/or bone and other tissues, and seven were deficient in other tissues. Accordingly, the posterior mutant teratomas lacking cartilage and/or bone differentiation are not always deficient in other components. Defective tissues varied in the mutant teratomas at day 8 compared to those at day 9. In contrast, no significant difference was observed in the tissues examined between wild-type and the mutant teratomas from the anterior embryonic portions (P>5, Table 1). Although formation of ectodermal derivatives except nervous tissues was remarkably reduced in the anterior teratomas of both genotypes at day 9, the reasons for this reduction are not clear. DISCUSSION In the present experiments it was clearly shown that tissue fragments of the T/T embryos developed into benign teratomas containing mature tissue derivatives from three germ layers. Frequency of differentiation of cartilage and/or bone was significantly lower in the posterior mutant teratomas, compared to the wild-type, while these tissues differentiated well in the anterior mutant teratomas. Bennett (58) showed that the somites of the T/T embryo were defective in the capacity for cartilage induction by reconstituting dissociated somites and neural tube. She also reported that the forelimb buds of the T/T embryos formed well differentiated cartilage. Thus there is a possibility, in our experiments, that the cartilage and/or bone formed in the mutant teratomas is derived from the lateral mesoderm and that the somitic mesoderm has no ability to form these tissues. It is difficult, however, to decide whether the cartilage and/or bone in the teratomas are derived from the somites or the lateral mesoderm. We cannot tell, therefore, whether this defect is a genuine effect of T-mutation. Lethality of the T/t locus mutants has been a primary concern of many investigators. Ephrussi (35) cultured the T/T embryos in organ culture beyond the stage of lethality in utero. Yanagisawa & Fujimoto (77) isolated cell lines from the T/T embryos. These results are all in accord with our present observations. Artzt & Bennett (72) grafted t /^ egg cylinders, in which no healthy mesoderm was observed, into the testes of adults. The teratomas formed contained no mesodermal derivatives. In vitro culture of blastocysts derived from

9 Effect oft-mutation on histogenesis 29 matings producing r vl8 homozygotes suggested that / w homozygotes are viable beyond the normal period of lethality of the mutants in utero (Wudl, Sherman & Hillman, 77). On the other hand, t 12, t* or? w5 embryos were lethal in both in vitro culture and grafting in ectopic sites (Wudl & Sherman, 76; Wudl et al. 77). Thus it is likely that the late-acting mutations (f wl8, T) behave in a different way from the early-acting mutations (t 12, t e, t w5 ). We cannot, however, exclude the possibility that some fraction of embryonic cells of the T/T mutants is lethal and that the other fraction of cells survives beyond the destined lethal stage. A summary of this work was presented in a preliminary form at the 8th International Congress of the International Society of Developmental Biologists in Tokyo, Japan, in August 77. We acknowledge with thanks Drs T. Miyake and T. Noguchi for their helpful advice and constant encouragement. We are also indebted to Miss C. Murosaka for looking after the mutant stock and technical assistance of histology. REFERENCES ARTZT, K. & BENNETT, D. (72). A genetically caused embryonal ectodermal tumor in the mouse. /. natn. Cancer Inst. 48, BENNETT, D. (58). In vitro study of cartilage induction in I/I 1 mice. Nature, Lond. 1, BLOOM, W. & FAWCETT. D. W. (75). A Textbook of Histology, 1th ed. Philadelphia: W. B. Saunders Company. CHESLEY, P. (35). Development of the short-tailed mutant in the house mouse. /. exp. Zool. 7, DIWAN, S. B. & STEVENS, L. C. (76). Development of teratomas from the ectoderm of mouse egg cylinders. /. natn. Cancer Inst. 57, EPHRUSSI, B. (35). The behavior in vitro of tissues from lethal embryos. /. exp. zool. 7, 7-4. GLUECKSOHN-SCHOENHEIMER, S. (44). The development of normal and homozygous Brachy {T/T) mouse embryos in the extraembryonic coelom of ihe chick. Proc. natn. Acad. Sci., U.S.A. 3, GROBSTEIN, C. (51). Intra-ocular growth and differentiation of the mouse embryonic shield implanted directly and following in vitro cultivation. J. exp. Zool. 116, ITO, S. & WINCHESTER, R. J. (63). Thefinestructure of gastric mucosa in the bat. /. Cell Biol. 16, LEVAK-SVAJGER, B. & SVAJGER, A. (74). Investigation on the origin of the definitive endoderm in the rat embryo. /. Embryol. exp. Morph. 32, LUFT, J. H. (61). Improvements in epoxy resin embedding methods. /. Biophys. Biochem. Cytol. 9, OTSUKA, N. (62). Histologisch-Entwicklungsgeschichtliche Untersuchungen an Mauthnerschen Zellen von Fischen. Z. Zellforsch. mikrosk. Anat. 58, SPJEGELMEN, M. (76). Electron microscopy of cell associations in T-locus mutants. In Embryogenesis in Mammals (ed. K. Elliot & M. O'Connor). Ciba Foundation Symposium 4, pp Amsterdam: Elsevier. STEVENS, L. C. (68). The development of teratomas from the intra-testicular grafts of tubal mouse eggs. /. Embryol. exp. Morph., STEVENS, L. C. & HUMMEL, K. P. (57). A description of spontaneous congenital testicular teratomas in strain 129 mice. /. natn. Cancer Inst., SVAJGER, A. & LEVAK-SVAJGER, B. (74). Regional developmental capacities of the rat embryonic endoderm at the head-fold stage. /. Embryol. exp. Morph. 32, EMB 5

10 3 H. FUJIMOTO AND K. O. YANAGISAWA WUDL, L. R. & SHERMAN, M. I. (76). In vitro studies of mouse embryos bearing mutations at the Tlocus: r* and t 12. Cell9, WUDL, L. R., SHERMAN, M. I. & HILLMAN, N. (77). Nature of lethality of t mutations in embryos. Nature, Lond. 27, YANAGISAWA, K. O. & FUJIMOTO, H. (77). Viability and metabolic activity of homozygous Brachyury (T) embryos. /. Embryol. exp. Morph. 4, YATES, F. (43). Contigency tables involving small numbers and the,\' 2 test. Supplement to Jl R. statist. Soc. 1, {Received 23 March 78, revised 3 October 78)