Morphogenesis of the placental membranes in the viviparous, placentotrophic lizard Chalcides chalcides (Squamata: Scincidae)

July 24, 2017 | Autor: Daniel Blackburn | Categoria: Development Studies, Embryology, Placenta, Reptiles, Lizards, Viviparity
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JOURNAL OF MORPHOLOGY 232:35–55 (1997)

Morphogenesis of Placental Membranes in the Viviparous, Placentotrophic Lizard Chalcides chalcides (Squamata: Scincidae) DANIEL G. BLACKBURN1* AND IAN P. CALLARD2 of Biology, Life Sciences Center, Trinity College, Hartford, Connecticut 06106 2Department of Biology, Boston University, Boston, Massachusetts 02215 1Department

ABSTRACT In the scincid lizard Chalcides chalcides, females ovulate small ova and supply most of the nutrients for development by placental means. The yolk is enveloped precocially by extraembryonic ectoderm and endoderm during the gastrula stage, establishing a simple bilaminar yolk sac placenta. The shell membrane begins to degenerate at this time, resulting in apposition of extraembryonic and maternal tissues. A true chorioplacenta has developed by the early pharyngula stage, as has a choriovitelline placenta and the first stages of an omphaloplacenta. Although the choriovitelline membrane disappears rapidly, the omphaloplacenta spreads to occupy the entire abembryonic pole. The yolk cleft is not confluent with the exocoelom, and no omphalallantoic placenta develops. By the limb-bud stage, an allantoplacenta has been established, with a mesometrial placentome composed of interdigitating ridges of chorioallantois and uterine mucosa. The discovery of five distinct placental arrangements in this species, three of which are transitory and two of which have not previously been recorded in reptiles, emphasizes the need for accounts that specify ontogenetic stages and the precise identity and composition of squamate placental membranes. Contrary to previous interpretations, the pattern of extraembryonic membrane development in C. chalcides is evolutionarily conservative, despite the presence of a reduced yolk mass and cytological specializations for nutrient transfer. Our observations indicate that substantial placentotrophy can evolve in squamates without major modifications of morphogenetic patterns. J Morphol 232:35–55, 1997. r 1997 Wiley-Liss, Inc. Chalcides chalcides, a viviparous lizard of the western Mediterranean region, is one of a very few nonmammalian amniotes known to have evolved specialized placental organs that provide most of the nutrients for fetal development (Yaron, ’85; Blackburn, ’93a). This species was the subject of the earliest gross and microscopic descriptions of reptilian placentae (Studiati, 1853; Giacomini, 1891), and the mature placentae have been studied from the standpoints of histology, ultrastructure, and immunocytochemistry (Ghiara et al., ’87; Angelini and Ghiara, ’91; Blackburn, ’93b; Paulesu et al., ’95). Unfortunately, information on early placental development in C. chalcides is extremely limited (Luckett, ’77a), preventing resolution of persistent questions about underlying morphor 1997 WILEY-LISS, INC.

genetic patterns as well as the identity and developmental origins of various constituent tissues. The most fundamental unresolved issue is whether Chalcides chalcides differs from typical squamates (lizards, amphisbaenians, and snakes) in the pattern of extraembryonic membrane development. In this species, females ovulate eggs that are very small by squamate standards (3 mm in diameter), in association with their extreme placentotrophy (Giacomini, 1891). Various reports have suggested that, in conjunction with the evolutionary reduction in yolk, extraembry-

*Correspondence to: Daniel G. Blackburn, Department of Biology, Life Sciences Center, Trinity College, Hartford, CT 06106.

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onic membrane development in C. chalcides has departed substantially from the typical squamate pattern (Weekes, ’27, ’35; ten CateHoedemaker, ’33; also see Boyd, ’42). A corollary of this view is that fetal contributions to the placentae differ substantially from those of other viviparous lizards and snakes. However, a recent reinterpretation of the morphology of the late-stage placenta (Blackburn, ’93b) suggested that the developmental pattern of the extraembryonic membranes in this species is similar to that of other squamates, specializations for placentotrophy notwithstanding. The present study draws on the histology of specimens from early development to test the hypothesis that Chalcides chalcides has evolved a novel pattern of extraembryonic membrane morphogenesis, in association with yolk reduction and placentotrophy. In addition, this paper represents the first detailed account with corroborative photomicrographs of early morphogenesis of the placental membranes in a viviparous lizard. As such, this investigation helps to establish a basis for future work on placental development, function, and evolution in squamates. EXTRAEMBRYONIC MEMBRANE DEVELOPMENT IN SQUAMATES

To understand the focus and significance of the present study, one must have some familiarity with the pattern of development of the extraembryonic membranes in lizards and snakes. Although squamates are like other Reptilia (i.e., chelonians, crocodilians, and birds) with regard to formation of the chorion and chorioallantois, they exhibit a unique pattern of yolk sac development. The squamate developmental pattern is often misrepresented in the scientific literature and is overlooked in the textbooks, which universally treat turtles as typical reptiles. However, aspects of the squamate pattern were documented over half a century ago by such pioneering researchers as Hanni Hrabowski (’26), Claire Weekes (’27, ’29, ’30, ’35), and Mary Boyd (’42). In recent years, details of yolk sac development have been corroborated and extended in work on both snakes (Hoffman, ’70; Baxter, ’87; Stewart, ’90) and lizards (Blackburn, ’85; Stewart, ’85; Villagran Santa Cruz, ’89; Stewart and Thompson, ’94, ’96). Several recent reviews, most with detailed diagrams, are also available (Yaron, ’85; Stewart and Blackburn, ’88; Blackburn, ’92, ’93b; Stewart, ’92, ’93).

The following summary is based chiefly on the garter snake Thamnophis sirtalis (Hoffman, ’70; Blackburn, ’85; Stewart and Blackburn, ’88; Blackburn et al., unpublished data) but applies to squamates in general. Early in development, extraembryonic ectoderm and endoderm spread over the surface of the yolk, enveloping it and forming a bilaminar yolk sac. In the embryonic hemisphere, mesoderm spreads between the ectoderm and endoderm, forming a trilaminar choriovitelline membrane. In these respects, squamates are like other reptiles. However, in a pattern unique to squamates, putative mesoderm invades the yolk mass as ‘‘intravitelline cells’’ at the sinus terminalis. These intravitelline cells penetrate, parallel to the yolk surface, as far as the abembryonic pole. An extracoelomic space, the ‘‘yolk cleft,’’ forms in association with the intravitelline cells, splitting off an abembryonic layer of yolk, the ‘‘isolated yolk mass.’’ The isolated yolk mass is an avascular structure that is lined externally by tissues of the bilaminar omphalopleure (ectoderm 1 endoderm). In T. sirtalis and in most other species studied, the yolk cleft becomes continuous with the exocoelom and is invaded by the allantois. The allantois and the overlying isolated yolk mass form an omphalallantoic membrane. In some species, however, a cellular partition excludes the allantois from the yolk cleft, and no omphalallantoic membrane forms (Stewart and Blackburn, ’88). Many aspects of the squamate pattern are poorly understood or controversial, including the following: 1) germ layer origins of the intravitelline cells; 2) definitive germ layer constituents at the abembryonic pole; 3) the extent to which choriovitelline membranes are common among squamates; 4) the origin of the partition that separates the exocoelom and yolk cleft; 5) developmental mechanisms by which intravitelline cells penetrate the yolk mass (yolk generally is regarded as difficult to penetrate—hence the cleavage pattern of macrolecithal eggs); 6) the functional significance of the squamate pattern and the membranes it produces; 7) modifications of the pattern associated with viviparity and placental formation; and 8) the functional and evolutionary significance of species differences. Unresolved questions about Chalcides chalcides include not only these issues but several others, including whether the follow-

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ing structures form during development: a choriovitelline membrane, intravitelline mesoderm, a yolk cleft, an isolated yolk mass, an omphalallantoic membrane, and an extracellular shell membrane (Blackburn, ’93b). The overarching issues are whether C. chalcides differs from other squamates in its pattern of development and the extent to which its specializations reflect the placentotrophic nutritional pattern by which the females supply nutrients to the embryos. By focusing on early morphogenesis of the placental membranes, our study is able to address such issues in C. chalcides and also to shed light on a developmental pattern that is a synapomorphy for squamates. MATERIALS AND METHODS

Pregnant, female Chalcides chalcides were obtained commercially and were sacrificed periodically over a period of 4 weeks during early gestation (mid-May through mid-June). Gravid reproductive tracts were excised, cut into separate incubation chambers, and fixed in 10% formalin. Following dehydration through an ethanol series, the tissue was embedded in paraffin and sectioned with microtomes at 6–9 µ. To facilitate sectioning of the hardened yolk-laden tissues, the paraffin blocks and steel knives were prepared by storage in a freezer (210°C) for at least 15 min, and the sectioning room was cooled to approximately 60°C. Sections were mounted on albumen-coated glass slides and stained either with hematoxylin and eosin or with a Periodic acid–Schiff (PAS) protocol (Humason, ’62). Embryonic stages were determined from the sectioned material in accord with Hubert’s (’85) system. Recognition of placental categories largely follows Stewart and Blackburn (’88). Photomicrographs were taken on Kodak T-Max 100 professional film using an Olympus BH-2 microscope equipped with S Plan Apochromatic objective lenses with correction collars. Magnifications reported herein are of the published micrographs, as determined by using parallel negatives of a stage micrometer and a photographic enlarger. Under the assumption that the mesometrium attaches to the oviduct along its dorsal aspect (Fox, ’77), overlying where the embryo itself develops, the terms dorsal and ventral are applied to the embryonic and abembryonic poles of the egg, respectively.

RESULTS

Postovulation The earliest developmental stage in the study series is represented by a longitudinally sectioned, gravid uterus in which no blastomeres were apparent. Evidence of fertilization was not observed in the eggs examined; however, some sections were lost in preparation. The yolk at this stage appears heterogeneous and contains droplets or granules of various sizes. Surrounding each yolk is a shell membrane that varies from 4–10 µ in thickness (Figs. 1, 3). The shell membrane is eosinophilic and PAS-positive. The pregnant uterus is distended at the site of each egg, forming ‘‘incubation chambers’’ (Giacomini, 1891) that are much larger in diameter than are the intervening segments. Components of the uterus wall are most easily discerned in the interembryonic segments (Fig. 2). In the interembryonic regions, as well as in adjacent regions of the incubation chambers (Fig. 1), the uterine mucosa is thrown up into folds that protrude into the uterine lumen. The uterine epithelium consists of a simple to pseudostratified layer of cuboidal and low columnar cells with basophilic cytoplasm. Nuclei of the epithelial cells are ovoid and typically are located towards the apical region of the cells. Epithelial cell apices are joined laterally via terminal bars, and luminal borders of many of the cells are strongly PAS-positive, probably indicating a substantial glycocalyx. Ciliated cells are common in some areas, but microvilli are not apparent. Basal to the epithelial cells lies a lamina propria of irregular connective tissue (Fig. 2), in which capillaries and larger vessels are abundant. Tubular glands occur sporadically in the lamina propria. Each gland exhibits cuboidal epithelial cells that are lightly basophilic and PAS-negative, surrounding a small central lumen that communicates with the uterine lumen. Two layers of smooth muscle surround the uterus, an inner circular layer and an outer longitudinal layer, the latter of which is lined by a monolayer of flattened adventitial cells of the visceral peritoneum. Collagenous fibers of the lamina propria penetrate into and between bundles of the circular muscle, forming distinct regions of endomysium and perimysium. In the area of an incubation chamber, the uterine components are attenuated (Fig. 3) and not always able to be distinguished as separate layers. The uterine epithelium

Fig. 1. Chalcides chalcides. Incubation chamber in early pregnancy at low magnification. The rounded spaces in the vitellus represent yolk droplets extracted during preparation. At the region photographed, uterine folds from an adjacent interembryonic segment project towards the vitellus. S, shell membrane; U, uterine tissue; V, vitellus (yolk). 3109. Fig. 2. Chalcides chalcides. Interembryonic segment of a pregnant uterus. L, lamina propria; M, muscle. 3480.

Fig. 3. Chalcides chalcides. An egg in the uterus during early pregnancy, prior to establishment of the omphalopleure. The flattened nature of uterine mucosa is more typical than in Fig. 1. S, shell membrane, U, uterine tissue; V, vitellus. 3248. Fig. 4. Chalcides chalcides. Degenerating egg in the uterus. Pieces of shell membrane (S) are randomly intermixed with the dark-staining yolk material. U, uterine tissue. 3224.

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ranges from squamous to cuboidal. Uterine glands and small blood vessels, each with constricted lumina, sometimes are evident. The lamina propria is thin in some areas, and the circular and longitudinal layers of smooth muscle often blend almost imperceptibly. One uterine area contained a degenerating egg (Fig. 4). Pieces of yolk were randomly interspersed with masses of shell membrane, the latter forming thick mats of material with strongly eosinophilic and lightly basophilic components. In this region, uterine epithelium and underlying tissues appear like that of the interembryonic segments. Late blastula/early gastrula In early pregnancy, the uterine lumen is lined primarily by a pseudostratified columnar epithelium (Fig. 5); however, in the abembryonic region, a simple cuboidal epithelium predominates. Except for occasional ciliated cells, the uterine epithelium lacks visible apical specializations. Immediately basal to the epithelial cells lie small blood vessels filled with erythrocytes. Scattered, tubular glands with constricted lumina are occasionally apparent in the thin lamina propria. During the late blastula stage, cells begin to spread over the surface of the vitellus such that by the early gastrula stage a bilaminar omphalopleure surrounds much of the yolk (Figs. 5, 6). The outermost cell population, the extraembryonic ectoderm, forms a layer of thin, flattened cells. Deep to this ectodermal layer lies a layer of yolk endoderm, consisting of irregularly shaped cells, each of which contains a large, elliptical nucleus with a prominent nucleolus. Cells of the yolk endoderm vary considerably in size, and some are embedded in the vitellus. In transverse sections, the extraembryonic cells commonly can be seen to extend between 40% and 70% around the periphery of the yolk; however, in sections through the center of the vitellus, the extraembryonic tissue surrounds the entire yolk to reach the abembryonic pole. Deep to the extraembryonic ectoderm, peculiar knots or clumps of cells are randomly distributed around the circumference of the egg, from the embryonic hemisphere to the abembryonic pole (Fig. 5). These cellular clumps appear to be sites of endodermal cell proliferation, some products of which have advanced into the body of the yolk itself.

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An intact shell membrane surrounds most of the egg (Fig. 5). However, in the embryonic pole of the egg, the membrane is thin or absent (Fig. 6), and in places appears to be broken and in the process of being shed. In some abembryonic regions where the shell membrane does not lie directly in contact with the yolk, a very thin and eosinophilic, acellular membrane, possibly the vitelline membrane, is visible on the yolk surface. Accumulations of scattered cells intermixed with eosinophilic material occasionally are found external to the shell membrane; presumably this material is of uterine origin. Late gastrula/early neurula Although the uterus during this period appears as in the preceding description, the extraembryonic tissues are in a more advanced condition. By the late gastrula stage, both extraembryonic ectoderm and endoderm entirely envelop the egg (Fig. 7), as shown by sagittal and parasagittal sections. Cells of the extraembryonic ectoderm range from squamous to low cuboidal in shape. Beyond the equatorial plane, the ectoderm is attenuated into a very thin, flattened layer of cells that extends as far as the abembryonic pole, overlying the polymorphic endodermal cells. Thin, mesodermal cells are apparent between the ectoderm and endoderm only in the vicinity of the embryo itself (Fig. 8). As in the previous developmental stage, clumps of endodermal cells of various shapes and sizes occur sporadically around the yolk periphery. As in the early gastrula, the thin shell membrane is intact abembryonically (Fig. 7) but thin or entirely absent in the embryonic pole (Fig. 8). In sections parallel to the longitudinal axis of the egg, as much as 25–50% of the yolk surface is devoid of shell membrane. Immediately beneath the gastrula, yolk appears (in the paraffin sections) to have been extracted, presumably reflecting its liquification at this early stage (Luckett, ’77a). Late neurula/early pharyngula By the neurula stage, no shell membrane is visible histologically in the dorsal hemisphere of the egg; rather, the membrane is represented as randomly folded pieces of tissue lying at the abembryonic pole. These pieces of membrane vary markedly in thickness, size, and staining properties, as if undergoing deterioration. In some specimens,

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Figures 5–8

LIZARD PLACENTAL DEVELOPMENT

a very thin remnant of the shell membrane appears to be present throughout the yolk sac region at the maternal-embryonic interface. Cells of the yolk endoderm have penetrated deep into the body of the yolk. These cells are often large and irregular and contain a large granular nucleus with a prominent nucleolus. In transverse paraffin sections of the early pharyngula, as much as 30–50% of the yolk area is filled with basophilic liquid rather than yolk granules and endodermal cells. This region tends to be concentrated under the embryo itself. Although it may be an artifact of preparation, this feature progressively increases in extent throughout the ontogenetic series. Most likely it reflects the ongoing process of yolk liquification (see Discussion), initiated earlier in development. Chorion The newly formed amniotic cavity of the pharyngula is small; however, the exocoelom is greatly enlarged, such that an extensive region of chorionic somatopleure lies exposed to the uterine lumen throughout the embryonic hemisphere, prior to expansion of the allantois (Fig. 9). Along most of its extent, the chorion is more or less apposed to the uterine epithelium, forming an anatomical ‘‘chorioplacenta’’ (sensu Stewart and Blackburn, ’88). No trace of a shell membrane is visible histologically in this region. The entire external surface of the chorion

Fig. 5. Chalcides chalcides. Early gastrula, abembryonic hemisphere. Extraembryonic ectoderm and endoderm have spread over the surface of the egg. Below a thin layer of extraembryonic ectoderm, endodermal cells (EN) are undergoing proliferation. The ectodermal layer is easily discernible in Fig. 6. S, shell membrane; U, uterine tissue. 3173. Fig. 6. Chalcides chalcides. Early gastrula stage, embryonic hemisphere. In apposition to the uterine tissue (U), the extraembryonic ectoderm (E) and endoderm (EN) form the placenta of the bilaminar yolk sac. The shell membrane has already been shed in this region. 3393. Fig. 7. Chalcides chalcides. Late gastrula stage, abembryonic hemisphere. Extraembryonic ectoderm (E) and endoderm (EN) surround the entire yolk at this early stage. A shell membrane separates the bilaminar omphalopleure from the uterine tissue. Note the constricted uterine gland (G). 3309. Fig. 8. Chalcides chalcides. Gastrula, lying at the mesometrial pole. B, blastoporal canal; M, mesoderm; N, neuroectoderm. The shell membrane is degenerating immediately dorsal to the embryo. 3127.

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consists of tightly packed, pseudostratified columnar and cuboidal cells of the extraembryonic ectoderm (Fig. 10). Between these cells and a PAS-positive basement membrane lies a basal layer of flattened cells; thus, the ectoderm consists of two cell layers. Deep to the basement membrane, an avascular layer of mesoderm, comprising a single layer of squamous cells, lines the exocoelom. Yolk sac The yolk sac is markedly different from that of the gastrula. Most of it is bounded externally by columnar and cuboidal cells of the extraembryonic ectoderm (Fig. 11); only in some regions towards the abembryonic pole do these cells appear squamous in shape. Due to absence of a shell membrane along most of the yolk sac, the epithelium lies directly apposed to the uterine epithelium. A basal layer of flattened cells lies deep to the columnar epithelial cells; because they lie superficial to the basement membrane, they are interpreted here as epithelial (ectodermal) as well. Within the yolk, enlarged endodermal cells extend a considerable distance into the body of the vitellus. Three distinct regions of omphalopleure can be recognized, definable by their constituent tissue layers (Figs. 11, 12). In dorsalventral sequence along the vitellus, away from the embryo, these constitute the trilaminar choriovitelline membrane, the omphaloplacental region (site of the bilaminar, isolated yolk mass), and the region of unspecialized bilaminar omphalopleure. These three regions will now be described in succession. Choriovitelline membrane. The choriovitelline membrane lies at the equatorial plane of the egg, ventral to the chorion (Fig. 12). This membrane is bounded ventrally (abembryonically) by the sinus terminalis and dorsally by the exocoelom, which separates the somatopleure from the yolk surface. The choriovitelline membrane is developmentally trilaminar, being formed of ectoderm, heavily vascularized mesoderm, and endoderm (Fig. 13). The ectoderm consists of an external layer of low, cuboidal cells and a deeper layer of thin, flattened cells. Beneath the bilayer of epithelial cells lie the squamous cells of the extraembryonic mesoderm. The mesodermal cells commonly are visible as two layers, apparently corresponding to the mesodermal cells of the somatopleure and splanchno-

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Figures 9–12

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pleure, which surround the exocoelom, dorsal to the choriovitelline membrane. The mesodermal vascularization derives from the vitelline vessels. The deepest layer of the choriovitelline membrane is represented by the enlarged cells of the yolk sac endoderm. In most areas the choriovitelline membrane is apposed to the uterine tissues, forming a choriovitelline placenta; however, portions of the choriovitelline membrane face dorsally into the uterine lumen (Figs. 12, 13). Omphaloplacental region. Ventral to the choriovitelline membrane lies the second region of the yolk sac. Here, the yolk sac is a bilaminar omphalopleure, consisting of extraembryonic ectoderm overlying a layer of developing isolated yolk mass and yolk cleft (Figs. 11, 12, 14). Consequently, it represents the embryonic contribution to the ‘‘omphaloplacenta’’ (sensu Stewart and Blackburn, ’88). The isolated yolk mass consists of yolk granules or droplets, interspersed with large, polymorphic cells of the yolk endoderm (Fig. 15). A yolk cleft separates the omphalopleure from the yolk sac proper. Lining the yolk cleft are thin intravitelline cells that appear very similar to the mesodermal cells that line the exocoelom. This region is avascular; the yolk cleft separates the isolated yolk mass and omphalopleure from the closest blood supply, the vitelline circulation (Figs. 12, 15). Superficial to the isolated yolk mass, the epithelium consists of columnar cells that sit atop a layer of basal epithelial

Fig. 9. Chalcides chalcides. Neurula, showing the newly established chorion (CH). EC, extraembryonic coelom; U, uterine tissue. 385. Fig. 10. Chalcides chalcides. Chorionic placenta in the neurula stage. Overlying the chorionic mesoderm (M), the chorionic ectoderm (E) consists of two layers, a superficial layer of columnar cells and a deeper layer of flattened cells. U, uterine tissue. 3338. Fig. 11. Chalcides chalcides. Yolk sac at the pharyngula stage. B, bilaminar omphalopleure beyond the yolk cleft; C, yolk cleft; I, isolated yolk mass and associated omphalopleure; ST, sinus terminalis; U, uterine tissue. In the region of the yolk cleft, the extraembryonic ectoderm, endoderm, and isolated yolk mass contribute to the omphaloplacenta. 389. Fig. 12. Chalcides chalcides. Yolk sac at the pharyngula stage. C, yolk cleft; I, isolated yolk mass and associated omphalopleure. As in Fig. 11, the bilaminar omphalopleure lies ventral to the yolk cleft; dorsal to it lie the sinus terminalis and the choriovitelline membrane. Adjacent to the yolk cleft, the vitellus is well vascularized. 389.

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cells. In occasional areas, the isolated yolk mass is separated by a space from the overlying ectoderm, probably as an artifact of preparation. Unspecialized bilaminar omphalopleure. By the early pharyngula stage, the yolk cleft extends no more than one-quarter of the way from the equatorial plane to the abembryonic pole. Ventral to the yolk cleft, the omphalopleure is not separated from the yolk itself; however, the band of intravitelline cells has begun to penetrate from the adjacent yolk cleft region (Figs. 11, 14, 16). This area represents the third and most extensive region of yolk sac that can be recognized at this stage, the unspecialized bilaminar omphalopleure. Like the yolk sac of the gastrula (Fig. 6), it is bounded by an avascular bilayer of ectoderm and endoderm (Figs. 15, 16). Throughout most of its length, the external epithelium of the bilaminar omphalopleure (including that of the omphaloplacenta) is represented by enlarged, columnar cells, like those overlying the isolated yolk mass (Figs. 14, 15, 16). In contrast, in some regions, the ectoderm consists of cuboidal to squamous cells like those of the entire omphalopleure of the gastrula (see Fig. 17). As in the limb bud stage, the regions of columnar and squamous epithelium do not intergrade but are sharply delineated (see Fig. 18). Uterus At the mesometrial pole of the egg, in the region of the chorioplacenta, the uterine epithelium consists of a single layer of cuboidal cells that overlie a highly vascularized stroma (Fig. 10). However, in the region of the yolk sac, the uterine epithelium consists of very low cuboidal cells and, in occasional regions, squamous cells (Figs. 15, 16). Throughout the uterus, glands are sparsely scattered in the lamina propria, and small blood vessels are abundant. Limb-bud stage No intact shell membrane is visible between the omphalopleure and the uterine epithelium at this stage; the membrane is represented by pieces lying at the abembryonic pole of the egg. The intravitelline mesoderm has penetrated the yolk as far as the abembryonic pole. Through expansion of the associated yolk cleft, the isolated yolk mass is separated from the vitellus in most areas

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Figures 13–16

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(Fig. 17). At some sites, the yolk cleft is very narrow, such that the isolated yolk mass is in close proximity to the vitellus. At the abembryonic pole, the bilaminar omphalopleure protrudes into small ridges or papillae that extend into the degenerating pieces of shell membrane (Fig. 18). The extraembryonic ectoderm on the surface of these papillae is enlarged into columnar cells that exhibit unusual, contorted shapes. As in the previous stage, the areas of enlarged cells adjoin regions of squamous epithelium of the bilaminar omphalopleure (Fig. 18). Placentome At the limb-bud stage, the entire embryonic pole of the egg is delimited externally by the chorioallantois (Fig. 19). This membrane is formed through fusion of the outer leaf of the allantoic membrane to the inner surface of the chorion. In some regions, the allantoic cavity is obliterated, and the inner and outer leaves of the allantois are in contact. No trace of shell membrane is evident in the embryonic pole; thus, fetal and maternal tissues are in direct apposition. The amnion closely surrounds the embryo and is not fused to the inner leaf of the allantois. Along most of its length, the chorioallantois is thrown up into deep folds that interdigitate with folds in the uterine mucosa, a region known as the placentome (Fig. 19). The surface cells of the chorionic epithelium

Fig. 13. Chalcides chalcides. Choriovitelline placenta, frontal section, pharyngula stage. This placenta lies at the equatorial plane of the egg. A low columnar epithelium of extraembryonic ectoderm (E) overlies the abundant vitelline vasculature (arrowheads) of the placenta. EN, yolk endodermal cell; U, uterine tissue; V, vitellus. 3305. Fig. 14. Chalcides chalcides. Developing omphaloplacenta, frontal section, pharyngula stage. C, developing yolk cleft; E, ectoderm of the omphalopleure; I, developing isolated yolk mass with associated endoderm; U, uterine tissue. 3230. Fig. 15. Chalcides chalcides. The definitive omphaloplacenta, frontal section. C, yolk cleft; E, ectoderm; I, isolated yolk mass with associated endoderm; U, uterine tissue; V, vitellus. Thin intravitelline cells line both sides of the yolk cleft. The surface of the vitellus is not yet vascularized in this region. 3199. Fig. 16. Chalcides chalcides. The developing omphaloplacenta, in frontal section, ventral to the yolk cleft. A thin bilayer of intravitelline mesoderm (arrows) is separating a layer of yolk, the future isolated yolk mass, from the vitellus (V) proper. Spaces between the layers of intravitelline cells are beginning to coalesce into a yolk cleft. 3191.

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of the placentome are of the pseudostratified columnar type, and many bear prominent microvilli. The epithelial cells exhibit a basophilic cytoplasm, and, although they are tightly packed, they appear to be mononucleated (Fig. 20). In PAS-stained tissue, a prominent glycocalyx is evident at the cell apices. The chorioallantoic epithelium consists of two cell layers, for a layer of flattened cells lies at the base of the columnar cells. Beneath the epithelium lie abundant small blood vessels and the vascularized, mucoid connective tissue of the allantois. The allantoic blood vessels and small amounts of connective tissue penetrate into the chorioallantoic folds. The uterine epithelium of the placentome consists of simple columnar cells (Fig. 20). The nucleus of each cell can be located at any level, including apically. Glands with open lumina are abundant in the lamina propria. This region also contains an abundance of capillaries and other small blood vessels. Whether the degree of vascularity of the stroma exceeds that of previous stages was not apparent. Indirect evidence of maternal secretion and embryonic absorption was observed in the placentome. Eosinophilic, PAS-positive material occurs in the uterine lumen between the fetal and maternal tissues. Material with similar staining properties occasionally is evident in the lumina of the uterine glands. In addition, the apical cytoplasm of the cells of the chorionic epithelium stains PAS-positive, in contrast to the basophilic cytoplasm deeper in the cells. Paraplacentomal region Adjacent to the placentome lies a region of unspecialized allantoplacenta, in which the fetal-maternal interface is smooth and nonvillous (Fig. 21). Epithelium of the chorion consists of cuboidal cells that lie atop a basal layer of squamous cells. Microvilli are not evident. Allantoic capillaries and other small vessels lie immediately beneath the epithelium. In the apposing uterine tissue, the epithelium is simple and low cuboidal to squamous and overlies a vascularized stroma. Glands are not evident in this region. Yolk sac placenta In the limb-bud stage, much of the yolk of the vitelline mass appears to have become liquified and no longer occurs in the form of granules or droplets. Although the possibil-

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Fig. 17. Chalcides chalcides. Frontal section of the yolk sac. The developing yolk cleft (asterisks) is becoming continuous around the periphery of the egg. 373. Fig. 18. Chalcides chalcides. Abembryonic pole of the egg. The bilaminar omphalopleure extends into ridges or papillae that protrude into degenerating pieces of shell membrane (S). Note the abrupt transition be-

tween columnar and squamous epithelium of the omphalopleure. 3184. Fig. 19. Chalcides chalcides. Topographic relationships at the limb-bud stage. A placentome, formed by interdigitating chorioallantoic (CA) and uterine (U) tissue, occupies the mesometrial (dorsal) pole. A, amniotic cavity. 339.

Fig. 20. Chalcides chalcides. Placentome, limb-bud stage. Protrusions of hypertrophic chorioallantoic tissue (CA) interdigitate with ridges of uterine mucosa (U). 3155. Fig. 21. Chalcides chalcides. Paraplacentomal region, showing apposition of the highly vascularized chorioallantois (CA) to the lining of the uterus (U). 3307.

Fig. 22. Chalcides chalcides. Topographic relationships in the limb-bud stage. A thin septum of tissue (arrowhead) separates the exocoelom from the yolk cleft (C), thereby excluding the allantois (AL) from the cleft. The tissue lying superficial to the yolk cleft constitutes the omphaloplacental region. YS, yolk sac. 3230. Fig. 23. Chalcides chalcides. Omphaloplacenta following depletion of the isolated yolk mass. Note the vascularization on the surface of the yolk (top of micrograph). C, yolk cleft; E, extraembryonic ectoderm; I, intravitelline cells; U, uterine tissue. 3145.

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ity of artifact cannot be discounted, large areas of the yolk sac appear to be devoid even of yolk endoderm. However, the vitellus is surrounded by a continuous border of vacuolated endodermal cells that contain tiny droplets of presumed yolk material (Fig. 22). Deep to these cells, the yolk is heavily vascularized. No shell membrane is visible around the egg, although small pieces of degenerating membrane are found at the abembryonic pole in some specimens. Through confluence of the yolk cleft across the yolk at the abembryonic pole, the bilaminar omphalopleure has been converted into the mature omphaloplacenta (Fig. 17). At this developmental stage, the choriovitelline membrane has been obliterated through expansion of the exocoelom. A thin, cellular partition separates the yolk cleft from the exocoelom (Fig. 22). As a result, the allantois is excluded from the yolk cleft. This partition appears to consist primarily of enlarged intravitelline cells, although on its dorsal face contributions are made by cells lining the exocoelom. In some areas, the omphalopleure appears as described in the late pharyngula and consists of isolated yolk mass, yolk endoderm, and a bilayered epithelium (Fig. 17). However, in the late limb-bud stage, the isolated yolk mass has regressed over extensive areas (Fig. 23). As a result, the epithelium of the omphalopleure lies in proximity to the intravitelline cells that line the yolk cleft. Definitive endodermal cells are no longer evident, although these cells could account for some of the small nuclei basal to the epithelium. The uterine tissue of the omphaloplacenta appears much like that of the previous ontogenetic stage. The epithelium consists of low cuboidal cells that overlie capillaries and other small blood vessels (Fig. 23). No uterine glands are evident in this region. DISCUSSION

In most viviparous squamates, nutrients for embryonic development are provided by the yolk, although the placental membranes can supplement vitelline supplies. Consequently, the ovulated yolk in viviparous squamates typically is large, being similar in size to the offspring at birth and greater in dry mass (Blackburn, ’94). Chalcides chalcides, in which females ovulate yolks of about 3 mm in diameter (Giacomini, 1891), is one of a very few squamates with substantial placentotrophy (Ghiara et al., ’87). Because C.

chalcides has converged evolutionarily on the reproductive pattern of therian mammals (ten Cate-Hoedemaker, ’33; Blackburn, ’92) and its placental membranes are highly specialized, this species has figured prominently in interpretations of placental diversity and evolution (Weekes, ’30; Kasturirangan, ’51; Bauchot, ’65; Angelini and Ghiara, ’91; Blackburn, ’93a). From early descriptions, several researchers have questioned whether Chalcides chalcides has evolved a novel pattern of extraembryonic membrane morphogenesis. For example, Weekes (’27) inferred from Giacomini’s (1891) account that invasion of mesoderm into the yolk, a prerequisite for formation of an isolated yolk mass and an omphaloplacenta (Stewart, ’93); does not occur in this species. Likewise, in his detailed monograph on the mature placentae, ten Cate-Hoedemaker (’33) questioned whether a yolk cleft ever forms in C. chalcides. However, according to other interpretations, a yolk cleft and isolated yolk mass develop but disappear through progressive advance of the exocoelom (Boyd, ’42), or, alternatively, a cleft arises that remains separated from the exocoelom (Luckett, ’77b). Most recently, a study based on ten Cate-Hoedemaker’s (’33) histological material of the near-term placenta inferred that a reduced yolk cleft and omphaloplacenta are still present at the end of development (Blackburn, ’93b). This study predicted that examination of the developing extraembryonic membranes in C. chalcides would reveal a pattern of morphogenesis similar to that of other squamates. The pattern of extraembryonic membrane development in Chalcides chalcides is important to an understanding of its placentation. If the extraembryonic membranes do not develop conventionally in this species, then the resultant placentae may be neither structurally equivalent nor functionally related to those of other squamates, including other Chalcides. Furthermore, if an evolutionary reduction in yolk (as is represented in C. chalcides) requires fundamental modifications of developmental patterns, then such a pattern may be serving as a substantial constraint on the evolution of placentotrophy. The fact that only three of the more than 100 viviparous clades of squamates are known to have evolved substantial placentotrophy (Blackburn, ’92, ’93a; Stewart and Thompson, ’93) remains an ongoing puzzle, into

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which the study of C. chalcides may provide some insight. Shell membrane Acellular membrane that surrounds the yolk in early ontogeny seems to occur in two layers: the outermost is probably the shell membrane, and the deeper layer is either a portion of the shell membrane or the vitelline membrane. While presumed to be a common squamate feature, this acellular structure is seldom mentioned in developmental studies (Rathke, 1839; Hrabowski, ’26); however, the term vitelline membrane occasionally has been applied mistakenly to the choriovitelline membrane, a very different structure. In Chalcides chalcides, the deeper membrane is a very thin, eosinophilic structure that is visible on the yolk surface only in regions where the overlying membrane is pulled away artifactually. As in most other viviparous squamates (e.g., Yaron, ’85; Guillette, ’92; Blackburn, ’93a), the shell membrane is very thin. The membrane degenerates rapidly, starting at the embryonic pole; thus, by the late gastrula stage, much of the dorsal hemisphere may be free of membrane. The shell membrane persists longer in the abembryonic hemisphere, but, by the pharyngula stage, much or all of it has been shed. It accumulates at the abembryonic pole of the egg, where it appears to undergo further deterioration, possibly with the aid of epithelium of the omphalopleure. As a consequence of the early loss of the membrane, extraembryonic and uterine tissues are apposed for most of gestation. Loss of the membrane so early in development is highly unusual among viviparous squamates and may be, in part, a mechanical consequence of the swelling of the egg (Blackburn, ’93a). However, such an explanation does not account for the fact that membrane loss always begins at the embryonic hemisphere. The thinning of the membrane in selected areas raises the possibility that proteases, perhaps of uterine origin, assist in its degeneration. Giacomini (1891) considered that no tertiary investments were deposited in Chalcides chalcides (cf. Blackburn, ’93b). The present investigation leaves no doubt that a shell membrane is deposited, and is shed, where it accumulates at the abembryonic pole.

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Mobilization of yolk The timing and mechanism of yolk mobilization are relevant to an understanding of extraembryonic membrane function. Nevertheless, the process has received almost no attention in the squamate literature. During the gastrula and neurula stages, extraembryonic endodermal cells of Chalcides chalcides are found deep in the vitellus. The peculiar cell clumps that lie around the yolk periphery appear to be sites of endodermal proliferation, sites that give rise to the cells that invade the yolk. Within the vitellus, the endodermal cells tend to accumulate around yolk droplets which, from the presence of material in their cytoplasm, they may be digesting. That the vitelline mass undergoes rapid dissolution is suggested by the fact that progressively more of it is represented as a basophilic fluid in paraffin sections. In the late gastrula, the area of yolk liquification is confined to the region beneath the embryonic disk, and yet in the pharyngula as much as 50% of the sectioned vitellus is fluid-filled. Given the rapid depletion of yolk, the placental membranes may be supplying organic nutrients relatively early in development. Whether the products of yolk digestion are transported back to the embryo via both the vitelline circulation and the vitellointestinal duct is unknown; however, yolk particles can be found in the latter in late development (D.G.B., personal observation). In addition, as noted by Luckett (’77a), the subgerminal cavity is lined with enlarged cells that appear to be involved in the process of yolk absorption. Germ layer origins The germ layer concept provides a valuable framework within which to interpret the developmental origins and movement of cell populations in squamates. Broad consensus exists as to the germ layer origins of most cellular components of the squamate placentae. However, the derivation of intravitelline cells has been subject to some discussion (Yaron, ’85; Mossman, ’87), although most researchers have considered them to be of mesodermal origin (Stewart, ’93). In Chalcides chalcides, the squamous intravitelline cells are virtually indistinguishable from cells of the extraembryonic mesoderm, including those lining the exocoelom. Moreover, the two cell populations are contiguous, and no nearby cell population is morpho-

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logically similar to either. Therefore, derivation of the intravitelline cells from indisputable mesodermal cells in C. chalcides seems likely. Debate also has focused on whether the squamate omphalopleure ventral to the sinus terminalis is developmentally bilaminar (ectoderm 1 endoderm) (e.g., Weekes, ’27; Hoffman, ’70; Baxter, ’87; Stewart, ’92, ’93) or whether mesoderm penetrates between the two layers, forming a trilaminar (choriovitelline) membrane (Weekes, ’35; Boyd, ’42). Interpretation is complicated by the presence of two layers of cells overlying the endoderm, which have sometimes been interpreted as ectoderm 1 mesoderm (Weekes, ’35). However, as Hoffman (’70) noted in the garter snake, Thamnophis sirtalis, the position of these two layers superficial to a basal lamina indicates them to be epithelial (ectodermal). This study indicates that a bilayered epithelium occurs in the chorion, the choriovitelline membrane, and the bilaminar omphalopleure and, as has previously been observed (Ghiara et al., ’87; Angelini and Ghiara, ’91), in the chorioallantois. The bilaminar omphalopleure contains no cells that are morphologically similar to those of the choriovitelline mesoderm. Our study suggests that the omphalopleure of Chalcides chalcides consists solely of ectoderm and endoderm, as has been suggested for squamates in general (Stewart and Blackburn, ’88). Bilaminar omphalopleure In Chalcides chalcides, extraembryonic ectoderm and endoderm spread rapidly over the yolk surface and have entirely enveloped the yolk by the late gastrula stage. The resulting bilaminar omphalopleure establishes the first placental arrangement to form in C. chalcides. The omphalopleure lies apposed to the uterine mucosa, being separated from it at the gastrula stage only by the vestigial shell membrane, which is gone by the pharyngula stage. Formal recognition of the apposed uterine tissue and bilaminar omphalopleure (prior to formation of the isolated yolk mass) as a placental arrangement seems not to have been suggested previously in squamates. Nevertheless, the tissue complex satisfies the operational (anatomical) criteria adopted for other squamate placentae (see Stewart and Blackburn, ’88; Blackburn, ’93a). The extent of physiological exchange across the membranes is unknown. Morphol-

ogy of the bilaminar omphalopleure soon after its formation is not strongly suggestive of such exchange, in that the membrane is avascular and its surface of squamous cells offers no evidence of histotrophy; however, prior to invasion of the intravitelline mesoderm, the surface epithelium quickly is replaced by columnar cells that may well have absorptive capabilities. The surrounding uterine lumen at this stage does contain eosinophilic secretory material, mixed with degenerating cells presumed to be of uterine origin. In any case, definitive data on physiological exchange are lacking for most organs recognized as placentae in squamates, and exclusion of the bilaminar omphalopleure on such a basis would be arbitrary. How such a placenta might be classified is another matter, for the structure is neither chorionic (sensu Mossman, ’37), choriovitelline, nor omphaloplacental (Stewart and Blackburn, ’88). Flynn’s (’22) proposed term ‘‘metrioplacenta’’ for a similar structure in mammals has not been adopted, and a recent classification of placental types (Stewart and Blackburn, ’88) did not formally recognize such an arrangement as placental. Provisionally, the tissue arrangement is referred to here as the bilaminar yolk sac placenta. Choriovitelline placenta By the early pharyngula stage, the spread of vascularized mesoderm between the extraembryonic ectoderm and endoderm has established a choriovitelline membrane dorsal to the sinus terminalis. Where this membrane faces the uterine epithelium, it forms a choriovitelline placenta, as first noted by Luckett (’77a). The choriovitelline placenta is a transitory structure that disappears as the exocoelom splits the somatopleure from the splanchnopleure. Luckett (’77a) indicated that the choriovitelline placenta in Chalcides chalcides could be observed during the 15–50 somite stages, and our observations indicate that it is no longer present in the limb-bud stage. Until the chorioallantois develops, the choriovitelline membrane represents the only vascularized extraembryonic tissue available for gas exchange with the uterus. The vitelline and uterine circulatory systems are separated by low cuboidal epithelia which offer a thin barrier to diffusion. Morphology, therefore, suggests that the choriovitelline placenta has the potential to play a role in gas exchange in early development.

LIZARD PLACENTAL DEVELOPMENT

Chorioplacenta A true chorioplacenta apparently has not been described before in squamates, perhaps because morphogenesis of the placental membranes has not been studied in sufficient detail. A chorionic placenta does occur among mammals, where it can be the site of significant nutrient transfer (Mossman, ’37, ’87; Angelini and Ghiara, ’84). While noting that such a placenta had not been described, Stewart and Blackburn (’88) extended the concept of chorioplacentation to squamates on theoretical grounds, under the implicit assumption of a common pattern of extraembryonic membrane morphogenesis. An anatomical chorioplacenta does form in Chalcides chalcides by the pharyngula stage. Due to loss of the shell membrane, the chorionic somatopleure is exposed to the uterine lumen and apposed to the uterine mucosa. The surface epithelium of the (avascular) chorion consists of columnar and cuboidal epithelium. In addition, uterine glands are active during the time of its occurrence. Therefore, histology is consistent with the possibility of histotrophic transfer. Given the presence of a chorioplacenta in early development in C. chalcides, its development is to be expected in other viviparous squamates as well between the time of amnion formation and establishment of the chorioallantois. Since allantoic development may be precocial in squamates (Lemus and Badinez, ’67; Hubert, ’85), this period may be relatively brief, perhaps accounting for the fact that this placenta has not been described previously. Omphaloplacental development As invasion of the intravitelline cells and formation of the yolk cleft begin to separate an isolated yolk mass and its overlying bilaminar omphalopleure from the vitellus proper, the resulting membrane forms the embryonic contribution to the omphaloplacenta. Given known patterns of squamate development (Hoffman, ’70; Blackburn, ’93a; Stewart, ’93), the choriovitelline membrane presumably precedes initiation of the omphaloplacenta in Chalcides chalcides; certainly in the early pharyngula the latter is still very limited in extent. Observations on the pharyngula suggest the following sequence of events at any given site around the yolk periphery: spreading of extraembryonic ectoderm and endoderm over the yolk surface (Fig. 6); invasion into the yolk of the intravi-

51

telline cells (Fig. 16); formation of small spaces between the cells (Fig. 14); coalescence of the spaces into a cleft that separates a band of yolk from the vitellus (Fig. 17); and expansion of the width of the yolk cleft (Fig. 15). In addition, transformation of the epithelium of the bilaminar omphalopleure from squamous to cuboidal and columnar (Fig. 18) proceeds in advance of the invasion of the intravitelline cells. Because these events proceed in a proximal to distal (dorsal to ventral) direction, all of these stages of omphaloplacental development can be seen within a single conceptus of appropriate age (Figs. 11, 12). By the limb-bud stage, the process is complete, and the omphaloplacenta occupies the ventral hemisphere of the egg (Fig. 23). A persistent question about the squamate omphaloplacenta is whether any uterine substances absorbed by the avascular omphalopleure could make their way into the embryonic circulation. By the limb-bud stage in Chalcides chalcides, the entire surface of the yolk sac is heavily vascularized. The vitelline capillaries are separated from the omphalopleure by the yolk cleft (Figs. 17, 23), offering a potential if circuitous route for nutrient transport into the embryo. A similar situation has been described in the placentotrophic skink Pseudemoia entrecasteauxii (Stewart and Thompson, ’96). As noted above, the columnar cells lining the omphalopleure at the abembryonic pole extend into the degenerating shell membrane, taking on unusual shapes (Fig. 18). These epithelial extensions almost certainly represent precursors to the omphalopleuric papillae seen later in development (see Blackburn, ’93b). Significantly, where columnar epithelium occurs, the shell membrane is much depleted, whereas the membrane persists in those areas where squamous epithelium lines the omphalopleure (Fig. 18). The enlarged epithelial cells of the omphalopleure probably contribute to degeneration of the shell membrane, perhaps through phagocytosis. Because a cellular partition separates the yolk cleft from the exocoelom as of the limbbud stage (Fig. 22), the allantois is unable to enter the yolk cleft; thus, formation of an omphalallantoic placenta is prevented. Such a partition also has been seen in the Australian lizard Niveoscincus metallicus, in which the question of its origin has been raised (Stewart and Thompson, ’94; also see Stew-

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art and Thompson, ’96). In Chalcides chalcides, the partition is lined dorsally with mesoderm of the exocoelom and ventrally with intravitelline cells, and from its appearance it probably is derived from yolk endoderm, where the yolk sac abuts the chorion. Our observations of yolk sac ontogeny confirm previous suggestions (Luckett, ’77b; Blackburn, ’93b) that Chalcides chalcides develops a yolk cleft that is separated from the exocoelom. In addition, these observations resolve questions about the origins and identity of tissues at the abembryonic pole in late development (ten Cate-Hoedemaker, ’33; Yaron, ’85). Yolk of the isolated yolk mass is depleted by the limb bud stage (Fig. 23). Thus, for the omphaloplacenta of the limb-bud stage to regress into the rudimentary structure seen in the near-term fetus (Blackburn, ’93b) would simply require shrinkage of the omphaloplacenta through abembryonic migration of the cellular partition and a corresponding increase in the area given over to chorioallantois (see below). Some previous inferences are therefore corroborated: that the allantois never fully passes around the ventral side of the yolk sac (Giacomini, 1891; Blackburn, ’93b), that the omphaloplacenta is in regression by late development (ten Cate-Hoedemaker, ’33), and that a yolk cleft and bilaminar omphalopleure develop and persist, at least in reduced form, until the time of parturition (Blackburn, ’93b; cf. Weekes, ’27, ’35; ten Cate-Hoedemaker, ’33). Allantoplacenta By the limb-bud stage, a chorioallantoic placenta has developed that contains most of the characteristic features previously described in Chalcides chalcides in late development (see Angelini and Ghiara, ’91; Blackburn, ’93b). The placentome exhibits the structure of an organ that functions in histotrophic transfer, with active uterine glands secreting an eosinophilic, PAS-positive material that may be absorbed by the microvillate epithelium of the chorion. Ultrastructural examination has provided corroborative evidence for uptake by the chorionic epithelium as well as evidence for secretion by the surface epithelium of the uterus (Angelini and Ghiara, ’91). The infolding of the chorionic and endometrial tissues greatly increases the surface area across which maternal-fetal transfer can occur. By the time of parturition, the uterine epithelial cells are larger than in the limb-bud stage, and the

placentomal infolding is deeper and more elaborate (Blackburn, ’93b). We are unable to confirm whether initiation of the uterine villi precedes establishment of those of the chorion, such as was suggested by Giacomini (1891). In the paraplacentomal region, the surface of the chorion is lined by a cuboidal epithelium. As shown in Chalcides ocellatus (Ghiara et al., ’87), the epithelium is bilayered, the deeper layer consisting of squamous cells. The uterine epithelium in this region is low cuboidal to squamous. Both membranes are highly vascularized, and the uterine component is aglandular. The diffusion distance between allantoic and uterine capillaries therefore is considerably smaller than in the placentome, as one would expect of a respiratory organ. However, a comparison of the early chorion with that of late development indicates that this diffusion distance decreases as development proceeds. By late gestation, the epithelia overlying the capillaries on both sides of the placental interface are so thin as to be difficult to distinguish histologically (Angelini and Ghiara, ’91; Blackburn, ’93b), accounting for the mistaken inference (ten Cate-Hoedemaker, ’33) that they are eroded. The paraplacentomal region of Chalcides chalcides is anatomically generalized, being similar to the entire allantoplacenta in viviparous squamates that are relatively lecithotrophic (Yaron, ’85; Blackburn, ’93a). Placentae with this morphology commonly are regarded as functioning in gas exchange. However, ultrastructural examination of a similar placental arrangement in C. ocellatus has revealed some evidence of uterine exocytosis and chorionic endocytosis (Angelini and Ghiara, ’91). Thus, although the capability for gas exchange undoubtedly exists, histotrophic transfer cannot be ruled out. Interspecific comparisons Although studies focusing on early morphogenesis of extraembryonic membranes in lizards and snakes are rare, available information indicates that Chalcides chalcides shares a variety of basic developmental features with other squamates. The movement of extraembryonic ectoderm and endoderm around the yolk in this species conforms to the basic squamate pattern (e.g., see Virchow, 1892; Hrabowski, ’26; Hoffman, ’70; Stewart, ’93), as does invasion of intravitelline cells into the yolk (see Stewart and

LIZARD PLACENTAL DEVELOPMENT

Blackburn, ’88). Likewise, the yolk cleft forms between bands of intravitelline cells in C. chalcides, as has been observed in most squamates that have been examined (Hoffman, ’70; Yaron, ’85; Stewart, ’93; cf. Boyd, ’42; Parameswaran, ’62). The resulting omphalopleure and isolated yolk mass are essentially like those described in a wide variety of squamates, oviparous and viviparous alike (Stewart, ’92, ’93). In addition, as discussed above, the germ layer origins of problematic cell populations (intravitelline cells, omphalopleuric components) appear to be similar in C. chalcides and other squamates. A choriovitelline membrane such as occurs in Chalcides chalcides has been described in various other lizards (Lemus and Badinez, ’67; Stewart, ’85; Stewart and Presch, ’92) and snakes (Baxter, ’87; Stewart, ’90; Jones and Baxter, ’91). Most likely, this membrane is characteristic of squamates in general and simply has been overlooked in some species due to its transitory existence. Such is probably the case for the chorion (and, in viviparous forms, the chorioplacenta) as well, given the similarities in amniogenesis among squamates (see Fisk and Tribe, ’49; Hubert, ’85). Likewise, a chorioallantois seems to be universal among squamates (Yaron, ’85; Blackburn, ’93a), being a synapomorphy for amniotes. A few other features of C. chalcides are not universal but nonetheless do occur in other squamates. For example, formation of the yolk cleft by coalescence of small spaces between the intravitelline layers appears to be somewhat similar to that described in Lacerta by Hrabowski (’26). Likewise, an omphalallantoic membrane may be common among snakes, but its absence in C. chalcides is like the situation in several lizards (Stewart and Blackburn, ’88; Stewart, ’93). Almost all of the unusual developmental features of Chalcides chalcides are specializations associated with placentotrophy. One such feature is the rapid enclosure of the yolk by the bilaminar omphalopleure, which occurs precocially in the presomite stages. In other viviparous squamates, this envelopment is not completed until well after amniogenesis and establishment of the chorioallantois (e.g., Hrabowski, ’26; Boyd, ’42; Parameswaran, ’62; Hoffman, ’70). Precocial envelopment in C. chalcides may partly reflect the fact that the smaller yolk volume requires less time to enclose by the spreading ectoderm and endoderm. However, it

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may also have functional consequences, either in aiding the uptake of water and nutrients by the egg (Luckett, ’77b), or in facilitating yolk digestion through the invasion of the vitellus by the endoderm. Complete loss of the shell membrane so early in development also is unusual for squamates, having been observed elsewhere in species of Mabuya that ovulate eggs even smaller than those of Chalcides chalcides (Blackburn et al., ’84; Blackburn and Vitt, ’92). Such loss probably is necessary if the egg is to increase in volume through the uptake of nutrients, and it facilitates nutrient uptake by allowing close apposition of uterine and extraembryonic tissues. Whether shell membrane loss is a functional consequence of the swelling of the egg in these forms remains to be determined; histological evidence offered herein suggests that extraembryonic ectoderm may take an active role in its degeneration. Another unusual feature of Chalcides chalcides is the placentome, formed of interdigitating tissues lined with secretory (uterine) and absorptive (chorioallantoic) epithelia. Although common among eutherians (Mossman, ’87), placentomes are known elsewhere only in a few unrelated viviparous clades of squamates that exhibit an extreme form of placentotrophy (Blackburn, ’92; Blackburn and Vitt, ’92; Stewart and Thompson, ’93, ’96; but also see Stewart and Thompson, ’94). Our observations have several evolutionary implications. As shown by the interspecific comparisons, Chalcides chalcides has conformed closely to a pattern of extraembryonic membrane development that is common to squamates. Indeed, the specializations that contribute to placentotrophy in C. chalcides represent consequences of its small egg size (shell membrane loss, precocial envelopment of the yolk) as well as histological specializations that have been superimposed onto a basic squamate pattern. The similarities between C. chalcides and other species may have been obscured by such specializations but mainly have been unappreciated because of the scarcity of information on early development in squamates. Our findings also suggest that substantial placentotrophy can evolve in squamates without major modifications of morphogenetic patterns. Evolutionary reduction of the yolk mass might be expected to be associated with evolutionary changes in morpho-

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genesis for at least two reasons: 1) the yolk provides a substrate for extraembryonic membrane expansion, and 2) penetration of the yolk by intravitelline cells forms the isolated yolk mass at the abembryonic pole. In eutherian mammals, evolutionary loss of the yolk mass seems to have involved fundamental alterations in the formation of the yolk sac, which develops out of the blastocyst rather than from tissue that envelops a yolk mass (Luckett, ’77b). Chalcides chalcides reveals the evolutionary retention of an ancestral morphogenetic pattern despite the presence of a greatly reduced yolk mass. Consequently, evolutionary constraints imposed by morphogenetic patterns probably do not account for the scarcity of extreme placentotrophy among squamates. Future placental work As one of the first detailed accounts of the histology of reptilian placental membranes during early development, this study offers a perspective that may prove useful in future work. In Chalcides chalcides, cell populations and placental membranes undergo dramatic transformations within short developmental periods. Thus, the epithelium of the abembryonic omphalopleure changes from simple squamous to columnar (overlying the isolated yolk mass) to a papillated bilayer of cells overlying the yolk cleft. Likewise, extraembryonic tissue at the egg equator is transformed from a bilaminar omphalopleure (avascular) to choriovitelline (vascularized) to chorionic (avascular) to chorioallantoic (secondarily vascularized), with the attendant, striking changes in its surface epithelium and cellular components. Past studies of placental anatomy in reptiles have not always met standards that are applied routinely in the mammalian literature. Given the dramatic ontogenetic changes and regional variation that can exist within a single squamate species, future anatomical accounts warrant detailed descriptions that designate topographic regions, ontogenetic stages, and cytological components. Specification of the particular extraembryonic membranes under investigation is also essential, given that at least six distinct types of placental arrangements can occur: choriovitelline, omphaloplacental, chorioallantoic, and omphalallantoic (Stewart and Blackburn, ’88) as well as placentae formed from the chorion and from the bilaminar yolk sac. That four of these placental types have been shown to be highly transitory emphasizes the need for descriptions that

specify the developmental stages of embryos being described. Because this paper focuses on early morphogenesis, it does not deal with developmental changes that occur in Chalcides chalcides between the limb-bud stage and the time of birth. For example, given Giacomini’s (1891) mention of elongated, omphalopleuric papillae that interlace with those of the uterus, detailed development of the most elaborate features of the omphaloplacentae may have yet to be described. How these papillae regress into the small structures present just before parturition (Blackburn, ’93b) also is unknown, as are aspects of the establishment of the chorioallantois and the placentome. Likewise, details of the fate of the isolated yolk mass and regression of the yolk cleft await further examination. Over a century ago, Ercole Giacomini (1891) recognized the potential of studies on viviparous lizards to an understanding of placental development and evolution in other amniotes. Conclusions reached herein tend to corroborate Giacomini’s vision. Placental research on Chalcides chalcides is of value not only because of the striking specializations for nutrient transfer in this species but because these specializations clearly have evolved within the framework of a developmental pattern that is common to squamates. ACKNOWLEDGMENTS

This work was facilitated by a three-year faculty research grant from Trinity College (to D.G.B.). Dr. Lora Miller helped with the translation of early literature in Italian. LITERATURE CITED Angelini, F., and G. Ghiara (1984) Reproductive modes and strategies in vertebrate evolution. Boll. Zool. 51: 121–203. Angelini, F., and G. Ghiara (1991) Viviparity in squamates. In G. Ghiara (ed): Symposium on the Evolution of Terrestrial Vertebrates. Selected Symposia and Monographs U.Z.I., Vol. 4. Modena: Mucchi, pp. 305– 334. Bauchot, R. (1965) La placentation chez les reptiles. Ann. Biol. 4:547–575. Baxter, D.C. (1987) Placentation in the Viviparous Lined Snake, Tropidoclonion lineatum: Ontogeny of the Extraembryonic Membranes and Histochemistry of Placental Tissues. M.Sc. thesis, University of Tulsa, Tulsa, OK. Blackburn, D.G. (1985) The Evolution of Viviparity and Matrotrophy in Vertebrates, With Special Reference to Reptiles. Ph.D. thesis, Cornell University, Ithaca, NY. Blackburn, D.G. (1992) Convergent evolution of viviparity, matrotrophy, and specializations for fetal nutrition in reptiles and other vertebrates. Am. Zool. 32:313– 321.

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