J. Anat. (1978), 125, 1, pp. 85-99 With 22 figures Printed in Great Britain
Postnatal development of the epidermis in a marsupial, Didelphis virginiana WILLIAM J. KRAUSE, J. HARRY CUTTS AND C. R. LEESON Department of Anatomy, University of Missouri, Columbia, Missouri 65201
(Accepted 1 December 1976) INTRODUCTION
The North American opossum (Didelphis virginiana) has a remarkably short gestation period, which lasts only 121 days. Immediately after birth the young crawl into the maternal marsupium, each attaches to a nipple, and they continue their development within the protection of the pouch until just after weaning. The newborn opossum measures 14 mm in length and weighs approximately 0-5 g. It is immature in appearance: in contrast to well developed forelimbs, the hind limbs are paddle-like and show only the initial stages of digit formation. Visceral structures such as the lung (Krause & Leeson, 1973, 1975; Krause, Cutts & Leeson, 1976), liver (Cutts, Leeson & Krause, 1973; Krause, Cutts & Leeson, 1975) and alimentary canal (Krause, Cutts & Leeson, 1976a,b, 1977) also show immature features at birth, and undergo an extensive period of postnatal development and differentiation. Previous work on the opossum integument has been concerned primarily with the distribution of vibrissae (Lyne, 1959), tylotrich (hair) follicles (Mann, 1968), cutaneous nerve receptors (Montagna & MacPherson, 1973) and the intraepidermal innervation of the snout (Munger, 1965). Although the pouch young opossum has been used in immunological studies involving skin allographs (LaPlante, et al. 1966; LaPlante et al. 1969), very little information is available about either the development or adult structure of the epidermis in the opossum. The present study is concerned with the postnatal development of the epidermis of the opossum. MATERIALS AND METHODS
Eighty opossums (Didelphis virginiana) were used. The pouch young opossums were divided into the following 15 groups according to their snout-rump lengths: 1-5, 2 5, 3.5, 4 0, 4-5, 5.5, 6-0, 6-5, 7 0, 7 5, 8-0, 10 0, 12-0, 13-0 and 20-0 cm. Four adults also were used. The animals were killed by decapitation. Blocks of skin, mostly from the dorsal and lateral walls of the trunk, were excised. Some animals in the youngest groups (15-3-5 cm) were serially sectioned and the skin of all regions examined. For light microscopy, segments of skin were fixed in either Bouin's solution or 10 % buffered neutral formalin. Tissues were processed routinely, embedded in paraffin, sectioned at 6 ,um, and subjected to the following staining procedures: haematoxylin and eosin, Masson's trichrome, periodic acid-Schiff, Wilder's reticular stain, and van Gieson. Additional blocks of tissue were fixed at 0 'C in 3 5 % glutaraldehyde buffered with 0-1 m phosphate to a pH of 7'3. The tissues were washed in buffer, osmicated for 2
hours in 1 0 % osmium tetroxide, and embedded in Epon-812. Thick sections of this
86 W. J. KRAUSE, J. H. CUTTS AND C. R. LEESON material were cut at 0 5-3-0 ,um, and stained with toluidine blue. After clearing in xylene, they were examined by light microscopy. Thin sections of the tissue embedded in Epon-812 were mounted on uncoated grids and stained with uranyl acetate and lead citrate (Reynolds, 1963). The sections were examined in a RCA EMU-3F electron microscope operated at 50 kV. Tissues taken for scanning electron microscopy were fixed as those for transmission electron microscopy and post-osmicated in 1 0 % osmium tetroxide at 0 °C for 2 hours. The specimens were dehydrated in ethanol and transferred to amyl acetate prior to critical point drying. The specimens were dried by liquid CO2 substitution, during which time a bleed valve was opened and closed until amyl acetate could not be detected at the time when the valve was open, indicating complete substitution by CO2. The dried tissues were placed on spinner stubs and coated with a goldpalladium alloy to a depth of approximately 20 nm in a vacuum evaporator. The specimens were viewed in a Cambridge Steroscan Mark II electron microscope operated at 20 kV. Light microscopy was used to determine epidermal thickness and epidermal mitotic activity at various stages of development. Thickness was determined from five random measurements on each of five sections from each animal. Three animals were studied ateach stage. The measurements were made by means of a filar micrometer eyepiece so orientated that the reference line of the micrometer lay along the base of the epidermis. The thickness reported represents the depth of the epithelium in the interval between either established or developing hair follicles. Counts of nmitotic figures were made on the same sections. The total number of epithelial cells and mitotic figures were counted in each section: mitotic figures were classed as those in the epithelium proper and those in (developing) hair follicles. RESULTS
The epidermis of the newborn opossum (1I4 cm) is about 43 ,um thick. It has a basal layer of cuboidal cells and a spinosal layer of fusiform cells (Fig. 1). The epidermal-dermal interface is irregular. Dermal papillae are not present. The external Fig. 1. A section of newborn opossum epidermis taken from the back. The epidermal-dermal interface appears irregular. A light staining periderm (arrows), covered with densely staining bacteria, lies immediately above the forming keratinizing layer. Cells of the granulosa, spinosum, and germinal layer are clearly seen. Epon-812-toluidine blue. x 450. Fig. 2. A section of epidermis taken from the snout region of a newborn opossum shows the early development of vibrissae. Haematoxylin and eosin. x 250. Fig. 3. The external surface of the newborn opossum epidermis is covered by a squamous epithelium (periderm). Cells of the periderm show discrete nuclei and distinct cell boundaries. x 200. Fig. 4. Increased magnification of the surface of the periderm shows a small colony of bacteria (arrow) on the external surface. Newborn opossum. x 500. Fig. 5. A junction between two surface cells of the periderm (P) shows an extensive implication of adjacent cell membranes. Numerous bacteria (B) are shown on the external surface. Immediately subjacent to the surface cells lies the forming keratinizing layer (K) that shows considerable electron density. A portion of a cell from the granulosa that contains irregular, dense, keratohyalin granules (arrows) is shown at bottom right. Newborn opossum. x 12000. Fig. 6. Bacteria occasionally lie within the cells of the periderm. The plasmalemma of surface cells, and of elements of the forming keratinizing layers (K), shows considerable electron density. Numerous filaments also are found in cells of the forming keratinizing layer. Newborn opossum. x 24000.
Postnatal development of opossum epidernmis
88 W. J. KRAUSE, J. H. CUTTS AND C. R. LEESON 88 surface often shows areas covered by bacteria which stain intensely with toluidine blue. The epidermis covering the dorsal and lateral body wall does not show hair follicles. Developing vibrissae, however, were observed in the snout region (Fig. 2). The epidermis of the snout region is thicker than that covering the trunk. The surface layer of the epidermis consists of a partially keratinized layer of squamous cells (the periderm). The external surface of cells of the periderm shows distinct cell boundaries and discrete nuclei (Figs. 3, 4). Scattered colonies of spherical bacteria are seen (Fig. 4). Ultrastructurally, cells of the periderm appear less electron-dense than elements of the underlying layer (Figs. 5, 6); adjacent plasma membranes show extensive implications rather than desmosomes (Fig. 5). The plasma membranes in both periderm and underlying keratinizing layer show increased electron density and thickness. In addition, cells of the keratinizing layer contain numerous filaments (Fig. 6). Bacteria were frequently observed on the surface of the periderm, and on occasions within the cells The stratum granulosum shows large, irregular, electron-dense kerato(Figs. 5, 6). hyalin granules (Figs. 5, 7). The spinous layer shows large fusiform cells with numerous free ribosomes and occasional profiles of granular endoplasmic reticulum. Mitochondria are scarce and the Golgi complexes are poorly developed. Scattered bundles of tonofilaments are present. There are numerous desmosomes between adjacent cells of the spinosum (Fig. 7). The germinal layer consists of cuboidal cells, the basal cell membranes of which form an irregular interface with the dermis (Fig. 8). The basal cell membrane shows numerous hemidesmosomes and lies on a delicate basal lamina. The cytoplasm of basal cells is characterized by numerous free ribosomes. The epidermis of the 2 5 cm opossum (7 days postnatum) is thicker than that of the newborn, due primarily to an increase in the depth of the spinous layer (Fig. 9). The epidermis covering the dorsal and lateral body wall shows initial development of hair follicles. The distinct squamous cells of the surface layer (periderm) observed in the newborn are lost by the end of the first postnatal week. The external surface of the epidermis at this age appears irregular and the cells are without nuclei, and there are scale-like regions that are thought to be sites of sloughing squamous cells (Fig. Elements of the keratinizing layer show a further increase in electron density (Fig. Cells of the granulosa and spinosum show what are thought to be membrane-coating granules. In addition, the fusiform cells of the spinosum show an increase in the number of bundles of tonofilaments (Figs. 11, 12). Cells of the germinal layer show fewer free ribosomes than those of the newborn, and bundles of tonofilaments are a more prominent feature (Fig. 12). in the 4-0 cm opossum days), The epidermis attains its greatest thickness (58 and shows further development and differentiation of hair follicles (Fig. 13). The increase in thickness of the epidermis, as before, is due primarily to an increase in the thickness of the spinous layer. Bacteria still are present on the external surface (Fig. 14). Tonofilaments often appear directly associated with the keratohyalin granules. The basal cell membrane of the germinal layer remains irregular and shows numerous hemidesmosomes (Fig. 15). Cells of the germinal layer show a continued
and filaments. Fig. 7. Cells of the granulosa show large, dense, keratohyalin granules (arrows) Cells of the spinosum (S) show numerous free polyribosomes, scattered tonofilaments and desmosomes. Newborn opossum. x 7000. arrows) Fig. 8. The germinal layer of the epidermis lies on a delicate basal lamina (largeopossum.
and the basal cell membrane shows numerous hemidesmosomes (arrows). Newborn x 10000.
Postnatal development of opossum epidermis
90 W. J. KRAUSE, J. H. CUTTS AND C. R. LEESON increase in tonofilaments, which are often disposed in relation to hemidesmosomes and desmosomes. Tonofilaments and polyribosomes remain prominent features of the spinosum. Langerhans' cells are frequently observed in the spinous layer at this stage of development. They appear irregular in shape and have numerous processes that extend between adjacent cells of the spinous layer. They are devoid of desmosomal attachments to adjacent cells of the spinosum. The nucleus is irregular in shape and varies considerably in electron density. The cells of Langerhans also lack the bundles of tonofilaments that characterize the fusiform cells of the spinous layer. In the 5-5 cm opossum (28 days) scattered hair shafts are beginning to emerge (Fig. 16). By the 7 0 cm stage (38 days) hairs are well established, but the epidermal surface remains irregular and continues to show areas of sloughing. The epidermis at 8 5 cm (49 days) is thinner than before and hairs are readily visible to the naked eye. The decrease in depth of the epidermis is due primarily to thinning of the spinous layer (Fig. 17). Developing hair follicles continue to be a prominent feature at the 10 cm (60 day) stage, while the epidermis is thinner still (Fig. 18). By the 12 cm stage (72 days), the epidermis is only 20 thick, and the opossums are well furred. The keratinizing zone has several layers. Numerous desmosomes are present in the spinosum and granulosum (Fig. 19). Amorphous, electron-dense bodies, thought to be degenerating desmosomes, are present in the keratinizing layer (Fig. 19). The germinal layer consists of cells with numerous bundles of tonofilaments. The ultrastructural features of the germinal and spinous layers are similar to those in younger animals. The epidermis of the adult opossum is only 14 thick and consists of a thin Malpighian layer and a desquamating keratinized layer (Figs. 20, 21). Cells of the germinal layer continue to show bundles of tonofilaments in relation to hemidesmosomes and desmosomes. As in all previous stages, the epidermal-dermal interface is irregular (Fig. 20). Keratohyalin granules continue to be a prominent feature of the thin, discontinuous granular layer (Fig. 21). Mitotic activity in the epidermis is highest in the newborn and decreases to the adult level after 4 weeks (Fig. 22). Hair follicles begin to develop at the end of the first week. They continue to develop and differentiate throughout the period of increasing epidermal thickness and during most of the subsequent period of decreasing thickness (Fig. 22). Mitotic activity in hair follicles gradually increases from the 2 5 cm stage (7 days postnatum), reaching a peak of activity at 6-0 cm (32 days), and then gradually decreases until the 13 cm stage, when the activity is similar to that observed in the adult. The rapid increase in epidermal thickness coincides with the first population of hair follicles. Later follicles form during the time of decreasing thickness. Not all the mitoses in the epidermis are confined to the basal layer. This is especially
Fig. 9. The epidermis of the 2-5 cm opossum (7 days postnatum) shows an increased thickness of the spinous layer. Papillae are not present. Haematoxylin and eosin. x 350. Fig. 10. The external surface of the epidermis shows what appears to be sloughing squames. 7 days old opossum. x 150.
Fig. 11. Keratinizing, granulosa and spinous layers from the epidermis of a 2-5 cm opossum. Cells of the spinous layer contain increased numbers of bundles of tonofilaments, and what are thought to be membrane-coating granules (arrows). x 2500. Fig. 12. Cells of the germinal and lower spinous layers show increased bundles of tonofilaments and numerous polyribosomes. 2-5 cm opossum. x 6000.
Postnatal development ofopossum epidermis
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Postnatal development of opossum epidermis 93 true during the period of increasing thickness of the epidermis (i.e. between the 1-5 and 4 0 cm stages) when approximately one third of the mitotic figures are within the spinous layer. Occasionally mitoses could even be found within one or two cell diameters of the surface. However, as the epithelial layer became progressively thinner fewer and fewer mitotic figures could be found beyond the basal layers, and from 5-5 cm onward all mitoses were in basal cells. DISCUSSION
Already relatively thick at birth, the epidermis of the North American opossum continues to increase in thickness throughout the first period of pouch life, reaching a maximum (58 ,um) at the 18th postnatal day. This increase is the result mainly of an expansion of the stratum spinosum, where additional cells are derived not only from the basal layer, but also from the mitotic activity of the spinosal cells themselves. By the end of the first week the stratum corneum shows an increase in electron density, and the epidermis of the back and lateral body walls shows initial development of hair follicles: hair shafts are seen by the fourth week of development. After the 18th day the epidermis undergoes a progressive thinning due mainly to a decrease in the spinous layer. Mitosis of spinosal cells is no longer a feature after the 18th day, and this layer becomes gradually thinned. Ultimately the epidermis of the adult opossum measures only 14 ,um in depth, and consists primarily of a thin Malpighian layer and a desquamating, keratinized surface layer. A similar sequence of development has been reported in the epidermis of the Australian opossum, Trichosurus (Lyne, 1970; Lyne, Henrikson & Hollis, 1970). In this species the epidermis undergoes a marked increase in thickness during the first 3 weeks of postnatal life, and subsequently undergoes a progressive thinning. As in Didelphis, the epidermis of the adult Trichosurus measures only about 15 ,um in depth. The period of rapid development of the epidermis, in both species, coincides with the development of the first population of hair follicles. Similarly, both species show continued differentiation and development of hair follicles during the period of decreasing thickness of the epidermis. An increase in epidermal thickness prior to and/or during the period of follicular differentiation also occurs in eutherian forms (Hanson, 1947; Lyne & Heideman, 1959). Throughout the first 7 days of postnatal life the epidermis of the opossum bears a surface layer of simple, partially keratinized squamous cells that form the periderm. An epidermal surface layer of similar nature has been described in the newborn of two Australian marsupials, the native cat, Dasyrus viverinus (Hill & Hill, 1955), and the brush-tailed opossum, Trichosurus vulpecula (Lyne et al. 1970). Although consisting of non-keratinized cells, a somewhat analogous layer of simple squamous
Fig. 13. The epidermis of the 4-0 cm opossum, which shows an increase in thickness of the spinous layer. A developing hair follicle (F) is shown. Epon-812. Toluidine blue. x 350. Fig. 14. Scattered bacteria (B) continue to be observed on the surface of the epidermis of the 4-0 cm opossum. Tonofilaments and membrane-coating granules (arrows) remain. prominent features of the spinous layer. Keratohyalin granules of the granulosa are shown. x 8000.
Fig. 15. The germinal layer of the epidermis shows a continued increase in tonofilaments (f). The epidermal-dermal interface remains irregular and numerous hemidesmosomes (arrows) are observed. 4 0 cm opossum. x 10000.
W. J. KRAUSE, J. H. CUTTS AND C. R. LEESON
Postnatal development of opossum epidermis
epithelial cells is present on the epidermal surface of several fetal eutherian mammals also, including the rat and mouse (Hanson, 1947; Bonneville, 1968), sheep (Lyne & Hollis, 1971) and man (Breathnach & Wyllie, 1965; Holbrook & Odland, 1975). In marsupials the periderm is believed to provide a protective barrier that prevents desiccation during the first few days after birth. The periderm is no longer present in Didelphis after the 7th day of postnatal life. The fine structure of the epidermis of the opossum appears similar to that described in Trichosurus (Henrikson, 1969). The keratinizing layer of the epidermis in Didelphis shows numerous desmosome-like structures that may represent degenerating desmosomes. Similar structures have been reported in the keratinizing layers of the guineapig (Snell, 1965) and in the mouse, where they have been termed squamosomes (Allen & Potten, 1975; 1976). The basal membrane of the germinal layer shows numerous hemidesmosomes. Throughout the postnatal period the epidermal-dermal interface appears irregular, but there is no development of dermal ridges or papillae, even at the 4 0 cm stage when the epidermis has attained its greatest depth. Presumably, in the absence of such structures, the thickness of the adult epidermis represents the maximum that can be supported by interchange of nutrients between the epithelium and the underlying vascular dermis. If this is so, failure to develop dermal ridges and papillae would necessitate a thinning of the epidermis after the formation of the first hair follicles. Because of the primitive nature of the developing lungs early in postnatal development (Krause & Leeson, 1973, 1975; Krause, Cutts & Leeson, 1976), it might be thought that the epidermis contributes to respiratory exchange, as occurs in some amphibian forms. However, the presence at birth of a periderm and a forming keratinizing layer, and a progressive increase in thickness of the epidermis during the early period of postnatal life, makes this unlikely. It is more probable that the thick epidermis serves to prevent dehydration of the tiny organism during the first weeks of postnatal life. Block (1960) has reported that non-sterile incisions made in the skin of the opossum prior to the 6th day of birth fail to heal. Such wounds become grossly infected, gangrenous and result, ultimately, in death of the animals within 2-3 days of wounding. In contrast, sterile wounds or wounds made later than the 6th day, undergo complete helaling. This difference has been related to the development of the haemopoietic tissues which, prior to six days, are insufficiently developed to permit the mounting of an inflammatory response. Thus, in addition to protecting the newborn from desiccation, the periderm and thick epidermis of the young opossum may serve as a mechanical barrier to infection during the period of haemopoietic immaturity. This is almost certainly a matter of considerable importance in view of the large numbers Fig. 16. The external surface of the epidermis of a 55 cm opossum. An emerging hair is shown near the centre of the micrograph. x 600. Fig. 17. A section through the epidermis of an 8-5 cm opossum. Epon-812. Toluidine blue. x 500. Fig. 18. A section through the skin of the 10 cm opossum (60 days). Numerous developing hair follicles are shown. Haematoxylin and eosin. x 250. Fig. 19. The keratinizing layer and portions of the granular and spinous layers from the epidermis of a 12 cm opossum. Numerous desmosomes are shown between cells of the granulosa and spinosum (small arrows). What appear to be degenerate desmosomes (large arrows) are found between elem^nts of the keratinizing layer. x 20000.
W. J. KRAUSE, J. H. CUTTS AND C. R. LEESON
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Fig. 20. The epidermis from an adult opossum. Epon-812-Toluidine blue. x 500. Fig. 21. The epidermis of the adult opossum consists of a thin Malpighian layer and a desquamating cornified layer. A portion of a cell from the granulosa also is shown (arrows). x 7500.
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Postnatal development ofopossum epidermis
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Fig. 22. Development of the epidermis in the postnatal opossum. Thickness of -the epidermis is represented by the vertical bars. The thin horizontal line represents mitotic activity within the epidermis and the bold line mitoses associated with hair follicles.
of bacteria found on the surface of the epidermis up to the 18th day of pouch life. Such organisms presumably were the source of infection in the non-sterile wounds studied by Block (1960). The literature contains few studies dealing with skin transplantation in the opossum. LaPlante et al. (1966, 1969) have reported that maternal allografts to pouch young less than 12 days of age were accepted as permanent grafts, whereas 17 days old animals supported a viable maternal graft only for 80-93 days. Similar grafts placed on animals 3 months of age or older were regularly rejected within the time period observed for grafts between adult animals. Lymphoid organs are established in the opossum by the 5th-6th postnatal day, and pouch young opossums are able to form antibodies against S. typhosa at 8 days (LaVie, Rowlands & Block, 1963). Thus it would appear that immuno-competence as regards rejection of foreign tissue is attained at a later stage of development than is that component responsible for formation of circulating antibody. While the ultimate basis for acceptance or rejection of a graft undoubtedly resides in the immune response, other local factors may be implicated. It is interesting that permanent grafts can be established when the epidermis is actively increasing in thickness, whereas at 3 months grafts behave as if they were being placed in adult skin. Grafts made into animals 17 days of age, while persisting for some time, did not become permanent and were ultimately rejected. This is the time at which we have found the epidermis to reach its greatest thickness, after which it begins to thin. Thus alterations in nutritional support, in diffusion of antigen, and in the chalone content of the epidermis, need to be considered as factors affecting graft acceptance or rejection in the early stages of development of the opossum skin. SUMMARY
At birth the epidermis of the opossum is 43 ,um thick and consists of a basal layer of columnar cells, an intermediate layer of fusiform cells, a layer of incompletely cornified cells and a single surface layer of cells (the periderm). The latter shows central nuclei and distinct cell boundaries. Adjacent surface cells are contiguous and 7
W. J. KRAUSE, J. H. CUTTS AND C. R. LEESON
show extensive interdigitations of the lateral cell membranes. The periderm is lost during the first week of postnatal development. The epidermis attains its greatest thickness (58 ,sm) at the 4 0 cm stage (18 days postnatum), and this is due primarily to an increase in the thickness of the spinous layer. After this the epidermis thins to 14 1am in the adult. The epidermis of the adult consists of a thin Malphighian layer and a desquamating cornified layer. Hair follicles begin to differentiate at the 2'5 cm stage (7 days postnatum). They continue to differentiate and develop while the epidermis is increasing, and then decreasing, in thickness. The young are fully furred prior to the time they first venture from the protection of the pouch. REFERENCES ALLEN, T. D. & POTTEN, C. S. (1975). Desmosomal form, fate and function in mammalian epidermis. Journal of Ultrastructure Research 51, 94-105. ALLEN, T. D. & POTTEN, C. S. (1976). Ultrastructural site variations in mouse epidermal organization. Journal of Cell Science 21, 341-359. BLOCK, M. (1960). Wound healing in the newborn opossum (Didelphis virginiana). Nature 187, 340-359. BONNEVILLE, M. A. (1968). Observations on epidermal differentiation in the fetal rat. American Journal of Anatomy 123, 147-164. BREATHNACH, A. S. & WYLLIE, L. M. (1965). Fine structure of cells forming the surface layer of the epidermis in human fetuses at fourteen and twelve weeks. Journal of Investigative Dermatology 45, 179-189. CuTTs, J. H., LEESON, C. R. & KRAUSE, W. J. (1973). The postnatal development of the liver in a marsupial, Didelphis virginiana. I. Light microscopy. Journal of Anatomy 115, 327-346. HANSON, J. (1947). The histogenesis of the epidermis in the rat and mouse. Journal ofAnatomy 81, 174-197. HENRIKSON, R. C. (1969). Observations on the fine structure of the epidermis of an Australian possum (Trichosurus vulpecula). Journal of Anatomy 104, 409. HILL, J. P. & HILL, W. C. 0. (1955). The growth stages of the pouch young of the native cat (Dasyurus viverinus) together with observations on the anatomy of the newborn young. Transactions of the Zoological Society 28, 349-452. HOLBROOK, K. A. & ODLAND, G. F. (1975). The fine structure of developing human epidermis: light, scanning, and transmission electron microscopy of the periderm. Journal of Investigative Dermatology 65, 16-38. KRAUSE, W. J. & LEESON, C. R. (1973). The postnatal development of the respiratory system of the opossum. 1. Light and scanning electron microscopy. American Journal of Anatomy 137, 337-356. KRAUSE, W. J. & LEESON, C. R. (1975). Postnatal development of the respiratory system of the opossum. II. Electron microscopy of the epithelium and pleura. Acta anatomica 92, 28-44. KRAUSE, W. J., CuTmrs, J. H. & LEESON, C. R. (1975). The postnatal development of the liver in a marsupial, Didelphis virginiana. II. Electron microscopy. Journal of Anatomy 120, 191-205. KRAUSE, W. J., Cur1s, J. H. & LEESON, C. R. (1976). Type II pulmonary epithelial cells of the newborn opossum lung. Americal Journal of Anatomy 146, 181-188. KRAUSE, W. J., CUrrs, J. H. & LEESON, C. R. (1976a). The postnatal development of the alimentary canal in the opossum. I. Oesophagus. Journal of Anatomy 122, 293-314. KRAUSE, W. J., Currs, J. H. & LEESON, C. R. (1976b). The postnatal development of the alimentary canal in the opossum. II. Stomach. Journal of Anatomy 122, 499-519. KRAUSE, W. J., CUTTS, J. H. & LEESON, C. R. (1977). The postnatal development of the alimentary canal in the opossum. lll. Small intestine and colon. Journal of Anatomy 123, 21-45. LAPLANTE, E. S., BURRELL, R. G., WATNE, A. L. & ZIMMERMANN, B. (1966). Permanent survival of skin allografts applied to the pouch young of the opossum. Surgical Forum 17, 200-201. LAPLANTE, E. S., BURRELL, R. G., WATNE, A. L., TAYLOR, D. L. & ZIMMERMANN, B. (1969). Skin allograft studies in the pouch young of the opossum. Transplantation 7, 67-72. LAVIE, M. F., ROWLAND, D. T. & BLOCK, M. (1963). Antibody formation in embryos. Science 140, 1219-1220. LYNE, A. G. (1959). The systematic and adaptive significance of the vibrissae in the marsupialia. Proceedings of the Zoological Society of London 133, 79-1 33. LYNE, A. G. (1970). The development of hair follicles in the marsupial Trichosurus vulpecula. Australian Journal of Biological Sciences 23, 1241-1253. LYNE, A. G. & HEIDEMAN, M. J. (1959). The prenatal development of skin and hair in cattle (Bos taurus L.). Australian Journal ofBiological Sciences 12, 72-95. LYNE, A. G., HENRIKSON, R. C. & HOLLIS, D. E. (1970). Development of the epidermis of the marsupial Trichosurus vulpecula. Australian Journal ofBiological Sciences 23, 1067-1075.
Postnatal development ofopossum epidermis
LYNE, A. G. & HOLLIS, D. E. (1971). Ultrastructural changes in Merino sheep epidermis during foetal development. Journal of Anatomy 108, 211. MANN, S. L. (1968). The tylotrich (hair) follicle of the American opossum. Anatomical Record 160,171-180. MONTAGNA, W. & MACPHERSON, E. (1973). Similarities in cutaneous nerve receptors. Archives of Dermatology 107, 383-385. MUNGER, B. L. (1965). The intraepidermal innervation of the snout skin of the opossum. A light and electron microscopic study, with observations on the nature of Merkel's Tastzellen. Journal of Cell Biology 26, 79-98. REYNOLDS, E. S. (1963). The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. Journal of Cell Biology 17, 208-212. SNELL, R. S. (1965). The fate of epidermal desmosomes in mammalian skin. Zeitschrift fiur Zellforschung und mikroskopische Anatomie 66, 471-487.