Diastematomyelia. (doubling of the tube) was found in several animals. The process of cavitation was completed by the joining of severall small, focal cavities.
Arch. Histol. Cytol., Vol. 52, No. 2 (1989) p. 95-108
The
Morphogenesis
of the
Tail
in Monodeiphis
domes
Kenton
J. ZEHR,
Department U.S.A. Received
Bryce
of Anatomy,
October
L. MUNGER
The Milton
S. Hershey
and
Posterior
Tube
and
ticus
Terrell
Medical
Neural
E. JONES
Center,
The Pennsylvania
State
University,
Hershey,
Pennsylvania,
25, 1988
Summary. The process of secondary neuralation has been studied in the Brazilian opossum, Monodelphis domesticus. Secondary neuralation in this mammal was found to have qualities of secondary neuralation that were present in both the chick and the mouse. In this study, four stages of secondary neuralation were found beginning with the medullary cord stage. Other stages in the differentiation of the secondary neural tube were: differentiation of the neuroepithelium, cavitation of the medullary cord, and proliferation of the neural tube. The process of secondary neuralation proceeded in a rostral-to-caudal direction and was found to be independent of age. Diastematomyelia (doubling of the tube) was found in several animals. The process of cavitation was completed by the joining of severall small, focal cavities in a rostral-to-caudal direction. The most distinctive feature of secondary neuralation in this animal was the finding of axons within the secondary neural tube, a feature not characteristic of either the chick or the mouse.
development. The neural tube in rostral regions of the body forms by closure and fusion of the neural folds (for review see KARFUNKEL,1974), while the caudal neural tube forms by a process involving removal of cells from a solid core of mesenchymal cells (for review of chicken, see CRILEY, 1969; SCHOENWOLF, 1979; and for mouse SCHOENWOLF,1984). The process of hollowing of a solid cord of cells can be found in earlier, less comprehensive reviews by BRAUN (1882) who described canalization of a "medullarstrang" or sold cord in sheep and mice, and by STREETER (1919) and KERNOHAN(1925) who described the development of the filum terminale and the "ventriculus terminalis" in human embryos. Secondary neuralation in the chick embryo consists of four definable stages (CRILEY, 1969; SCHOENWOLF and DELONGO,1980) :1) segregation of medullary cord cells; 2) formation of a medullary cord; 3) cavitation of the mesodermal cord; and 4) coalescence of multiple lumina. Only two stages have been recognized in the mouse :1) formation of a medullary rosette and 2) cavitation (SCHOENWOLF,1984). Secondary neuralation in the opossum differed from both the mouse and chick, possessing features of both. Our interest in the process of neuralation of the Brazilian opossum, Monodelphis domesticus, was based on ongoing studies on the development of the peripheral sensory nervous system (JONES and MUNGER, 1985) where precocious development can be documented in the cranial part of the newborn pup. The hind quarters, on the contrary, are not functional at birth and the process of secondary neuralation occurs predominately after birth. The objectives of the present study were to provide a detailed description of posterior neural tube development in the short-tailed opossum, and to correlate development of the adjacent sclerotomes and myotomes with the secondary neural tube.
The formation of the mammalian neural tube involves two entirely different morphogenetic processes referred to as primary and secondary neuralation. Primary neuralation is the process of forming a hollow neural tube by closure of the neural folds. while secondary neuralation is the process of forming a neural tube by hollowing a solid cord of mesodermal cells. The two portions of the neural tube formed by these processes differ in relative position and have been designated by CRILEY (1969) as the anterior neural tube formed by neural fold fusion and the posterior neural tube as that portion formed by cavitation of mesoderm. HOLMDAHL(1925a, b) was among the first to recognize two different morphogenetic events in the formation of the neural tube, and suggested that these two processes were a general principle of vertebrate 95
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MATERIALS
AND
METHODS
Monodelphis domesticus females were bred according to the recommendations outlined by FADEM et al. (1982). and FADEM(1985) used successfully in our laboratory (JONES and MUNGER, 1985). Ten days following the initial contact between the male and female, the male was removed from the cage. Beginning on the 14th day following mating, potentially pregnant females were checked every 6-8h until birth; birth was designated day 0. Postnatal pups were collected beginning at day 0. A total of 24 animals constituted the base line material for normal development. For electron microscopy, we concentrated on pups of 0 and 1 day (10 specimens), 3 and 5 days (2 specimens each) and 8 days (2 specimens). Following removal of the pups from the mothers, animals were anesthetized with Nembutal (50mg/ml) intraperitoneal injections of 0.05ml. The trunk was fixed by immersion for both light and electron microscopy in 10% neutral buffered f ormalin for the former and in Karnovsky's fixative (KARNOVSKY,1965) for the latter. Electron microscopy: Following rinsing, the tissue was post-fixed in 2.0% osmium tetroxide. Standard dehydration and embedding procedures were followed using Durcupan (Fluka AG, Chemische Fabrik, 9470 Buchs, Buchs SG/Switzerland) as the embedding medium. As orientation was a critical step, the spinal cord was cut perpendicular to the long axis. Relative position within the spinal cord was achieved by use of 1.0um semithin sections beginning at the rostral end of the tail. The tail was divided into proximal, middle and distal segments. This sections of appropriate areas were cut at 60-90nm and stained with lead citrate and uranyl acetate. Light microscopy: For light microscopy, the pelvis and attached tail was fixed by immersion in 10% neutral buffered formalin, and paraffin embedded. Serial sections through the entire tail were cut at 8um and stained with the silver procedure of SEVIER and MUNGER (1965).
RESULTS
A wide range of developmental stages were observed in the secondary neural tube in the ages studied; the proximal and distal tail were at different developmental stages. In this study, we recognized four stages of development, similar, but also slightly different from those described in chick and mouse (SCHOENWOLF, 1979, 1984). We have been able to confirm the finding that the stage of development was independent of age; i.e., the major events of secondary neuralation progressed rapidly with distinctly different stages sometimes found in different parts of the same animal. The development of the secondary neural tube in the Brazilian opossum was best divided into four stages:1) medullary cord, 2) differentiation of neuroepithelium, 3) cavitation and 4) proliferation of the neural tube. Several of these events were common to both the chick and the mouse and will be treated in detail in the discussion. The stage of development found in animals of comparable ages was not consistent. Of the ten 0-1 day specimens investigated, one was in the early medullary cord stage prior to cavitation, two were undergoing cavitation in the distal two-thirds of the tail, and seven were in the proliferative stage. In the latter case, occasionally the distal neural tube was still undergoing cavitation, based on the analysis of serial sectioning.
Medullary
cord
stage
The secondary neural tube was present throughout the entire length of the tail dorsal to the notochord in postnatal opossum pups. The medullary cord was recognized in semithin sections as a randomly arranged cluster of cells with no apparent polarity. Examples of medullary cords were most frequent in 0 and 1 day animals or in the distal segments of slightly older animals (Fig. 1 A, B). The medullary cord was well delineated from the surrounding mesenchyme even though a complete basal lamina was not present. Cells throughout the solid mass were irregular in shape with numerous small cytoplasmic processes. Definitive axons could not be recognized by either
Fig. 1 A and B. Medullary cord stage. Cells forming the medullary cord were unremarkable and could not be identified as future neuroblasts. Medullary cord cells were well delineated from the surrounding mesenchyme but were not found to have a continuous basal lamina. A. A semithin section through the proximal one-third of the tail of a neonate. The box in A represents the area depicted in B. NT notocord, MC medullary cord, astrick adjacent mesenchymal cells.
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B
A Fig.
1.
Legend
on the
opposite
page.
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light or electron microscopy. Many of the cells had a foamy appearance due to their content of vacuoles. The nuclei were spherical in shape, ranging from large nuclei (average 5.5um, n=30, from 7 micrographs), with a tendency to stain lightly, to small nuclei (average 2.7um, n=30, from 12 micrographs) with a tendency toward dark staining. Neither pyknotic nuclei nor signs of degeneration in the cell parenchyma were apparent in the medullary cord stage. Mitotic figures were not observed. Some intercellular spaces were present throughout the cord, but intercellular junctions were not observed in our preparations prior to the beginning of differentiation of the neural tube. At postnatal days 0-1, lateral to the medullary cord, two principal coccygeal nerve trunks (as identified in other tailed animals such as the dog, see MILLER et al., 1965) extended caudally from the lumbosacral plexus into the proximal tail. Each trunk split in the proximal tail forming dorsal and ventral coccygeal nerve bundles cupped by the four segmented myotomes. The distal half of the tail contained only the ventral bundles and a single lateral myotome. Neither myotome nor skin were innervated at this stage. The nerve bundles contained few presumptive Schwann cells. Differentiation
of
neuroepithelium
Differentiation of the neuroepithelium occurred in a circumferential manner beginning at the lateral aspects of the secondary neural tube (Fig. 2a, b). Differentiation was in a dorsal and ventral direction (Fig. 2A, arrows). Differentiating cells from each side made contact at the ventral border before the dorsal border. Differentiation of the neuroepithelium also proceeded in a rostral-to-caudal direction as determined by serial sections of the tail. The lateral differentiating neuroblast cells were distinctly separate from the central cell mass of undifferentiated cells (Fig. 2B, arrows). Cells forming the neuroepithelium were initially oriented circumferentially in a single cell layer. In regions where the neuroepithelium was two or more cells thick, the cells were
radially oriented (Fig. 3) with respect to the longitudinal axis of the secondary neural tube. The differentiating neuroblast cells were homogeneous in their staining properties with elongated nuclei, but the central population of cells maintained the heterogeneous appearance similar to that depicted in Figure lb. Dense junctional complexes were found joining all adjacent neuroepithelial cells at their apical border (Fig. 3, arrows) with small intercellular junctions occasionally observed between adjacent neuroblasts at the basal border. Although mitochondria were found at both the basal and apical poles of neuroblasts, the greatest concentration was at the apical border. A well defined and continuous basal lamina was always present between the developing neuroepithelium and the surrounding connective tissue. In contrast, a well developed basal lamina was not found in regions were medullary cord cells apposed the surrounding connective tissue. Diastematomyelia (doubling of the neural tube) was noted in several neonates. Diastematomyelia was seen during differentiation of the neuroepithelium, and the "accessory" neural tube contained medullary cord cells (Fig. 4A, B). In these specimens, groups of undifferentiated cells appeared to bulge from the differentiating secondary neural tube. Cells of the accessory tube were partially separated from the secondary neural tube by the secondary neural tube basal lamina and fibroblasts. Cells of the accessory tube were never surrounded by a basal lamina (Fig. 5). Diastematomyelia was not confined to or preferentially found in rostral or caudal portions of the developing neural tube.
Cavitation
The transition from the neuroepithelial stage to the formation of a lumen involved clearing of the residual central medullary cord cells. In regions with a partially occluded lumen (Fig. 6A, B), many pyknotic nuclei were apparent in the central cell population. The central population of cells appeared to be undergoing autolysis. The lumen was filled with vacuoles and disorganized cellular debris. Some neurepithelial
Fig. 2 A and B. Differentiation of neuroepithelium. B represents the the semithin pictured in A. B was taken from the area contained neuroepithelial cells can be seen in the lower, right-hand corner of the B. (N). Formation of the neuroepithelial cells was in a circumferential of the cord. The arrows in A. illustrate the direction of differentiation the seperation of the residual medullary cord cells and the developing
consecutive thin section taken following within the box of A. Differentiating area within the box of A (astrick) and in manner beginning at the lateral margin of these cells. The arrows in B depict neuroepithelial cells.
Morphogenesis
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99
A
B Fig.
2.
Legend
on the
opposite
page.
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Fig. 3. The cells of the neuroepithelium were easily distinguished from the cells of the medullary cord. Neuroepithelial cells were joined by cellular junctions at both the apical (arrows) and basal border. At the time of neuroepithelial differentiation, cavitation had not begun and resuidual medullary cord cells remained. M medullary cord cells, N neuroepithelial cells.
Morphogenesis
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Tube
101
B
A Fig. 4. Diastematomyelia approximately the same during
neuroepithelium
during Figure
the cavitation 5.
was found in several level within the proximal development,
stage
animals. one-third
and did not persist.
of secondary
neuralation.
A and B were taken of the tail. Doubling
In fact, The
cells contained vacuoles at the luminal border along with numerous mitochondria. An occasional band of microfilaments was located at the apices of the neuroblast in cavitating regions. Neuroepithelial cells had filopodia extending into the incipient lumen. Prior to this stage, the cells were without cytoplasmic extensions. Cavitation in the posterior neural tube proceeded in a rostral-to-caudal direction, and multiple cavitation at the level of primary and secondary neuralation overlap, a prominent feature in the chick, was not observed. However, cavitating areas were observed caudal to areas of solid neuroepithelial and medullary cord cells, suggesting that cavitation in this opossum was a focal event occurring along the rostral-to-caudal axis of the tube. The joining of these focal cavities effectively hollowed the posterior neural tube in a rostral-to-caudal direction.
area
no example contained
Proliferation hypertrophy
from of the
of spinal within
of the
the
cord
different cord was doubling
animals at found only was
box of B is illustrated
neural
tube
found in
by hyperplasia
and
Concurrent with the end of cavitation and extending to postnatal day 3, a proliferation of neuroblasts and masisive growth of the secondary neural tube was observed. Many neuroblasts were seen in metaphase adjacent to the forming lumen. Just before the time of maximum hypertrophy and hyperplasia of the secondary neural tube, axonal processes were seen penetrating the lateral portions of the developing neural tube in rostral-to-caudal directions observed initially by light microscopy and verified by electron microscopy (Fig. 7). Axonal processes within the posterior neural tube were first observed at the level of the last sacral dorsal root ganglia. Initially, they were seen in bundles of up to ten axons. Additionally, large axons extended ventrally to form the anterior commissure. At postnatal day 3, numerous axons displaced the neuroblasts at the lateral and ventral aspects of the proliferating neural tube, occupying the outer one-fourth of the neural tube. The central lumen was completely obliterated by the apposition
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Fig. 5. Diastematomyelia was found to involve only medullary cord cells. These cells were similar to the cells seen in the original medullary cord. Developing neuroepithelial cells can be seen in the adjacent secondary neural tube. N neuroepithelial cells, D medullary cord cells within the doubled neural tube.
Morphogenesis
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Tube
103
B
A Fig. 6 A and B. A fortuitous section through the secondary neural tube. The lumen is partially degenerating cells from the original medullary cord. In this section, both the dorsal and ventral been formed by the joining of neuroepithelial cells from opposite sides.
occluded by bouder have
104
Fig.
K. J. ZEHR, B. L. MUNGER and T. E. JONES:
7.
Proliferation
tube on postnatal axons is unknown.
of the neural
tube.
Axons
were
found
in the lateral
day 2 and 3. The axons increased in number a Axons, N neuroepithelial cells.
over
margins
the next
of the developing
few days.
The
function
secondary of these
Morphogenesis
of Opossum
Neural
Tube
105
neuroepithelial (Fig. 8). The neural tube thus closed as documented in the chick by DESMOND and SCHOENWOLF(1985) and DESMOND (1986). As serial paraffin sections were followed caudally, the number of axonal profiles within the posterior neural tube decreased. The anterior commissure was not present below the midpoint of the tail, and the cross sectional area occupied by axons in the lateral margins was diminished by approximately 50%. By day 3, myotomes were differentiated into individual muscle groups containing numerous myofibrils and myotubules. Growth cones pierced the muscle fascicles. The sclerotome consisted of a cartilage matrix surrounding the secondary neural tube and the notochord. Developing vertebrae had well delineated segments corresponding to future vertebral bodies (Fig. 8).
DISCUSSION
The present study documents a unique mammalian model of secondary neuralation as well as the segmental innervation and differentiation of both neural and mesodermal components of the tail. Caudal regions of the nervous system in Monodelphis domesticus were extremely immature in the neonate. At birth, the stage of differentiation of the secondary neural tube can be correlated with a Hamburger-Hamilton (HAMBURGERand HAMILTON,1951) stage 18-19 chick. In the chick, secondary neuralation and closure of the caudal neuropore occur concomitantly and begin at approximately stage 14-16 (SCHOENWOLF et al., 1985). In the mouse, secondary neuralation and closure of the posterior neuropore begin on gestational day 9.510.0 with formation of a medullary plate and cavitation on day 11.0-12.0 (SCHOENWOLF,1984). In this Brazillian opossum, the medullary cord and the beginning of cavitation can be seen in the neonate. Therefore, if secondary neuralation is used as a yardstick for equating the neonate Monodelphis domesticus with other animals in which secondary neuralation has been studied, this opossum is developmentally equivalent to a day 11.0-12.0 mouse. In terms of somites, the 11.0-12.0 day mouse has 45 somites (SCHNEIDERand NORTON, 1979), the equivalent days for the chick would be 3.5-4.0 days (HAM BURGERand HAMILTON, 1951; PATTEN, 1951), and for the rat 13.5 days (SCHNEIDERand NORTON, 1979). In the opossum, by counting the number of vertebral bodies and the number of identifiable somites in the tail, the neonate opossum has 42-45 somites. However, equating different animals based on single
Fig. 8. Complete occlusion of the lumin was accomplished by apposition of the neuroepithelial cells. Axons can be seen in the lateral areas of the developing tube (arrows). Apposition of the neuroepithelial cells occurred only after complete cavitation of the medullary cord. V vertebral body.
developmental events or processes must be guarded, even though it seems to be the best procedure to date. For example, if another yardstick were used and development of the facial region, particularly development of the vibrissae, were compared for the mouse, rat and opossum, the neonate opossum would be equivalent to a day 13.5 mouse or a 14.5-15.0 day rat (JONES and MUNGER, 1986). A possible factor contributing to the apparent discrepancy in timing may be found in differences in time between fertilization of the ovum and the initiation of development (implantation, blastula formation). These times have been noted for the rat and mouse (SCHNEIDER and NORTON, 1979) but not for the opossum. The earliest stage of differentiation of the secondary neural tube observed in the neonate opossum was the medullary cord stage identified by the presence of undifferentiated mesodermal cells similar to that described in the chick (CRILEY, 1969; JELINEK et al., 1969; KLIKA and JELINEK, 1969; SCHOENWOLF, 1979; SCHOENWOLFand DELONGO, 1980) and in the
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mouse (SCHOENWOLF,1979). In neonatal opossums, where the beginning of differentiation of the medullary cord was taking place at distal regions of the tail, it was possible to observe different steps of the neuralation process up to cavitation within the same embryo in serial paraffin sections. The process of neuralation progresses very rapidly following the formation of the medullary cord. In fact, in some of our specimens, different stages of development were seen in different regions of the tail; i.e., the beginning of formation of neuroepithelial cells in rostral regions; whereas, medullary cord cells were still present in more caudal regions. It is welml known that neuralation proceeds in a craniocaudal direction in the chick and mouse, and we were able to confirm this directional differentiation in the opossum. In the present study, we did not find the characteristic overlap in the transition between primary and secondary neural tube which has been described in the chick (SCHOENWOLFand DELONGO,1980). In this regard, this marsupial shared features found in the mouse; e.g., both demonstrate a continuous neural tube with no distinctive overlap zone between primary and secondary neural tubes. The process of cavitation in Monodelphis domesticus was by a rostral-to-caudal coalescence of focal cavities which have formed following the development of neuroepithelial cells. A distinct basal lamina was present only in association with neuroepithelial cells. The formation of the neuroepithelial cells began at the lateral margins of the medullary cord and joined those of the opposite side at the ventral border of the tube first. Figure 2B clearly shows the preference for closure of the ventral border before the dorsal. In secondary neural tubes that had both neuroepithelial and undifferentiated cells, a continuous basal lamina was found only subjacent to neuroepithelial cells. This would indicate that the neuroepithelial cells were capable of contributing to and supporting a basal lamina, but the undifferentiated cells of the medullary cord were not. A distinct basal lamina was absent in the medullary cord stage. Intercellular junctions have been described at the apical aspect of neuroepithelial cells in the secondary neural tube of the chick (CRILEY, 1969; SCHOENWOLF, 1979; SCHOENWOLFand DELONGO,1980; in particular see SCHOENWOLF and KELLEY, 1980) and mouse (SCHOENWOLF,1984). In the chick, these junctions were recessed from the apex of the cell. Intercellular junctions observed in Monodelphis domesticus were located at the juxtaluminal contact between adjacent cells and contained central densities. The exact
nature of the junctions was not determined as gap junctions could be distinguished from tight junctions best by freeze-fracture. However, it is noteworthy that in the mouse all junctions apposing the lumen in the secondary neural tube were found to be of the gap variety (SCHOENWOLFand KELLEY, 1980). SCHOENWOLFand DELONGO (1980) questioned the fate of the central cells masses in chick embryos. They postulated that the cells were removed by one of two processes; elongation and lateral migration into the peripheral cell population, thus becoming part of the developing neural tube proper; or caudal migration away from areas undergoing cavitation, whereby they undergo degeneration and necrosis. Their observations favored the first process since local evidence of cell death and necrosis was not found. Other literature (CRILEY, 1969; KLIKA and JELINEK, 1969; SCHOENWOLF,1978) did not observe localized pyknosis or necrosis in areas undergoing cavitation. The present study is not in agreement with the above chick data. This study consistently demonstrated degenerating and necrotic cells in the incipient central cavity of the medullary cord (Fig. 6). Further, the degenerating cells were found in areas undergoing cavitation and in particular in areas between focal cavities. In the mouse, cellular debris was evident (compare Figs. 22, 24 and 25 in SCHOENWOLF,1984 with Fig. 6 in this paper). LEMIRE (1969) and HUGHES and FREEMAN (1974) did note degenerative processes occurring in human embryos. In this regard, Monodelphis domesticus would seem to be similar to the mouse or human. We were unable to find evidence for significant incorporation of the central cells into the developing neuroepithelium. Apical junctional complexes were consistently observed between adjacent neuroblasts from the beginning of differentiation to the end of cavitation. Because of the presence of these dense junctions and evidence of central degeneration, the present authors feel that large scale lateral migration of central cells into the developing neural tube did not occur in the opossum. It appears that the primary mechanism for the removal of the central cell mass is by degeneration in this Brazilian opossum. It should also be noted that in an argument presented by SCHOENWOLF and KELLEY (1980) in the chick, the presence of annular junctions would be expected if lateral migration of central cells were involved in the mechanism of cavitation since these junctions have been considered to be the degradative form of gap junctions (LARSEN, 1977). None were seen (SCHOENWOLF and KELLEY, 1980). Early investigators (STREETER, 1919; KERNOHAN,
Morphogenesis
1925) found duplication of the caudal tube in developing embryos, but they were not sure if the condition was normal or pathological. LEMIRE (1969), HUGHES and FREEMAN (1974), and DRYDEN (1980a, b) concluded that the frequency of observed diastematomyelia in secondary neuralation indicated that it was a normal variation in development, but could predispose the embryo to posterior neural tube defects. In the present study, the group of cells that were displaced from the developing neural tube were not surrounded by basal lamina and did not show evidence that this initial structural deformation would develop into a differentiated duplicate cord. The aberrant cells of the diastematomyelia did not develop further and showed some signs of degeneration. The potential significance of these cells could not be determined. The presence of axons within the secondary neural tube even extending into the distal tail was a totally unexpected finding. Previous studies on chick and mouse have not reported the presence of such descending axons within the secondary neural tube. The major source of axons (motor and sensory) to all tail spinal segments were the paired lateral dorsal and ventral coccygeal nerves (as identified in the dog, MILLER et al., 1964). The tail lacks dorsal root ganglia, and axons within the secondary neural tube would thus presumptively be motor in nature. The function and ultimate fate of these nerves is currently under study. However, the presence of axons within the tail secondary neural tube mimics the events of differentiation within the primary neural tube and thus strengthens the argument that the peripheral cells within the secondary neural tube are in fact developing neuroblasts. REFERENCES BRAUN, M.: Entwickelungsvorgange am Schwanzend bei einigen Saugethieren mit Berucksichtigung der Verhaltnisse beim Menschen. Arch. Anat. Physiol., Anat. Abt. 6: 207-241 (1882). CRILEY, B. B.: Analysis of the embryonic sources and mechanisms of development of posterior levels of chick neural tubes. J. Morphol. 72: 465-501 (1969). DESMOND, M. E.: Evaluation of the roles of intrinsic and extrinsic factors in occlusion of the spinal neurocoele during rapid brain enlargement in the chick embryo. J Embryol. Exp. Morphol. 97: 25-46 (1986). DESMOND, M. E. and G. C. SCHOENWOLF: Timing and positioning of occlusion of the spinal neurocele in the chick embryo. J. Comp. Neurol. 235: 479-487 (1985). DRYDEN, R. J.: Spinal bifida in chick embryos: ultrastructure of open neural defects in the transitional region between primary and secondary modes of neural
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Tube
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tube formation. In: (ed. by) T. V. N. PERSAUD: Advances in the study of birth defects. Vol. 4. MTP Press Limited, Lancaster, 1980a (p. 75-100). -: Duplication of the spinal cord: a discussion of the possible embryogenesis of diplomyelia. Devel. Med. Child Neurol. 22: 234-243 (1980b). FADEM, B. H.: Evidence for the activation of female reproduction by males in a marsupial, the gray shorttailed opossum (Monodelphis domestica). Biol. Reprod. 33: 112-116 (1985). FADEM, B. H., G. L. TURPIN, E. MALINIAK, J. L. VAN DE BERG and V. HAYSSEN: Care and breeding of the gray, short-tailed opossum (Monodelphis domestics). Lab. Anim. Sci. 32: 405-409 (1982). HAMBURGER, V. and H. L. HAMILTON: A series of normal stages in the development of the chick embryo. J. Morphol. 88: 49-92 (1951). HOLMDAHL,D. E.: Die erste Entwicklung des Korpers bei den Vogeln and Saugetieren, inkl. dem Menschen, besonders mit Ri cksicht auf die Bildung des Ri ckenmarks, des Zoloms and der entodermalen Kloake, nebst einem Exkurs fiber die Entstehung der Spina bifida in der Lumbosakralregionen. I; II-V. Gegenbaurs Morphol. Jahrb. 54: 333-384; 55: 112-208 (1925a, b). HUGHES, A. F. and R. B. FREEMAN: Comparative remarks on the development of the tail cord among higher vertebrates. J. Embryol. Exp. Morphol. 32: 355363 (1974). JELINEK, R., V. SEICHERT and E. KLIKA: Mechanism of morphogenesis of caudal neural tube in the chick embryo. Fol. Morphol. (Praha) 17: 355-367 (1969). JONES, T. E. and B. L. MUNGER: Early differentiation of the afferent nervous system in glabrous snout skin of the opossum (Monodelphis domesticus). Somatosens. Res. 3: 169-184 (1985). -: Comparative sequential maturation of vibrissae in the mouse, rat, and opossum. Soc. Neurosci. Abstr. 12(1): 126 (1986). KARFUNKEL, P.: The mechanisms of neural tube formation. Int. Rev. Cytol. 38: 245-271 (1974). KARNOVSKY, M. J.: A formaldehyde-glutaraldehyde fixative of high osmolarity for use in electron microscopy. J. Cell Biol. 27: 137A (1965). KERNOHAN, J. W.: The ventriculus terminals: it's growth and development. J. Comp. Neurol. 38: 107-125 (1925). KLIKA, E. and R. JELINEK: The sturcture of the end and tail bud of the chick embryo. Fol. Morphol. (Praha) 17: 29-40 (1969). LARSEN, W. J.: Structural diversity of gap junctions. A review. Tiss. Cell 9: 373-394 (1977). LEMIRE, R. J.: Variations in development of the caudal neural tube in human embryos (Horizons XIV-XXI). Teratology 2: 361-370 (1969). MILLER, M. E., G. C. CHRISTENSEN and H. E. EVANS: Anatomy of the dog. W. B. Saunders Co., Philadelphia, 1965. PATTEN, B. M.: Early embryology of the chick. 4th ed. McGraw-Hill Book Co., Inc., New York, 1951.
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SCHNEIDER, B. F. and S. NORTON : Equivalent ages in rat, mouse and chick embryos. Teratology 19: 273-278 (1979). SCHOENWOLF, G. C.: An SEM study of posterior spinal cord development in the chick embryo. In: (ed. by) R. P. BECKER and O. JOHARI: Scanning Electron Microscopy/ 1978/II. SEM Inc., AMF O'Hare, 1978(p. 739-746) -: Observations on closure of the neuropores in the chick embryo. Amer. J. Anat. 155: 445-465 (1979). -: Histological and ultrastructural studies of secondary neuralation in mouse embryos. Amer. J. Anat. 169: 361-376 (1984). SCHOENWOLF, G. C. and J. DELONGO: Ultrastructure of secondary neuralation in the chick embryo. Amer. J. Anat. 158: 43-63 (1980). SCHOENWOLF,G. C. and R. D. KELLEY: Characterization of intercellular junctions in the caudal portion of the developing neural tube of the chick embryo. Amer. J. Anta. 158: 29-41 (1980).
SCHOENWOLF, G. C., N. B. CHANDLER and J. L. SMITH: Analysis of the origins and early fates of neural crest cells in caudal regions of avian embryos. Devel. Biol. 110: 467-479 (1985). SEVIER, A. C. and B. L. MUNGER: A technical note. A silver method for paraffin sections of neural tissue. J. Neuropathol. Exp. Neurol. 24: 130-135 (1965). STREETER, G. L.: Factors involved in the formation of the filum terminale. Amer. J. Anat. 25: 1-11 (1919).
Dr. Terrell E. JONES Department of Anatomy Milton S. Hershey Medical Center The Pennsylvania State University P. O. Box 805 Hershey, Pennsylvania 17033 U.S.A.
