Anatomy and Embryology

Three-dimensional (3-D) reconstruction from microscopic images represents a useful tool for the study of biological structures in embryology and developmental biology. However, it is usually necessary to cope with many difficulties connected with the preparation of specimens. In order to minimize mutual displacement of structures in successive sections, the applicability of non-deparaffinized tissue sections for 3-D reconstruction was tested. Chicken embryos were fixed and stained in toto with eosin and then embedded in paraffin. About 30-μm-thick non-deparaffinized serial sections were used for obtaining initial data for 3-D reconstruction of larger stacks of embryonic bodies using either fluorescence or confocal microscope. The same sections served for both collecting optical serial sections of mesonephros as source images for its 3-D reconstruction, and immunohistochemical detection of fibronectin, laminin and vimentin. It was found that sections with retained paraffin preserve the mutual spatial relationships of tissue components as well as provide an excellent differentiation of structure. It makes the process of 3-D reconstruction easier. The localization of the products of immunohistochemical reactions demonstrated the co-localization of fibronectin and laminin in basal laminas and the presence of vimentin in glomeruli and mesenchymal tissue. The use of non-deparaffinized sections represents a less time consuming and more effective alternative to thin histological sections for the purpose of 3-D reconstruction, and enables further application of material.

The articular capsules between the thoracic vertebrae, which have physiologically different functions from those of other levels of the vertebrae, have yet to be subjected to neuro-anatomical and fine structural analysis. In the present study, we analyzed serial frozen sections of decalcified thoracic vertebrae in human fetuses, and identified the articular capsule tissue with its unique distribution of elastic fibers. The fine structure of the elastic fibers was studied by transmission electron microscopy. In the early-stage fetus, the fibrous membrane forming the lateral intervertebral articular capsule contained abundant thin elastic fibers consisting of microfibrils. In the late-stage fetus, the lateral capsule of fibrous membrane was occupied by thick elastic fibers. A medial articular capsule, namely the ligamenta flava, contained numerous thick elastic fibers in both early and late-stage fetuses. The distributional differences in nerve fibers between early and late-stage fetuses were determined by immunostaining, using antibodies raised against protein gene product 9.5 (PGP 9.5; ubiquitin carboxyl-terminal hydrolase). Innervation by PGP 9.5 immunoreactive fibers was limited to the areas of the articular capsules near the blood vessels, which may indicate their functional relation with blood flow. No PGP 9.5 immunoreactive fibers were found in the ligamenta flava of the late-stage fetus. Innervation might be directly involved in the development of the intervertebral articular capsules in normal human fetuses.

Twenty-six embryos (6–11 mm) of stage 15 (approximately 33 days) were studied in detail and graphic reconstructions of three of them were prepared. Characteristic features of this stage include closed lens vesicles, presence of nasal pits, and retinal pigment. The neuromeric pattern is still visible. Each cerebral hemisphere is limited by the torus hemisphericus internally and by the di-telencephalic sulcus externally. The medial (diencephalic) eminence of the basal nuclei (previously misinterpreted by others as the lateral) had appeared in stage 14, and the lateral eminence, which is telencephalic, is now distinguishable. The amygdaloid body in stages 14 and 15 is derived from the medial eminence. The hippocampal thickening is identifiable in the dorsomedial part of the cerebral hemisphere. Medial and basal forebrain bundles are developing. The olfactory eminence is visible. Future olfactory bulb and tubercle possess an intermediate layer. The wall of the diencephalon presents five longitudinal zones: epithalamus, dorsal thalamus, ventral thalamus, subthalamus, and hypothalamus. The primordium of the epiphysis cerebri is beginning in the more advanced embryos. The sulcus limitans ends rostrally at the midbrain (M1) and is not continuous with the hypothalamic sulcus. Hence the alar/basal distinction does not arise in the forebrain. In the roof of the midbrain (M2) the mesencephalic evagination already noticed at stage 14 is characteristic. It is suggested that it may function as a temporary circumventricular organ. The precursors of some new tracts are identifiable: habenulo-interpeduncular, medial tectobulbar, and mamillotegmental fibres. Commissures include the supramamillary, that of the superior colliculi, and (in some embryos) the first fibres of the posterior commissure. Nuclei include the habenular, mamillary, and probably subthalamic. The cerebellum, the beginning of which was already noted at stages 13 and 14, consists of (1) a rostral part that arises from the alar plate of the isthmic segment and will form the superior medullary velum and part of the corpus cerebelli; and (2) a caudal part that develops from rhombomere 1. The involvement of the isthmic segment, first elucidated with stage 14, has not been observed in previous reports. All cranial nerves except the olfactory and optic are present in the more advanced embryos.

Die Mißbildungen der Extremitäten werden eingeteilt in regellose und systematische. Die regellosen Mißbildungen werden durch Faktoren außerhalb der Extremitätenanlage hervorgerufen, sie sind unpaar, nicht erbbedingt. Die systematischen Mißbildungen sind an bestimmte Territorien gebunden, sind vielfach paarig, stets erbbedingt. An oberer und unterer Extremität sind vier Mißbildungsterritorien zu unterscheiden, je zwei an Unterarm und Unterschenkel, je zwei an Hand und Fuß. Die Grenze zwischen den beiden Hand- und Fußterritorien entspricht einer Ursegmentgrenze. Systematische territoriale Abweichungen, welche dem Anschein nach vereinzelt vorkommen, sind trotzdem erbbedingt.

 We have used carbocyanine dye tracing techniques in conjunction with photoconversion and electronmicroscopy to examine the prenatal development of the central and peripheral processes of those vestibular ganglion cells projecting to the cerebellum. Developmental changes in the number of vestibular ganglion cells were assessed in paraffin-embedded material by nucleolar counting. In agreement with the results of parvalbumin staining, afferents to the cerebellum from the vestibular ganglion pursued a superficial course during early fetal life (E13 to E15). From E16 to E19, this superficial position was progressively lost and vestibulocerebellar fibres were seen to be directed towards the ventricular surface (prospective posterior/inferior vermis). The change in the course of vestibular afferents to the cerebellum coincided with a profound reduction in the number of ganglion cells which could be retrogradely labelled from the cerebellar anlage (mean ±SD: E16–2040±1130; E19–510±440). During that same period the total number of vestibular ganglion cells rose to peak at a mean of 9200 at E19, although there was a subsequent decline to an average of 4660 at P0. This population size was maintained through to adult life (4600). We also examined the development of connections between the vestibular gangion and the vestibular apparatus. Peripheral processes of vestibular ganglion cells invaded the macula utricle and saccule and cristae of the semicircular canals from E13. We found that the peripheral vestibular ganglion cell processes themselves did not show any significant morphological changes from E16 to E21, but the sensory epithelium itself adopts a mature pseudostratified appearance by E21. This suggests that the loss of vestibular ganglion cells from E19 to birth is not related to major morphological changes in the peripheral axons, at least as revealed by carbocyanine dye labelling of these from the cerebellum, but may be associated with differentiation of the sensory epithelium to the mature pseudostratified form. Electronmicroscopy of photoconverted vestibulocerebellar fibres showed that at E14 these afferents were grouped in tight bundles of up to 20 axons. No particular association with the superficially placed external granular layer cells was found at that age. By E16 photoconverted vestibulocerebellar axons were no longer as tightly bundled and could be seen coursing more ventrally through the cerebellar anlage. The findings indicate that vestibulocerebellar fibres are not likely to physically facilitate external granular layer migration, since they do not attain a particularly close structural association with those cells. The observed developmental changes in the number of vestibular ganglion cells projecting to the cerebellum and the total number of vestibular ganglion cells suggests that changes in the course of vestibulocerebellar fibres are associated at first with retraction of cerebellar afferents, and subsequently with developmental cell death in the ganglion.

The development of stereociliary attachment to the tectorial membrane was investigated in the mouse cochlea using transmission and scanning electron microscopy. At the 18th gestational day, only the major tectorial membrane can be identified covering the greater epithelial ridge and the inner hair cells in all turns. At the 19th gestational day, the minor tectorial membrane was first seen in the basal turn, over the outer hair cells. During early stages of development, the stereocilia of hair cells were surrounded by a loose fibrillar material underneath the tectorial membrane. After the 10th postnatal day, the outer hair cells' stereocilia were attached to Kimura's (or Hardesty's) membrane, while inner hair cells' stereociliary bundles were attached to the undersurface of the tectorial membrane near the Hensen's stripe. Between the 10th and the 14th postnatal days, the space between the inner hair cells and the first row of outer hair cells widened by virtue of the growth of the heads of pillar cells, and the inner hair cells' stereocilia were displaced towards the Hensen's stripe. After the 14th postnatal day, the inner hair cells' stereociliary bundles detached from the tectorial membrane, while the outer hair cells' stereocilia remained attached to it. The tip-link system, which connects the tips of the stereocilia to the next tallest stereocilia, is present at birth in the outer hair cells. The marginal pillar, that anchored the tectorial membrane to the underlying organ of Corti during development, first appeared on the 6th postnatal day and disappeared on the 14th–15th postnatal day. The present data together with other reports support the idea that although some structures, such as hair cells' stereocilia and innervation, are already formed early during development, the cochlear microarchitecture is not fully developed morphologically and ready to function normally until the end of the second postnatal week in the mouse.