Apoptosis and proliferation in the trigeminal placode.

The neurogenic trigeminal placode develops from the crescent-shaped panplacodal primordium which delineates the neural plate anteriorly. We show that, in Tupaia belangeri, the trigeminal placode is represented by a field of focal ectodermal thickenings which over time changes positions from as far rostral as the level of the forebrain to as far caudal as opposite rhombomere 3. Delamination proceeds rostrocaudally from the ectoderm adjacent to the rostral midbrain, and contributes neurons to the trigeminal ganglion as well as to the ciliary ganglion/oculomotor complex. Proliferative events are centered on the field prior to the peak of delamination. They are preceded, paralleled and, finally, outnumbered by apoptotic events which proceed rostrocaudally from non-delaminating to delaminating parts of the field. Apoptosis persists upon regression of the placode, thereby exhibiting a massive "wedge" of apoptotic cells which includes the postulated position of the "ventrolateral postoptic placode" (Lee et al. in Dev Biol 263:176-190, 2003), merges with groups of lens-associated apoptotic cells, and disappears upon lens detachment. In conjunction with earlier work (Washausen et al. in Dev Biol 278:86-102, 2005) our findings suggest that apoptosis contributes repeatedly to the disintegration of the panplacodal primordium, to the elimination of subsets of premigratory placodal neuroblasts, and to the regression of placodes.

Diversity in mammalian chiasmatic architecture: ipsilateral axons are deflected at glial arches in the prechiasmatic optic nerve of the eutherian Tupaia belangeri.

Permanent ipsilaterally projecting axons approach the chiasmatic midline in rodents but are confined to lateral parts of the optic chiasm in marsupials. Hence, principally different mechanisms were thought to underlie axon pathway choice in eutherian (placental) and marsupial mammals. First evidence of diversity in eutherian chiasmatic architecture came from studies in the newborn and adult tree shrew Tupaia belangeri (Jeffery et al. [1998] J. Comp. Neurol. 390:183-193). Here, as in marsupials, ipsilaterally projecting axons do not approach the midline. The present study aims to clarify how the developing tree shrew chiasm is organized, how glial cells are arranged therein, and the extent to which the tree shrew chiasm is similar to that of marsupials or other eutherians. By using routinely stained serial sections as well as immunohistochemistry with antibodies against glial fibrillary acidic protein, vimentin, and medium-molecular-weight neurofilament protein, we investigated chiasm formation from embryonic day 18 (E18) to birth (E43). From E22 onward, ipsilaterally projecting axons diverged from contralaterally projecting axons in prechiasmatic parts of the optic nerve. They made sharp turns when arriving at glial arches found at the transition from the optic nerve to the chiasm. Thus, during the ingrowth period of axons, Tupaia belangeri and marsupials have specialized glial arrays in common, which probably help to deflect ipsilaterally projecting axons to lateral parts of the chiasm. Our observations provide new evidence of diversity in eutherian chiasmatic architecture and identify Tupaia belangeri as an appropriate animal model for studies on the mechanisms underlying axon guidance in the developing chiasm of higher primates.

Development of starburst cholinergic amacrine cells in the retina of Tupaia belangeri.

"Starburst" cholinergic amacrines specify the response of direction-selective ganglion cells to image motion. Here, development of cholinergic amacrines was studied in the tree shrew Tupaia belangeri (Scandentia) by immunohistochemistry with antibodies against choline acetyltransferase (ChAT) and neurofilament proteins. Starburst amacrines expressed ChAT much earlier than previously thought. From embryonic day 34 (E34) onward, orthotopic and displaced subpopulations segregated from a single cluster of immunoreactive precursor cells. Orthotopic starburst amacrines rapidly took up positions in the inner nuclear layer. Displaced starburst amacrines were first arranged in a monocellular row in the inner plexiform layer, and, with a delay of 1 week, they descended to the ganglion cell layer. Conversely, dendritic stratification of displaced amacrines slightly preceded that of orthotopic ones. Starburst amacrines expressed the medium-molecular-weight neurofilament protein (NF-M) from E34 to postnatal day 11 (P11) and coexpressed alpha-internexin from E36.5 to P11. Consequently, neurofilaments composed of alpha-internexin and NF-M may stabilize developing dendrites of starburst amacrines. During the first 2 postnatal weeks, subpopulations of anti-NF-M-labeled ganglion cells costratified with the preexisting dendritic strata of starburst amacrines in the ON sublamina, OFF sublamina, or both. Hence, anti-NF-M-labeled ganglion cells may include direction-selective ones. Thereafter, NF-M and alpha-internexin proteins disappeared from starburst amacrines, and NF-M immunoreactivity was lost in the dendrites of ganglion cells. Our findings suggest that NF-M and alpha-internexin are important for starburst amacrines and ganglion cells to recognize each other and, thus, contribute to the formation of early developing retinal circuits in the inner plexiform layer.

Expression patterns of erythropoietin and its receptor in the developing spinal cord and dorsal root ganglia.

Recombinant human erythropoietin (EPO) is neuroprotective in animal models of adult spinal cord injury, and reduces apoptosis in adult dorsal root ganglia after spinal nerve crush. The present work demonstrates that spinal cord and dorsal root ganglia share dynamic expression patterns of EPO and its receptor (EPOR) during development. C57Bl mice from embryonic days (E) 8 (E8) to E19 were studied. In spinal cord and dorsal root ganglia, EPOR expression in all precursor cells preceded the expression of EPO in subsets of neurons. On E11, EPO-immunoreactive spinal motoneurons and ganglionic sensory neurons resided adjacent to EPOR-expressing radial glial cells and satellite cells, respectively. From E12 onwards, EPOR-immunoreactivity decreased in radial glial cells and, transiently, in satellite cells. Simultaneously, large-scale apoptosis of motoneurons and sensory neurons started, and subsets of neurons were labelled by antibodies against EPOR. Viable neurons expressed EPO and EPOR. Up to E12.5, apoptotic cells were EPOR-immunopositive, but variably EPO-immunonegative or EPO-immunopositive. Thereafter, EPO-immunonegative and EPOR-immunopositive apoptotic cells predominated. Our findings suggest that EPO-mediated neuron-glial and, later, neuron-neuronal interactions promote the differentiation and/or the survival of subsets of neurons and glial cells in central as well as in peripheral parts of the embryonic nervous system. Correspondingly, expression of phospho-Akt-1/protein-kinase B extensively overlapped expression sites of EPO and EPOR, but was absent from apoptotic cells. Identified other sites of EPO and/or EPOR expression include radial glial cells that transform to astrocytes, cells of the floor plate and notochord as well as neural crest-derived boundary cap cells at motor exit points and cells of the primary sympathetic chain.

Apoptosis and proliferation in developing, mature, and regressing epibranchial placodes.

Epibranchial placodes and rhombencephalic neural crest provide precursor cells for the geniculate, petrosal, and nodose ganglia. In chick embryos and in Tupaia belangeri, apoptosis in rhombomeres 3 and 5 helps to select premigratory precursor cells and to segregate crest cell streams derived from the even-numbered rhombomeres. Much less is known about the patterns and functions of apoptosis in epibranchial placodes. We found that, in Tupaia belangeri, combined anlagen of the otic placode and epibranchial placode 1 transiently share a primordial low grade thickening with post-otic epibranchial placodes. Three-dimensional reconstructions reveal complementary, spatially, and temporally regulated apoptotic and proliferative events that demarcate the otic placode and epibranchial placode 1, and help to individualize three pairs of epibranchial placodes in a rostrocaudal sequence. Later, rostrocaudal waves of proliferation and apoptosis extend from dorsal to ventral parts of the placodes, paralleled by the dorsoventral progression of precursor cell delamination. These findings suggest a role for apoptosis during the process of neuroblast generation in the epibranchial placodes. Finally, apoptosis eliminates remnants of the placodes in the presence of late invading macrophages.

Rhombomere-specific patterns of apoptosis in the tree shrew Tupaia belangeri.

Whether rhombomere-specific patterns of apoptosis exist in the developing hindbrain of vertebrates is under debate. We have investigated the sequence of apoptotic events in three-dimensionally reconstructed hindbrains of Tupaia belangeri (8- to 19-somite embryos). Apoptotic cells were identified by structural criteria and by applying an in situ tailing technique to visualize DNA fragmentation. Seven rhombomeres originated from three pro-rhombomeres. Among pre-migratory neural crest cells in the dorsal thirds of the neural folds, the earliest apoptotic concentrations appeared in the developing third rhombomere (r3). Dorsal apoptotic maxima then persisted in r3, extended from r3 to r2, and also arose in r5. Transverse apoptotic bands increased the total amount of apoptotic cells in odd-numbered rhombomeres first in r3 and, with a delay, also in r5. This sequence of apoptotic events was paralleled by an approximate rostrocaudal sequence of neural crest cell delamination from the even-numbered rhombomeres. Thus, large-scale apoptosis in r3 and r5 helped to establish crest-free zones that segregated streams of migrating neural crest cells adjacent to r2, r4, and r6. The sequence of apoptotic events observed in the dorsal thirds of rhombomeres matches that reported for the chick embryo. Other shared features are apoptotic peaks in the position of a circumscribed ventricular protrusion of fusing parts of the neural folds in r1 and r2, and Y-shaped apoptotic patterns composed of apoptotic maxima in the dorsal and lateral thirds of r1, r2, and r3. These rhombomere-specific patterns of apoptosis may therefore represent a conserved character, at least in amniotes.

Use of "reference series" to realign histological serial sections for three-dimensional reconstructions of the positions of cellular events in the developing brain.

The present study demonstrates how, predominantly by external fiducials, histological serial sections used to reconstruct patterns of individually marked cellular events in large organs or whole embryos can be realigned with the help of "reference series". Resin-embedded embryos were cut at 1 microm and consecutive sections were alternately placed on two sets of slides. For cytological diagnosis and acquisition of embryonic contours, stained sections of the first series, termed "working series", were scanned with the x 100 objective using "Huge Image", a recently established image acquisition system. For acquisition of the contours of the resin block, adjacent unstained sections of the second series, termed "reference series", were scanned with the x 5 objective. Thereafter, "hybrid sections" were created which combined vectorized embryonic contours and cellular events taken from the working series with vectorized block contours taken from the reference series. For realignment, consecutive "hybrid sections" were matched by best-fit of the block contours. Stacks of realigned "hybrid sections" were shaped like truncated pyramids and, thus, reflected repeated "trimming" of the resin block during the sectioning procedure. Among 266 "hybrid sections" at intervals of 8 microm, needed to reconstruct the brain of a 15-day-old embryo of Tupaia belangeri (Scandentia), internal fiducials were required five times for realigning a total of six adjacent truncated pyramids. Application of this method provided realistic reconstructions of the positions of apoptotic cells in the entire developing brain without the need of secondary introduction of external fiducials.

Calretinin immunoreactivity in the prenatally developing olfactory systems of the tree shrew Tupaia belangeri.

The prenatal patterns of calretinin immunoreactivity were studied in the olfactory systems of Tupaia belangeri. We investigated the peripheral and primary central parts of the vomeronasal system and of the main olfactory system from the 19th to the 43rd (last) day of gestation and compared the findings with the known calretinin immunoreactivity patterns in adult T. belangeri and the published data on other mammals. The onset of calretinin immunoreactivity was noted in the main olfactory system on the 23rd day of gestation and, in the vomeronasal system, on the 25th day of gestation: single precursors of receptor cells with calretinin immunoreactive perikarya and processes were observed in both epithelia. Their neuronal identity was proven by olfactory marker protein immunoreactivity. On the 42nd day of gestation, almost all receptor cells and nerve fibers, many interneurons and projecting cells were calretinin immunoreactive in the main olfactory and in the vomeronasal systems. In contrast to the intensive calretinin labeling previously observed in virtually all vomeronasal epithelial cells of adult T. belangeri, among developing receptor cells a population of intensively labeled, basally located perikarya was distinguishable from a population of less intensively stained, more apically located ones. In the main olfactory epithelium of fetal T. belangeri, calretinin immunoreactive receptor cells occurred in the middle layers. Whereas in the vomeronasal sensory epithelium differently reacting layers of receptor cells might represent the two known subfamilies of receptor cells, in the main olfactory epithelium the differing calretinin expression in the layers of the epithelium, most probably, did not reflect known subfamilies of odour receptor cells. Transiently, ectopic calretinin immunoreactive receptor cells were observed in the future non-sensory epithelium of the vomeronasal organ.

Calretinin and FMRFamide immunoreactivity in the nervus terminalis of prenatal tree shrews (Tupaia belangeri).

The distribution and development of FMRFamide- and calretinin-immunoreactive neurons were investigated in the nervus terminalis of prenatal tree shrews from gestation day 19 onwards. The first FMRFamide-immunoreactive cells were observed medially in the olfactory epithelium on gestation day 20. From gestation day 23 onwards, the migrating nervus terminalis ganglion cells showed FMRFamide calretinin immunoreactivity. The distribution pattern of FMRFamide- and calretinin-immunoreactive cells was similar along the migratory route and in the ganglion of the terminal nerve. However, most probably calretinin and FMRFamide were expressed in separate neuronal populations. For the first time in a mammal, FMRFamide and calretinin are reported to occur in the migrating perikarya and neuronal processes of the nervus terminalis during prenatal development. The results suggest (i) an early activation of the rostral FMRFamide-immunoreactive migratory stream comparable to that described for the GnRH-immunoreactive part of the terminal nerve in other mammals and possibly (ii) an involvement of calretinin in mechanisms of cell migration and outgrowth of neuronal processes in the terminal nerve during the studied period.

High resolution scanning and three-dimensional reconstruction of cellular events in large objects during brain development.

Detailed knowledge of the spatial and temporal interactions of distinct cellular events and of the genes involved in their regulation is a precondition for the understanding of morphogenetic and pathogenetic processes. Here, how patterns of cellular events in large objects can be visualized with the help of the image acquisition system 'Huge Image' is demonstrated. Huge images are composed of a multitude of small images scanned with the highest light microscopical resolution. The system is equipped with a programmable autofocus device and permits precise and rapid cytological diagnosis. A vector-based three-dimensional (3-D) reconstruction method which, in future projects, will be combined with 'Huge Image', is applied to visualize dynamic interactions between macrophages and the occurrence of apoptotic neuroepithelial cells in the early developing forebrain of Tupaia belangeri (Scandentia). Proportionally correct meshwire surfaces of small and large objects are generated independently of each other. The combined reconstruction of cellular events and large embryonic surfaces can be carried out from only subsets of histological serial sections, and, compared with volume-based systems, with a much lower need for memory. The practicability of our approach is compared with recent other methods used to demonstrate apoptotic patterns.