Christoph Redies

Morphologic fate of diencephalic prosomeres and their subdivisions revealed by mapping cadherin expression.

The expression of four cadherins (cadherin‐6B, cadherin‐7, R‐cadherin, and N‐cadherin) was mapped in the diencephalon of chicken embryos at 11 days and 15 days of incubation and was compared with Nissl stains and radial glial topology. Results showed that each cadherin is expressed in a restricted manner by a different set of embryonic divisions, brain nuclei, and their subregions. An analysis of the segmental organization based on the prosomeric model indicated that, in the mature diencephalon, each prosomere persists and forms a coherent domain of gray matter extending across the entire transverse dimension of the neural tube, from the ventricular surface to the pial surface. Moreover, the results suggest the presence of a novel set of secondary subdivisions for the dorsal thalamus (dorsal, intermediate, and ventral tiers and anteroventral subregion). They also confirm the presence of secondary subdivisions in the pretectum (commissural, juxtacommissural, and precommissural). At most of the borders between the prosomeres and their secondary subdivisions, changes in radial glial fiber density were observed. The diencephalic brain nuclei that derive from each of the subdivisions were determined. In addition, a number of previously less well‐characterized gray matter regions of the diencephalon were defined in more detail based on the mapping of cadherin expression. The results demonstrate in detail how the divisions of the early embryonic diencephalon persist and transform into mature gray matter architecture during brain morphogenesis, and they support the hypothesis that cadherins play a role in this process by providing a framework of potentially adhesive specificities. J. Comp. Neurol. 421:481–514, 2000.

CADHERIN EXPRESSION IN THE RETINA AND RETINOFUGAL PATHWAYS OF THE CHICKEN EMBRYO

The expression of two calcium‐dependent adhesion molecules of the cadherin superfamily (cadherin‐6B and cadherin‐7) was mapped in the embryonic neural retina and retinofugal pathways of the chicken embryo and compared with the expression of R‐cadherin, N‐cadherin, and B‐cadherin, studied previously. Whereas B‐cadherin is only found in Müller glia, the other four cadherins are each expressed by specific subpopulations of retinal neurons. For example, different (but partly overlapping) populations of bipolar cells express R‐cadherin, cadherin‐6B, and cadherin‐7. Cadherin‐6B and cadherin‐7 are also expressed by subsets of amacrine cells. In the inner plexiform layer, cadherin‐6B and cadherin‐7 immunoreactivities are restricted to specific sublaminae associated with synapsin‐I‐positive nerve terminals. In addition, cadherin‐6B and cadherin‐7 are expressed by a subset of ganglion cells that project to several retinorecipient nuclei forming part of the accessory optic system (e.g., nucleus of the basal optic root and external pretectal nucleus). Together with their connecting fiber tracts, these nuclei also express cadherin‐6B and cadherin‐7 in their neurons and neuropile. The expression patterns of the two cadherins overlap but show distinct differences. Some other visual nuclei express cadherin‐7 but not cadherin‐6B. The expression patterns differ from those previously described for N‐ and R‐cadherin. Together, these results demonstrate that cadherins could provide a system of adhesive cues that specify developing retinal circuits and other functional connections and subsystems in the embryonic chicken visual system. J. Comp. Neurol. 396:20–38, 1998.

Cadherin-Defined Segments and Parasagittal Cell Ribbons in the Developing Chicken Cerebellum.

In the developing chicken cerebellar cortex, three cadherins (Cad6B, Cad7, and R-cadherin) are expressed in distinct parasagittal segments that are separated from each other by ribbons of migrating interneurons and granule cells which express R-cadherin and Cad7, respectively. The segment/ribbon pattern is respected by the expression of other types of molecules, such as engrailed-2 and SC1/BEN/DM-GRASP. The cadherin-defined segments contain young Purkinje cells which are connected to underlying nuclear zones expressing the same cadherin, thereby forming parasagittal cortico-nuclear zones of topographically organized connections. In addition, R-cadherin-positive mossy fiber terminals display a periodic pattern in the internal granular layer. In this layer, Cad7 and R-cadherin are associated with synaptic complexes. These results suggest that cadherins play a pivotal role in the formation of functional cerebellar architecture by providing a three-dimensional scaffold of adhesive information.

Blocking N-Cadherin Function Disrupts the Epithelial Structure of Differentiating Neural Tissue in the Embryonic Chicken Brain

The cell adhesion molecule N-cadherin is ubiquitously expressed in the early neuroepithelium, with strongest expression in the ependymal lining. We blocked the function of N-cadherin during early chicken brain development by injecting antibodies against N-cadherin into the tectal ventricle of embryos at 4–5 d of incubation [embryonic day 4 (E4)–E5]. N-cadherin blockage results in massive morphological changes in restricted brain regions. At approximately E6, these changes consist of invaginations of pieces of the ependymal lining and the formation of neuroepithelial rosettes. The rosettes are composed of central fragments of ependymal lining, surrounded by an inner ventricular layer and an outer mantle layer. Radial glia processes are radially arranged around the ependymal centers of the rosettes. The normal layering of the neural tissue is thus preserved, but its coherent epithelial structure is disrupted. The observed morphological changes are restricted to specific brain regions such as the tectum and the dorsal thalamus, whereas the ventral thalamus and the pretectum are almost undisturbed. At E10–E11, analysis of late effects of N-cadherin blockage reveals that in the dorsal thalamus, gray matter is fragmented and disorganized; in the tectum, additional layers have formed at the ventricular surface. Together, these results indicate that N-cadherin function is required for the maintenance of a coherent sheet of neuroepithelium in specific brain regions. Disruption of this sheet results in an abnormal morphogenesis of brain gray matter.

Combinatorial expression of cadherins in the tectum and the sorting of neurites in the tectofugal pathways of the chicken embryo

The expression of four cadherins (N-cadherin, R-cadherin, cadherin-6B and cadherin-7) was mapped in the developing tectal system of the chicken embryo from four to 19 days of incubation. Each of the cadherins is expressed in a restricted fashion in specific tectal layers, with partial overlap between the cadherins. In some layers, subpopulations of neurons differentially express the cadherins, e.g., in the stratum griseum centrale. Double labeling demonstrates that many of the projection neurons in this layer co-express at least two cadherins. Fibers of the efferent (tectofugal) pathways originating in these neurons also differentially express the cadherins, most prominently at around 1 1 days of incubation. While the different subpopulations of cadherin-expressing projection neurons are dispersed and mixed within the tectum, their neurites sort out and fasciculate according to which cadherin they express, as they collect in the major output of the tectum, the brachium colliculi superioris. From here, cadherin-expressing fascicles follow separate paths to their respective target areas, some of which also express the respective cadherins, in a matching fashion. We propose that the preferentially homophilic binding of cadherins provides a potential adhesive basis for the sorting and selective fasciculation of specific subpopulations of neurites, similar to the well-established sorting and aggregation of cells expressing cadherins. The combinatorial expression of cadherins by the tectal projection neurons may contribute to the complexity and specificity of functional connections in this system.

Expression of cadherin‐8 mRNA in the developing mouse central nervous system

The expression of cadherin‐8 was mapped by in situ hybridization in the embryonic and postnatal mouse central nervous system (CNS). From embryonic day 18 (E18) to postnatal day 6 (P6), cadherin‐8 expression is restricted to a subset of developing brain nuclei and cortical areas in all major subdivisions of the CNS. The anlagen of some of the cadherin‐8‐positive structures also express this molecule at earlier developmental stages (E12.5–E16). The cadherin‐8‐positive neuroanatomical structures are parts of several functional systems in the brain. In the limbic system, cadherin‐8‐positive regions are found in the septal region, habenular nuclei, amygdala, interpeduncular nucleus, raphe nuclei, and hippocampus. Cerebral cortex shows expression in several limbic areas at P6. In the basal ganglia and related nuclei, cadherin‐8 is expressed by parts of the striatum, globus pallidus, substantia nigra, entopeduncular nucleus, subthalamic nucleus, zona incerta, and pedunculopontine nuclei. A third group of cadherin‐8‐positive gray matter structures has functional connections with the cerebellum (superior colliculus, anterior pretectal nucleus, red nucleus, nucleus of posterior commissure, inferior olive, pontine, pontine reticular, and vestibular nuclei). The cerebellum itself shows parasagittal stripes of cadherin‐8 expression in the Purkinje cell layer. In the hindbrain, cadherin‐8 is expressed by several cranial nerve nuclei.

Restricted expression of cadherin-8 in segmental and functional subdivisions of the embryonic mouse brain.

We have cloned full‐length cDNA of a novel mouse cadherin (“mCad8”). The deduced amino acid sequence of the mature form of mCad8 shows 98.2% identity with the sequence of human cadherin‐8. The expression of mCad8 was studied by in situ hybridization in mouse embryos of 9.5–14 days gestation (E9.5‐E14). Results show that mCad8 expression is restricted to particular subdivisions of the early central nervous system (CNS) and to the thymus. In the CNS, mCad8 expression was observed from E11.5. In the telencephalon, mCad8 is expressed by the ventricular layer of the ganglionic eminence, by cortical areas, and by cells at the caudato‐pallial angle. In the diencephalon, the margins of one mCad8‐positive area correspond to the borders of the ventral thalamic neuromere, as confirmed by mapping the expression of gene regulatory proteins (Dlx‐2, Pax‐6, and Gbx‐2). In the rhombencephalon, two large groups of mCad8‐expressing cells were seen in the pons and in an area of the lateral basal plate of the myelencephalon. These groups of cells extend from the intermediate zone to the mantle zone at E12.5 and later form the anlage of the pontine and the facial nuclei. In conclusion, the expression of mCad8 reflects, in part, the neuromeric organization of the early embryonic CNS. In the mantle layer, mCad8 is expressed by developing gray matter structures, such as brain nuclei, suggesting a role for mCad8 in brain morphogenesis. Dev. Dyn. 208:178–189, 1997.

Development of cadherin‐Defined parasagittal subdivisions in the embryonic chicken cerebellum

The expression of three cadherins (cadherin‐6B, cadherin‐7, and R‐cadherin) was analyzed by immunohistochemistry at early to intermediate stages of chicken cerebellar development (4.5–12 days of incubation [E4.5–E12]). Expression was first detected at approximately E5. On the cerebellar surface, expression of cadherin‐6B and cadherin‐7 is initially observed in transverse domains that subsequently elongate into parasagittal stripes. The sequence of emergence, the borders, and the orientation of these expression domains suggest a parcellation of the cerebellum into distinct medial, lateral, and caudal embryonic subdivisions. These subdivisions relate to histologic features, to the expression of gene regulatory proteins, and, possibly, to patterns of clonal restriction. Individual cadherin‐expressing cell clusters can be observed to split into cortical and nuclear subdivisions, which are connected by nerve fibers expressing the same cadherin, thus establishing the parasagittal corticonuclear connectivity pattern found at later developmental stages. Our results suggest that cadherins may play a role in the transition from the early embryonic to the later functional organization of the cerebellum by providing a scaffold of potential adhesive cues. J. Comp. Neurol. 401:367–381, 1998.

H19 and Igf2 are expressed and differentially imprinted in neuroectoderm-derived cells in the mouse brain

Abstract Igf2 and H19 are reciprocally imprinted genes that are closely linked and coexpressed in tissues of mesodermal and endodermal origin. Here we report that coexpression of these genes is also found in specific fetal tissues of neuroectodermal origin, that is in the ventral midline region of both the hindbrain and spinal cord. For cells of neuroectodermal origin, complete absence of Igf2 and H19 transcription was previously described. Analysis of allele-specific expression of both Igf2 and H19 in the ventral midline region of the hindbrain shows that H19 is expressed monoallelically, with the paternal allele being silent, whereas Igf2 is expressed biallelically. Furthermore, we observed a strong influence of the parental species background, in that the Mus musculus allele was always expressed at higher levels than the M. spretus allele. This was observed when the M. spretus allele was contributed by the mother or by the father. An analysis of Igf2 methylation by bisulphite genomic sequencing provided no clear answer as to whether Igf2 expression and methylation are linked in a tissue of neuroectodermal origin. Taken together, our results provide novel information on H19 and Igf2 expression and imprinting patterns in the fetal mouse brain. In addition, they indicate that some aspects of Igf2 regulation in cells of neuroectodermal origin do not follow the pattern that exists in mesoderm- and endoderm-derived tissues. Apart from the ventral midline region, H19 and Igf2 were found to be coexpressed in the ectodermally derived Rathke’s pouch and in some circumventricular organs of the brain, such as the organum vasculosum of the lamina terminalis (OVLT) and the pineal gland.

EXPRESSION OF THE CELL ADHESION MOLECULE AXONIN-1 IN NEUROMERES OF THE CHICKEN DIENCEPHALON

Axonin‐1/TAG‐1, a member of the immunoglobulin (Ig) superfamily of adhesion molecules, has been shown to be selectively expressed by a subset of neurons and fiber tracts in the developing nervous system of vertebrates. Axonin‐1/TAG‐1 is thought to play a role in the outgrowth, guidance, and fasciculation of neurites. In the present study, we map the expression of axonin‐1 in the diencephalon of the chicken brain at early and intermediate stages of development [2–8 days of incubation; embryonic day (E)2–E8] by immunohistochemical methods. Results show that axonin‐1 is first expressed at about E2.5 by postmitotic neurons scattered throughout most of the diencephalon. During the neuromeric stage of brain development (about E3–E5), axonin‐1+ nerve cell bodies are predominantly found in two neuromeric subdivisions: 1) in the alar plate of the precommissural pretectum and dorsal thalamus and 2) in the posterior preoptic region of the hypothalamus. The axonin‐1+ fiber bundles emerging from these areas grow across segmental boundaries. For example, axonin‐1+ neurites originating in the dorsal thalamus cross the zona limitans intrathalamica at a right angle to project to the striatum. Later, the axonin‐1+ neuromere areas give rise to particular axonin‐1+ gray and white matter structures. Most of these structures correspond to the structures described to express TAG‐1 in rodents. In conclusion, axonin‐1 can be used as a marker to study aspects of the transition from the early neuromeric structure to the mature anatomy of the chicken brain.J. Comp. Neurol. 381:230‐252, 1997.