Morphological domains of Lewis-X/FORSE-1 immunolabeling in the embryonic neural tube are due to developmental regulation of cell surface carbohydrate expression.

The Lewis-X (LeX) carbohydrate epitope, recognized by the FORSE-1 monoclonal antibody (mAb), shares expression boundaries with neural regulatory genes and may be involved in patterning the neural tube by creating domains of differential cell adhesion. The present experiments focus on the question of what determines the expression pattern of LeX in embryonic rat brain. Comparisons of FORSE-1-positive glycolipid and protein antigens in embryonic, early postnatal, and adult tissues show that the LeX epitope is carried primarily by glycolipids during embryonic development and by a proteoglycan and glycoproteins in postnatal and adult tissue. Immunohistochemistry using FORSE-1 and an antibody to the proteoglycan phosphacan, which carries LeX, shows that the distribution of LeX is more restricted than phosphacan. These observations suggest that the precise spatial regulation of FORSE-1 binding in the embryonic forebrain is due to the expression pattern of the LeX carbohydrate on glycolipids, rather than to the transcriptional regulation of a carrier protein.

Changing patterns of expression and subcellular localization of TrkB in the developing visual system.

Neurotrophins play important roles in the survival, differentiation, and maintenance of CNS neurons. To begin to investigate specific roles for these factors in the mammalian visual system, we have examined the cellular localization of the neurotrophin receptor trkB within the developing cerebral cortex and thalamus of the ferret using extracellular domain-specific antibodies. At prenatal ages (gestation is 41 d), trkB-immunostained fibers were observed in the internal capsule and as two distinct fascicles within the intermediate zone of the cerebral cortex. The staining of these fiber tracts declined with increasing age, whereas soma and dendrite staining of cortical neurons was first evident in early postnatal life and increased during subsequent development. Staining of subplate neurons [by prenatal day 5 (P5)] was followed by staining of cortical layer 5 neurons (at P10). By P31, trkB immunoreactivity was particularly prominent in layers 3 and 5 but was absent from subplate neurons. Staining included cells, especially pyramidal neurons, in all cortical layers by P45, and this pattern was maintained into adulthood. The optic tract and fibers within the lateral geniculate nucleus (LGN) were also strongly trkB immunoreactive at prenatal ages. Cellular staining of a subset of LGN neurons, those within the C-layers and perigeniculate nucleus, was apparent by P10 and maintained until P45, when the adult pattern of highly trkB-immunoreactive neurons in all layers of the LGN first appeared. The pattern of trkB immunoreactivity suggests that specific subsets of cortical and thalamic neurons may respond to neurotrophins such as brain-derived neurotrophic factor and/or NT-4/5 at discrete developmental times and locations. The appearance of trkB on axon fibers early in development and then on cell bodies and dendritic processes later is consistent with roles for both long-range and local, including autocrine and/or paracrine, delivery of neurotrophins in cell survival and maturation.

FORSE-1, an antibody that labels regionally restricted subpopulations of progenitor cells in the embryonic central nervous system, recognizes the Le(x) carbohydrate on a proteoglycan and two glycolipid antigens.

A key problem in nervous system development is how distinct subpopulations of progenitor cells give rise to different adult brain structures. The labeling pattern of the FORSE-1 antibody subdivides the neuroepithelium of the embryonic forebrain into domains resembling those of certain transcription factors, suggesting that the FORSE-1 epitope may be involved in the specification of development compartments. Therefore, it is important to determine the identity of the antigen(s) recognized by FORSE-1. On immunoblots, FORSE-1 recognizes a single, high-molecular-weight species, which we have identified as phosphacan, a brain-specific chondroitin sulfate proteoglycan that binds neural cell adhesion molecules. This identification is based on cross-immunoprecipitations and immunoblotting using an anti-phosphacan antibody and FORSE-1. FORSE-1 also recognizes two major neutral glycolipids in embryonic brain. The FORSE-1 epitope is sensitive to endo-beta-galactosidase, suggesting that the epitope corresponds to a carbohydrate moiety. Moreover, immunoprecipitates of the proteoglycan bearing the FORSE-1 epitope bind antibodies that recognize the Le* carbohydrate, and immunostaining patterns of embryonic brain sections by FORSE-1 and a known anti-Le* antibody are identical. Finally, purified FORSE-1 specifically recognizes Le*-containing glycoconjugates in ELISAs. The pattern of FORSE-1 labeling, the identification of its epitope as Le*, which has implicated in cell adhesion, and the presence of Le* on phosphacan suggest that this carbohydrate epitope may play a role in adhesive interactions important for proliferation, cell migration, or axon guidance.

Regulation of neurotrophin receptors during the maturation of the mammalian visual system.

Cell division, cell death, and remodeling of connections are major features of the construction of the mammalian CNS. We have begun to address the role of neurotrophins in these events through characterization of the expression of their receptors in the developing ferret visual system. By use of chemical cross-linking of iodinated neurotrophins, proteins corresponding to trkB, trkC, and p75 were identified as receptors for brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) throughout development. BDNF was also cross-linked to a truncated form of trkB that lacks the tyrosine kinase domain (trkB. T1) in retinal target tissues and in cortex. At the earliest developmental age examined (E24), the ratio of full-length to truncated trkB is > > 1 in the retinal target tissues, LGN and superior colliculus. During the ensuing period of retinal ganglion cell death and segregation into eye-specific layers, the amount of truncated trkB increases markedly relative to full-length trkB. By P27, truncated trkB is the predominant receptor for BDNF in the retinal target tissues and this pattern is maintained into adulthood. Within all subdivisions of visual cortex including the ventricular zone (VZ), intermediate zone (IZ), and cortical plate (CP), similar profiles of bands are observed. The developmental increase in abundance of truncated trkB relative to full-length occurs earliest in the VZ, with a major increase between E30 and P3. In the IZ, this shift to a predominance of truncated trkB occurs between P15 and P30, while in the CP the shift is even further delayed, not occurring until after P30. Within each subdivision of cortex, the shift to a predominance of truncated trkB occurs at times that correlate with the onset of cell death and maturation of axonal connections. This study demonstrates that members of the trk family, previously identified in the CNS on the basis of mRNA transcripts, are present as receptors with specific binding affinities for BDNF and NT-3. Moreover, the correspondence between the developmental shift from full-length to truncated trkB and the critical periods for cell fate determination, cell death, and axonal remodeling suggests an important role for neurotrophic factors in the development of the visual system.

Nerve growth factor receptor immunoreactivity is transiently associated with the subplate neurons of the mammalian cerebral cortex.

Nerve growth factor and its receptor (NGFR) are known to be present in diverse embryonic and neonatal central nervous system tissues, including the cerebral cortex. However, the identity of the cortical cells expressing NGFR immunoreactivity has not been established. We have used immunolabeling coupled with [3H]thymidine autoradiography to identify such cells in ferret and cat brain. Polyclonal antibodies raised against a synthetic peptide corresponding to a conserved amino acid sequence of the NGFR were used for this purpose. Western (immunologic) blot analyses show that these antibodies specifically recognize NGFR and precursor proteins. In both species, NGFR immunoreactivity is primarily associated with the early generated and transient subplate neuron population of the developing neocortex, as indicated by the following evidence: the immunoreactive cells (i) are located directly beneath the developing cortical plate, (ii) frequently have the inverted pyramid shape characteristic of subplate neurons, and (iii) can be labeled by an injection of [3H]thymidine on embryonic day (E) 28, a time when only subplate neurons are being generated. Intense NGFR immunostaining is seen on the cell bodies of these neurons as early as E30, several days after their last round of cell division, and this immunostaining remains strong for approximately 3 weeks. The NGFR immunoreactivity begins to decline around E52 and has disappeared from the region altogether by E60, at which time subplate neurons begin to die. The cellular localization and timing of expression suggest that the NGFR may play a role in the maintenance of subplate neurons and in the maturation of the cerebral cortex.