Surface and Cytoskeletal Markers of Rostrocaudal Position in the Mammalian Nervous System

To identify cell surface molecules that define position in the mammalian nervous system, we previously characterized the binding of two monoclonal antibodies, ROCA1 and ROCA2, to adult rat sympathetic ganglia and intercostal nerves. The binding of ROCA1 is highest in rostral ganglia and nerves and declines in a graded manner in the caudal segments. ROCA2 labels the same cells in ganglia and nerves as ROCA1, but not in a position-selective manner. We now show by immunoblot analysis that ROCA1 recognizes two antigens in membrane/cytoskeletal fractions of peripheral nerves and ganglia: (1) a Triton X-100-insoluble, 60 kDa protein and (2) a Triton x-100- insoluble, 26 kDa protein. The 60 kDa protein is expressed at higher levels in rostral than in caudal intercostal nerves, and is identified as the intermediate filament protein peripherin. In contrast, it is the ROCA1 epitope on the 26 kDa protein, and not the protein itself, that is preferentially visualized immunohistochemically in rostral nerves and ganglia. We suggest that the ROCA1 epitope on the 26 kDa protein is masked in sections of caudal nerves and ganglia. Amino acid sequence data obtained from the affinity-purified 26 kDa protein indicate significant homology with human CD9, a cell surface protein implicated in intercellular signaling in hematopoietic cells. These results suggest that intermediate filament gene expression and epitope masking on the cell surface may be involved in functions related to position in the nervous system

CD9, a Major Platelet Cell Surface Glycoprotein, is a ROCA Antigen and Is Expressed in the Nervous System

We previously generated a monoclonal antibody (mAb), ROCA1, which binds preferentially to rostral versus caudal sympathetic ganglia and intercostal nerves. Two other mAbs, ROCA2 and B2C11, bind to the same structures but not in rostrocaudal gradients. All three mAbs recognize a 26 kDa cell surface protein. Amino acid sequence data obtained from the affinity purified 26 kDa protein showed some homology with human CD9, a tetraspan protein implicated in intercellular signaling in hematopoietic cells. Using the PCR, we obtained cDNA clones representing the entire rat CD9 coding sequence from sciatic nerve and sympathetic ganglia. ROCA1, ROCA2, and B2C11 each immunoprecipitate a 26 kDa protein from CHO cells stably transfected with one of the clones, demonstrating that the ROCA cell surface antigen is indeed rat CD9. We find that CD9 mRNA is widely expressed, with particularly high levels present in a number of neural tissues. In situ hybridization demonstrates that peripheral neurons and Schwann cells, as well as adrenal chromaffin cells express CD9 mRNA. Consistent with immunoblot analyses showing that, unlike the ROCA1 epitope, the 26 kDa protein is not expressed in a rostrocaudal gradient, we find similar levels of rat CD9 mRNA in rostral and caudal intercostal nerves. In developing postnatal rat sciatic nerve, CD9 mRNA levels are coordinately regulated with the expression of myelin genes. These results provide another example of a cell surface protein expressed by both hematopoietic and neural cells, and suggest a role for CD9 in intercellular signaling in the nervous system

FORSE-1: A Positionally Regulated Epitope in the Developing Rat Central Nervous System

We designed a protocol to identify cell surface molecules expressed in restricted spatial patterns in the developing central nervous system (CNS) that might be regulated by regionally restricted transcription factors. The immunogen was a membrane fraction from NT2/D1 embryocarcinoma cells that were induced to differentiate into neurons and upregulate Hox gene expression in response to retinoic acid. One monoclonal antibody (mAb), FORSE-1, specifically labels the rostral rat CNS from the earliest stages. Staining is observed in the rostral but not caudal neural folds of the embryo prior to neural tube closure. Staining is enriched in the forebrain as compared to the rest of the CNS, until E18. Between E11.5 and E13.5, only certain areas of the telencephalon and diencephalon are labeled. Later, up to E17.5, FORSE-1 labeling is specifically restricted to the telencephalon, where a correlation with mitotic activity is apparent: the ventricular zone labels with FORSE-1, while the cortical plate is negative. The staining of the neuroepithelium is intensified by acetone fixation, which also reveals, between E11.5 and E13.5, a dorsoventrally restricted, FORSE-1- positive region of the spinal cord. After E18, the entire CNS is labeled, through adulthood. The mAb labels the surfaces of dissociated, living cells. Other, non-CNS areas of FORSE-1 labeling are nasal and otic placodes, nasal epithelium, nasal glands, and early (E9.5–10.5) endoderm. mAb FORSE-1 recognizes an epitope present on both a high- molecular-weight (> 200 kDa) proteoglycan from embryonic and early postnatal brain, and on a 80 kDa doublet that is restricted to the CNS in the adult. These findings suggest the FORSE-1 antigen as a candidate cell surface molecule for mediating regional specification from the earliest stages of CNS development

Floor plate-derived neuropilin-2 functions as a secreted semaphorin sink to facilitate commissural axon midline crossing

Commissural axon guidance depends on a myriad of cues expressed by intermediate targets. Secreted semaphorins signal through neuropilin-2/plexin-A1 receptor complexes on post-crossing commissural axons to mediate floor plate repulsion in the mouse spinal cord. Here, we show that neuropilin-2/plexin-A1 are also coexpressed on commissural axons prior to midline crossing and can mediate precrossing semaphorin-induced repulsion in vitro. How premature semaphorin-induced repulsion of precrossing axons is suppressed in vivo is not known. We discovered that a novel source of floor plate-derived, but not axon-derived, neuropilin-2 is required for precrossing axon pathfinding. Floor plate-specific deletion of neuropilin-2 significantly reduces the presence of precrossing axons in the ventral spinal cord, which can be rescued by inhibiting plexin-A1 signaling in vivo. Our results show that floor plate-derived neuropilin-2 is developmentally regulated, functioning as a molecular sink to sequester semaphorins, preventing premature repulsion of precrossing axons prior to subsequent down-regulation, and allowing for semaphorin-mediated repulsion of post-crossing axons

Crossing the Border: Molecular Control of Motor Axon Exit

Living organisms heavily rely on the function of motor circuits for their survival and for adapting to ever-changing environments. Unique among central nervous system (CNS) neurons, motor neurons (MNs) project their axons out of the CNS. Once in the periphery, motor axons navigate along highly stereotyped trajectories, often at considerable distances from their cell bodies, to innervate appropriate muscle targets. A key decision made by pathfinding motor axons is whether to exit the CNS through dorsal or ventral motor exit points (MEPs). In contrast to the major advances made in understanding the mechanisms that regulate the specification of MN subtypes and the innervation of limb muscles, remarkably little is known about how MN axons project out of the CNS. Nevertheless, a limited number of studies, mainly in Drosophila, have identified transcription factors, and in some cases candidate downstream effector molecules, that are required for motor axons to exit the spinal cord. Notably, specialized neural crest cell derivatives, referred to as Boundary Cap (BC) cells, pre-figure and demarcate MEPs in vertebrates. Surprisingly, however, BC cells are not required for MN axon exit, but rather restrict MN cell bodies from ectopically migrating along their axons out of the CNS. Here, we describe the small set of studies that have addressed motor axon exit in Drosophila and vertebrates, and discuss our fragmentary knowledge of the mechanisms, which guide motor axons out of the CNS

The molecular basis of retinotectal topography

Over 50 years have passed since Roger Sperry formulated a simple model of how visual space, as seen by the retina, can be projected onto the brain in a two-dimensional, topographic map during development. Sperry posited a set of two orthogonal gradients in the retina that gives each cell a positional identity. He further suggested that these molecules could be used to match up with complementary gradients in the target field of the retinal projection, the tectum. While some investigators hold that the existence of such molecules may not be necessary to establish retinotectal maps, recent work has identified several cell surface proteins whose distributions are of the type predicted by Sperry. An unexpected twist comes from culture assays demonstrating that inhibitory activities on tectal membranes can guide the growth of processes from retinal neurons. Moreover, the expression patterns of several enzymes and three transcription factors suggest that these proteins are candidates for regulatory agents in the determination of cell position in the retina. In addition, results from perturbation experiments support the candidacy of two of the enzymes, and a new mutant screen has uncovered several as yet unidentified genes that are required for establishment of the proper retinotectal map. A number of these results were presented at a recent meeting on neurospecificity held in Cargese, Corsica and sponsored by NATO and NSF

A Monoclonal Antibody That Defines Rostrocaudal Gradients in the Mammalian Nervous System

Spinal cord axons display a rostrocaudal, positional bias in their innervation of sympathetic ganglia and inter-costal skeletal muscles. In an effort to examine the molecular basis of this positional specificity, we used the cyclophosphamide immunosuppression method to produce monoclonal antibodies that bind preferentially to rostral ganglia. The staining distribution of one of these antibodies, ROCA1, has been analyzed using a novel histological method. A graded decline in binding is observed along the chain of adult rat sympathetic ganglia, as well as in the nerves innervating intercostal muscles. The antigen is identified on immunoblots as a 65 kd protein, whose distribution corresponds to the pattern found histologically. Surprisingly, ROCA1 appears to bind to glial cells, implying rostrocaudal, molecular differences in their surfaces