Using two-dimensional gel electrophoresis we previously identified membrane-associated proteins that are upregulated over the course of neurogenesis. One of these, TOAD-64 (Turned On After Division, 64 kDa), is expressed immediately after neuronal birth and is dramatically downregulated in the adult. The gene encoding TOAD-64 has now been cloned, and its sequence shows homology to the unc-33 gene from C. elegans, mutations in which lead to aberrations in axon outgrowth. Northern and in situ hybridization show that TOAD-64 mRNA is enriched in the nervous system and is developmentally regulated in parallel with the protein. The expression of the TOAD-64 protein and gene coincident with initial neuronal differentiation and the downregulation when the majority of axon growth is complete suggests a role in axon elaboration. Three additional lines of evidence support this possibility: TOAD-64 is upregulated following neuronal induction of P19 and PC12 cells; the protein is found in lamellipodia and filopodia of growth cones; and axotomy of the sciatic nerve induces reexpression. While the sequence of TOAD-64 lacks a signal sequence and therefore is likely to encode a cytoplasmic protein, biochemical experiments demonstrate that the protein is tightly, but noncovalently, associated with membranes. The data presented here suggest that TOAD-64 could be a central element in the machinery underlying axonal outgrowth and pathfinding, perhaps playing a role in the signal transduction processes that permit growing axons to choose correct routes and targets.

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Between embryonic day 14 (E14) and embryonic day 21 (E21), the rat neopallium develops from a relatively homogeneous band of mitotic precursor cells into a complex laminated structure containing diverse classes of neurons. In order to identify some of the molecular components underlying this process, 2-dimensional PAGE was used to compare proteins expressed before cortical neurons are born (E14) with those expressed during neurogenesis and neuronal migration (E17 and E21). This approach has permitted the identification of 15 proteins that show greater than 3-fold changes in their rate of accumulation between E14 and E21. Six proteins show consistent up-regulation, ranging from 3.2- to 10.7-fold. Five proteins show consistent down-regulation ranging from 9- to 22-fold. Four proteins that appear at E21 are not detectable on fluorograms of E14 cortex, even after long exposures, and thus are up-regulated more than 200-fold from E14 to E21 and may be considered to appear de novo. The molecular weights and isoelectric points of most of these 15 suggest that they are previously unreported, developmentally regulated proteins. Comparisons of gels of cortex to gels of lung and heart suggest that several of these proteins are enriched in brain relative to non-neural tissues. This analysis also indicates that, despite the large morphogenic changes observed during this developmental period, few proteins (less than 3%) among the total spectrum analyzed show large changes in their rates of synthesis.

Hematopoietic stem cells (HSC) are clonogenic cells that can self‐renew and give rise to both lymphoid and hematopoietic systems throughout the life span. Using cDNA subtraction, we have identified a set of genes selectively expressed in mouse adult HSC as opposed to its direct microenvironment, whole bone marrow. Some of these genes were found to be expressed in the subventricular zone of fetal and adult brains, one of the regions of continious neurogenesis. Using cDNA microarray techniques we confirmed gene expression in mouse neurospheres, a population enriched for neural stem cells, and down‐regulation in differentiated cells. We propose that transcripts enriched in several types of stem cells define a functionally conserved group of genes likely to include the regulatory genes involved in self‐renewal of stem cells. To address the self‐renewal potential of stem cell associated genes we have used retroviral transfer into highly purified HSC and primitive neural progenitor cells.

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