Abstract. Neocortical neuroblast cell lines were used to clone G-protein-coupled receptor (GPCR) genes to study signaling mechanisms regulating cortical neurogenesis. One putative GPCR gene displayed an in situ expression pattern enriched in cortical neurogenic regions and was therefore named ventricular zone gene-! (vzg-1). The vzg-1 cDNA hybridized to a 3.8-kb mRNA transcript and encoded a protein with a predicted molecular mass of 41--42 kD, confirmed by Western blot analysis. To assess its function, vzg-1 was overexpressed in a cell line from which it was cloned, inducing serum-dependent "cell rounding." Lysophosphatidic acid (LPA), a bioactive lipid present in high concentrations in serum, reproduced the effect seen with serum alone. Morphological responses to other related phospholipids or to thrombin, another agent that induces cell rounding through a GPCR, were not observed in vzg-1 overexpressing cells. Vzg-1 overexpression decreased the EC50 of both cell rounding and Gi activation in response to LPA. Pertussis toxin treatment inhibited vzg-l-dependent LPA-mediated Gi activation, but had no effect on cell rounding. Membrane binding studies indicated that vzg-1 overexpression increased specific LPA binding. These analyses identify the vzg-1 gene product as a receptor for LPA, suggesting the operation of LPA signaling mechanisms in cortical neurogenesis. Vzg-1 therefore provides a link between extracellular LPA and the activation of LPAmediated signaling pathways through a single receptor and will allow new investigations into LPA signaling both in neural and nonneural systems.CRITICAL event in the formation of the mammalian cerebral cortex is the ordered generation of its neurons from a discrete proliferative region overlying the cerebral ventricles, the ventricular zone (vz) t (6), (Fig. 1). In most mammalian species, neurogenesis occurs during fetal life when the vz can be delineated by histological stains, or by brief pulses of 5-Bromo-2'-deoxyuridine (BrdU) or [3H]thymidine, which identify neuroblasts undergoing S-phase (58,64,67). Cortical neuroblasts display a stereotyped change in their morphology that is linked to their proliferation. During S-phase of the cell cycle, vz neuroblasts appear bipolar, with the cell body at the super-

The murine genome contains approximately 70 protocadherin (Pcdh) genes. Many are expressed in the nervous system, suggesting that Pcdhs may specify neuronal connectivity. Here, we analyze the 22 contiguous genes of the Pcdh-gamma cluster. Individual neurons express subsets of Pcdh-gamma genes. Pcdh-gamma proteins are present in most neurons and associated with, but not confined to, synapses. Early steps in neuronal migration, axon outgrowth, and synapse formation proceed in mutant mice lacking all 22 Pcdh-gamma genes. At late embryonic stages, however, dramatic neurodegeneration leads to neonatal death. In mutant spinal cord, many interneurons are lost, but sensory and motor neurons are relatively spared. In cultures from mutant spinal cord, neurons differentiate and form synapses but then die. Thus, Pcdh-gamma genes are dispensable for at least some aspects of connectivity but required for survival of specific neuronal types.

The specificity of interactions between neurons is believed to be mediated by diverse cell adhesion molecules, including members of the cadherin superfamily. Whereas mechanisms of classical cadherin adhesion have been studied extensively, much less is known about the related protocadherins (Pcdhs), which together make up the majority of the superfamily. Here we use quantitative cell aggregation assays and biochemical analyses to characterize cis and trans interactions among the 22-member γ-Pcdh family, which have been shown to be critical for the control of synaptogenesis and neuronal survival. We show that γ-Pcdh isoforms engage in trans interactions that are strictly homophilic. In contrast to classical cadherins, γ-Pcdh interactions are only partially Ca 2+ -dependent, and their specificity is mediated through the second and third extracellular cadherin (EC) domains (EC2 and EC3), rather than through EC1. The γ-Pcdhs also interact both covalently and noncovalently in the cis-orientation to form multimers both in vitro and in vivo. In contrast to γ-Pcdh trans interactions, cis interactions are highly promiscuous, with no isoform specificity. We present data supporting a model in which γ-Pcdh cistetramers represent the unit of their adhesive trans interactions. Unrestricted tetramerization in cis, coupled with strictly homophilic interactions in trans, predicts that the 22 γ-Pcdhs could form 234,256 distinct adhesive interfaces. Given the demonstrated role of the γ-Pcdhs in synaptogenesis, our data have important implications for the molecular control of neuronal specificity.cadherin | calcium-dependent | synaptogenesis | recognition | trafficking

Although extracellular application of lysophosphatidic acid (LPA) has been extensively documented to produce a variety of cellular responses through a family of specific G protein-coupled receptors, the in vivo organismal role of LPA signaling remains largely unknown. The first identified LPA receptor gene, lp A1͞vzg-1͞ edg-2, was previously shown to have remarkably enriched embryonic expression in the cerebral cortex and dorsal olfactory bulb and postnatal expression in myelinating glia including Schwann cells. Here, we show that targeted deletion of lp A1 results in approximately 50% neonatal lethality, impaired suckling in neonatal pups, and loss of LPA responsivity in embryonic cerebral cortical neuroblasts with survivors showing reduced size, craniofacial dysmorphism, and increased apoptosis in sciatic nerve Schwann cells. The suckling defect was responsible for the death among lpA1 (؊/؊) neonates and the stunted growth of survivors. Impaired suckling behavior was attributable to defective olfaction, which is likely related to developmental abnormalities in olfactory bulb and͞or cerebral cortex. Our results provide evidence that endogenous lysophospholipid signaling requires an lp receptor gene and indicate that LPA signaling through the LP A1 receptor is required for normal development of an inborn, neonatal behavior.

SUMMARY The 22 γ-protocadherins (γ-Pcdhs) potentially specify thousands of distinct homophilic adhesive interactions in the brain. Neonatal lethality of mice lacking the Pcdh-γ gene cluster has, however, precluded analysis of many brain regions. Here, we use a conditional Pcdh-γ allele to restrict mutation to the cerebral cortex and find that, in contrast to other central nervous system phenotypes, loss of γ-Pcdhs in cortical neurons does not affect their survival or result in reduced synaptic density. Instead, mutant cortical neurons exhibit severely reduced dendritic arborization. Mutant cortices have aberrantly high levels of protein kinase C (PKC) activity and of phosphorylated (inactive) myristoylated alanine-rich C-kinase substrate, a PKC target that promotes arborization. Dendrite complexity can be rescued in Pcdh-γ mutant neurons by inhibiting PKC, its upstream activator phospholipase C, or the γ-Pcdh binding partner focal adhesion kinase. Our results reveal a distinct role for the γ-Pcdhs in cortical development and identify a signaling pathway through which they play this role.