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-

Summary Extrinsic signals controlling generation of neocortical neurons during embryonic life have been difficult to identify. In this study we demonstrate that the dorsal forebrain meninges communicate with the adjacent radial glial endfeet and influence cortical development. We took advantage of Foxc1 mutant mice with defects in forebrain meningeal formation. Foxc1 dosage and loss of meninges correlated with a dramatic reduction in both neuron and intermediate progenitor production and elongation of the neuroepithelium. Several types of experiments demonstrate that retinoic acid (RA) is the key component of this secreted activity. In addition, Rdh10 and Raldh2 expressing cells in the dorsal meninges were either reduced or absent in the Foxc1 mutants and Rdh10 mutants had a cortical phenotype similar to the Foxc1-null mutants. Lastly, in utero RA treatment rescued the cortical phenotype in Foxc1 mutants. These results establish RA as a potent, meningeal-derived cue required for successful corticogenesis.

The growth-factor-like phospholipid lysophosphatidic acid (LPA) mediates a wide variety of biological functions. We recently reported the cloning of the first G-protein-coupled receptor for LPA, called ventricular zone gene-1 (vzg-1/lpA1/edg-2) because its embryonic central nervous system (CNS) expression is restricted to the neocortical ventricular zone (Hecht et al. [1996] J. Cell Biol. 135:1071-1083). Vzg-1 neural expression diminishes at the end of the cortical neurogenetic period, just before birth. Here, we have investigated the subsequent reappearance of vzg-1 expression in the postnatal murine brain, by using in situ hybridization and northern blot analyses. Vzg-1 expression was undetectable by in situ hybridization at birth, but reappeared in the hindbrain during the 1st postnatal week. Subsequently, expression expanded from caudal to rostral, with peak expression observed around postnatal day 18. At all postnatal ages, vzg-1 expression was concentrated in and around developing white matter tracts, and its expansion, peak, and subsequent downregulation closely paralleled the progress of myelination. Double-label in situ hybridization studies demonstrated that vzg-1-expressing cells co-expressed mRNA encoding proteolipid protein (PLP), a mature oligodendrocyte marker, but not glial fibrillary acidic protein (GFAP), an astrocyte marker. Consistent with this, vzg-1 mRNA expression was reduced by 40% in the brains of jimpy mice, which exhibit aberrant oligodendrocyte differentiation and cell death. Together with our characterization of vzg-1 during cortical neurogenesis, these data suggest distinct pre- and postnatal roles for LPA in the development of neurons and oligodendrocytes and implicate lysophospholipid signaling as a potential regulator of myelination.

The human glycoprotein hormone ot-subunit gene is expressed in two quite dissimilar tissues, the placenta and anterior pituitary. Tissue-specific expression is determined by combinations of elements, some ofwhich are common and others of which are specific to each tissue. In the placenta, a composite enhancer confers specific expression. It contains four protein-binding sites: two cyclic AMP (cAMP) response elements that bind CREB, a trophoblast-specific element that binds TSEB, and a sequence motif, AGATAA, that matches the consensus binding site for a family of transcription factors termed the GATA-binding proteins. In pituitary gonadotropes, the cAMP response elements remain important for expression, TSEB is absent, and elements further upstream participate in tissue-specific expression. Here we establish a regulatory role for the GATA element in both the placenta and pituitary by demonstrating that a mutation of this element decreases ot-subunit gene expression 15-fold in JEG-3 human placental cells and 2.5-fold in otT3-1 mouse pituitary gonadotropes. In JEG-3 cells, human GATA-2 (hGATA-2) and hGATA-3 are highly expressed and both proteins bind to the ot-subunit gene GATA element. In otT3-1 cells, the GATA motif is bound by mouse GATA-2 (mGATA-2) and an mGATA-4-related protein. Cotransfection of hGATA-2 or hGATA-3 into otT3-1 cells activates the ft-subunit gene threefold. These studies establish a role for the GATA-binding proteins in placental and pituitary ft-subunit gene expression, significantly expanding the known target genes of GATA-2, GATA-3, and perhaps GATA-4.

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