One major function of the hypothalamus is to maintain homeostasis by modulating the secretion of pituitary hormones. The paraventricular (PVN) and supraoptic (SON) nuclei are major integration centers for the output of the hypothalamus to the pituitary. The bHLH-PAS transcription factor SIM1 is crucial for the development of several neuroendocrine lineages within the PVN and SON. bHLH-PAS proteins require heterodimerization for their function. ARNT, ARNT2, and BMAL1 are the three known general heterodimerization partners for bHLH-PAS proteins. Here. we provide evidence that Sim1 and Arnt2 form dimers in vitro, that they are co-expressed in the PVN and SON, and that their loss of function affects the development of the same sets of neuroendocrine cell types within the PVN and SON. Together, these results implicate ARNT2 as the in vivo dimerization partner of SIM1 in controlling the development of these neuroendocrine lineages. (C) 2000 Elsevier Science Ireland Ltd. All rights reserved.

The secreted protein sonic hedgehog (Shh) is required to establish patterns of cellular growth and differentiation within ventral regions of the developing CNS, The expression of Shh in the two tissue sources responsible for this activity, the axial mesoderm and the ventral midline of the neural tube, is regulated along the anteroposterior neuraxis, Separate cis-acting regulatory sequences have been identified which direct Shh expression to distinct regions of the neural tube, supporting the view that multiple genes are involved in activating Shh transcription along the length of the CNS, We show here that the activity of one Shh enhancer, which directs reporter expression to portions of the ventral midbrain and diencephalon, overlaps both temporally and spatially with the expression of Sim2, Sim2 encodes a basic helix-loop-helix (bHLH-PAS) PAS domain containing transcriptional regulator whose Drosophila homolog, single-minded, is a master regulator of ventral midline development. Both vertebrate and invertebrate Sim family members were found sufficient for the activation of the Shh reporter as well as endogenous Shh mRNA, Although Shh expression is maintained in Sim2(-/-) embryos, it was determined to be absent from the rostral midbrain and caudal diencephalon of embryos carrying a dominant-negative transgene that disrupts the function of bNLH-PAS proteins. Together, these results suggest that bHLH-PAS family members are required for the regulation of Shh transcription within aspects of the ventral midbrain and diencephalon.

Control of cell proliferation is essential to generate the defined form of a multi-cellular organism. While much is known about the regulators for cell cycle progression, relatively little is known about the start of growth arrest. Growth arrest (GO) is defined as a cell in a metabolically active but proliferation-quiescent state (reviewed in Baserga (1985) The Biology of Cell Reproduction), typically induced by serum starvation in vitro. Using subtractive hybridization, Schneider et al. (Cell 54 (1988) 787) identified six genes (Gas1 through Gas6) whose expressions are upregulated in serum-deprived NIH3T3 cells. Among the Gas genes. Gas1 is the only one that can cause growth arrest when expressed in cultured cell (Cell 70 (1995) 595: int. J. Cancer 9 (1998, 569). Here, we describe for the first time the expression pattern of Gas1 during mouse embryogenesis. Our data reveal that Gas1 is expressed in many regions that the cells are actively proliferating and suggest that it may have other roles during development than negatively regulating cell proliferation. Furthermore, we have cloned the chick GAS1 gene and documented the similarity and divergence of Gas1 gene expression patterns between the two species. (C) 2001 Elsevier Science Ireland Ltd. All rights reserved.

The bHLH-PAS transcription factor SIM1 is required for the development of the paraventricular nucleus (PVN) of the hypothalamus. Mice homozygous for a null allele of Sim1 (Sim1(-/-)) lack a PVN and die perinatally. In contrast, we show here that Sim1 heterozygous mice are viable but develop early-onset obesity, with increased linear growth, hyperinsulinemia and hyperleptinemia. Sim1(+/-) mice are hyperphagic but their energy expenditure is not decreased, distinguishing them from other mouse models of early-onset obesity such as deficiencies in leptin and melanocortin receptor 4. Quantitative histological comparison with normal littermates showed that the PVN of Sim1(+/-) mice contains on average 24% fewer cells without a selective loss of any identifiable major cell type. Since acquired lesions in the PVN also induce increased appetite without a decrease in energy expenditure, we propose that abnormalities of PVN development cause the obesity of Sim1(+/-) mice. Severe obesity was described recently in a patient with a balanced translocation disrupting SIM1. Pathways controlling the development of the PVN thus have the potential to cause obesity in both mice and humans.

Postnatal cerebellum development involves the generation of granule cells and Bergmann glias (BGs). The granule cell precursors are located in the external germinal layer (EGL) and the BG precursors are located in the Purkinje layer (PL). BGs extend their glial fibers into the EGL and facilitate granule cells' inward migration to their final location. Growth arrest specific gene 1 (Gas1) has been implicated in inhibiting cell-cycle progression in cell culture studies (G. Del Sal et al., 1992, Cell 70, 595-607). However, its growth regulatory function in the CNS has not been described. To investigate its role in cerebellar growth, we analyzed the Gas1 mutant mice. At birth, wild-type and mutant mice have cerebella of similar size; however, mature mutant cerebella are less than half the size of wild-type cerebella. Molecular and cellular examinations indicate that Gas1 mutant cerebella have a reduced number of granule cells and BG fibers. We provide direct evidence that Gas1 is required for normal levels of proliferation in the EGL and the PL, but not for their differentiation. Furthermore, we show that Gas1 is specifically and coordinately expressed in both the EGL and the BGs postnatally. These results support Gas1 as a common genetic component in coordinating EGL cell and BG cell proliferation, a link which has not been previously appreciated. (C) 2001 Academic Press.

During eye development, retinal pigmented epithelium (RPE) and neural retina (NR) arise from a common origin, the optic vesicle. One of the early distinctions of RPE from NR is the reduced mitotic activity of the RPE. Growth arrest specific gene 1 (Gas1) has been documented to inhibit cell cycle progression in vitro (G. Del Sal et al., 1992, Cell 70, 595-607). We show here that the expression pattern of Gas1 in the eye supports its negative role in RPE proliferation. To test this hypothesis, we generated a mouse carrying a targeted mutation in the Gas1 locus. Gas1 mutant mice have microphthalmia. Histological examination revealed that the remnant mutant eyes are ingressed from the surface with minimal RPE and lens, and disorganized eyelid, cornea, and NR. Analysis of the Gas1 mutant indicates that there is overproliferation of the outer layer of optic cup (E10.5) immediately after the initial specification of the RPE. This defect is specific to the ventral region of the RPE. Using molecular markers for RPE (Mi and Tyrp2) and NR (Math5), we demonstrate that there is a gradual loss of AV and Tyrp2 expression and an appearance of Math5 expression in the mutant ventral RPE region, indicating that this domain becomes respecified to NR. This "ectopic" NR develops as a mirror image of the normal NR and is entirely of ventral identity. Our data not only support Gas1's function in regulating cell proliferation, but also uncover an unexpected regional-specific cell fate change associated with dysregulated growth. Furthermore, we provide evidence that the dorsal and ventral RPEs are maintained by distinct genetic components. (C) 2001 Academic Press.

The dorsal-ventral polarity of the somite is controlled by antagonistic signals from the dorsal neural tube/surface ectoderm, mediated by WNTs, and from the ventral notochord, mediated by sonic hedgehog (SHH). Each factor can act over a distance greater than a somite diameter in vitro, suggesting they must limit each other's actions within their own patterning domains in vivo. We show here that the growth-arrest specific gene 1 (Gas1), which is expressed in the dorsal somite, is induced by WNTs and encodes a protein that can bind to SHH. Furthermore, ectopic expression of Gas1 in presomitic cells attenuates the response of these cells to SHH in vitro. Taken together, these data suggest that GAS1 functions to reduce the availability of active SHH within the dorsal somite.

The mouse genome contains two Sim genes, Sim1 and Sim2. They are presumed to be important for central nervous system (CNS) development because they are homologous to the Drosophila single-minded (sun) gene, mutations in which cause a complete loss of CNS midline cells. In the mammalian CNS, Sim2 and Sim1 are coexpressed in the paraventricular nucleus (PVN). While Sim1 is essential for the development of the PVN (J. L. Michaud, T. Rosenquist, N. R. May, and C.-M. Fan, Genes Dev. 12:3264-3275, 1998), we report here that Sim2 mutant has a normal PVN. Analyses of the Sim1 and Sim2 compound mutants did not reveal obvious genetic interaction between them in PVN histogenesis. However, Sim2 mutant mice die within 3 days of birth due to lung atelectasis and breathing failure. We attribute the diminished efficacy of lung inflation to the compromised structural components surrounding the pleural cavity, which include rib protrusions, abnormal intercostal muscle attachments, diaphragm hypoplasia, and pleural mesothelium tearing. Although each of these structures is minimally affected, we propose that their combined effects lead to the mechanical failure of lung inflation and death. Sim2 mutants also develop congenital scoliosis, reflected by the unequal sizes of the left and right vertebrae and ribs. The temporal and spatial expression patterns of Sim2 in these skeletal elements suggest that Sim2 regulates their growth and/or integrity.