Using a floxed allele of the obligatory Ldb1 coregulator of Lhx gene function, we extended our analysis of transcriptional controls exerted by LIM-homeodomain factors on the development of a diverse array of structures in the nascent forebrain. Nkx2.1-Cre mediated deletion of Ldb1 in the Major Ganglionic Eminence (MGE) affected development of a number of meuronal precursors derived from this structure. Our study has shown that Ldb1 plays an essential role in the formation of several important nuclei in this forebrain region. Some of the Ldb1/Nkx2.1-Cre mutants survive to adulthood because deletion of Ldb1 is restricted to populations of cells derived from the Nkx2.1 lineage. This has provided us with an opportunity to analyze behavioral consequences resulting from a loss of Nkx2.1 lineage-derived neurons in the forebrain. We detected a defect in the nesting behavior of these mutants. Furthermore, a close examination of the mutant hypothalamus showed that Ldb1 plays an essential role in the formation of several important nuclei in this region of the forebrain, including the arcuate nucleus, the ventromedial nucleus (VMH), and the paraventricular nucleus. Nkx2.1-Cre mediated deletion of Ldb1 resulted in a loss of NPY+ and POMC+ neurons in the arcuate nucleus and a disorganization of the VMH. We observed that the Ldb1/Nkx2.1-Cre mutants accumulate more fat, resulting in characteristic distortions of the body shape. This is consistent with earlier reports pointing to a role of the ventral-medial hypothalamus in the regulation of energy balance and metabolism. Several members of the Lhx gene family whose action is mediated by Ldb1 are also expressed in the developing hypothalamus. We are currently analyzing mutants that lack the function of these genes in an effort to determine their contribution to the development of the hypothalamus. The discovery of transcription factor-mediated reprogramming of somatic cells to an induced pluripotent stem (iPS) cell state has created a powerful technological advance toward cell and tissue regeneration and cell replacement therapy. A prominent goal of iPS technology is the derivation of disease-specific iPS cells, allowing for the study of cell-based disease pathology and the identification of novel drug therapies. Smith-Lemli-Opitz syndrome (SLOS) is a recessive syndrome caused by a spectrum of mild to severe mutations of the 7-dehydrocholesterol reductase (DHCR7) gene. This enzyme catalyses the penultimate step in endogenous cholesterol synthesis. Hence, DHCR7 mutations significantly impair intrinsic cholesterol supply, resulting in multiple malformations and central nervous system dysfunction. DHCR7 mutant mice die soon after birth, limiting the utility of animal models for the study of this disorder. In SLOS patients, cholesterol supplementation can alleviate some symptoms, yet dietary cholesterol fails to cross the blood-brain-barrier. Thus, there is currently no established therapeutic regimen to address behavioral and learning problems in these patients. Neural derivatives produced from SLOS patient-derived iPS cells would represent ideal tools for studying disease pathology and identifying new therapies. We have gained proficiency in generating human iPS cells, verifying their pluripotency and subjecting them to neural differentiation programs. The lab utilizes viral vectors encoding the transcription factors Sox2, Oct-4 and Klf4 to reprogram fibroblasts derived from skin biopsies of SLOS patients or healthy individuals to the iPS cell state. We have been able to generate a number of iPS cell lines from skin fibroblasts of SLOS patients collected by F.D. Porter, MD, PhD, at NICHD. These fibroblasts carry either mild to classical or severe DHCR7 mutations. These iPS lines have been thoroughly analyzed for cell surface marker and pluripotent gene expression, germ layer differentiation and karyotypic integrity. Cognitive and behavioral impairments suggest functional deficits in both neural progenitor and neuronal populations of the cerebral cortex of SLOS patients. We are therefore in the process of generating neural progenitors as well as forebrain and dopaminergic neurons from DHCR7 mutant and control iPS lines. Differentiation, progenitor proliferation, synaptic activity and sterol synthesis will be analyzed in the iPS-derived derivates at various stages of neural differentiation to characterize impairments of sterol metabolism. In support of an ongoing NICHD intramural drug trial, we will also test the effect of the HMG-CoA reductase inhibitor simvastatin on sterol levels in these cells. If successful, this study will advance our understanding of the cellular basis of the SLOS disorder and open novel avenues of drug therapy.

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