Mammalian Developmental Genetics and Stem Cells

利用LHX基因功能必需的LDB1辅助调节因子的FLOG等位基因，我们扩展了对LIM同源结构域因子在新生前脑中各种结构的发育中所起的转录控制作用的分析。Nkx2.1-Cre介导的Ldb1在主要神经节隆起(MGE)的缺失影响了许多源自该结构的中枢神经前体的发育。我们的研究表明，LDB1在这一前脑区几个重要核团的形成中起着至关重要的作用。一些Ldb1/Nkx2.1-Cre突变体存活到成年，因为Ldb1的缺失仅限于来自Nkx2.1谱系的细胞群体。这为我们提供了一个机会来分析前脑Nkx2.1谱系衍生神经元丢失所造成的行为后果。我们在这些突变体的筑巢行为中发现了一个缺陷。此外，对突变的下丘脑的仔细研究表明，LDB1在前脑这一区域几个重要核团的形成中起着重要作用，包括弓状核、腹内侧核(VMH)和室旁核。Nkx2.1-Cre介导的LDB1缺失导致弓状核NPY+和POMC+神经元的丢失和VMH的紊乱。我们观察到，Ldb1/Nkx2.1-Cre突变体积累了更多的脂肪，导致了典型的体型扭曲。这与早期关于下丘脑腹侧内侧在能量平衡和新陈代谢调节中的作用的报道是一致的。Lhx基因家族的几个成员也在发育中的下丘脑表达，其作用由LDB1介导。我们目前正在分析缺乏这些基因功能的突变体，以努力确定它们对下丘脑发育的贡献。 转录因子介导的体细胞重编程为诱导多能干细胞(IPS)状态的发现，为细胞和组织再生以及细胞替代治疗创造了强大的技术进步。IPS技术的一个突出目标是衍生疾病特异性iPS细胞，允许研究基于细胞的疾病病理学和识别新的药物疗法。Smith-Lemli-Opitz综合征(SLOS)是由7-脱氢胆固醇还原酶(DHCR7)基因轻微到严重突变引起的一种隐性综合征。这种酶催化内源性胆固醇合成的倒数第二步。因此，DHCR7突变显著损害内在胆固醇供应，导致多发性畸形和中枢神经系统功能障碍。DHCR7突变小鼠在出生后不久就会死亡，这限制了动物模型对这种疾病的研究。在SLOS患者中，补充胆固醇可以缓解一些症状，但饮食中的胆固醇无法通过血脑屏障。因此，目前还没有确定的治疗方案来解决这些患者的行为和学习问题。SLOS患者来源的iPS细胞产生的神经衍生物将成为研究疾病病理学和确定新疗法的理想工具。我们已经熟练地生成了人类iPS细胞，验证了它们的多能性，并对它们进行了神经分化计划。该实验室利用编码转录因子Sox2、Oct-4和Klf4的病毒载体，将来自SLOS患者或健康人皮肤活检的成纤维细胞重新编程为iPS细胞状态。我们已经能够从NICHD的F.D.Porter，MD，PhD收集的SLOS患者的皮肤成纤维细胞中培养出许多iPS细胞系。这些成纤维细胞携带轻微到经典或严重的DHCR7突变。对这些iPS系进行了彻底的细胞表面标记和多能基因表达、胚层分化和核型完整性的分析。认知和行为障碍表明SLOS患者大脑皮层的神经前体和神经元群体都存在功能缺陷。因此，我们正在从DHCR7突变株和对照iPS系中产生神经前体细胞以及前脑和多巴胺能神经元。在神经分化的不同阶段，将分析iPS衍生产物的分化、祖细胞增殖、突触活性和类固醇合成，以表征类固醇代谢的损害。为了支持正在进行的NICHD室壁内药物试验，我们还将测试HMG-CoA还原酶抑制剂辛伐他汀对这些细胞中固醇水平的影响。如果成功，这项研究将促进我们对SLOS障碍的细胞学基础的理解，并开辟药物治疗的新途径。

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.