Mammalian Developmental Genetics And Animal Models

哺乳动物分子遗传学部分对控制哺乳动物胚胎植入后发育的关键步骤的基因进行了功能分析。转录因子是实施身体计划的一连串分子事件中的主要参与者。我们小组使用功能缺失的方法详细研究了LIM/同源域(LHX)类转录因子的功能。 多年来，我们的研究表明，LIM同源结构域蛋白参与了早期的模式事件、神经系统的发育和器官组装的协调。最近，我们发现了两个基因家族，分别命名为LDB和SSDP，它们编码lhx基因作用的必需辅因子。 目前的工作主要集中在LIM同源结构域蛋白LHX8和LHX2以及LIM结构域结合蛋白LDB1在胚胎发育中的功能。之前，我们产生了一种LIM-Homeobox基因Lhx8零缺失的基因敲除小鼠。我们对突变小鼠的初步分析揭示了Lhx8在味觉发育中的重要作用。由于该基因在发育中的腹侧前脑中也大量表达，我们最近检测了Lhx8基因定向零突变的小鼠的大脑。在这些基因敲除突变体中，腹侧端脑胆碱能神经元的发育严重受损。因此，这些小鼠缺乏基底核，而基底核是大脑皮层胆碱能神经元的主要来源。此外，皮质下前脑其他几个区域的胆碱能神经元的数量也减少了，包括尾壳核、隔核和大细胞视前核。我们的标记分析表明，在Lhx8基因功能缺失的情况下，祖细胞会形成。然而，该基因对于腹侧端脑中大多数胆碱能神经元的终末分化是必不可少的。端脑胆碱能投射神经元的丢失是阿尔茨海默病等神经退行性疾病的标志，可能在这些患者的认知障碍中发挥作用。因此，Lhx8缺失突变小鼠构成了一个在记忆和认知背景下研究胆碱能投射神经元功能的有价值的模型。 另一项正在进行的研究涉及LIM-Homeobox基因LHX2在大脑发育中的功能。我们最初的LHX2基因敲除研究表明，该基因对眼睛发育和端脑的形成是必不可少的。最近，我们观察到，在零突变胚胎中，腹侧间脑和垂体的发育也受到了损害，这表明LHX2在前脑发育中还有重要的额外作用。 该科还继续对LIM同源结构域蛋白质及其相关辅因子形成的蛋白质复合体进行功能评价。我们发现的SSDP辅助因子被认为与许多转录因子形成复合体，这些转录因子在胚胎发育的早期阶段是活跃的。脊椎动物SSDP基因家族有几个密切相关的成员，而果蝇SSDP基因是独一无二的。此外，该基因的几个突变等位基因的存在使果蝇系统对全基因组范围内寻找其转录受SSDP及其相关蛋白调控的下游基因具有吸引力。由于果蝇SSDP基因的功能缺失突变会在幼虫早期致死，因此我们选择将研究重点放在幼虫的三龄后期，也就是在幼虫期之前。这项分析是与NIDDK的Brian Oliver博士的实验室合作进行的，使用的是果蝇基因组扩增子微阵列，目前正在评估由此产生的丰富数据，以努力确定SSDP介导的转录调控的直接靶标。 在一个不相关的项目中，我们的部门已经在无精子症患者中发现了SYCP3基因的单核苷酸缺失，该缺失导致减数分裂过程中染色体配对所需的蛋白质被截断。这项研究代表了我们对早期减数分裂停止作为非梗阻性无精子症原因的分子基础的临床理解的重要进展。我们的实验表明，截短的蛋白质主要干扰野生型等位基因的功能，从而导致男性不育。另一个单独的项目导致了FoxP4的鉴定，FoxP4是一种叉头转录因子，参与胚胎发育、细胞周期调节和肿瘤发生。 我们团队还参与了一系列不同的合作项目，旨在通过基因敲除揭示基因功能。这些研究表明：(1)D5多巴胺受体参与血压调节；(2)加压素受体1a是B细胞受体信号的负调节因子；(3)一氧化氮是成年哺乳动物脑内细胞增殖的负调节因子；(4)糖原储存疾病Ib型缺乏的糖-6-磷酸转运体是一种重要的免疫调节蛋白。 最后，在几本专著中，我总结了(A)目前关于参与脑下垂体形成的基因的知识立场，以及(B)关于干细胞研究的国际考虑。

The Section on Mammalian Molecular Genetics carries out a functional analysis of genes that control key steps of post-implantation development in the mammalian embryo. Transcription factors are major players in the cascade of molecular events that implement the body plan. Our group uses a loss-of-function approach to study in detail the function of the LIM/homeodomain (Lhx) class of transcription factors. Over the years, our studies have shown that LIM-homeodomain proteins are involved in early patterning events, in the development of the nervous system, and in orchestrating organ assembly. More recently, we identified two gene families, termed Ldb and Ssdp, respectively, that encode obligatory cofactors of Lhx gene action. Current work is focused on functions of the LIM-homeodomain proteins Lhx8 and Lhx2 and the LIM-domain-binding protein Ldb1 in embryonic development. Previously, we generated a strain of knockout mice with a null-deletion of the LIM-homeobox gene Lhx8. Our initial analysis of the mutant mice revealed an important role for Lhx8 in development of the palate. Since this gene is also abundantly expressed in the developing ventral forebrain, we recently examined the brain of mice with a targeted null mutation of the Lhx8 gene. Development of cholinergic neurons in the ventral telencephalon is severely impaired in these knockout mutants. As a result, these mice lack the nucleus basalis, a major source of cholinergic neuron input to the cerebral cortex. In addition, the number of cholinergic neurons in several other regions of the subcortical forebrain including the caudate putamen, septum, and magnocellular preoptic nucleus is reduced. Our marker analysis revealed that progenitor cells form in the absence of Lhx8 gene function. However, the gene is essential for terminal differentiation of the majority of cholinergic neurons in the ventral telencephalon. The loss of telencephalic cholinergic projection neurons is a hallmark of neurodegenerative disorders such as Alzheimer's disease and is likely to play a role in the cognitive impairments of these patients. For this reason, the Lhx8 null mutant mouse constitutes a valuable model for functional studies of cholinergic projection neurons in the context of memory and cognition. Another ongoing study concerns the function of the LIM-homeobox gene Lhx2 in brain development. Our initial Lhx2 knockout study had shown that this gene is essential for eye development and the formation of the telencephalon. More recently we observed that development of the ventral diencephalon and pituitary is also impaired in the null mutant embryo, indicating an important additional role of Lhx2 in forebrain development. The Section also continued its functional evaluation of protein complexes formed by LIM-homeodomain proteins and their associated cofactors. The Ssdp co-factors that we discovered are thought to form complexes with many transcription factors active during early stages of embryonic development. Whereas the vertebrate Ssdp gene family has several closely related members, the Drosophila ssdp gene is unique. Moreover, the existence of several mutant alleles of this gene makes the Drosophila system attractive for a genome wide search for downstream genes whose transcription is regulated by Ssdp and associated proteins. Since loss-of-function mutations in the Drosophila ssdp gene cause lethality at early pupal stages we chose to focus our search on the late third instar larval stage which precedes the pupal stage. The analysis was carried out in collaboration with the laboratory of Dr. Brian Oliver at NIDDK, using Drosophila genomic amplicon micro arrays, and the resulting wealth of data is currently being evaluated in an effort to identify direct targets of ssdp-mediated transcriptional regulation. In an unrelated project, our Section has identified in azoospermic patients a single nucleotide deletion in the SYCP3 gene that causes a truncation of a protein required for chromosomal pairing during meiosis. This study represents an important advance in our clinical understanding of the molecular basis of early meiotic arrest as a cause of non-obstructive azoospermia. Our experiments suggest that the truncated protein dominantly interferes with the function of the wild type allele and thus causes male infertility. Another separate project led to the identification of FoxP4, a forkhead transcription factor involved in embryonic development, cell cycle regulation and oncogenesis. Our group has also been involved in a diverse array of collaborative projects, aimed at revealing gene functions via knockout. These studies have shown that (1) the D5 dopamine receptor is involved in blood pressure regulation; (2) the vasopressin receptor 1a is a negative regulator of B cell receptor signaling; (3) nitric oxide acts as a negative regulator of cell proliferation in the adult mammalian brain; and (4) the glycose-6-phosphate transporter, deficient in glycogen storage disease type Ib, is an important immuno-modulatory protein. Finally, in a couple of monographs, I have summarized (a) the current stand of knowledge with regard to genes that participate in the formation of the pituitary gland and (b) international considerations with respect to stem cell research.