Mammalian Developmental Genetics And Animal Models

哺乳动物发育遗传学和动物模型

• 批准号: 6811582
• 负责人: HEINER WESTPHAL
• 金额: --
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6811582
• 关键词: cell growth regulation; cofactor; developmental genetics; developmental neurobiology; disease /disorder model; functional /structural genomics; gene expression; gene targeting; homeobox genes; laboratory mouse; mammalian embryology; prosencephalon; protein structure function; transcription factor

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.

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关键步骤的基因进行功能分析。转录因子是执行身体计划的一系列分子事件的主要参与者。我们的小组使用功能丧失方法来详细研究 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早期模式事件、神经系统的发育和协调器官组装。最近，我们确定了两个基因家族，分别称为 Ldb 和 Ssdp，它们编码 Lhx 基因作用的必需辅助因子。 目前的工作重点是 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。此前，我们培育了一种 LIM-同源盒基因 Lhx8 零缺失的敲除小鼠品系。我们对突变小鼠的初步分析揭示了 Lhx8 在上颚发育中的重要作用。由于该基因在发育中的腹侧前脑中也大量表达，因此我们最近检查了具有 Lhx8 基因靶向无效突变的小鼠的大脑。在这些敲除突变体中，腹侧端脑胆碱能神经元的发育受到严重损害。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输入的主要来源。此外，皮质下前脑其他几个区域（包括尾壳核、隔膜和大细胞视前核）的胆碱能神经元数量减少。我们的标记分析表明，祖细胞是在缺乏 Lhx8 基因功能的情况下形成的。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至关重要。端脑胆碱能投射神经元的丧失是阿尔茨海默病等神经退行性疾病的标志，并且可能在这些患者的认知障碍中发挥作用。因此，Lhx8 无效突变小鼠构成了记忆和认知背景下胆碱能投射神经元功能研究的有价值的模型。 另一项正在进行的研究涉及 LIM 同源框基因 Lhx2 在大脑发育中的功能。我们最初的 Lhx2 敲除研究表明，该基因对于眼睛发育和端脑的形成至关重要。最近，我们观察到在无效突变胚胎中腹侧间脑和垂体的发育也受到损害，这表明 Lhx2 在前脑发育中具有重要的附加作用。 该科还继续对 LIM-同源域蛋白及其相关辅因子形成的蛋白复合物进行功能评估。我们发现的 Ssdp 辅因子被认为与胚胎发育早期阶段活跃的许多转录因子形成复合物。尽管脊椎动物 Ssdp 基因家族有几个密切相关的成员，但果蝇 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得果蝇系统对于全基因组搜索下游基因（其转录受Ssdp和相关蛋白调节）有吸引力。由于果蝇 ssdp 基因中的功能丧失突变会导致蛹早期的致死性，因此我们选择将研究重点放在蛹阶段之前的第三龄幼虫阶段晚期。该分析是与 NIDDK 的 Brian Oliver 博士实验室合作进行的，使用果蝇基因组扩增子微阵列，目前正在评估由此产生的大量数据，以确定 ssdp 介导的转录调控的直接目标。 在一个不相关的项目中，我们的部门在无精子症患者中发现了 SYCP3 基因中的单核苷酸缺失，该缺失导致减数分裂期间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滞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，这是一种参与胚胎发育、细胞周期调节和肿瘤发生的叉头转录因子。 我们的团队还参与了一系列旨在通过基因敲除揭示基因功能的合作项目。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细胞受体信号传导的负调节因子； (3)一氧化氮在成年哺乳动物大脑中充当细胞增殖的负调节剂； (4) Ib型糖原累积病中缺乏的6-磷酸糖转运蛋白是一种重要的免疫调节蛋白。 最后，在几本专着中，我总结了（a）目前有关参与脑下垂体形成的基因的知识状况和（b）干细胞研究方面的国际考虑。