Mammalian Developmental Genetics & Animal Models Of Dis

哺乳动物发育遗传学

• 批准号: 7198240
• 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/7198240
• 关键词: cell growth regulation; cofactor; developmental genetics; developmental neurobiology; disease /disorder model; functional /structural genomics; gene expression; gene targeting; genetically modified animals; homeobox genes; laboratory mouse; mammalian embryology; molecular genetics; pluripotent stem cells; prosencephalon; protein structure function; transcription factor

The Section on Mammalian Molecular Genetics (SMMG) studies the molecular genetics of embryonic development. Our interest is focused on developmental controls exerted by LIM-homeodomain transcription factors and their cofactors, and by Wnt pathway regulators. In a separate project we describe our current efforts to reverse the differentiated state of somatic cells to a state of pluripotency. Members of the Lhx gene family encode LIM-homeodomain transcription factors that control important aspects of embryonic development. Studies on the mechanism of Lhx gene function led to our discovery of two obligatory cofactors that mediate the action of the LIM-homeodomain proteins. The first of these, Ldb1, interacts physically and genetically with Lhx gene products. Each Lhx gene family examined so far is dependent on Ldb1 cofactor activity. We found that embryos carrying Ldb1 null mutations exhibit severe early developmental defects. Even the in vitro differentiation of embryonic stem cells is severely affected by the lack of Ldb1 activity. This prompted us to ask whether Ldb1 is an obligatory interaction partner of each of the diverse array of Lhx gene products during mammalian development. To this end, our current efforts are directed toward examining Ldb1 action in specific regions of the developing embryo. If Ldb1 is indeed required to render each of the individual Lhx gene products active, selective Ldb1 gene inactivation should allow us to investigate the concerted activity of Lhx gene products in targeted areas of the developing embryo that lack Ldb1 activity. The main tool of our current protocol is a mouse mutant in which we inserted a floxed Ldb1 gene that can be inactivated in cells targeted by the Cre recombinase. If, as postulated, Ldb1 is obligatory for all LIM-homeodomain factor activity, the resulting phenotypes are expected to provide much needed information on the combined action of individual Lhx genes in specific target fields of the developing embryo. We plan to target an array of organotypic regions. In the first experiment of this kind we used a transgenic line that expresses Cre under the control of an enhancer element of the Nestin gene to delete the Ldb1 gene in the developing central nervous system. Our analysis of the resulting mutant revealed a defect in the specification of Purkinje cells in the developing cerebellum. Two Lhx genes, Lhx1 and Lhx5, are prominently expressed in the cerebellum of wild type embryos. It thus seemed likely that the products of these two genes were inactive in cells lacking Ldb1 activity. And indeed, when we generated mouse mutants with null deletions of both Lhx1 and Lhx5, we were able to copy the Purkinje cell phenotype, indicating that specific aspects of cerebellar development are controlled by the interaction of Ldb1 with these two LIM-homeodomain transcription factors. Ssdp1 is a second cofactor of Lhx transcriptional activity originally detected in our laboratory. Like Ldb1 it has been physically and genetically linked to Lhx gene action. In a recent collaboration with Dr. Sasaki and his colleagues in Japan we showed that Ssdp1 is an essential component of Ldb1-Lhx1 transcriptional activity during head formation in the mouse embryo. We presently examine the spectrum of Ssdp target genes in an effort to describe the extent of developmental controls exerted by this cofactor. Members of the Dkk family act as inhibitors of the canonical Wnt pathway during embryogenesis. The importance of Dkk1 in mediating transcriptional activity of LIM-homeodomain proteins and other transcription factors during head induction was demonstrated in our previous experiments. Our studies of the past year have uncovered an exciting new aspect of Dkk action. We studied mutant mice that lack the function of Dkk2 and noted that this gene controls the integrity of the ocular surface epithelium via regulation of the canonical Wnt pathway. In the absence of Dkk2 function, the epithelium of the cornea that covers the ocular surface is completely transformed into a stratified epithelium containing hair follicles and sebaceous glands. Stem cells that maintain corneal integrity throughout life reside in the limbus of the eye. It is here that Dkk2 is prominently expressed. This suggests that the selection and fate of these stem cells is tightly controlled by Wnt ligands and their regulators. Novel efforts are directed toward reprogramming somatic cells to an embryonic state of unlimited growth and multifaceted differentiation potential. In a direct search for a suitable source of reprogramming activity several laboratories, including our own, fused mouse ES cells with somatic cells and were able to demonstrate that markers of pluripotency, notably the transcription factor Oct4, were reactivated in the somatic nucleus of the resulting hybrid cells. The task ahead is to create conditions whereby the ES cell nucleus changes the epigenetic state of the hybrid cell but is prevented from participating in the formation of daughter cell nuclei. Pretreatment of the ES cell with inhibitors of DNA synthesis prior to fusion or mechanical removal of the ES cell nucleus from the hybrid cell are among the most promising of a host of attempts to generate a cell hybrid that will selectively replicate the somatic cell genome in a reprogrammed epigenetic state so that multipotent progeny cells can be propagated. In terms of potential benefits of this approach for future avenues of stem cell therapy, the line of thinking is as follows. Expose a patient?s somatic cell to factors that bring it back to a pluripotent stage comparable to that of an embryonic stem cell. Unlike most cells in our body, this cell would replicate fast and could be propagated under suitable in vitro culture conditions to generate a large number of progeny cells, all retaining pluripotency. When subjected to suitable differentiation protocols, these cells would give rise to a wide variety of differentiated cells available for use in tissue replacement protocols. Most important, these reprogrammed cells carry the patient?s own set of histocompatibility antigens on their surface, thus drastically reducing the risk of graft rejection, and they are not derived from an embryo, hence not subject to ethical concerns that restrict present human embryonic cell research. Clearly, the importance of experiments aimed at restoring stemness to somatic cells cannot be overstated.

哺乳动物分子遗传学 (SMMG) 部门研究胚胎发育的分子遗传学。我们的兴趣集中在 LIM 同源域转录因子及其辅助因子以及 Wnt 通路调节因子所施加的发育控制上。在一个单独的项目中，我们描述了我们目前将体细胞的分化状态逆转为多能性状态的努力。 Lhx 基因家族的成员编码 LIM 同源域转录因子，控制胚胎发育的重要方面。对 Lhx 基因功能机制的研究使我们发现了介导 LIM 同源结构域蛋白作用的两个必需辅助因子。其中第一个是 Ldb1，与 Lhx 基因产物在物理和遗传上相互作用。迄今为止检查的每个 Lhx 基因家族都依赖于 Ldb1 辅因子活性。我们发现携带 Ldb1 无效突变的胚胎表现出严重的早期发育缺陷。 Ldb1 活性的缺乏甚至会严重影响胚胎干细胞的体外分化。这促使我们询问 Ldb1 是否是哺乳动物发育过程中各种 Lhx 基因产物的必需相互作用伙伴。为此，我们目前的努力旨在检查 Ldb1 在发育胚胎特定区域的作用。如果 Ldb1 确实需要使每个单独的 Lhx 基因产物活化，选择性 Ldb1 基因失活应该使我们能够研究 Lhx 基因产物在缺乏 Ldb1 活性的发育胚胎的目标区域中的协同活性。我们当前协议的主要工具是小鼠突变体，其中我们插入了 floxed Ldb1 基因，该基因可以在 Cre 重组酶靶向的细胞中失活。如果正如假设的那样，Ldb1 对于所有 LIM 同源域因子活性都是必需的，那么由此产生的表型有望提供关于单个 Lhx 基因在发育胚胎的特定目标区域中的联合作用的急需信息。我们计划针对一系列器官型区域。在第一个此类实验中，我们使用了在 Nestin 基因增强子元件控制下表达 Cre 的转基因系，以删除发育中的中枢神经系统中的 Ldb1 基因。我们对所得突变体的分析揭示了发育中小脑中浦肯野细胞规格的缺陷。两个 Lhx 基因 Lhx1 和 Lhx5 在野生型胚胎的小脑中显着表达。因此，这两个基因的产物在缺乏 Ldb1 活性的细胞中似乎是失活的。事实上，当我们产生 Lhx1 和 Lhx5 均缺失的小鼠突变体时，我们能够复制浦肯野细胞表型，表明小脑发育的特定方面是由 Ldb1 与这两个 LIM 同源域转录因子的相互作用控制的。 Ssdp1 是我们实验室最初检测到的 Lhx 转录活性的第二个辅助因子。与 Ldb1 一样，它在物理和遗传上与 Lhx 基因的作用有关。在最近与 Sasaki 博士及其日本同事的合作中，我们发现 Ssdp1 是小鼠胚胎头部形成过程中 Ldb1-Lhx1 转录活性的重要组成部分。我们目前检查了 Ssdp 靶基因的范围，以描述该辅助因子发挥的发育控制程度。 Dkk 家族成员在胚胎发生过程中充当经典 Wnt 通路的抑制剂。我们之前的实验证明了 Dkk1 在头部诱导过程中介导 LIM 同源域蛋白和其他转录因子转录活性中的重要性。我们过去一年的研究揭示了 Dkk 行动的一个令人兴奋的新方面。我们研究了缺乏 Dkk2 功能的突变小鼠，并注意到该基因通过调节经典 Wnt 通路来控制眼表上皮的完整性。在缺乏 Dkk2 功能的情况下，覆盖眼表的角膜上皮完全转变为含有毛囊和皮脂腺的复层上皮。终生维持角膜完整性的干细胞位于眼睛的边缘。 Dkk2 正是在这里显着表达。这表明这些干细胞的选择和命运受到 Wnt 配体及其调节因子的严格控制。 新的努力旨在将体细胞重新编程为无限生长和多方面分化潜力的胚胎状态。在直接寻找合适的重编程活性来源的过程中，包括我们自己的实验室在内的多个实验室将小鼠 ES 细胞与体细胞融合，并能够证明多能性标记物，特别是转录因子 Oct4，在所得杂交细胞的体细胞核中被重新激活。未来的任务是创造条件，使 ES 细胞核改变杂交细胞的表观遗传状态，但阻止其参与子细胞核的形成。在融合之前用 DNA 合成抑制剂对 ES 细胞进行预处理，或者从杂交细胞中机械去除 ES 细胞核，是产生细胞杂交体的众多尝试中最有希望的一种，该细胞杂交体将选择性地以重编程的表观遗传状态复制体细胞基因组，从而可以繁殖多能子代细胞。就这种方法对未来干细胞治疗途径的潜在好处而言，思路如下。将患者的体细胞暴露于使其恢复到与胚胎干细胞相当的多能阶段的因素中。与我们体内的大多数细胞不同，这种细胞可以快速复制，并且可以在合适的体外培养条件下繁殖，产生大量后代细胞，并且全部保留多能性。当接受合适的分化方案时，这些细胞将产生多种可用于组织替代方案的分化细胞。最重要的是，这些重编程的细胞在其表面携带患者自己的一组组织相容性抗原，从而大大降低了移植物排斥的风险，并且它们不是源自胚胎，因此不受限制当前人类胚胎细胞研究的伦理问题的影响。显然，旨在恢复体细胞干性的实验的重要性怎么强调都不为过。