Mammalian Developmental Genetics And Animal Models Of Human Diseases

哺乳动物发育遗传学和人类疾病动物模型

• 批准号: 7594110
• 负责人: HEINER WESTPHAL
• 金额: $ 209.56万
• 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/7594110
• 关键词: Ablation; Adult; Alkaline Phosphatase; Alleles; Animal Model; Anterior; Area; Axon; Birth; Cell Communication; Cell Cycle; Cell Differentiation process; Cell Lineage; Cell Nucleus; Cells; Cerebellum; Code; Complex; Cytoplasm; Defect; Development; Distal; Dorsal; Embryo; Embryonic Development; Enzymes; Event; Family; Forebrain Development; Future; Gene Targeting; Generations; Genes; Genetic Transcription; Globus Pallidus; Growth; Hippocampus (Brain); Homeobox Genes; Homeodomain Proteins; Homeostasis; In Vitro; Interneurons; Intracellular Transport; Knock-out; Life; Ligands; Limb Bud; Limb Development; Limb structure; Link; Localized; Mediating; Mesenchymal; Movement; Mus; Mutant Strains Mice; Mutation; N-terminal; Neocortex; Neurons; Neurotransmitters; Nuclear; Nuclear Translocation; Numbers; Organ; Organism; Organogenesis; Pattern; Phase; Phenotype; Phosphorylation; Play; Positioning Attribute; Posture; Process; Purkinje Cells; Receptor Protein-Tyrosine Kinases; Research Project Grants; Role; Signal Pathway; Signal Transduction; Spinal; Staging; Stem cells; Structure; Telencephalon; Tissues; Transcriptional Regulation; Transgenes; Tyrosine; Tyrosine Kinase Inhibitor; Ventricular; Western Blotting; Work; body system; cell motility; cholinergic neuron; cofactor; developmental genetics; established cell line; granule cell; homeodomain; human disease; loss of function; member; mutant; progenitor; programs; research study; src-Family Kinases; transcription factor

This Section is focused on key developmental controls exerted by LIM-homeodomain (LIM-HD) transcription factors as the mouse embryo enters the phase of post-gastrulation assembly of organ systems. There is mounting evidence that the LIM-HD proteins play a key role in lineage specification by activating downstream genes that define the ground state of organ identity. The assembly of newly formed cell lineages into complex tissue structures depends on precise patterns of cell migration and cell-cell communication that involve all major signaling pathways. The transcriptional activity of LIM-HD proteins is mediated by two families of obligatory cofactors, termed Ldb and Ssdp, that become part of the LIM-HD complex controlling target gene transcription. In the work described below, we have analyzed different scenarios of tissue assembly in the developing mouse embryo. We discovered an intricate interplay between transcriptional regulation and secreted signal exchanges that accompanies organogenesis. Purkinje cells are major components of the neuronal network of the cerebellum essential for fine control of movement and posture. During embryonic development, these cells play an important role in controlling the proliferation of progenitors of granule cells, the other major type of neurons in the cerebellum. In this study we describe the process that controls differentiation of Purkinje cells from earlier precursors. We noted that two closely related LIM-homeobox genes, Lhx1 and Lhx5, are expressed in the developing Purkinje cells soon after they exit the cell cycle and migrate out of the cerebellar ventricular zone. Double mutant mice lacking function of both Lhx1 and Lhx5 show a severe reduction in the number of Purkinje cells. In addition, targeted inactivation of Ldb1, which encodes an obligatory cofactor for all LIM-homeodomain proteins, resulted in a similar phenotype. This experiment provides conclusive evidence that these transcription regulators are essential for controlling Purkinje cell differentiation in the developing mammalian cerebellum. In a separate project, we joined our collaborators to investigate the role of the Lhx1/5 transcriptional apparatus in generating and maintaining the functional identity of a group of interneurons in the developing spinal chord. The establishment of a dorsal inhibitory neurotransmitter program in these interneurons was found to be critically dependent on an interaction of Lhx1/5 with members of the Pax family of homeobox genes. In order to study the role of Lhx genes in early limb development we targeted their obligatory cofactor Ldb1. A conditional knockout allele was generated by inserting two LoxP fragments into the Ldb1 locus flanking coding axons. Transgenes that target the Cre enzyme to the developing limb bud were used to delete Ldb1 function at he earliest possible stage of limb development. Patterning and growth during vertebrate limb development is controlled by three signaling centers that regulate limb patterning and growth along the proximo-distal, antero-posterior and dorso-ventral limb axes, respectively. Interactions among these signaling centers coordinate limb development. We found that Ldb1 plays a central role in the mechanism that coordinates these interactions via its interaction with distinct LIM homeodomain transcription factors, encoded by the Lhx genes Lhx2, Lhx9 and Lmx1b. The resulting transcription complexes integrate the signaling events that link limb patterning and outgrowth along all three axes. Simultaneous loss of Lhx2 and Lhx9 function resulted in patterning and growth defects along the anterior-posterior and the proximal-distal limb axis. Similar but more severe phenotypes were observed when the activities of all three factors, Lmx1b, Lhx2 and Lhx9, were significantly reduced by removing their required cofactor Ldb1. Thus, we have uncovered a previously unappreciated function of the dorsal limb specific Lmx1b gene in regulating anterior-posterior and proximal-distal limb patterning and outgrowth. A detailed analysis of signaling events during early limb development revealed that Lhx2, Lhx9 and Lmx1b play redundant roles in regulating the responsiveness of mesenchymal cells to Fgf8 and Shh ligands by maintaining their progenitor cell states. Lhx6 and Lhx8 are two closely related LIM-homeodomain genes whose patterns of expression often overlap in the developing ventral telencephalon. We have previously generated an Lhx8 null allele. The phenotype of mutant embryos that lack Lhx8 function has revealed an important role for this gene in the development of cholinergic neurons in the telencephalon. More recently, we have also studied embryos that lack Lhx6 function. Their phenotype allowed us to conclude that this gene is essential for the generation and positioning of major sub-types of GABAergic neurons present in the neocortex, the hippocampus, and several sub-cortical areas of the telencephalon. The Lhx6 loss-of-function allele expresses placental alkaline phosphatase (PLAP) which enabled us to follow the development and fate of Lhx6-competent interneurons in the Lhx6 null embryos. To analyze whether Lhx6 and Lhx8 cooperate during the development of the telencephalon, we have generated mutants lacking the function of both of these genes. Our analysis of these double mutants revealed a specific defect in the formation of the globus pallidus, indicating that Lhx6 and Lhx8 indeed play additional and redundant roles in the assembly of the ventral telencephalon. In addition to the analysis of Lhx6/Lhx8 double mutants, we have also generated an Ldb1/Nkx2.1-Cre conditional mutant. In this mutant, Ldb1, and thereby the function of all Lhx genes who depend on this cofactor, is inactivated in cells that normally express Lhx6 and Lhx8. The phenotype of this conditional mutant resembles that of the Lhx6/Lhx8 double knockout mutant. We conclude that Lhx6 and Lhx8, in conjunction with Ldb1 are essential regulators of forebrain development. Ssdp1 is another essential cofactor mediating the action of LIM-homeodomain (LIM-HD) proteins in mouse development. Its ablation causes severe developmental defects in the embryo. However, Ssdp1 is predominantly found in the cytoplasm of cells, and the molecule does not contain a nuclear localization domain. So, how does it regulate transcriptional events in the nucleus? To find out, we analyzed the intracellular transport of Ssdp1 in established cell lines grown in vitro. We observed that Ssdp1 localizes predominantly to the cytoplasm of 293T cells but is translocated to the nucleus when co-transfected with Lck, a member of the Src family of non-receptor tyrosine kinases. The Src tyrosine kinase inhibitor PP2 blocks the nuclear translocation of Ssdp1. Western blot analysis shows that co-expression of Ssdp1 and Lck in 293T cells induces Ssdp1 phosphorylation. Mutation of the N terminal tyrosine residues 23 and 25 of Ssdp1 markedly reduces both the phosphorylation and the nuclear localization of Ssdp1. Lck enhances the transcriptional activity of Ssdp1 in the context of known components of a LIM-HD/cofactor complex. We propose that phosphorylation involving N-terminal tyrosine residues of Ssdp1 is a fast and efficient means of regulating the transcriptional activity of LIM-HD complexes.

本节重点关注lim同源结构域（LIM-HD）转录因子在小鼠胚胎进入器官系统原肠胚后组装阶段时发挥的关键发育控制作用。越来越多的证据表明，LIM-HD蛋白通过激活定义器官身份基态的下游基因，在谱系规范中发挥关键作用。新形成的细胞系组装成复杂的组织结构依赖于涉及所有主要信号通路的细胞迁移和细胞间通讯的精确模式。LIM-HD蛋白的转录活性由两个家族的强制性辅助因子介导，称为Ldb和Ssdp，它们成为LIM-HD复合体控制靶基因转录的一部分。在下面的工作中，我们分析了发育中的小鼠胚胎中组织组装的不同情况。我们发现在器官发生过程中，转录调控和分泌信号交换之间存在复杂的相互作用。