Developmental Genetics in the Zebrafish Danio rerio

斑马鱼斑马鱼的发育遗传学

• 批准号: 8736891
• 负责人: Igor B Dawid
• 金额: $ 106.5万
• 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/8736891
• 关键词: Affect; Alanine; Anterior; Antisense Oligonucleotides; BTB/POZ Domain; Binding; Biological; Biological Assay; Biological Models; Biological Process; Brain region; Branchial arch structure; Cell Nucleus; Cell fusion; Cells; Chromatin; Circadian Rhythms; Co-Immunoprecipitations; Commissure; Cues; Cultured Cells; DNA Microarray Chip; Defect; Development; Developmental Cell Biology; Dimerization; Embryo; Embryonic Development; Family; Family member; Fishes; Gene Expression; Gene Expression Profile; Gene Mutation; Gene Targeting; Genes; Genetic; Genome; Giant Cells; Homologous Gene; Homologous Protein; Human; Imaging Techniques; Individual; Inherited; Laboratories; Life; Light; Literature; Mediating; Membrane; Mesoderm; Methodological Studies; Methods; Microtubules; Modeling; Molecular; Molecular Genetics; Monomeric GTP-Binding Proteins; Mutagenesis; Names; Neural Crest; Neural Crest Cell; Nuclear; Oligonucleotides; Organ; Pathway interactions; Pattern; Phenotype; Phosphorylation; Pineal gland; Pituitary Gland; Process; Proline; Property; Prosencephalon; Protein Family; Protein Secretion; Proteins; Regulation; Reporter; Reporting; Retina; Role; Signal Pathway; Signal Transduction Pathway; Site; Staging; Stem cells; Structure; System; TFAP2A gene; Time; Tissues; Tyrosine Phosphorylation; Vertebrates; Work; Xenopus laevis; Zebrafish; attenuation; base; developmental genetics; embryo tissue; gene discovery; gene function; human disease; interest; knock-down; lens; member; migration; mutant; neural plate; novel; overexpression; precursor cell; prevent; programs; protein transport; research study; solute; transcription factor; zebrafish development

A focus of interest in this laboratory has been the study of genes that are involved in the formation of the neural crest and of its derivatives such as the pharyngeal arches. We found that the BTB-domain containing protein Kctd15 that is first expressed in the embryo in the neural plate border, is an important factor in regulating the domain in which neural crest forms. Overexpression of Kctd15 strongly inhibits neural crest specification, while attenuation of Kctd15 expression leads to expansion of the neural crest precursor domain. In contrast, we have shown that anterior placodal domains are expanded after Kctd15 overexpression, supporting the idea that Kctd15 limits the extent of the neural crest domain and prevents incursion of the placodal domain by neural crest precursor cells. Recently we have found that Kctd15 inhibits the activity of transcription factor AP-2, a factor well known to be required at several stages of neural crest formation. Mechanistic studies on the interaction between AP-2 and Kctd15 have been carried out. Ap-2 is a major regulator of neural crest formation, being involved in the initial specification of neural crest cells as well as in later stages such as their migration and differentiation into multiple derivatives. Kctd15 is capable of binding AP-2 in co-immunoprecipitation experiments, and inhibits the activation of an AP-2 reporter both in cultured cells and in zebrafish embryos. In studying the mechanism of this inhibition we found that Kctd15 does not affect the level of AP-2 in the cell, does not inhibit dimerization of AP-2 which is required for activity, and does not affect nuclear localization of AP-2. Also, AP-2 remains bound to its cognate sites in chromatin in the presence of Kctd15. To probe the mechanism further we studied the activation domain of AP-2. Kctd15 binds to the activation domain, and specifically requires proline 59 for this interaction. An AP-2 mutant in which this proline is changed to alanine (P59A) is active in the reporter assay but cannot bind Kctd15, and its activity is not inhibited by Kctd15. We conclude that Kctd15 inhibits AP-2 activity by specific binding to its activation domain which precludes its function. Among genes identified in our DNA microarray studies as differentially expressed in the pineal gland we noted a gene encoding a homolog of the Unc119 protein family. Whereas two Unc119 homologs are known in humans, the gene we isolated is the third family member noted in zebrafish; accordingly we named this protein Unc119c. The unc119c gene is specifically expressed in the pineal, with a low level of expression in the retina. In fish the pineal is a photosensitive organ and shows many similarities in gene expression to the retina. Unc119 proteins interact with small GTPases of the Arl3 family, and we have shown that Unc119c binds to Arl3l2 when both are coexpressed in heterologous cells. Using morpholino antisense oligonucleotide (MO) mediated knock-down of Unc119c expression we found that this protein is required for the formation of the habenular commissure (HC) in the zebrafish. The HC crosses the midline in close proximity to the pineal gland. Knock-down of the Unc119c binding partners Arl3l1 or Arl3l2 also affect HC formation. We hypothesized that Unc119c might be involved in protein trafficking or secretion of a guidance factor involved in HC formation. Based on the literature and expression pattern we picked Wnt4a as a candidate target gene; Wnt4a has been reported by others to be required for HC formation, a fact confirmed in our experiments. We found that Wnt4a accumulation and secretion from cultured HEK293T cells is stimulated by Unc119c and Arl3l1/2. Thus we propose that Unc119c is required in the pineal gland to stimulate Wnt4a secretion, while Wnt4a in turn acts as a guidance cue for the HC as it traverses the zebrafish forebrain. The yolk syncytial layer (YSL) of the zebrafish embryo arises during early stages of development. It is a multinucleated syncytium that is generated by the collapse of membranes between cells adjoining the yolk layer. The YSL is essential for multiple steps in embryo development, for example mesoderm induction and the establishment of the dorsoventral axis. In spite of its importance the molecular mechanisms underlying YSL formation have not been fully elucidated at this time. We have shown that the zebrafish homolog of the protein named solute carrier family 3 member 2 (Slc3a2) is expressed specifically in the YSL, and is required for its normal development. When the expression of Slc3a2 is reduced by morpholino oligonucleotide-mediated knockdown, YSL development becomes abnormal, showing clustering of the yolk syncytial nuclei, an enhanced level of cell fusion, and the disruption of microtubule networks that normally have a highly ordered structure in the YSL. The artificial introduction of a constitutively active version of the small GTPase RhoA leads to a similar YSL phenotype as that generated by Slc3a2 knockdown. Conversely, the reduction of RhoA activity rescues the Slc3a2 knockdown phenotype. Likewise, inhibition of Rock, a downstream effector of RhoA, also rescues the Slc3a2 phenotype. In addition, Slc3a2 knockdown strongly inhibits tyrosine phosphorylation of c-Src. Consistent with this observation, overexpression of a constitutively active Src rescues the phenotype generated by the reduction in Slc3a2 expression. These observations suggest that the signaling pathway that regulates YSL formation involves the inhibition of RhoA/Rock by Slc3a2 as a consequence of the phosphorylation of c-Src. We suggest that this signal transduction pathway modulates microtubule dynamics in the developing YSL. Loss of microtubule structure then causes the abnormalities in the arrangement of yolk syncytial nuclei and in the regulation of cell fusion that are observed in Slc3a2 knockdown embryos. These studied shed light on more general aspects of the regulation of cell fusion, a process that has many roles in different biological circumstances.

该实验室的关注重点是对与咽弓等神经rest及其衍生物形成有关的基因的研究。我们发现，含有最初在神经板边界的胚胎中表达的蛋白KCTD15的BTB域是调节神经rest形成的结构的重要因素。 KCTD15的过表达强烈抑制神经rest规范，而KCTD15表达的衰减会导致神经Crest前体域的扩展。相比之下，我们已经表明，在KCTD15过表达后扩展了前部位座结构域，支持KCTD15限制了神经Crest结构域的程度，并防止神经犯罪前体细胞侵袭了位置域。最近，我们发现KCTD15抑制了转录因子AP-2的活性，这是在神经rest形成的几个阶段所需的一个因子。已经进行了有关AP-2和KCTD15之间相互作用的机械研究。 AP-2是神经rest形成的主要调节剂，参与了神经rest细胞的初始规范以及后期的阶段，例如迁移并分化为多个衍生物。 KCTD15能够在共免疫沉淀实验中结合AP-2，并抑制培养细胞和斑马鱼胚胎中AP-2报告剂的激活。在研究这种抑制作用的机制时，我们发现KCTD15不会影响细胞中的AP-2水平，不会抑制AP-2的二聚化，而AP-2的二聚化，并且不会影响AP-2的核定位。同样，在KCTD15存在下，AP-2仍与其染色质中的同源位点结合。为了进一步探测机制，我们研究了AP-2的激活结构域。 KCTD15与激活结构域结合，特别需要脯氨酸59进行这种相互作用。将这种脯氨酸更改为丙氨酸（p59a）的AP-2突变体在报告基因测定中活跃，但不能结合KCTD15，并且KCTD15不会抑制其活性。我们得出的结论是，KCTD15通过特异性结合其活化域抑制了AP-2活性，从而抑制其功能。 在我们的DNA微阵列研究中鉴定出的基因在松果腺中差异表达，我们注意到编码UNC119蛋白家族的同源物的基因。尽管在人类中已知两个UNC119同源物，但我们分离的基因是斑马鱼中指出的第三个家庭成员。因此，我们命名了该蛋白UNC119C。 UNC119C基因在松果体中特异性表达，在视网膜中表达较低。在鱼类中，松果是一种光敏器官，在基因表达与视网膜的表达中显示了许多相似之处。 UNC119蛋白与ARL3家族的小GTPase相互作用，我们已经表明，当两者在异源细胞中共表达时，UNC119C与ARL3L2结合。使用吗啡反义寡核苷酸（MO）介导的UNC119C表达的敲低敲低，我们发现该蛋白在斑马鱼中形成了Habenular Compsure（HC）所必需的蛋白质。 HC靠近松果体横穿中线。 UNC119C结合伙伴ARL3L1或ARL3L2的敲除也影响HC的形成。我们假设UNC119C可能参与蛋白质运输或与HC形成有关的引导因素的分泌。根据文献和表达模式，我们选择了Wnt4a作为候选靶基因。其他人报告了HC形成需要WNT4A，这是我们实验中证实的事实。我们发现，UNC119C和ARL3L1/2刺激了培养的HEK293T细胞的Wnt4a积累和分泌。因此，我们建议在松果体中需要UNC119C刺激Wnt4a分泌，而Wnt4a又可以作为HC的指导提示，因为它遍历了斑马鱼前脑。 斑马鱼胚胎的蛋黄合层（YSL）在发育的早期阶段出现。它是一种多核合胞体，是由靠近蛋黄层的细胞之间的膜塌陷而产生的。 YSL对于胚胎发育的多个步骤至关重要，例如中胚层诱导和背腹轴的建立。尽管其重要性，但目前尚未完全阐明YSL形成的分子机制。我们已经表明，称为溶质载体家族3成员2（SLC3A2）的蛋白质的斑马鱼同源物在YSL中专门表达，并且是其正常发育所必需的。当形态寡核苷酸介导的敲低降低SLC3A2的表达时，YSL发育变得异常，显示蛋黄合胞细胞核的聚类，细胞融合的增强水平，并且微管网络的破坏通常在YSL中具有高度有序的结构。小型GTPase RhoA的组成型活性版本的人工引入与SLC3A2敲低产生的YSL表型相似。相反，RhoA活性的减少挽救了SLC3A2敲低表型。同样，RhoA的下游效应子岩石的抑制也挽救了SLC3A2表型。此外，SLC3A2敲低强烈抑制C-SRC的酪氨酸磷酸化。与这种观察结果一致，对组成型活性SRC的过表达挽救了SLC3A2表达的降低产生的表型。这些观察结果表明，调节YSL形成的信号传导途径涉及SLC3A2抑制RhoA/Rock，这是由于C-SRC的磷酸化。我们建议该信号转导途径调节发育中的YSL中的微管动力学。然后，微管结构的丧失会导致蛋黄合核核的排列异常以及在SLC3A2敲低胚胎中观察到的细胞融合的调节。这些研究阐明了细胞融合调节的更一般方面，该过程在不同的生物学环境中具有许多作用。