Regionalization of the Vertebrate Endoderm

描述（由申请人提供）：我们的长期目标是利用斑马鱼模型系统的优势来研究脊椎动物内胚层是如何区域化的，以便内分泌和消化器官，包括胰腺，被指定并在正确的位置发育。我们的研究结果可能最终促进实验指导人类干细胞在体外分化为特定的胰腺细胞类型。这种方法为糖尿病和胰腺癌的移植治疗提供了希望。我们的总体策略是测试已知在神经外胚层区域化中重要的分子机制对内胚层区域化也很重要的假设。为了支持这一假设，我们已经确定了在神经外胚层和中胚层区域化中起重要作用的分泌信号分子维甲酸（retinoic acid， RA）对内胚层区域化至关重要。因此，RA是胰腺特化所必需的，外源性RA具有引起前内胚层形成大量异位胰腺细胞的显著能力。我们提出了具体的目标：(1)确定四足脊椎动物胰腺规格是否需要RA；(2)探讨斑马鱼胰腺分化过程中RA信号转导的分子和细胞机制；(3)验证Hox和Parahox基因对内胚层的区域化假说；(4)研究Wnt和FGF信号分子在内胚层区域化中的作用。RA信号可以在内胚层接收，在那里它们可以细胞自主地指定胰腺，或者可以在邻近组织接收，然后向内胚层发送二次信号。我们将使用细胞移植方法来区分这些假设。Hox基因和相关的Parahox基因编码进化上保守的转录因子。脊椎动物的Hox基因分布于外胚层和中胚层，经常是RA的靶点。我们拥有所有斑马鱼Hox基因的cdna，这将使我们能够使用功能获得和功能丧失方法（Mrna错误表达和morpholino敲低）来确定Hox基因是否也对内胚层区域化起作用。最后，我们将结合敲除、显性阴性和错误表达的方法来研究其他与神经外胚层后分化有关的分泌信号分子，如FGFs和wnt，是否也参与内胚层区域化。

DESCRIPTION (provided by applicant): Our long-term objective is to use the advantages of the zebrafish model system to investigate how the vertebrate endoderm is regionalized such that endocrine and digestive organs, including the pancreas, are specified and develop in the correct location. The results of our studies may ultimately facilitate experiments to direct human stem cells to differentiate into specific pancreatic cell types in vitro. Such approaches offer promise for transplantation therapies for diabetes and pancreatic cancers. Our general strategy is to test the hypothesis that molecular mechanisms known to be important in regionalization of the neural ectoderm are also important for endoderm regionalization. In support of this hypothesis, we have established that the secreted signaling molecule retinoic acid (RA), which plays an important role in regionalization of both neural ectoderm and mesoderm, is critical to endoderm regionalization. Thus, RA is required for specification of the pancreas, and exogenous RA has the remarkable capacity to cause anterior endoderm to form large numbers of ectopic pancreatic cells. We propose specific aims to: (1) Determine whether RA is required for pancreas specification in tetrapod vertebrates; (2) Investigate the molecular and cellular mechanism of RA signal transduction during zebrafish pancreas specification; (3) Test the hypothesis that Hox and Parahox genes regionalize the endoderm; and (4) Investigate the roles of Wnt and FGF signaling molecules in regionalization of the endoderm. RA signals may be received in the endoderm where they act cell-autonomously to specify pancreas, or alternatively may be received in adjacent tissues, which then send secondary signals to the endoderm. We will use a cell transplantation approach to distinguish between these hypotheses. Hox genes, and related Parahox genes, encode evolutionarily conserved transcription factors. The vertebrate Hox genes regionalize ectoderm and mesoderm and are frequently RA targets. We are in the unique position of having cDNAs to all the zebrafish Hox genes, this will allow us to use gain and loss-of-function approaches (Mrna mis-expression and morpholino knockdown) to determine whether Hox genes also act to regionalize the endoderm. Finally, we will use a combination of knockdown, dominant-negative, and mis-expression approaches to investigate whether other secreted signaling molecules implicated in posteriorizing the neural ectoderm, FGFs and Wnts, are also involved in endoderm regionalization.