视黄酸信号抑制斑马鱼原始髓性细胞发生的分子机制研究

Zebrafish primitive myelopoiesis is controlled by a genetic network comprising many genes. It is known that gata4/5/6 (gata4, gata5 and gata6) lie at the top of the genetic cascade to control the primitive myelopoiesis by regulating expressions of scl and lmo2, the master regulators of hemangioblasts, in the rostral blood island of the zebrafish embryo. Retinoic acid (RA) is an important morphogen. It also plays crucial roles in myelopoiesis. Our newly published researches and unpublished data demonstrated that RA signaling acts downstream of gata4/5/6 to suppress zebrafish primitive myelopoiesis by not only restricting the expressions of scl and lmo2 but also working downstream of pu.1, the key transcription factor of the precursors of primitive myeloid cells, to prevent the precursors of the primitive myeloid cells from differentiating into macrophages and granulocytes, respectively. To further understand the molecular mechanisms underlying RA controlling the primitive myelopoiesis in detail, we will perform studies on investigating how RA signaling suppresses the expressions of scl and lmo2, and how RA signaling acts downstream of pu.1 to prevent the precursors of the primitive myeloid cells from differentiating into macrophages and granulocytes. We will also be going to identify new DEAB (4-diethylamino-benzaldehyde, an inhibitor of aldehyde dehydrogenases that are responsible for RA synthesis) sensitive aldehyde dehydrogenases that mediate the roles of gata4/5/6 in controlling the primitive myelopoiesis, and to reveal the molecular mechanisms that gata4/5/6 restrict the expressions of the DEAB sensitive aldehyde dehydrogenases to control the primitive myelopoiesis. Accomplishing our research goals, we will reveal the molecular mechanisms underlying suppression of zebrafish primitive myelopoiesis by retinoic acid signaling in the genetic network.

斑马鱼的原始髓性细胞发生受到由多个基因组成的遗传网络的调控。已知Gata4/5/6位于遗传网络最上游，它们通过调控scl和lmo2在胚胎前部血岛的表达控制原始髓性细胞发生。视黄酸（RA）是一种重要的形态发生素，在髓性细胞发生中也发挥着关键作用。我们已有的工作基础揭示RA信号位于Gata4/5/6下游，既通过限制scl和lmo2表达抑制斑马鱼原始髓性细胞发生，也可通过作用于髓性细胞前体标识基因pu.1下游抑制原始髓性细胞前体向成熟髓性细胞分化。本申请将通过回答RA信号如何调控scl和lmo2的表达、以何种方式作用于哪些pu.1下游基因抑制原始髓性细胞前体向成熟髓性细胞分化、有哪些醛脱氢酶基因通过合成RA介导了Gata4/5/6对原始髓性细胞发生的调控以及Gata4/5/6是如何通过抑制这些醛脱氢酶基因的表达控制原始髓性细胞发生等问题，深入阐明RA信号调控斑马鱼胚胎原始髓性细胞发生的分子机制。

研究表明RA信号不能通过直接作用于scl和lmo2的调控序列抑制二者的表达。借助于基因表达芯片和microRNA深度测序的高通量分析，我们发现RA信号除可通过调控meis2b等编码基因的表达进而影响原始髓性细胞发生外，还可通过调控非编码RNA如miRNA-210-5p的表达影响原始髓性细胞的发生。.meis2b和miRNA-210-5p在进化上均高度保守，过表达meis2b抑制原始髓性细胞的发生，但不影响血液血管祖细胞以及原始髓性前体细胞标识基因（spi1b）的表达，提示meis2b作用于spi1b下游影响原始髓性细胞的分化。.miRNA-210-5p同时受Gata4/5/6调控，敲降gata4/5/6上调miRNA-210-5p的表达。过表达miRNA-210-5p抑制原始髓性细胞的发生，下调spi1b的表达，但却上调血液血管祖细胞的标识基因的表达。这一结果表明miRNA-210-5p在介导Gata4/5/6调控血液血管祖细胞向原始髓性细胞前体的分化过程中扮演着重要角色。进一步分析发现，foxj1b、slc3a2和csf3r作用于miRNA-210-5p的下游。过表达foxj1b、slc3a2和csf3r可以拯救因敲降gata4/5/6或过表达miRNA-210-5p而导致的原始髓性细胞发生的抑制。与此一致的是，虽然敲除miRNA-210-5p不影响斑马鱼原始髓性细胞发生，但其下游基因foxj1b和slc3a2在胚胎中的表达显著上调，敲除miRNA-210-5p可以部分拯救因敲降gata4/5/6而受抑制的原始髓性细胞发生。.研究未能发现斑马鱼中存在除aldh1a2外的参与抑制原始髓性细胞发生的可合成RA的新醛脱氢酶基因。Gata4/5/6不能通过直接抑制aldh1a2的表达调控原始髓性细胞的发生。rbp1a在进化上高度保守，受RA直接调控，敲降gata4/5/6也上调rbp1a的表达。Rbp1a是RA前体视黄醇的结合蛋白，可降低RA前体的水平，从而使得敲降gata4/5/6后因aldh1a2表达的上升而导致的RA水平上升得到缓冲。.研究还揭示了Ncor1和Ncor2在原始髓性细发生中发挥着关键但不相同的作用、亚硝酸盐和RA信号影响起源于血液血管祖细胞的心内膜向房室瓣膜特化、研发了一种基于结构识别的新型基因组编辑工具。