【作者】 李奎；

【导师】 赵庆顺；

【作者基本信息】 南京大学 ， 生物学， 2011， 博士

【摘要】 核受体对基因表达的主动抑制在脊椎动物早期发育中发挥着十分重要的作用。这种主动抑制作用是核受体通过招募核辅阻遏物及其他复合物实现的。NCoR是最早发现的核辅阻遏物,它能够抑制视黄酸受体介导的基因表达。已有的研究表明,NCoR对小鼠晚期的器官发生起着重要的作用。而NCoR是否影响胚胎的早期发育尚不清楚。在本研究中,我们发现当在斑马鱼中敲落ncor时,胚胎呈现出增强的内源性视黄酸信号并导致神经外胚层出现后部化的现象。过表达或敲落ncor则使斑马鱼的后脑长度出现相应的变短或变长,而这变化与减弱或加强内源性视黄酸信号引起的胚胎表型相一致。这些结果表明NCoR对视黄酸信号的主动抑制是早期后脑图式形成所必需的。作为NCoR的同源基因,SMRT介导的基因沉默在脊椎动物的发育及代谢中也起着十分重要的作用。已有的研究表明,SMRT是小鼠前脑发育、抗炎症信号正常工作所必需的。但对于SMRT是否通过抑制视黄酸信号而影响早期胚胎发育,如前后轴的形成,尚未有报道。通过在斑马鱼中敲落smrt,我们发现SMRT同样是胚胎抑制内源性视黄酸信号所必需的。尽管敲落smrt并未影响后脑和早期神经嵴的图式形成,但我们却发现SMRT通过抑制h0x家族基因的表达,进而影响躯干部和尾部的前后轴形成。在斑马鱼中,多能性的造血干细胞通过一系列的分化步骤,可分化为红细胞前体、髓性细胞前体及内皮细胞前体。其中红细胞前体最终可分化为红细胞,而髓性前体细胞则分化为粒细胞、巨噬细胞及单核细胞。在敲除NCoR的小鼠里,原始红细胞生成并不受影响而永久的红细胞生成则比正常小鼠要少。迄今,有关NCoR和SMRT是否影响斑马鱼胚胎的原始血液发生尚未有报道。通过在斑马鱼中敲落ncor和smrt,我们发现它们影响了斑马鱼原始髓性细胞的生成,但不影响原始红细胞的生成。在敲落smrt的斑马鱼胚胎中,粒细胞的标记基因mpx及巨噬细胞／单核细胞的标记基因l-plastin的表达水平都出现极大的降低。但在敲落ncor的斑马鱼胚胎中,只有mpx的表达水平出现明显的降低。通过分析smrt敲落胚胎细胞的细胞生物学变化,我们发现这种髓性细胞缺失的现象并不是由髓性细胞的凋亡或增殖减少引起的,而是由于影响了早期血液发生前体和原始髓性前体细胞的分化造成的。在敲落smrt的斑马鱼中过表达spil或scl/lmo2然后研究髓性细胞缺失的表型是否能被拯救,我们证明了smrt发挥的作用的平行于scl和lmo2,并和它们一起作用于spil进而影响原始髓性前体细胞生成。在敲落ncor的斑马鱼胚胎中,决定早期血液发生的转录因子如：scl、lmo2、etsrp、hhex、gata2、draculin、c/ebpα以及spil的表达均为未发生变化。故NCoR似乎仅仅是粒细胞生成所必需而不影响其他髓性细胞的生成。综上所述,核辅阻遏物NCoR和SMRT是斑马鱼前后轴图式形成及原始髓性细胞发生所必需的。更多还原

【Abstract】 Active repression of gene expression mediated by unliganded nuclear receptors plays crucial roles in early development of vertebrates. NCoR is the first identified corepressor that can repress RA inducible gene transcription in the absence of RA. Previously, NCoR was reported to be required for late-stage organogenesis in mouse but whether NCoR can affect RA-responsive early embryonic patterning is unknown. In this study, we report that knocking down ncor elevates endogenous RA signaling in zebrafish embryos and posteriorizes the neural ectoderm. Overexpressing or knocking down ncor in zebrafish embryos alters the length of hindbrain in a manner similar to decreasing or increasing RA signaling in embryos, respectively. Our results demonstrate that NCoR is essential for early hindbrain patterning by actively repressing retinoid signaling. As a close relative of NCoR, SMRT mediate target genes silencing in developmental systems and homeostasis. Previously, SMRT was reported to be required for forebrain development and anti-inflammatory signaling pathways in mouse but whether SMRT affects RA-responsive early embryonic patterning, such as A-P axis, is unknown. By knocking down smrt in zebrafish embryos, we found that unlike ncor, smrt plays no significant role in hindbrain and early neural crest patterning though it is involved in repressing retinoid signaling like ncor. Interestingly, our results showed that zebrafish SMRT is essential to the A-P specification of trunk and tail in zebrafish embryos through repressing hox gene expressions. In zebrafish, pluripotent hematopoietic stem cells generate common myeloid progenitor cells, erythroid progenitor cells and endothelial progenitor cells through progressive commitment and differentiation steps. Terminally myeloid progenitor cells differentiate to granulocyte and macrophage/monocyte lineages whereas erythroid progenitor cells differentiate to erythroid lineages. In NCoR knockout mice, definitive erythropoiesis is impaired whereas primitive erythropoiesis is normal. However, whether NCoR or/and SMRT play roles in primitive hematopoiesis in zebrafish is still unknown. By knocking down ncor or smrt in zebrafish embryos, we showed that the corepressors affect myelopoiesis differently rather than erythropoiesis. In smrt morphants, the expression of either mpx or l-plsatin, marker genes of granulocyte and macrophage/monocyte, respectively, is greatly reduced. However, in ncor morphants, only mpx expression is reduced. Analyzing smrt morphants, we demonstrated that the loss of myeloid cells in smrt-depleted embryos is not attributed to decreased proliferation or extensive apoptosis of myeloid cells, but due to affecting the specification of the earlier hematopoietic precursors and primitive myelopoietic precursors. By rescuing the phenotype in smrt morphants through forced expression of spil or scl/lmo2, we revealed that smrt acts parallel of scl/lmo2 and upstream of spil during primitive myelopoiesis. However, none of the early transcriptional factors including scl, lmo2, etsrp, hhex, gata2, draculin, c/ebpa and spil has changed their expression patterns in ncor morphants. The results suggest that NCoR is not required to the formation of myeloid progenitor but is crucial for granulogenesis. Taken together, our results demonstrated that NCoR and SMRT are essential for zebrafish A-P axis patterning and primitive myelopoiesis.更多还原