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鱼类C1q-like和Mtmr8基因的表达及功能分析
Alternative TitleExpression and Functional Analysis of C1q-like and Mtmr8 in Fish
梅洁
Subtype博士
Thesis Advisor桂建芳
2009-05-31
Degree Grantor中国科学院水生生物研究所
Place of Conferral水生生物研究所
KeywordC1q-like因子 滤泡细胞 皮质颗粒 凋亡 原始生殖细胞 斑马鱼 血细胞生成 Mtmr8 Actin纤维组装 肌肉发生 血管发生
Abstract在脊椎动物中,含有C1q结构域的C1q家族蛋白已有一些报道,但他们的生物学功能还不是很清楚。首先,我们鉴定了一个银鲫C1q-like因子(CagOC1q-like),它已被确认为一种皮质颗粒组分。免疫荧光定位显示CagOC1q-like特异在滤泡表皮细胞上表达,并在完全长成的卵母细胞上同皮质颗粒一起。而且受精后,它会排出到卵黄周隙和卵膜上。由于它是第一个被发现在卵母细胞周围的滤泡细胞上表达的C1q家族成员,它被用于滤泡细胞凋亡和缺失的检测中。卵母细胞成熟和排出阶段,滤泡细胞凋亡和缺失的整个细胞学过程可以清楚地通过CagOC1q-like抗体免疫标记滤泡细胞以及TUNEL标记凋亡滤泡细胞共标记来观察。 C1q-like因子在彩鲫(CaOC1q)和银鲫(CagOC1q)中分子特征几乎相同。然而,它在成熟卵子中的转录水平却有差异,前者的转录水平明显高于后者。有趣的是,CagOC1q在卵子排出后至心跳期之间几乎检测不到表达,而CaOC1q的表达水平相对比较高。整体RNA原位杂交显示心跳时CaOC1q特异在胚胎的生殖脊处表达,而CagOC1q几乎没有表达。为了研究C1q-like因子在胚胎发育过程中的作用,我们设计了反义吗琳代低聚核甘酸C1q-like MO作用于CaOC1q和CagOC1q的翻译起始位点。C1q-like的敲降会造成彩鲫胚胎PGCs的大量减少,而银鲫胚胎PGCs数目并没有明显变化。本研究或许能为揭示C1q家族成员在生殖和发育过程中的功能提供新的线索。 我们进一步在模式生物斑马鱼中来研究C1q-like的功能。通过对斑马鱼的基因组,cDNA和EST数据的全面搜索,我们找到了和CaOC1q-like factor同源性最高的基因,斑马鱼C1q-like,并对它做进一步的功能研究。C1q-like的转录和翻译在斑马鱼胚胎发育早期呈广泛的分布,随后主要在中后脑和眼睛部位表达。体外研究表明,在EPC细胞中,C1q-like能通过抑制caspase 3/9的活性来抑制ActD和CHX诱导的凋亡。进一步通过morpholino调节的敲降技术来探讨C1q-like在胚胎发育过程中的生理功能。与注射对照morpholino的胚胎相比,C1q-like敲降胚胎的头部及其软骨发生呈现明显的缺陷。这些缺陷是由于p53诱导的凋亡造成的,体外转录的C1q-like mRNA或p53 MO共注射能挽救这些缺陷可以验证。TUNEL分析揭示在C1q-like敲降胚胎中产生大量的凋亡细胞,而且caspase 3/9的活性也有两倍以上的升高,这些可以被p53 MO共注射所抑制。荧光定量PCR显示,一些凋亡调节因子如p53, mdm2, p21, Bax 和 caspase 3在C1q-like敲降胚胎中有明显上调,而hbae1则显著下调。在斑马鱼胚胎中,敲降C1q-like会减少血红素产物并且造成中脑静脉及其它脑部血管组装缺陷。有趣的是,斑马鱼胚胎用UV诱导损伤会促使C1q-like mRNA的表达上调,但仅仅C1q-like的过表达不足以抵抗UV诱导的损伤。另外,用嗜水气单胞菌感染胚胎也会促使C1q-like mRNA的表达上调,C1q-like敲降胚胎的存活率显著下降。这些数据表明,在斑马鱼胚胎发育过程中,特别是脑部发育,C1q-like可能起着保护作用,抵抗p53依赖性和caspase 3/9调节的凋亡。C1q-like应该是斑马鱼胚胎发育过程中一个新的细胞存活调节因子。 最近的研究表明,至少有四个人类肌管素(myotubularin)家族基因的突变会造成神经性肌肉紊乱,如MTM1, MTMR1, 2和13。但是它们的信号通路和调节机制还不清楚。这里,我们报道了斑马鱼Mtmr8在胚胎肌肉发生过程中所扮演的新角色。在体节发生早期,Mtmr8主要在眼部和体节部位表达。morpholino敲降造成的Mtmr8功能缺失会导致严重的体节发生缺陷。并且我们发现在Mtmr8敲降的胚胎中,Akt的磷酸化水平明显上升。接着我们探讨了Mtmr8蛋白中PH/G结构域的功能。仅仅PH/G结构域的缺失并不会造成明显的胚胎发育缺陷,但同时加入PI3K的抑制剂LY294002后会产生明显的缺陷表型,这表明PH/G结构域对于Mtmr8的功能非常重要。我们进一步观察了Mtmr8和PI3K在actin纤维组装和肌肉发生过程中的协同作用。加入LY294002后,Mtmr8及其PH/G结构域缺失的胚胎中actin细胞骨架会进一步去组装。另外,我们揭示Mtmr8通过Hedgehog,并以非细胞自动方式来调节actin组装。以上这些数据显示Mtmr8与PI3K共同作用调节斑马鱼actin组装和肌肉发生,而且Hedgehog途径参与其中。这为研究MTM家族成员的生理功能提供新的线索。 胚胎的血管和肌肉形态发生系统是紧密协调的,且最近的研究揭示一些信号通路同时控制这两个系统,如Hedgehog和PI3K途径。在前面的研究中,我们揭示了Mtmr8通过Hedgehog和PI3K/Akt途径来调节早期肌肉发生的功能作用。Mtmr8稍后在血管中的表达暗示了它在斑马鱼胚胎发生中有更广泛的功能。Mtmr8敲降造成严重的血管发生缺陷,包括节间血管生成的阻滞和躯干背部动脉生成的阻断。而背部动脉是造血干细胞生成的地方,这也造成血细胞生成的减少。此外,Mtmr8敲降胚胎的背部动脉的内皮细胞呈现出大量的减少,通过动脉标记基因EphrinB2a的减少或消失表达来指示。而Mtmr8敲降胚胎的EphrinB2a表达缺陷可通过低浓度PI3K抑制剂处理或dnPKA和Vegf mRNA共注射来挽救。而且,当用PI3K或Hedgehog途径的抑制剂处理野生型胚胎时,它们会消除动脉标记基因的表达,但Mtmr8反而迁移至动脉处表达。这些数据表明,Mtmr8可能通过抑制PI3K的活性,从而对动脉的分化起着保护作用,且作为Hedgehog/PI3K/VEGF级联信号控制动静脉命运过程中的一个新组分。
Other AbstractC1q family proteins with C1q domain have been reported in vertebrates, but their biological roles are currently unknown. In this study, a C1q-like factor, designated Carassius auratus gibelio C1q-like factor (CagOC1q-like), was identified as a cortical granules component. Immunofluorescence localization revealed that the C1q family member was specifically expressed in follicular epithelial cells, and associated with cortical granules in fully grown oocytes. Moreover, it was discharged to the perivitelline space and egg envelope upon fertilization. As it is the first identified C1q family member that is expressed in follicular cells that surround oocyte, CagOC1q-like was applied to detection of follicular cell apoptosis and deletion. The entire cytological process of follicular cell apoptosis and deletion was clearly seen from double visualizations of follicular cells with CagOC1q-like immunofluorescence and apoptotic follicular cells labeled by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) during oocyte maturation and ovulation. The molecular characterizations of C1q-like factor were almost the same in gonochoristic color crucian carp (abbreviated CaOC1q) and gynogenetic gibel crucian carp(abbreviated CagOC1q), in previous studies. However, difference transcription level is found between CaOC1q and CagOC1q in mature eggs. The former was much higher than the latter. Interestingly, the transcription level of CagOC1q was almost under detection, compared to relatively high level of CaOC1q between ovulation and cardiopalmus stage. RNA in situ hybridization on cardiopalmus stage embryos revealed the specific expression of CaOC1q in the genital ridge, where little expression of CagOC1q was found. In order to study the role of C1q-like factor during embryo development, a morpholino antisense oligonucleotide (MO) was designed complementary to the putative translation initiation start site of both CaOC1q and CagOC1q mRNA. And the knockdown of C1q-like resulted in loss of PGCs in color crucian carp, whereas no obvious change of PGCs in gibel crucian carp. Perhaps our present studies will give new clue about how C1q family makes its function in reproduction and development. We further study the functions of C1q-like in model organism, zebrafish. Through comprehensive searches of C1qDC (C1q-domain-containing) gene in zebrafish genome, cDNA and EST databases, we discovered zebrafish C1q-like, the the closest homologue of CaOC1q-like factor. The transcription and translation of C1q-like display a uniform distribution in early embryos, and are restricted to mid-hind brain and eye in later embryos. In vitro studies showed that C1q-like could inhibit the apoptosis induced by ActD and CHX in EPC cells, through repressing caspase 3/9 activities. Moreover, its physiological roles were studied by morpholino-mediated knockdown in zebrafish embryogenesis. In comparison with control embryos, the C1q-like knockdown embryos display obvious defects in the head and craniofacial development mediated through p53-induced apoptosis, which was confirmed by the in vitro transcribed C1q-like mRNA or p53 MO co-injection. TUNEL assays revealed extensive cell death, and caspase 3/9 activity measurement also revealed about two folds increase in C1q-like morphant embryos, which was inhibited by p53 MO co-injection. Real-time quantitative PCR showed the upregulation expression of several apoptosis regulators such as p53, mdm2, p21, Bax and caspase 3, and down-regulation expression of hbae1 in the C1q-like morphant embryos. Knockdown of C1q-like in zebrafish embryos decreased hemoglobin production and impaired the organization of mesencephalic vein and other brain blood vessels. Interestingly, exposure of zebrafish embryos to UV resulted in an increase in mRNA expression of C1q-like, whereas over-expression of C1q-like was not enough resist to the damage. Furthermore, C1q-like transcription was up-regulated in response to pathogen Aeromonas hydrophila, and embryo survival significantly decreased in the C1q-like morphants after exposure to the bacteria. The data suggested that C1q-like might play an antiapoptotic and protective role in inhibiting p53-dependent and caspase 3/9-mediated apoptosis during embryogenesis, especially in the brain development, and C1q-like should be a novel regulator of cell survival during zebrafish embryogenesis. It has been shown that mutations in at least four myotubularin family genes (MTM1, MTMR1, 2 and 13) are causative for human neuromuscular disorders. However, the pathway and regulative mechanism remain unknown. Here, we reported a new role for Mtmr8 in neuromuscular development of zebrafish. Firstly, we cloned and characterized zebrafish Mtmr8, and revealed the expression pattern predominantly in the eye field and somites during early somitogenesis. Using morpholino knockdown, then, we observed that loss-of-function of Mtmr8 led to defects in somitogenesis. Subsequently, the possible underlying mechanism and signal pathway were examined. We first checked the Akt phosphorylation, and observed an increase of Akt phosphorylation in the morphant embryos. Furthermore, we studied the PH/G domain function within Mtmr8. Although the PH/G domain deletion by itself did not result in embryonic defect, addition of PI3K inhibitor LY294002 did give a defective phenotype in the PH/G deletion morphants, indicating that the PH/G domain was essential for Mtmr8’s function. Moreover, we investigated the cooperation of Mtmr8 with PI3K in actin filament modeling and muscle development, and found that both Mtmr8-MO1 and Mtmr8-MO2+LY294002 led to the disorganization of the actin cytoskeleton. In addition, we revealed a possible participation of Mtmr8 in the Hedgehog pathway, and cell transplantation experiments showed that Mtmr8 worked in a non-cell autonomous manner in actin modeling. The above data indicate that a conserved functional cooperation of Mtmr8 with PI3K regulates actin filament modeling and muscle development in zebrafish, and reveal a possible participation of Mtmr8 in the Hedgehog pathway. Therefore, this work provides a new clue to study the physiological function of MTM family members. Embryonic morphogenesis of vascular and muscular systems is tightly coordinated, and recent studies revealed that some pathways, such as Hedgehog and PI3K, control both systems. In previous studies, we have revealed the function of Mtmr8 in regulating early muscle development through hedgehog and PI3K/Akt pathway. Later expression of Mtmr8 in the vasculature, suggests that zebrafish Mtmr8 may have more extensively functions during embryo development. Knockdown of Mtmr8 by morpholino resulted in severe defects in vascular development, including retardation in intersegmental vessel development and interruption of trunk dorsal aorta where hematopoietic stem cells arise, that might lead to a reduction in hematopoiesis. Moreover, Mtmr8 morphants showed loss of arterial endothelial cell identity in dorsal aorta, as indicated by the reduced or absent expression of the arterial marker EphrinB2a, which was effectively rescued by low concentration of PI3K inhibitor, and by over-expression of dnPKA mRNA or Vegf mRNA. Furthermore, the expression of Mtmr8 was especially migrated to the artery when treated with specific inhibitors of both PI3K and Hedgehog pathway, which abolished the expression of arterial marker. These data demonstrated that Mtmr8 might play a protect role in arterial differentiation through repressing the activity of PI3K, representing a novel element of the Hedgehog/PI3K/VEGF signaling cascade that controls arterial/venous fate.
Pages135
Language中文
Document Type学位论文
Identifierhttp://ir.ihb.ac.cn/handle/342005/12424
Collection学位论文
Recommended Citation
GB/T 7714
梅洁. 鱼类C1q-like和Mtmr8基因的表达及功能分析[D]. 水生生物研究所. 中国科学院水生生物研究所,2009.
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