细胞内视黄酸信号传递系统

视黄酸对基因表达的调控与肿瘤细胞的分化、胚胎的发育以及疾病的发生关系密切．视黄酸的基因调控作用是通过视黄酸信号传递系统实现的．视黄酸信号传递系统包括视黄酸、细胞液视黄醇（酸）结合蛋白、视黄酸细胞核受体及视黄酸反应元件等．视黄酸信号传递系统自成一体系,在这一系列调控的级联反应中存在着多级反馈调控环节,而且该系统还与视黄酸配体以外的信号系统相联系．     

视黄酸缺乏对斑马鱼心脏房室分化的影响

目的 通过化学遗传学方法建立视黄酸缺乏的斑马鱼模型,探讨视黄酸缺乏对斑马鱼胚胎心脏前后轴发育即房室分化的影响.方法 在斑马鱼胚胎孵育的5 hpf,用不同浓度梯度的视黄醛脱氢酶2抑制剂DEAB(1×10-6、5×10-6、10×10-6、25×10-6 mol/L)处理斑马鱼胚胎,实时观察斑马鱼胚胎发育的全过程.通过给予斑马鱼胚胎外源性视黄酸,观察其对DEAB的拮抗作用.应用胚胎整体原位杂交观察视黄酸缺乏对心脏特异基因vmhc和amhc表达的影响.结果 斑马鱼胚胎的生存率随着DEAB处理浓度的增加而降低,当DEAB浓度≥5×10-6 mol/L时,斑马鱼的畸胎率达100%.5×10-6 mol/L DEAB的致畸作用能够被1×10-9mol/L外源性视黄酸所拮抗.整体原位杂交结果显示视黄酸缺乏会导致斑马鱼胚胎心脏房室分化异常,表现为vmhc表达细胞的范围增大,amhc表达细胞的范围缩小.结论 通过外源性DEAB处理能有效地建立视黄酸缺乏的斑马鱼模型,DEAB影响胚胎发育存在剂量依赖性.视黄酸在斑马鱼心脏前后轴发育过程中起重要调控作用,心脏发育早期视黄酸缺乏会抑制心房的发育而支持心室的发育,出现房室分化异常.     

外源性视黄酸对斑马鱼心血管系统发育的影响

目的观察不同浓度外源性视黄酸对斑马鱼早期胚胎和心血管系统发育的影响,为进一步研究视黄酸影响斑马鱼心脏前后轴(A-P轴)发育的分子机制提供形态学依据。方法选择斑马鱼胚胎孵育的3,6,9·5,12h四个时间点,用不同浓度视黄酸(1×10-6,1×10-7,4×10-8,1×10-8mol/L)处理斑马鱼胚胎,在解剖显微镜下实时观察斑马鱼胚胎心脏发育的全过程和视黄酸对斑马鱼心脏发育的影响。并采用胚胎整体原位杂交技术观察flk-1mRNA在斑马鱼胚胎的表达。结果1×10-6mol/L视黄酸可导致斑马鱼胚胎表现出多系统的严重畸形,胚胎很快死亡。在胚胎孵育的9·5、12h给与10-7~10-8mol/L浓度的视黄酸,胚胎只表现出心血管系统的畸形,其他系统无明显异常。胚胎整体原位杂交显示视黄酸对flk-1mRNA在斑马鱼胚胎血管的表达没有影响。结论视黄酸影响斑马鱼胚胎心脏发育有剂量依赖性和严格的时间窗,视黄酸影响心脏前后轴发育的关键时间是原肠胚晚期。视黄酸处理组胚胎的循环缺陷主要为心脏发育异常所致。10-7~10-8mol/L浓度视黄酸在9·5、12h处理斑马鱼胚胎可以作为研究心脏发育调控机制的动物模型。     

视黄酸对中枢神经元的作用

视黄酸是维生素A的活性代谢物,广泛分布于发育组织和成年组织中,能促进细胞分化,阻止细胞增殖和程序化死亡,是哺乳动物胚胎发育和脑发育的重要形态发生素及营养因子。视黄酸通过细胞核受体（RARs,RXRs及ROR）介导调节基因表达。它可以抑制c-myc癌基因或下调相关的N-myc基因的表达,促使人或鼠的瘤细胞分化,最令人感兴趣的是视黄酸可提高多种神经元种群ChATmRNA水平,促使细胞向胆碱能神经元方向分化,并且可能参与中枢神经系统神经损伤的修复。     

细胞视黄酸结合蛋白与肿瘤相关研究

细胞视黄酸结合蛋白(CRABP)在细胞质介导视黄酸与细胞核上视黄酸核受体之间互相作用,同时促进视黄酸代谢酶类的活性,增强视黄酸的转化速率。在脊椎动物细胞中,CRABP包含两种高度同源蛋白质:CRABP1和CRABP2。CRABP属于低分子量的蛋白质,种属间高度保守。现有研究表明在许多肿瘤中存在CRABP基因异常甲基化,基因表达水平较正常组织有差别;CRABP基因异常甲基化与部分肿瘤患者低生存率存在相关性,CRABP基因可以成为常见的肿瘤诊断以及判断预后的分子靶标。     

全反式视黄酸通过RARα对铁调节蛋白2的基因表达的影响

目的：通过体外肝细胞培养来研究全反式视黄酸（atRA）对铁调节蛋白2（IRP2）的影响。方法：体外实验将大鼠原代肝细胞按Seglent法分离后,随机分为严重缺乏组 A、边缘性缺乏组B、正常生理组C、治疗剂量组D、RARα阻抑组E,分别给予0、0.5、1.0、50μmol/L全反式视黄酸和50μmol/L全反式视黄酸+10μmol/L RO培养96h。用逆转录—聚合酶链反应（RT-PCR法）检测肝脏的IRP2 mRNA的表达。结果：大鼠全反式视黄酸缺乏时,肝脏IPR2 mRNA表达增强（P＜0.05）,补充全反式视黄酸后,IPR2 mRNA表达减弱,但阻抑RARα后,VA这种作用减弱。结论：全反式视黄酸可通过RARα改变IRP2 mRNA的转录来影响铁代谢。     

视黄酸通路的启示——从心脏发育到心肌谱系定向分化

多潜能干细胞具有无限增殖的能力,并能够分化为心肌细胞,因此在心脏再生方面拥有巨大潜力.胚胎发育过程为干细胞定向分化提供了重要线索,在过去的几年中,通过操控心脏发育关键通路,在心肌定向分化方面取得了重要进展,但是现有的分化方法仍不能稳定地诱导心肌细胞,表明现有的通路不能有效解决这些问题.视黄酸(RA)通路在心脏发育过程中发挥重要作用,RA缺失会导致心房变小、心室小梁减少、心肌壁增厚且细胞间连接松散.在体外心肌定向分化过程中,RA多用于促进多潜能干细胞向心房分化.但从RA通路基因敲除小鼠的表型来看,除了调控心肌亚型分化,RA在多个发育阶段发挥重要作用.深入解析RA在心肌分化各阶段的作用机制,将有助于获得高质量的心肌细胞.同时,研究RA在心内膜和心外膜分化中的作用机制也有助于解释RA通路敲除小鼠的心脏异常.总之,从RA在胚胎发育中的作用来看,需要更多的体外研究来揭示RA在心肌谱系分化中的作用.本文综述了RA通路在心脏发育的心肌分化过程中的作用,并探讨了需要解决的问题.     

异视黄酸对人肝癌细胞的诱导分化作用

10μmol/L的异视黄酸(13顺视黄酸,IRA)可使培养的人肝癌细胞株SMMC-7721中肝癌标志酶γ-GT逐日下降,而肝细胞分化酶TAT明显增加,峰值从对照的第4天延至第6天。这些作用基本上和视黄酸(全反式视黄酸,RA)相仿,唯IRA培养后期(第6、8天)的作用强于RA。用ABC免疫组织化学法发现:IRA还可使该细胞生成的甲胎蛋白(AFP)减少,尤其在培养的第4天作用最为明显,仅为对照细胞的1/3—1/4。并且,在IRA培养4天后,细胞表面的纤维连接蛋白Fn增高至对照的6.7—8.2倍。这些结果提示IRA能使肝癌细胞某些恶性表型转变,向正常细胞的方向诱导分化。     

qPCR array检测分析 C57BL/6小鼠胚胎后肢发育基因表达谱

目的:胚胎生育过程中因肢体发育异常造成的出生缺陷比率不低,其相关基因表达模式尚不明确。本实验通过建立实时定量PCR芯片(Real-time quantitative polymerasechain reaction array,qPCR array)检测方案,研究C57BL/6品系小鼠后肢发育相关基因的表达谱。方法:以同源异形盒基因家族(Hox)、Wnt5a、配对同源结构域基因(Pitx1)、成纤维生长因子(Fgf8)、音猬因子(Shh)等小鼠肢体发育相关的重要基因制作基因检测表达谱,以C57BL/6品系怀孕雌鼠为材料,取胚胎肢芽发育的四个关键时期(E10.5,E11.5,E12.5,E13.5)的胎鼠后肢,利用qPCR array方案检测表达谱中基因的相对表达水平差异。结果:通过已建立的qPCR array检测了C57BL/6品系小鼠胚胎后肢发育时期Hox家族、Wnt5a、Pitx1、Fgf8、Shh等基因的表达差异。以E10.5为对照,检测出在后肢发育时期基因呈三种表达模式,即Hoxb6、Hoxb8、Hoxc8、Hoxc9、Hoxc10、Hoxd9和Shh基因的表达水平呈上调;Hoxa11、Hoxa13、Hoxc12、Hoxc13、Hoxd13等基因表达出现下调;Hoxc9、Hoxc10、Hoxc11、Hoxd9、Hoxd12、Fgf8和Pitx1等基因的相对表达量呈先上调后下调的曲线表达模式,且有少部分基因在小鼠后肢发育时期表达水平无明显变化。结论:Hox家族、Wnt5a、Pitx1、Fgf8、Shh等基因在小鼠后肢发育时期表达,并且表达模式存在明显差异。     

Targeted disruption of Aldh1a1 (Raldh1) provides evidence for a complex mechanism of retinoic acid synthesis in the developing retina

Genetic studies have shown that retinoic acid (RA) signaling is required for mouse retina development, controlled in part by an RA-generating aldehyde dehydrogenase encoded by Aldh1a2 (Raldh2) expressed transiently in the optic vesicles. We examined the function of a related gene, Aldh1a1 (Raldh1), expressed throughout development in the dorsal retina. Raldh1(-/-) mice are viable and exhibit apparently normal retinal morphology despite a complete absence of Raldh1 protein in the dorsal neural retina. RA signaling in the optic cup, detected by using a RARE-lacZ transgene, is not significantly altered in Raldh1(-/-) embryos at embryonic day 10.5, possibly due to normal expression of Aldh1a3 (Raldh3) in dorsal retinal pigment epithelium and ventral neural retina. However, at E16.5 when Raldh3 is expressed ventrally but not dorsally, Raldh1(-/-) embryos lack RARE-lacZ expression in the dorsal retina and its retinocollicular axonal projections, whereas normal RARE-lacZ expression is detected in the ventral retina and its axonal projections. Retrograde labeling of adult Raldh1(-/-) retinal ganglion cells indicated that dorsal retinal axons project to the superior colliculus, and electroretinography revealed no defect of adult visual function, suggesting that dorsal RA signaling is unnecessary for retinal ganglion cell axonal outgrowth. We observed that RA synthesis in liver of Raldh1(-/-) mice was greatly reduced, thus showing that Raldh1 indeed participates in RA synthesis in vivo. Our findings suggest that RA signaling may be necessary only during early stages of retina development and that if RA synthesis is needed in dorsal retina, it is catalyzed by multiple enzymes, including Raldh1.     

Retinoic acid synthesis controlled by Raldh2 is required early for limb bud initiation and then later as a proximodistal signal during apical ectodermal ridge formation

We present evidence for the existence of two phases of retinoic acid (RA) signaling required for vertebrate limb development. Limb RA synthesis is under the control of retinaldehyde dehydrogenase-2 (Raldh2) expressed in the lateral plate mesoderm, which generates a proximodistal RA signal during limb outgrowth. We report that Raldh2(-/-) embryos lack trunk mesodermal RA activity and fail to initiate forelimb development. This is associated with deficient expression of important limb determinants Tbx5, Meis2, and dHand needed to establish forelimb bud initiation, proximal identity, and the zone of polarizing activity (ZPA), respectively. Limb expression of these genes can be rescued by maternal RA treatment limited to embryonic day 8 (E8) during limb field establishment, but the mutant forelimbs obtained at E10 display a significant growth defect associated with a smaller apical ectodermal ridge (AER), referred to here as an apical ectodermal mound (AEM). In these RA-deficient forelimbs, a ZPA expressing Shh forms, but it is located distally adjacent to the Fgf8 expression domain in the AEM rather than posteriorly as is normal. AER formation in Raldh2(-/-) forelimbs is rescued by continuous RA treatment through E10, which restores RA to distal ectoderm fated to become the AER. Our findings indicate the existence of an early phase of RA signaling acting upstream of Tbx5, Meis2, and dHand, followed by a late phase of RA signaling needed to expand AER structure fully along the distal ectoderm. During ZPA formation, RA acts early to activate expression of dHand, but it is not required later for Shh activation.     

Novel retinoic acid generating activities in the neural tube and heart identified by conditional rescue of Raldh2 null mutant mice

Retinoid control of vertebrate development depends upon tissue-specific metabolism of retinol to retinoic acid (RA). The RA biosynthetic enzyme RALDH2 catalyzes much, but not all, RA production in mouse embryos, as revealed here with Raldh2 null mutants carrying an RA-responsive transgene. Targeted disruption of Raldh2 arrests development at midgestation and eliminates all RA synthesis except that associated with Raldh3 expression in the surface ectoderm of the eye field. Conditional rescue of Raldh2(-/-) embryos by limited maternal RA administration allows development to proceed and results in the establishment of additional sites of RA synthesis linked to Raldh1 expression in the dorsal retina and to Raldh3 expression in the ventral retina, olfactory pit and urinary tract. Unexpectedly, conditionally rescued Raldh2(-/-) embryos also possess novel sites of RA synthesis in the neural tube and heart that do not correspond to expression of Raldh1-3. RA synthesis in the mutant neural tube was localized in the spinal cord, posterior hindbrain and portions of the midbrain and forebrain, whereas activity in the mutant heart was localized in the conotruncus and sinus venosa. In the posterior hindbrain, this novel RA-generating activity was expressed during establishment of rhombomeric boundaries. In the spinal cord, the novel activity was localized in the floorplate plus in the intermediate region where retinoid-dependent interneurons develop. These novel RA-generating activities in the neural tube and heart fill gaps in our knowledge of how RA is generated spatiotemporally and may, along with Raldh1 and Raldh3, contribute to rescue of Raldh2(-/-) embryos by producing RA locally.     

Uncoupling of retinoic acid signaling from tailbud development before termination of body axis extension

During the early stages of body axis extension, retinoic acid (RA) synthesized in somites by Raldh2 represses caudal fibroblast growth factor (FGF) signaling to limit the tailbud progenitor zone. Excessive RA down-regulates Fgf8 and triggers premature termination of body axis extension, suggesting that endogenous RA may function in normal termination of body axis extension. Here, we demonstrate that Raldh2-/- mouse embryos undergo normal down-regulation of tailbud Fgf8 expression and termination of body axis extension in the absence of RA. Interestingly, Raldh2 expression in wild-type tail somites and tailbud from E10.5 onwards does not result in RA activity monitored by retinoic acid response element (RARE)-lacZ. Treatment of wild-type tailbuds with physiological levels of RA or retinaldehyde induces RARE-lacZ activity, validating the sensitivity of RARE-lacZ and demonstrating that deficient RA synthesis in wild-type tail somites and tailbud is due to a lack of retinaldehyde synthesis. These studies demonstrate an early uncoupling of RA signaling from mouse tailbud development and show that termination of body axis extension occurs in the absence of RA signaling.     

Role of retinoic acid during forebrain development begins late when Raldh3 generates retinoic acid in the ventral subventricular zone

Retinoic acid (RA) synthesized by Raldh3 in the frontonasal surface ectoderm of chick embryos has been suggested to function in early forebrain patterning by regulating Fgf8, Shh, and Meis2 expression. Similar expression of Raldh3 exists in E8.75 mouse embryos, but Raldh2 is also expressed in the optic vesicle at this stage suggesting that both genes may play a role in early forebrain patterning. Furthermore, Raldh3 is expressed later in the forebrain itself (lateral ganglionic eminence; LGE) starting at E12.5, suggesting a later role in forebrain neurogenesis. Here we have analyzed mouse embryos carrying single or double null mutations in Raldh2 and Raldh3 for defects in forebrain development. Raldh2(-/-);Raldh3(-/-) embryos completely lacked RA signaling activity in the early forebrain, but exhibited relatively normal expression of Fgf8, Shh, and Meis2 in the forebrain. Thus, we find no clear requirement for RA in controlling expression of these important forebrain patterning genes, but Raldh3 expression in the frontonasal surface ectoderm was found to be needed for normal Fgf8 expression in the olfactory pit. Our studies revealed that later expression of Raldh3 in the subventricular zone of the LGE is required for RA signaling activity in the ventral forebrain. Importantly, expression of dopamine receptor D2 in E18.5 Raldh3(-/-) embryos was essentially eliminated in the developing nucleus accumbens, a tissue lying close to the source of RA provided by Raldh3. Our results suggest that the role of RA during forebrain development begins late when Raldh3 expression initiates in the ventral subventricular zone.     

Rescue of cytochrome P450 oxidoreductase (Por) mouse mutants reveals functions in vasculogenesis, brain and limb patterning linked to retinoic acid homeostasis

Cytochrome P450 oxidoreductase (POR) acts as an electron donor for all cytochrome P450 enzymes. Knockout mouse Por(-/-) mutants, which are early embryonic (E9.5) lethal, have been found to have overall elevated retinoic acid (RA) levels, leading to the idea that POR early developmental function is mainly linked to the activity of the CYP26 RA-metabolizing enzymes (Otto et al., Mol. Cell. Biol. 23, 6103-6116). By crossing Por mutants with a RA-reporter lacZ transgene, we show that Por(-/-) embryos exhibit both elevated and ectopic RA signaling activity e.g. in cephalic and caudal tissues. Two strategies were used to functionally demonstrate that decreasing retinoid levels can reverse Por(-/-) phenotypic defects, (i) by culturing Por(-/-) embryos in defined serum-free medium, and (ii) by generating compound mutants defective in RA synthesis due to haploinsufficiency of the retinaldehyde dehydrogenase 2 (Raldh2) gene. Both approaches clearly improved the Por(-/-) early phenotype, the latter allowing mutants to be recovered up until E13.5. Abnormal brain patterning, with posteriorization of hindbrain cell fates and defective mid- and forebrain development and vascular defects were rescued in E9.5 Por(-/-) embryos. E13.5 Por(-/-); Raldh2(+/-) embryos exhibited abdominal/caudal and limb defects that strikingly phenocopy those of Cyp26a1(-/-) and Cyp26b1(-/-) mutants, respectively. Por(-/-); Raldh2(+/-) limb buds were truncated and proximalized and the anterior-posterior patterning system was not established. Thus, POR function is indispensable for the proper regulation of RA levels and tissue distribution not only during early embryonic development but also in later morphogenesis and molecular patterning of the brain, abdominal/caudal region and limbs.     

Retinoic-acid signalling in node ectoderm and posterior neural plate directs left-right patterning of somitic mesoderm

Somitogenesis requires bilateral rhythmic segmentation of paraxial mesoderm along the antero-posterior axis. The location of somite segmentation depends on opposing signalling gradients of retinoic acid (generated by retinaldehyde dehydrogenase-2; Raldh2) anteriorly and fibroblast growth factor (FGF; generated by Fgf8) posteriorly. Retinoic-acid-deficient embryos exhibit somite left-right asymmetry, but it remains unclear how retinoic acid mediates left-right patterning. Here, we demonstrate that retinoic-acid signalling is uniform across the left-right axis and occurs in node ectoderm but not node mesoderm. In Raldh2(-/-) mouse embryos, ectodermal Fgf8 expression encroaches anteriorly into node ectoderm and neural plate, but its expression in presomitic mesoderm is initially unchanged. The late stages of somitogenesis were rescued in Raldh2(-/-) mouse embryos when the maternal diet was supplemented with retinoic acid until only the 6-somite stage, demonstrating that retinoic acid is only needed during node stages. A retinoic-acid-reporter transgene marking the action of maternal retinoic acid in rescued Raldh2(-/-) embryos revealed that the targets of retinoic-acid signalling during somitogenesis are the node ectoderm and the posterior neural plate, not the presomitic mesoderm. Our findings suggest that antagonism of Fgf8 expression by retinoic acid occurs in the ectoderm and that failure of this mechanism generates excessive FGF8 signalling to adjacent mesoderm, resulting initially in smaller somites and then left-right asymmetry.