囊胚形成的基因表达与调控(英文)

囊胚形成是胚胎早期发育过程中一个重要阶段 ,涉及几个重要的生理事件 ,即细胞融合 (compaction ,亦称致密化作用 )、囊胚腔出现、囊胚腔扩张及滋养层和内细胞团的分化。在细胞间连接蛋白的作用下 ,各种细胞间连接方式逐步建立起来 ,在合子型基因组表达调控下 ,促进了最终囊胚的形成。细胞间连接蛋白和细胞粘附相关蛋白参与组建各种细胞间连接 ,参与细胞融合、囊胚腔形成、滋养层分化和囊胚扩张等过程。通过顶部的紧密连接、侧部的缝隙连接和桥粒 ,建立起细胞的连接复合体。在人胚胎 8 细胞之前 ,卵裂球细胞界限明显 ,可能以中间连接方式相互作用 ;8 细胞期发生致密化作用 ,通过紧密连接将细胞分成顶部和基部 ,使得胚胎处于半封闭状态 ,促进胚胎内部积液 ,形成囊胚腔。细胞融合的同时也产生缝隙连接。桥粒最初出现在人胚胎达到 3 2 细胞阶段 ,桥粒连接参与囊胚腔形成以及在囊胚扩张时维持滋养层的稳定性。桥粒由一些跨膜粘蛋白组成 ,包括参与细胞内粘附的桥粒子和桥粒球以及一些细胞质内蛋白 (如desmoplakins,plakoglobin ,plakophilin) ,由细胞内蛋白质形成空斑结构并介导细胞角蛋白丝固定。对植入前牛胚胎的研究表明 ,只有DcII,DcIII和plako三种桥粒蛋白参与桥粒组建。在鼠囊胚中DcII的表达部位位于     

囊胚形成的基因表达与调控

囊胚形成是胚胎早期发育过程中一个重要阶段,涉及几个重要的生理事件,即细胞融合（compaction,亦称致密化作用）、囊胚腔出现、囊胚腔扩张及滋养层和内细胞团的分化。在细胞间连接蛋白的作用下,各种细胞间连接方式逐步建立起来,在合子型基因组表达调控下,促进了最终囊胚的形成。细胞间连接蛋白和细胞粘附相关蛋白参与组建各种细胞间连接,参与细胞融合、囊胚腔形成、滋养层分化和囊腔扩张等过程。通过顶部的紧密连接、侧部的缝隙连接和桥粒,建立起细胞的连接复合体。在人胚胎8－细胞之前,卵裂球细胞界限明显,可能从中间连接方式相互作用;8－细胞期发生致密化作用,通过紧密连接将细胞分成顶部和基部,使得胚胎处于半封闭状态,促进胚胎内部积液,形成囊胚腔。细胞融合的同时也产生缝隙连接。桥粒最初出现在人胚胎达到32－细胞阶段,桥粒连接参与囊胚腔形成以及在囊胚扩张时维持滋养层的稳定性。桥粒由一些跨膜粒蛋白组成,包括参与细胞内粘附的桥粒子和桥粒球以及一些细胞质内蛋白（如desmoplakins,plakoglobin,plakophilin),由细胞内蛋白质形成空斑结构并介导细胞角蛋白丝固定。对植入前牛胚胎的研究表明,只有DcII,DcIII和plako三种桥粒蛋白参与桥粒组建。在鼠囊胚中DcII的表达部位位于滋养外胚层,对于滋养层的形成具有重要的作用。在牛中,直到桑椹胚期可能才出现紧密连接方式。人胚胎发育至囊胚期时,可检测到角蛋白－18基因的活跃表达,作为细胞骨架蛋白的角蛋白－18参与桥粒位点的细胞与细胞间“识别”的建立。缝隙连接蛋白连接子（Connexin43,Cx43)对于维持细胞融合以及之后囊胚的形成是十分必要的。细胞融合和囊胚腔形成还需要一些介导细胞粘附和滋养层细胞分化的分子表达,如整合素家族中的E－钙调素、链接子（caknin)和ZQ（一种细胞质带状咬合紧密连接相关蛋白）。Na/K-ATP酶基因及细胞因子、生长因子等可促进Na^ 浓度梯度变化,使水分进入囊胚内部,形成囊胚腔,并维持囊腔腔扩张状态。调节发育和分化的基因参与胚胎的发育和内细胞团和滋养外胚层细胞的分化,调节胚胎由未分化状态向分化状态过渡。另外,某些细胞因子对内细胞团细胞增殖有影响。在小鼠胚胎培养基中,添加IGF和EGF有利于胚胎从透明带孵出,TGF－α和EGF在小鼠胚胎发育中能分别通过自泌作用和旁分泌作用囊胚腔的扩张膨大。许多基因参与囊胚的发育和分化,如Pem基因调节早期胚胎细胞分化,可调节鼠早期胚胎由未分化状态向分化状态过渡,Oct-4表达与未分化表型有关,在早期胚胎发育中起转录调节作用。植入前胚胎发育基因调节鼠胚植入前卵裂速度的快慢,以及胚胎预后生存能力。植入前因子PIF在受精后妇女的血清中就能检测出来,其特点和功能与囊胚形成的关系仍需进一步研究。Rex-1编码锌指蛋白,可能是一个转录调节因子,它参与滋养层发育以及精子发生,它是研究内细胞团早期细胞命运决定的有用标志物,对于维持胚胎干细胞的未分化状态和全能性有作用,当其表达显著降低时,内细胞团将分化成胚层。总之,多种蛋白、因子参与囊胚形成的表达调控。通过基因调控的研究,将有助于培养液的研制以及通过测定一些标志基因的表达筛选健康的胚胎。     

小鼠胚胎致密化起始的调控机制

植入前小鼠胚胎的发育事件包括第一次卵裂、胚胎基因组激活、桑椹胚致密、囊胚形成。小鼠受精卵胚胎的致密化发生在8-细胞阶段晚期, 致密过程中, 胚胎卵裂球本身以及卵裂球之间发生了一系列的变化。这些变化包括卵裂球微绒毛以及胞质成分的极性化分布, 卵裂球之间形成特殊的胞间连接。致密化是哺乳动物胚胎发育过程中的第一个细胞分化事件, 即导致了内细胞团以及滋养外胚层的产生。植入后, 内细胞团将发育成为胚体, 滋养外胚层将发育成为胎盘等胚外组织。细胞粘附分子E-cadherin介导的胞间粘附起始了致密化。卵裂球发生粘附所需的组分在致密前已经存在, 但是直至8-细胞阶段晚期连接复合体才表现出明显的粘附活性。敲除E-cadherin基因, 发现母源性的E-cadherin足以介导致密。E-cadherin介导的胞间粘附是细胞粘附的第一步。文章综述了E-cadherin介导胞间粘附的具体过程以及蛋白激酶C(Protein kinase C, PKC)调控该过程的相关 机制。     

根毛的生长发育及其遗传基础

根毛是根系特异化表皮细胞外伸形成的管状凸起物,是植物吸收矿质养分和水分的重要器官。根毛的发育可分为根毛细胞命运决定、根毛起始、根毛顶端生长和根毛成熟等阶段。本文对根毛发育生长过程中的细胞形态及其生理生化变化进行了综述,并从根表皮细胞命运决定分子机理, EXPANSIN、bHLH和MYB等转录因子以及小G蛋白和生长素/乙烯等方面简要说明了根毛生长发育的遗传基础。     

蛋白激酶Cα在小鼠孤雌及四倍体胚胎早期发育过程中的分布和作用

为了观察蛋白激酶Cα(protein kinase Cα,PKCα在昆明白小鼠受精卵、孤雌激活和四倍体胚胎早期发育阶段的亚细胞定位和致密化进程中的表达变化,本实验利用免疫荧光化学染色与激光共聚焦显微镜观察相结合的方法,对受精卵、孤雌激活和四倍体胚胎早期发育阶段PKCα的表达进行了定位观察,并利用Western blot对三组胚胎致密化进程中PKCα的表达进行定量分析.结果显示,PKCα在上述三组胚胎发育的2-细胞期至囊胚期均有表达,虽然不同胚胎PKCα的分布在同一发育阶段存在差异,却表现出在各胚胎期主要分布于卵裂球核染色质内,以及在胚胎致密化开始,PKCα在卵裂球连接处发生重新分布的共同特点.此外,三组胚胎PKCα在致密化进程中的表达呈升高趋势,即致密化后的表达高于敛密化前.结果表明,PKCct对胚胎致密化的调节具有重要作用,其在8-细胞/4-细胞期的重新分布是胚胎进入桑椹胚期的必然事件,是胚胎致密化的前提,同时伴随蛋白表达增多.此外,PKCα在囊胚期发生了植入前的第二次重新分布.PKCα在三组胚胎各发育阶段表达情况各不相同,它对小鼠胚胎发育的影响体现在整个早期发育阶段.PKCα在小鼠受精卵早期发育阶段的两次重新分布可能与在致密化开始时启动的细胞黏附事件存在某种必然联系.     

皮层蛋白介导斑马鱼(Danio rerio)原肠化细胞运动

在胚胎发育过程中, 细胞运动对指导原肠期胚胎细胞的时空定位并决定其发育命运具有核心作用, 然而活体状态下原肠化过程中细胞运动的调控机制目前并不清楚. 微丝结合蛋白皮层蛋白(cortactin)是微丝核化过程的重要调控分子, 它通过激活微丝相关蛋白2/3复合物(Arp2/3 complex)促进微丝在细胞前导缘区域迅速组装, 从而直接作用于细胞运动. 为阐明斑马鱼(Danio rerio)原肠化细胞运动的分子调控机制, 本研究首先检测了皮层蛋白在斑马鱼胚胎发育过程的表达水平. Western blotting分析证明皮层蛋白在斑马鱼原肠期胚胎中大量表达; 整装胚胎抗体染色结果表明在斑马鱼原肠化过程中, 皮层蛋白主要分布于胚胎背侧胚盾区域的细胞中, 在发生活跃运动的上皮层细胞和下皮层细胞中含量较高;在亚细胞水平, 皮层蛋白和Arp2/3复合物共同定位于运动的皮层区域, 并在细胞连接处也有大量分布. 此外, 研究还发现皮层蛋白在发育中的中枢神经系统中表达量较高. 本研究结果首次表明皮层蛋白和Arp2/3复合物介导的微丝聚合参予了斑马鱼原肠化细胞运动, 并在中枢神经系统发育中扮演重要角色.     

应用乙二醇冷冻小鼠胚胎：优化和简化程序的探索

提高解冻胚胎的发育能力和简化冷冻解冻程序是胚胎冷冻研究的两大永恒的主题。尽管乙二醇（EG）广泛用于家畜胚胎冷冻,但很少用于冷冻小鼠和人胚胎。为数很少的以EG慢冻小鼠或人胚胎的研究均采用较为复杂的人胚冷冻程序,未见简化程序和用EG冷冻小鼠桑椹胚的报道。采用简单的牛胚胎冷冻程序研究了发育时期、EG浓度、平衡方法、添加蔗糖以及解冻后脱除EG等对小鼠胚胎冻后发育能力的影响。结果显示：（1）致密晚期桑椹胚冻后体外培养囊胚发育率（81.92%±2.24%）和孵出率（68.56％±2.43%）显著（P＜0.05)高于4-细胞、8-细胞胚胎和致密早期桑椹胚胎;（2）1.8mol/L EG冷冻小鼠致密晚期桑椹胚的囊胚发育和孵出率显著高于其它浓度;（3）在EG中平衡10min的冻后囊胚发育显著好于平衡5、20或30min;（4）两步平衡冷冻胚胎的囊胚发育率和孵出率显著高于一步平衡;（5）用EG冷冻小鼠胚胎无需添加蔗糖;（6）解冻后可不脱除EG;（7）冻后发育的早期囊胚和囊胚细胞数明显少于体内发育胚胎。因此,用EG冷冻小鼠胚胎的最佳方案为：致密晚期桑椹胚用1.8mol/L EG不添加蔗糖、两步平衡15min、以简单的牛胚胎冷冻程序冷冻解冻、解冻后不脱除EG直接培养或移植。     

小鼠着床前胚胎细胞发育命运

哺乳动物早期胚胎的发育机制一直是动物胚胎学和发育生物学领域中存在广泛争议的研究热点, 近年来, 争议的焦点主要集中在早期胚胎细胞的不同发育命运决定出现的阶段以及是否存在某种起关键作用的形态发生决定子等问题上.综述了近年来有关精子入卵位点、第二极体、胚胎形状、第二次卵裂的方向、细胞极性等因素对小鼠着床前胚胎细胞发育命运的影响.     

mED2-一个小鼠胚胎发育的新基因

克隆参与胚胎发育的新基因并研究其表达规律和功能是揭示胚胎发育的基因调控机理的重要途径。囊胚形成和原肠形成是哺乳动物胚胎发育过程中的两个关键阶段。囊胚阶段发生了胚胎的第一次分化,是细胞多能性和分化的一个转折点。此时涉及的基因活动,既有维持胚胎干细胞全能性或多能性的基因活动,又有按照预定发育模式参与胚胎定向分化的基因活动。原肠期是胚胎发育过程中的第二个关键转折点,涉及到3个胚层的形成和细胞命运决定等多种变化。在这个时期胚胎获得了胎儿原基的所有信息,新组织的产生和细胞迁移的再生组织与形态发生、细胞增殖、细胞分化、模式形成等存在着非常复杂而相互协调的关联。大多数细胞正由原来的多潜能逐渐向寡潜能发展,控制组织器官形态建成的基因正逐渐开启。这两个时期的基因表达图式、特征和种类会有很大的差异和变化,因此研究这两个时期的新基因的表达规律和功能,将是了解胚胎发育的基因调控机理的重要途径。文章以这两个时期胚胎为原始材料,利用减法杂交方法克隆到一新的小鼠胚胎基因mED2,对其进行了表达规律和生物学功能的初步分析。RT-PCR-Southern和原位杂交实验表明,mED2基因转录水平具有发育阶段的依赖性;随着发育过程的进行,其表达主要在胚神经系统和中胚层衍生的组织表达。mED2基因活性的knockdown对于合子的卵裂和植入前早期胚胎发育均有抑制作用。亚细胞定位实验表明,mED2基因编码的蛋白基本定位于细胞核膜及其临近的内膜细胞器（粗糙内质网和高尔基体）。根据生物信息学分析,mED2蛋白可能为一跨膜蛋白且与含有硫氧还蛋白结构域的蛋白有部分匹配。由此推测mED2基因参与了小鼠植入前早期胚胎发育,其基因产物可能通过蛋白之间的相互作用,即对蛋白进行后期修饰、折叠及行使分子伴侣等作用来活化或抑制其靶蛋白的活性,进而参与小鼠的早期胚胎发育。     

神经发育中的细胞周期时程

哺乳动物的神经发育经历一系列神经前体细胞的形态结构和机能改变,其细胞周期时程也呈现动态变化,从神经发生早期至后期,神经前体细胞的细胞周期时程逐渐延长,并与细胞发育命运转归有关,其调节因素包括周期蛋白复合体、Notch信号通路、原神经基因靶向蛋白、微管与分子马达蛋白等。细胞周期长度假说认为,细胞周期的长度影响到命运决定子的积累,因而决定细胞的命运。文章综述了相关的研究进展。     

Comparison of ES cell fate in sandwiched aggregates and co-cultured aggregates during blastocyst formation by monitored GFP expression

Markers and the means to detect them are required to monitor the fate of living cells. However, few suitable markers for living cells were known until a green fluorescent protein (GFP) was discovered. We have established mouse embryonic stem (ES) cell lines that express mutant GFP under the chicken beta-actin (CAG) promoter. Using these cell lines, we were able to follow the migration of ES cells during blastocyst formation both in sandwiching and coculture methods, even if only a single ES cell was used. Furthermore, the contribution of ES cells to the inner cell mass (ICM) was easily estimated at the blastocyst stage. We compared sandwiching with coculture aggregation relative to the contribution of the ES cell in the ICM, and the results indicated that there was no difference in the ratios of chimeric embryos having ICM contributed from cultured ES cells. Furthermore, an aggregated single ES cell was able to contribute three or four cells to the ICM at the blastocyst stage. Thus we conclude that one, instead of two, embryos is enough to make aggregation with ES cells, and a single ES cell attached to an embryo is enough to produce germline chimeras. Moreover, we could clearly observe single cell fate during blastocyst formation. This suggests that our established cell line can be used for monitoring single cell fate in vivo. In addition, we have shown that up to five doses of 30 sec of UV irradiation using GFP filters have no effect on the embryonic development.     

Keeping things quiet: roles of NuRD and Sin3 co-repressor complexes during mammalian development

Gene inactivation studies of mammalian histone and DNA-modifying proteins have demonstrated a role for many such proteins in embryonic development. Post-implantation embryonic lethality implies a role for epigenetic factors in differentiation and in development of specific lineages or tissues. However a handful of chromatin-modifying enzymes have been found to be required in pre- or peri-implantation embryos. This is significant as implantation is the time when inner cell mass cells of the blastocyst exit pluripotency and begin to commit to form the various lineages that will eventually form the adult animal. These observations indicate a critical role for chromatin-modifying proteins in the earliest lineage decisions of mammalian development, and/or in the formation of the first embryonic cell types. Recent work has shown that the two major class I histone deacetylase-containing co-repressor complexes, the NuRD and Sin3 complexes, are both required at peri-implantation stages of mouse development, demonstrating the importance of histone deacetylation in cell fate decisions. Over the past 10 years both genetic and biochemical studies have revealed surprisingly divergent roles for these two co-repressors in mammalian cells. In this review we will summarise the evidence that the two major class I histone deacetylase complexes in mammalian cells, the NuRD and Sin3 complexes, play important roles in distinct aspects of embryonic development.     

The LIM protein Ajuba is recruited to cadherin-dependent cell junctions through an association with alpha-catenin

Cell-cell adhesive events affect cell growth and fate decisions and provide spatial clues for cell polarity within tissues. The complete molecular determinants required for adhesive junction formation and their function are not completely understood. LIM domain-containing proteins have been shown to be present at cell-cell contact sites and are known to shuttle into the nucleus where they can affect cell fate and growth; however, their precise localization at cell-cell contacts, how they localize to these sites, and what their functions are at these sites is unknown. Here we show that, in primary keratinocytes, the LIM domain protein Ajuba is recruited to cadherin-dependent cell-cell adhesive complexes in a regulated manner. At cadherin adhesive complexes Ajuba interacts with alpha-catenin, and alpha-catenin is required for efficient recruitment of Ajuba to cell junctions. Ajuba also interacts directly with F-actin. Keratinocytes from Ajuba null mice exhibit abnormal cell-cell junction formation and/or stability and function. These data reveal Ajuba as a new component at cadherin-mediated cell-cell junctions and suggest that Ajuba may contribute to the bridging of the cadherin adhesive complexes to the actin cytoskeleton and as such contribute to the formation or strengthening of cadherin-mediated cell-cell adhesion.     

Inactivation of aPKClambda results in the loss of adherens junctions in neuroepithelial cells without affecting neurogenesis in mouse neocortex

In developing mammalian telencephalon, the loss of adherens junctions and cell cycle exit represent crucial steps in the differentiation of neuroepithelial cells into neurons, but the relationship between these cellular events remains obscure. Atypical protein kinase C (aPKC) is known to contribute to junction formation in epithelial cells and to cell fate determination for Drosophila neuroblasts. To elucidate the functions of aPKClambda, one out of two aPKC members, in mouse neocortical neurogenesis, a Nestin-Cre mediated conditional gene targeting system was employed. In conditional aPKClambda knockout mice, neuroepithelial cells of the neocortical region lost aPKClambda protein at embryonic day 15 and demonstrated a loss of adherens junctions, retraction of apical processes and impaired interkinetic nuclear migration that resulted in disordered neuroepithelial tissue architecture. These results are evidence that aPKClambda is indispensable for the maintenance of adherens junctions and may function in the regulation of adherens junction integrity upon differentiation of neuroepithelial cells into neurons. In spite of the loss of adherens junctions in the neuroepithelium of conditional aPKClambda knockout mice, neurons were produced at a normal rate. Therefore, we concluded that, at least in the later stages of neurogenesis, regulation of cell cycle exit is independent of adherens junctions.     

p38 MAPK Regulates Cavitation and Tight Junction Function in the Mouse Blastocyst

Blastocyst formation is essential for implantation and maintenance of pregnancy and is dependent on the expression and coordinated function of a series of proteins involved in establishing and maintaining the trans-trophectoderm ion gradient that enables blastocyst expansion. These consist of Na/K-ATPase, adherens junctions, tight junctions (TJ) and aquaporins (AQP). While their role in supporting blastocyst formation is established, the intracellular signaling pathways that coordinate their function is unclear. The p38 MAPK pathway plays a role in regulating these proteins in other cell types and is required for embryo development at the 8–16 cell stage, but its role has not been investigated in the blastocyst.

Hypothesis

p38 MAPK regulates blastocyst formation by regulating blastocyst formation gene expression and function.

Methods

Embryos were cultured from the early blastocyst stage for 12 h or 24 h in the presence of a potent and specific p38 MAPK inhibitor, SB 220025. Blastocyst expansion, hatching, gene family expression and localization, TJ function and apoptosis levels were analyzed.

Results

Inhibition of the p38 MAPK pathway reduced blastocyst expansion and hatching, increased tight junction permeability, affected TJP1 localization, reduced Aqp3 expression, and induced a significant increase in apoptosis.

Conclusion

The p38 MAPK pathway coordinates the overall events that regulate blastocyst formation.