C.elegans野生型和par突变体早期胚胎卵裂的观察

不对称分裂在动植物的发育中起到了非常重要的作用。Caenorhabditis elegans（C．elegans）胚胎最早的两次卵裂为研究控制不对称分裂的机制提供了很好的机会。用普通光学显微镜观察了野生型胚胎早期卵裂和par-1、par-2、par-3、par-4突变体胚胎的早期卵裂。野生型胚胎最早的分裂是不等的,产生了两个不同大小的子细胞。两个子细胞又以不同的方向进行第二次分裂。在C．elegans中任意一个par基因的缺失会使胚胎的第一次卵裂丧失不对称性。这会导致一些发育调控因子不能在特定的胚胎细胞中准确地定位,造成细胞分裂纺锤体方向的异常。par类基因参与不对称性的建立,这种不对称性决定了C．elegans身体的前后轴。     

斑马鱼胚胎第一次卵裂过程中胞内钙信号的研究

钙离子作为广泛存在的细胞内信使物质,在动物胚胎早期发育过程中扮演重要角色.为了研究钙离子在斑马鱼胚胎发育过程中的空间分布和浓度变化,采用Fluo-4和Indo-1作为钙离子指示剂,利用激光共聚焦和双波长荧光比例成像技术,对斑马鱼胚胎第一次卵裂过程中的钙信号进行了详细的跟踪观察.在第一次卵裂过程中,斑马鱼胚胎的动物极顶端首先出现高钙斑,然后在分裂沟部位出现高浓度的钙信号,这一信号在卵裂过程中持续存在.利用Indo-1双波长荧光比例成像对上述过程中钙离子的时空分布进行了定量测定,表明,胞内钙离子在卵裂开始之前是均匀分布的,随着分裂沟的出现,其附近区域的钙浓度显著升高,而胞内其他区域的钙浓度则保持不变.双波长荧光比例成像排除了荧光染料分布不均匀造成的干扰,为钙信号与胚胎分裂的密切关系提供了确凿的定量依据.     

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。     

斑马鱼ca15b的克隆及在原始生殖细胞中的特异表达

高保真PCR克隆获得了ca15b基因的全长,利用胚胎整体原位杂交等技术研究了ca15b基因在斑马鱼早期发育过程中的动态表达。结果发现,ca15b在斑马鱼早期发育过程中存在显著的原始生殖细胞（Primordial germ cell,PGC）特异表达模式。ca15b是一个母源性表达的基因:在分裂期的胚胎中,其mRNA集中分布于位于分裂沟的生殖质（Germ plasm）;从囊胚期开始,可以观察到其在PGC中的特异表达;在原肠胚中,其mRNA在体细胞中急剧降解,仅特异表达于PGC,这一表达特征一直持续到受精后1d的胚胎。将体外合成的包含5'UTR和3'UTR的ca15b全长mRNA注射到斑马鱼胚胎后,仅能增强原肠期之前胚胎中ca15b的整体杂交信号;在原肠胚期之后,注射的mRNA在体细胞中快速降解。这提示在ca15b mRNA上可能存在某种转录后调控其在早期胚胎体细胞中降解而在PGC中稳定存在的机制。       

斑马鱼ca15b的克隆及在原始生殖细胞中的特异表达简

高保真PCR克隆获得了ca15b基因的全长,利用胚胎整体原位杂交等技术研究了ca15b基因在斑马鱼早期发育过程中的动态表达。结果发现,ca15b在斑马鱼早期发育过程中存在显著的原始生殖细胞(Primordial germ cell,PGC)特异表达模式。ca15b是一个母源性表达的基因:在分裂期的胚胎中,其mRNA集中分布于位于分裂沟的生殖质(Germ plasm);从囊胚期开始,可以观察到其在PGC中的特异表达;在原肠胚中,其mRNA在体细胞中急剧降解,仅特异表达于PGC,这一表达特征一直持续到受精后1d的胚胎。将体外合成的包含5′UTR和3′UTR的ca15b全长mRNA注射到斑马鱼胚胎后,仅能增强原肠期之前胚胎中ca15b的整体杂交信号;在原肠胚期之后,注射的mRNA在体细胞中快速降解。这提示在ca15b mRNA上可能存在某种转录后调控其在早期胚胎体细胞中降解而在PGC中稳定存在的机制。     

果蝇发育中细胞决定和分化与基因表达环境

胚胎发育是个程序化的,复杂而有趣的生命现象。在胚胎发育中,不同细胞的分化和其 功能由基因决定,受到核内遗传物质的控制。而细胞的决定和分化则是在不同的细胞质对细胞核的不断作用下,才能逐步进行。核质之间的相互作用先建立特定的基因表达状态,从而选择性表达发育调控基因或分化基因。发育调控基因产物一旦进入胞质,就可改变原来的基因表达环境,使细胞核进入新的基因表达状态,选择表达新的发育调控基因。如果新的发育调控基因的产物再影响细胞核,改变原来的基因表达状态,其它的发育调控基因的表达就可使胚胎细胞进一步分化。在发育过程中,细胞质和细胞核的这个相互作用不断进行,使控制发育程序的不同基因群在特定的时空中表达,受精卵分裂产生的子细胞才能不断决定和逐步分化,最后形成组成个体所必须的各种细胞类型。     

荚果蕨胚胎发育的研究

利用石蜡切片法研究了荚果蕨（Matteuccia struthiopteris(L.) Todaro）胚胎发育过程。合子第一次分裂,分裂面垂直于原叶体纵轴且平行于颈卵器颈部;第二次分裂面平行于原叶体纵轴且垂直于颈卵器颈部;第三次分裂面同时平行于原叶体纵轴和颈卵器颈部。经多次分裂的球形胚胎,胚胎的外上和外下区域几乎同时分别发育出第一叶顶端细胞和第一根顶端细胞。随着发育的进行,它们分别斜向分裂产生第一叶原基和第一根原基。随后,第一叶原基迅速分裂,突破帽状体形成第一幼叶;而第一根原基的分裂速度稍慢,第一根发育速度稍慢于第一叶。     

基因表达谱分析牛早期胚胎发育过程的调控机理

早期胚胎的死亡会给畜牧业的发展带来巨大的经济损失, 特别是对于母牛生产来说更是如此, 因此研究早期胚胎发育过程具有极为重要的价值。文章从公共数据库GEO中选取了关于牛早期胚胎发育过程的一套基因表达谱数据, 通过显著性分析及聚类分析来研究牛早期胚胎发育过程中不同时期的基因组表达模式。结果表明: 整个牛早期胚胎发育过程大致可划分为3组不同的基因组表达模式阶段; 同时, 差显基因在不同时期表达量的波动情况表明8-细胞期和16-细胞期在牛的整个早期胚胎发育过程的重要性。另外, 通过进一步的功能注释和通路分析表明在牛胚胎早期发育不同阶段时期存在着一些重要因子及相关通路。     

林蛙卵收缩因子的进一步纯化(简报)

大家承认动物细胞的胞质分裂是由收缩环的微丝束收缩而引起的。那么微丝束又是在什么物质作用下诱发产生的呢?两栖类卵的第一次胞质分裂是单侧卵裂(unilateral cleavage),即先在动物极出现分裂沟,沟两端逐渐向下延     

植物胞质分裂发生机制

胞质分裂(cytohnesis)是指在同一细胞中在新形成的两个子核之间形成新的间隔,将母细胞一分为二的过程。胞质分裂存在于任何一种生命形式中,从单细胞的细菌到多细胞的真核生物都能进行胞质分裂。近些年由于细胞学方法的改进和研究材料增多等因素,使得对植物胞质分裂发生机制的研究取得了很大的进展。现对植物中不同类型的胞质分裂在细胞学、分子生物学方面的研究进展作一综述。     

Mutations in the par genes of Caenorhabditis elegans affect cytoplasmic reorganization during the first cell cycle

A dramatic reorganization of cytoplasm occurs during the first cell cycle in embryos of the nematode, Caenorhabditis elegans. We present here the results of a quantitative study of some of the events during this reorganization in wild-type embryos and in par mutant embryos. The par mutations define a set of genes required for cytoplasmic localization in early embryos. We show that par mutations lead to defects in several events of the reorganization. Mutations in all four of the par genes we studied lead to defects in pseudocleavage and asymmetric redistribution of cortical microfilaments. In addition, some of the par mutations affect streaming of cytoplasm, migration of the pronuclei, and asymmetric shortening of the embryo. We propose that the major function of the par genes might be to orchestrate this initial reorganization of cytoplasm.     

Cytoplasmic flow and the establishment of polarity in C. elegans 1-cell embryos

Early Caenorhabditis elegans embryos provide an excellent model for the study of developmental processes. Development can be studied by direct observation under the light microscope and can be perturbed using laser manipulations, drug inhibitor treatments, and genetic mutants. The first division of the C. elegans embryo is asymmetric, generating two daughter cells unequal in size and developmental fate. These distinct fates are generated by the partitioning of cytoplasmic determinants during the first mitotic cell cycle. Partitioning of these determinants is thought to be driven by cytoplasmic flow. Recent studies in C. elegans in the past year have identified a number of components necessary for this flow, giving us a clearer picture of the molecular mechanisms underlying developmental asymmetry.     

The Caenorhabditis elegans par-5 gene encodes a 14-3-3 protein required for cellular asymmetry in the early embryo.

The establishment of anterior-posterior polarity in the Caenorhabditis elegans embryo requires the activity of the maternally expressed par genes. We report the identification and analysis of a new par gene, par-5. We show that par-5 is required for asynchrony and asymmetry in the first embryonic cell divisions, normal pseudocleavage, normal cleavage spindle orientation at the two-cell stage, and localization of P granules and MEX-5 during the first and subsequent cell cycles. Furthermore, par-5 activity is required in the first cell cycle for the asymmetric cortical localization of PAR-1 and PAR-2 to the posterior, and PAR-3, PAR-6, and PKC-3 to the anterior. When PAR-5 is reduced by mutation or by RNA interference, these proteins spread around the cortex of the one-cell embryo and partially overlap. We have shown by sequence analysis of par-5 mutants and by RNA interference that the par-5 gene is the same as the ftt-1 gene, and encodes a 14-3-3 protein. The PAR-5 14-3-3 protein is present in gonads, oocytes, and early embryos, but is not asymmetrically distributed. Our analysis indicates that the par-5 14-3-3 gene plays a crucial role in the early events leading to polarization of the C. elegans zygote.     

SPD-3 is required for spindle alignment in Caenorhabditis elegans embryos and localizes to mitochondria

During the development of multicellular organisms, cellular diversity is often achieved through asymmetric cell divisions that produce two daughter cells having different developmental potentials. Prior to an asymmetric cell division, cellular components segregate to opposite ends of the cell defining an axis of polarity. The mitotic spindle rotationally aligns along this axis of polarity, thereby ensuring that the cleavage plane is positioned such that segregated components end up in individual daughter cells. Here we report our characterization of a novel gene required for spindle alignment in Caenorhabditis elegans. During the first mitosis in spd-3(oj35) embryos the spindle failed to align along the anterior/posterior axis, leading to abnormal cleavage configurations. spd-3(oj35) embryos had additional defects reminiscent of dynein/dynactin loss-of-function possibly caused by the mislocalization of dynactin. Surprisingly, we found that SPD-3GFP localized to mitochondria. Consistent with this localization, spd-3(oj35) worms exhibited slow growth and increased ATP concentrations, which are phenotypes similar to those described for other mitochondrial mutants in C. elegans. To our knowledge, SPD-3 is the first example of a link between mitochondria and spindle alignment in C. elegans.     

The C. elegans par-4 gene encodes a putative serine-threonine kinase required for establishing embryonic asymmetry

During the first cell cycle of Caenorhabditis elegans embryogenesis, asymmetries are established that are essential for determining the subsequent developmental fates of the daughter cells. The maternally expressed par genes are required for establishing this polarity. The products of several of the par genes have been found to be themselves asymmetrically distributed in the first cell cycle. We have identified the par-4 gene of C. elegans, and find that it encodes a putative serine-threonine kinase with similarity to a human kinase associated with Peutz-Jeghers Syndrome, LKB1 (STK11), and a Xenopus egg and embryo kinase, XEEK1. Several strong par-4 mutant alleles are missense mutations that alter conserved residues within the kinase domain, suggesting that kinase activity is essential for PAR-4 function. We find that the PAR-4 protein is present in the gonads, oocytes and early embryos of C. elegans, and is both cytoplasmically and cortically distributed. The cortical distribution begins at the late 1-cell stage, is more pronounced at the 2- and 4-cell stages and is reduced at late stages of embryonic development. We find no asymmetry in the distribution of PAR-4 protein in C. elegans embryos. The distribution of PAR-4 protein in early embryos is unaffected by mutations in the other par genes.     

The nonmuscle myosin regulatory light chain gene mlc-4 is required for cytokinesis, anterior-posterior polarity, and body morphology during Caenorhabditis elegans embryogenesis.

Using RNA-mediated genetic interference in a phenotypic screen, we identified a conserved nonmuscle myosin II regulatory light chain gene in Caenorhabditis elegans, which we name mlc-4. Maternally supplied mlc-4 function is required for cytokinesis during both meiosis and mitosis and for establishment of anterior-posterior (a-p) asymmetries after fertilization. Reducing the function of mlc-4 or nmy-2, a nonmuscle myosin II gene, also leads to a loss of polarized cytoplasmic flow in the C. elegans zygote, supporting models in which cytoplasmic flow may be required to establish a-p differences. Germline P granule localization at the time of cytoplasmic flow is also lost in these embryos, although P granules do become localized to the posterior pole after the first mitosis. This result suggests that a mechanism other than cytoplasmic flow or mlc-4/nmy-2 activity can generate some a-p asymmetries in the C. elegans zygote. By isolating a deletion allele, we show that removing zygotic mlc-4 function results in an elongation phenotype during embryogenesis. An mlc-4/green fluorescent protein transgene is expressed in lateral rows of hypodermal cells and these cells fail to properly change shape in mlc-4 mutant animals during elongation.     

The minibrain kinase homolog, mbk-2, is required for spindle positioning and asymmetric cell division in early C. elegans embryos

In the newly fertilized Caenorhabditis elegans zygote, cytoplasmic determinants become localized asymmetrically along the anterior-posterior (A-P) axis of the embryo. The mitotic apparatus then orients so as to cleave the embryo into anterior and posterior blastomeres that differ in both size and developmental potential. Here we describe a role for MBK-2, a member of the Dyrk family of protein kinases, in asymmetric cell division in C. elegans. In mbk-2 mutants, the initial mitotic spindle is misplaced and cytoplasmic factors, including the germline-specific protein PIE-1, are mislocalized. Our findings support a model in which MBK-2 down-regulates the katanin-related protein MEI-1 to control spindle positioning and acts through distinct, as yet unknown factors, to control the localization of cytoplasmic determinants. These findings in conjunction with work from Schizosaccharomyces pombe indicate a possible conserved role for Dyrk family kinases in the regulation of spindle placement during cell division.     

Brief cytochalasin-induced disruption of microfilaments during a critical interval in 1-cell C. elegans embryos alters the partitioning of developmental instructions to the 2-cell embryo

We are investigating the involvement of the microfilament cytoskeleton in the development of early Caenorhabditis elegans embryos. We previously reported that several cytoplasmic movements in the zygote require that the microfilament cytoskeleton remain intact during a narrow time interval approximately three-quarters of the way through the first cell cycle. In this study, we analyze the developmental consequences of brief, cytochalasin D-induced microfilament disruption during the 1-cell stage. Our results indicate that during the first cell cycle microfilaments are important only during the critical time interval for the 2-cell embryo to undergo the correct pattern of subsequent divisions and to initiate the differentiation of at least 4 tissue types. Disruption of microfilaments during the critical interval results in aberrant division and P-granule segregation patterns, generating some embryos that we classify as 'reverse polarity', 'anterior duplication', and 'posterior duplication' embryos. These altered patterns suggest that microfilament disruption during the critical interval leads to the incorrect distribution of developmental instructions responsible for early pattern formation. The strict correlation between unequal division, unequal germ-granule partitioning, and the generation of daughter cells with different cell cycle periods observed in these embryos suggests that the three processes are coupled. We hypothesize that (1) an 'asymmetry determinant', normally located at the posterior end of the zygote, governs asymmetric cell division, germ-granule segregation, and the segregation of cell cycle timing elements during the first cell cycle, and (2) the integrity or placement of this asymmetry determinant is sensitive to microfilament disruption during the critical time interval.     

Acto-myosin reorganization and PAR polarity in C. elegans

The symmetry-breaking event during polarization of C. elegans embryos is an asymmetric rearrangement of the acto-myosin network, which dictates cell polarity through the differential recruitment of PAR proteins. The sperm-supplied centrosomes are required to initiate this cortical reorganization. Several questions about this event remain unanswered: how is the acto-myosin network regulated during polarization and how does acto-myosin reorganization lead to asymmetric PAR protein distribution? As we discuss, recent studies show that C. elegans embryos use two GTPases, RHO-1 and CDC-42, to regulate these two steps in polarity establishment. Although RHO-1 and CDC-42 control distinct aspects of polarization, they function interdependently to regulate polarity establishment in C. elegans embryos.