Physical Blocking Neural Tube Closure Affects Radial Intercalation and Neural Crest Midline-directed Migration in Xenopus Dorsal Explants

神经管闭合缺陷(NTDs)是一种严重的先天畸形疾病,在新生儿中有千分之一的发病率.神经管融合前后,多种组织参与形态发生运动.神经管一经融合,神经嵴细胞就会向背侧中线方向产生单极突出并向此方向迁移形成神经管的顶部.与此同时,神经管从腹侧开始发生辐射状切入以实现单层化.在此,我们在非洲爪蟾的移植体中机械阻断神经管的闭合以检测其细胞运动及随后的图式形成.结果显示神经管闭合缺陷的移植体不能形成单层化的神经管,并且神经嵴细胞滞留在侧面区域不能向背侧中线迁移,而对神经前体标记基因的检测显示神经管的背腹图式形成并未受到影响.以上结果表明神经管的融合对于辐射状切入和神经嵴细胞向背侧中线方向的迁移过程是必需的,而对于神经管的沿背腹轴方向的图式形成是非必需的.

Temporal and Spatial Expression Patterns of Sox1 Gene in Xenopus laevis

克隆了非洲爪蟾的Sox1基因并研究了它在非洲爪蟾早期发育过程中的时空表达图式,比较了Sox1-3基因在发育的脑和眼中的表达图式.序列比对分析显示Sox1-3蛋白在其HMG框结构域具有高度的保守性.通过RT-PCR方法分析了Sox1基因在爪蟾早期不同发育时段的表达情况,结果显示Sox1基因从未受精卵到尾芽期均有表达,但表达强度有所差异.原位杂交结果显示,在早期卵裂阶段和囊胚期,Sox1基因主要在动物极表达;从神经板期开始,Sox1基因主要在中枢神经系统和眼原基中表达.在蝌蚪期,Sox1与Sox2、Sox3在脑部和眼睛的表达区域有所不同.对于爪蟾Sox1基因时空表达图式的研究将有助于阐明SoxB1基因家族在脊椎动物神经系统发生过程中的作用.

Cloning and developmental expression of the Xenopus Nkx6 genes

The evolutionarily conserved Nkx6 family transcription factors play important roles in the patterning of the central nervous system (CNS) and pancreas in vertebrates. In this study, we describe the cloning and expression patterns of the three Nkx6 family

Islet-1 is required for ventral neuron survival in Xenopus

Islet-1 is a LIM domain transcription factor involved in several processes of embryonic development. Xenopus Islet-1 (Xisl-1) has been shown to be crucial for proper heart development. Here we show that Xisl-1 and Xisl-2 are differentially expressed in th

Xenopus RCOR2 (REST corepressor 2) interacts with ZMYND8, which is involved in neural differentiation

Regulation of neuronal gene expression is critical to nervous system development. REST (RE1-silencing transcription factor) regulates neuronal gene expression through interacting with a group of corepressor proteins including REST corepressors (RCOR). Here we show that Xenopus RCOR2 is predominantly expressed in the developing nervous system. Through a yeast two-hybrid screen, we isolated Xenopus ZMYND8 (Zinc finger and MYND domain containing 8) as an XRCOR2 interacting factor. XRCOR2 and XZMYND8 bind each other in co-immunoprecipitation assays and both of them can function as transcriptional repressors. XZMYND8 is co-expressed with XRCOR2 in the nervous system and overexpression of XZMYND8 inhibits neural differentiation in Xenopus embryos. These data reveal a RCOR2/ZMYND8 complex which might be involved in the regulation of neural differentiation. (C) 2010 Elsevier Inc. All rights reserved.

Differential expression of the Brunol/CELF family genes during Xenopus laevis early development

The BRUNOL/CELF family of RNA-binding proteins plays important roles in post-transcriptional regulation and has been implicated in several developmental processes. In this study, we describe the cloning and expression patterns of five Brunol genes in Xenopus laevis. Among them, only Brunol2 is maternally expressed and the zygotic expression of the other four Brunol genes starts at different developmental stages. During Xenopus development, Brunol1, 4-5 are exclusively expressed in the nervous system including domains in the brain, spinal cord, optic and otic vesicles. Brunol2 and 3 are expressed in both the somatic mesoderm and the nervous system. Brunol2 is also extensively expressed in the lens. In transfected Hela cells, BRUNOL1, 2 and 3 proteins are localized in both the cytoplasm and the nucleus, while BRUNOL4 and 5 are only present in the cytoplasm, indicating their different functions.

Comparative study and expression analysis of the interferon gamma gene locus cytokines in Xenopus tropicalis

Using bioinformatics approach, the genome locus containing interleukin (IL)-22, IL-26, and interferon gamma (IFN-gamma) genes has been identified in the amphibian, Xenopus tropicalis. Like that in other vertebrates such as fish, birds, and mammals, the Xenopus IL-22, IL-26, and IFN-gamma are clustered in the same chromosome and the adjacent genes are conserved. The genomic structures of the Xenopus IL-22, IL-26, and IFN-gamma gene were identical to that of their mammalian counterparts. The Xenopus IL-22 and IL-26 genes contained five exons and four introns while the Xenopus IFN-gamma gene consisted of four exons and three introns. The Xenopus IL-22, IL-26, and IFN-gamma share 14.1-41.6%, 14.6-31.2%, and 23.7-36.5% identity to their counterparts in other species, respectively. Reverse-transcription polymerase chain reaction (PCR) and real-time quantitative PCR analyses revealed that the expression of IL-22, IL-26, and IFN-gamma genes was significantly upregulated after simulation with bacterial polyliposaccharide and/or synthetic double-stranded poly(I:C), suggesting these cytokines like those in other vertebrates play an important role in regulating immune response in Xenopus.

Environmental signals: Synthetic humic substances act as xeno-estrogen and affect the thyroid system of Xenopus laevis

According to outdated paradigms humic substances (HS) are considered to be refractory or inert that do not directly interact with aquatic organisms. However, they are taken up and induce biotransformation activities and may act as hormone-like substances. In the present study, we tested whether HS can interfere with endocrine regulation in the amphibian Xenopus laevis. In order to exclude contamination with phyto-hormones, which may occur in environmental isolates, the artificial HS 1500 was applied. The in vivo results showed that HS 1500 causes significant estrogenic effects on X. laevis during its larval development and results of semi-quantitative RT-PCR revealed a marked increase of the estrogenic biomarker estrogen receptor mRNA (ER-mRNA). Furthermore, preliminary RT-PCR results showed that the thyroid-stimulating hormone (TSH beta-mRNA) is enhanced after exposure to HS1500, indicating a weak adverse effect on T3/T4 availability. Hence, HS may have estrogenic and anti-thyroidal effects on aquatic animals, and therefore may influence the structure of aquatic communities and they may be considered environmental signaling chemicals. (c) 2005 Elsevier Ltd. All rights reserved.

非洲爪蟾血清白蛋白的分离纯化及胰蛋白酶抑制活性

通过凝胶过滤层析及两步阴离子交换层析, 从非洲爪蟾( Xenopus laevis ) 的血清中获得了其68 kDa 的血清白蛋白。与大蹼铃蟾血清白蛋白相似, 非洲爪蟾血清白蛋白也具有抑制胰蛋白酶的活性, 但其抑制活力 相对较低, 180 nmol/L 的非洲爪蟾血清白蛋白能抑制84 %的胰蛋白酶活性(30 nmol/L) 。经表面等离子共振法 获得了其与胰蛋白酶的结合动力学常数, 解离平衡常数KD = 1144 ×10 - 6 mol/L 。经Western blot 分析发现, 非洲 爪蟾的皮肤中也分布有血清白蛋白。推测两栖类动物血清白蛋白具有的胰蛋白酶抑制活性可能是其抵御天敌捕 食的一种防御措施。

Sox2基因3'非翻译区保守元件对基因表达的调控作用

转录因子Sox2是脊椎动物早期发育中最早表达的神经系统特异性基因之一,同时在干细胞的维持中也起着关键作用.通过生物信息学分析,作者发现在脊椎动物Sox2 mRNA 3'非翻译区中存在4段非常保守的富含AU的区域.将这些片段按照不同的组合克隆到GFP和荧光素酶两种报告基因载体中,在非洲爪蟾胚胎和培养细胞中检测了这些片段对报告基因表达的影响.结果显示,Sox2的3'UTR可影响报告基因的表达水平,特别是其中的保守片段2可显著提高报告基因的表达水平,表明Sox2 3'非翻译区有可能参与Sox2表达的转录后调控.

利用修饰的随机引物构建非洲爪蟾酵母双杂交定向cDNA文库

描述了一种新的构建cDNA文库的方法,其中用来合成cDNA第一链的随机引物5'-端被加上碱基d(AC),在cDNA双链合成后所添加的连接头的末端含有碱基d(GTCG).在cDNA舣链3'-端,连接头连接上后会形成一个完整的Sall酶切位点d(GTCGAC),而在5'-端几乎不会形成Sall位点.在Sall酶切后利用形成的3'-粘性末端与5'-EcoRI粘性末端一起将cDNA双链定向导入线性化的质粒载体,再通过转化细菌获得cDNA文库.利用此方法构建了一个不同发育时期非洲爪蟾胚胎酵母双杂交cDNA文库.检测了文库中空载体的比例,插入片段大小和不同基因的表达水平几个指标,都符合预期,但是同时发现定位于mRNA的3'-端的插入片段比例比较低.这种倾向性符合文献报道,应该是实验系统的倾向性,不影响进一步的酵母双杂交实验.这些数据证明成功地构建了定向非洲爪蟾酵母双杂交cDNA文库.

Evolutionary Conservation of Dazl Genomic Organization and its Continuous and Dynamic Distribution Throughout Germline Development in Gynogenetic Gibel Carp

To investigate germline development and germ cell specification, we identified a Dazl homolog (CagDazl) from gynogenetic gibel carp (Carassius auratus gibelio). Its cDNA sequence and BAC clone sequence analyses revealed the genomic organization conservation and conserved synteny of the Dazl family members and their neighborhood genes among vertebrates, especially in fish. Moreover, a polyclonal antibody specific to CagDazl was produced and used to examine its expression and distribution throughout germline development at protein level. Firstly, ovary-specific expression pattern of CagDazl was confirmed in adult tissues by RT-PCR and Western blot. In addition, in situ hybridization and immunofluorescence localization demonstrated its specific expression in germ cells, and both its transcript and protein were localized to germ plasm. Then, co-localization of CagDazl and mitochondrial cloud was found, confirming that CagDazl transcript and its protein are germ plasm component and move via METRO pathway during oogenesis. Furthermore, the CagDazl is abundant and continuous throughout germline development and germ cell specification including primordial germ cell (PGC) formation, oogonium differentiation, oocyte development, and embryogenesis, and the dynamic distribution occurs at different development stages. The data suggest that maternal CagDazl might play an important role in gibel carp PGC formation. Therefore, CagDazl is a useful and specific marker for tracing germ plasm and germ cell development in the gynogenetic gibel carp. In addition, in comparison with previous studies in sexual reproduction species, the continuous and dynamic distribution of CagDazl protein in the germ plasm throughout the life cycle seems to have significant implication in sex evolution of vertebrates. J. Exp. Zool. (Mol. Deu. Euol.) 312B:855-871, 2009. (C) 2009 Wiley-Liss, Inc.

Differential expression and dynamic changes of SOX3 during gametogenesis and sex reversal in protogynous hermaphroditic fish

SOX3 has been suggested to play significant roles in gametogenesis and gonad differentiation of vertebrates, but the exact cellular localization evidence is insufficient and controversial. In this study, a protogynous hermaphrodite fish Epinephelus coioides is selected to analyze EcSox3 differential expression and the expression pattern in both processes of oogenesis and spermatogenesis by utilizing the advantages that gonad development undergoes transition from ovary to intersexual gonad and then to testis, and primordial germ cells and different stage cells during oogenesis and spermatogenesis are synchronously observed in the transitional gonads. The detailed and clear immunofluoresence localization indicates that significantly differential expression and dynamic changes of Sox3 occur in the progresses of gametogenesis and sex reversal, and EcSOX3 protein exists in the differentiating primordial germ cells, oogonia, and different stage oocytes of ovaries, and also in the differentiating primordial germ cells and the Sertoli cells of testis. One important finding is that the EcSox3 expression is a significant time point for enterable gametogenesis of primordial germ cells because EcSOX3 is obviously expressed and localized in primordial germ cells. As EcSox3 continues to express, the EcSOX3-positive primordial germ cells develop toward oogonia and then oocytes, whereas when EcSox3 expression is ceased, the EcSOX3-positive primordial germ cells develop toward spermatogonia. Therefore, the current finding of EcSOX3 in the differentiating primordial germ cells again confirms the potential regulatory role in oogenesis and germ cell differentiation. The data further suggest that SOX3, as a transcription factor, might have more important roles in oogenesis than in spermatogenesis.

Cyclin A2 is differentially expressed during oocyte maturation between gynogenetic silver crucian carp and gonochoristic color crucian carp

Silver crucian carp (Carassius auratus gibelio) is a unique gynogenetic fish. Because of its specific genetic background and reproduction mode, it is an intriguing model system for understanding regulatory mechanism of oocyte maturation division. It keeps its chromosomal integrity by inhibiting the first meiotic division (no extrusion of the first pole body). The spindle behavior during oocyte maturation is significantly different from that in gonochoristic fish. The chromosomes are first arranged in a tripolar spindle, and then they turn around and are reunited mutually to form a normal bipolar spindle. A new member of the fish A-type cyclin gene, cyclin A2, has been isolated by suppression of subtractive hybridization on the basis of its differential transcription in fully-grown oocytes between the gynogenetic silver crucian carp and gonochoristic color crucian carp. There are 18 differing amino acids in the total 428 residues of cyclin A2 between the two forms of crucian carps. In addition, cDNAs of cyclin A1 and cyclin B have also been cloned from them. Thus two members of A-type cyclins, cyclin A1 and cyclin A2, are demonstrated to exist in fish, just as in frog, humans, and mouse. Northern blotting reveals that cyclin A2 mRNA is more than 20-fold and cyclin A1 mRNA is about 2-fold in fully grown oocytes of gynogenetic silver crucian carp compared to gonochoristic color crucian carp. However, cyclin B does not show such a difference between them. Western blot analysis also shows that the cyclin A2 protein stockpiled in fully grown oocytes of gynogenetic crucian carp is much more abundant than in gonochoristic crucian carp. Moreover, two different cyclin A2 expression patterns during oocyte maturation have been revealed in the two closely related crucian carps. For color crucian carp, cyclin A2 protein is translated only after hormone stimulation. For silver crucian carp, cyclin A2 protein can be detected throughout the process of maturation division. The different expression of cyclin A2 may be a clue to understanding the special maturation division of gynogenetic silver crucian carp.