Identification of up-regulated genes in amphioxus neurula and the expression of AmphiFABP

Amphioxus is a good model organism for understanding the origin and developmental mechanism of vertebrates owing to its important evolutionary position. During the developmental process of amphioxus embryo, the neurula is a crucial stage because of neural tube and notochord formation as well as somite emergence at this stage. In order to isolate genes up-regulated at the neurula stage, we constructed an 11-hour neurula subtracted cDNA library of amphioxus Branchiostoma belcheri and sequenced 204 ESTs representing 82 contigs. Comparative analysis revealed that 55% of those contigs were homologous to various known genes while 45% of them had no significant similarity to any known genes. Those observations imply that the un-identified ESTs might contain some new genes which are involved in the development of amphioxus neurula. Real-time quantitative PCR (RTqPCR) indicated that the expression levels of 14 genes are up-regulated after gastrulation among 20 assayed genes. Of those up-regulated genes, we further cloned and sequenced the full-length of fatty acid binding protein gene (AmphiFABP). The deduced protein sequence was similar to that of vertebrate brain FABP and heart FABP, and in situ hybridization displayed that AmphiFABP, similar to their vertebrate cognates, was expressed not only in nervous system but also in embryonic somite and gut, hinting a multifunctional property of AmphiFABP in amphioxus.     

鳙鱼ARISTICHTHYS NOBILIS早期发育的一些生物化学上的变化

本实验第一部分对鳙鱼卵及胚胎在不同的发育时期蛋白质合成的速度进行了研究,实验采用微量克氏定氮法,对鳙卵从未受精卵到受精卵、尾芽期和出膜期,共测定了十三个不同的发育时期。实验结果表明了在鱼的不同胚胎发育时期蛋白质合成速度有着明显的差异。 实验的第二部分对鳙鱼胚胎发育过程氨基酸组成和氨基酸含量进行了测定,其结果显示出鳙鱼卵和胚胎的各发育时期氨基酸的组分十分相似,但氨基酸的含量在各个时期是有差异的。     

Expression of somite segmentation genes in amphioxus: a clock without a wavefront?

In the basal chordate amphioxus (Branchiostoma), somites extend the full length of the body. The anteriormost somites segment during the gastrula and neurula stages from dorsolateral grooves of the archenteron. The remaining ones pinch off, one at a time, from the tail bud. These posterior somites appear to be homologous to those of vertebrates, even though the latter pinch off from the anterior end of bands of presomitic mesoderm rather than directly from the tail bud. To gain insights into the evolution of mesodermal segmentation in chordates, we determined the expression of ten genes in nascent amphioxus somites. Five (Uncx4.1, NeuroD/atonal-related, IrxA, Pcdhdelta2-17/18, and Hey1) are expressed in stripes in the dorsolateral mesoderm at the gastrula stage and in the tail bud while three (Paraxis, Lcx, and Axin) are expressed in the posterior mesendoderm at the gastrula and neurula stages and in the tail bud at later stages. Expression of two genes (Pbx and OligA) suggests roles in the anterior somites that may be unrelated to initial segmentation. Together with previous data, our results indicate that, with the exception that Engrailed is only segmentally expressed in the anterior somites, the genetic mechanisms controlling formation of both the anterior and posterior somites are probably largely identical. Thus, the fundamental pathways for mesodermal segmentation involving Notch-Delta, Wnt/beta-catenin, and Fgf signaling were already in place in the common ancestor of amphioxus and vertebrates although budding of somites from bands of presomitic mesoderm exhibiting waves of expression of Notch, Wnt, and Fgf target genes was likely a vertebrate novelty. Given the conservation of segmentation gene expression between amphioxus and vertebrate somites, we propose that the clock mechanism may have been established in the basal chordate, while the wavefront evolved later in the vertebrate lineage.     

文昌鱼tropomyosin基因的克隆、进化分析及其胚胎发育与成体中的表达模式

Tropomyosin是一种分布广泛而且在进化上十分保守的蛋白,是肌肉形成和收缩过程中重要的调节蛋白质。通过RT-PCR和RACE技术得到文昌鱼tropomyosin基因全长,编码一个含284个氨基酸残基的蛋白质,将文昌鱼Tropomyosin和在其他物种中的同源物进行比对建树,发现其在功能域上高度保守并且只有一个拷贝,符合动物分类学中各物种的进化地位。胚胎整体原位杂交实验得知,tropomyosin在文昌鱼早期发育的表达,最早从原肠胚末期神经胚早期开始,定位于分化中的中内胚层。到神经胚期,tropomyosin的表达出现在发育中的体节和脊索中。随着发育的进行,tropomyosin的表达稳定地集中在体节、脊索处。到72h幼虫阶段,tropomyosin的表达仍然在肌节内。成体的切片原位杂交结果显示,tropomyosin在肌节中的表达大幅度下调,而在神经管细胞、脊索和腮区腮瓣处仍然可以检测到明显的表达,在外胚层和表皮内没有发现杂交信号。研究结果表明,tropomyosin的表达与文昌鱼肌节、肌肉以及神经索的发生相关,参与文昌鱼胚胎躯体模式的构建,而且在成体的生命活动中发挥重要作用。     

EST analysis of mRNAs expressed in neurula of Chinese amphioxus

Amphioxus, a cephalochordate, is the closest living relative to the vertebrates. In order to investigate the molecular mechanisms of the early embryogenesis of amphioxus, we constructed a neurula embryo cDNA library of Chinese amphioxus (Branchiostoma belcheri tsingtauense) and generated 5235 expressed sequenced tags in the present study. The initial ESTs consisted of 638 clusters and 1855 singletons, which revealed approximately 2493 unique genes in the data set. Of these sequences, 35.52% ESTs matched to known genes, 12.76% matched to other ESTs, and 51.71% had no match to any known sequences in GenBank. Interestingly we found homologous genes related to neural development and human disease. Bioinformatic analysis showed the direct evidence that the gene homologue found only in vertebrates in previous studies also exists in the amphioxus genome. This study provides a preliminary view of the gene information involved in the development of neurula embryos of Chinese amphioxus and helps our understanding of vertebrate evolution at gene level.     

Curie-point pyrolysis, gas-liquid chromatography of xylan

Levels of nucleotides and sugar nucleotides in embryos of Bufo arenarum at the stages of morula, gastrula, and neurula have been measured. The total amounts of purine nucleoside diphosphates decreased from morula to gastrula, but increased sharply from gastrula to neurula. The levels of ADP followed this pattern, but those of GDP did not change significantly through the three stages. Purine nucleoside triphosphate levels, which had increased immediately after fertilization, remained almost constant through morula, gastrula, and neurula. As with the purine nucleoside diphosphates, the adenine nucleotide decreased from morula to gastrula, and increased from gastrula to neurula. In contrast, the level of GTP showed a sharp maximum at gastrula. The total pyrimidine nucleoside triphosphate did not change significantly from morula through neurula. As in previous stages of development, only uridine sugar nucleotides were detected. A sharp increase of the galactosyl ester of nucleotides was found at gastrula.     

Systematic investigation of Amphioxus (Branchiostoma floridae) microRNAs

MicroRNAs (miRNAs) participate in various biological processes via controlling gene activity. Amphioxus is the best available stand-in as the proximate invertebrate ancestor of the vertebrates. Here, we systematically investigated the miRNAs in amphioxus. First, we identified 245 candidate amphioxus miRNAs, in which 183 miRNAs were firstly reported. Second, we gave evidences to support a birth-and-death process of miRNA genes in some families and gave implications for the functional diversification of miRNA during evolution. Third, we identified 47 development-specific expression miRNAs. We found that only 19 miRNAs were expressed in all developmental stages, 16 miRNAs were neurula-specific and 13 miRNAs were larva-specific. In addition, these potential miRNA-targeting genes were mainly classified into development, muscle formation, cell adhesion, and gene regulation categories. Finally, we found 79 immune related genes targeted by 136 miRNAs in amphioxus. In conclusion, our results take an insight into both the function and evolution of the amphioxus miRNAs.     

Identification and expression of amphioxus beta-microseminoprotein (MSP)-like gene encoding an ancient and rapidly evolving protein in chordates

The cDNA encoding beta-microseminoprotein-like (beta-MSPL) was identified from the gut cDNA library of amphioxus. It contains a 336 bp open reading frame corresponding to a deduced protein of 111 amino acids and has eight cysteines conserved and located at the same positions as those in the vertebrate beta-MSPs. At amino acid level, it shares 12-20% similarity to the vertebrate beta-MSPs, and seems lacking the signal peptide at the N-terminus. This not only confirms that beta-MSP is a rapidly evolving protein during phylogeny, but also provides further data on the degree of diversity between species of this protein. RT-PCR and Northern blotting show that amphioxus beta-MSPL is expressed in all tissues examined, suggesting that beta-MSPL plays a fundamental role. However, in situ hybridization reveals that positive hybridization signals were present in all blastomeres of the embryos from 4-cell to gastrula stages, while its expression is restricted exclusively to notochord, somites and primitive gut in neurulae and larvae, and disappears in the ectoderm including the neural tube differentiated from the ectoderm. This suggests that beta-MSPL is possibly involved in the differentiation of ectoderm during embryonic development of cephalochordate amphioxus though it is ubiquitously expressed in embryos prior to gastrula stage and in the adult animal.     

Morphogenesis of extraembryonic membranes and placentation in Mabuya mabouya (Squamata, Scincidae)

Topological and histological analyses of Mabuya mabouya embryos at different developmental stages showed an extraembryonic membrane sequence as follows: a bilaminar omphalopleure and progressive mesodermal expansion around the whole yolk sac at gastrula stages; mesodermal split and formation of an exocoelom in the entire embryonic chamber at neurula stages; beginning of the expansion of the allantois into the exocoelom to form a chorioallantoic membrane at pharyngula stages; complete extension of the allantois into the exocoelom between limb-bud to preparturition stages. Thus, a placental sequence could be enumerated: bilaminar yolk sac placenta; chorioplacenta; allantoplacenta. All placentas are highly specialized for nutrient absorption from early developmental stages. The bistratified extraembryonic ectoderm possesses an external layer with cuboidal cells and a microvillar surface around the whole yolk sac, which absorbs uterine secretions during development of the bilaminar yolk sac placenta and chorioplacenta. During gastrulation, with mesodermal expansion a dorsal absorptive plaque forms above the embryo and several smaller absorptive plaques develop antimesometrially. Both structures are similar histologically and are active in histotrophic transfer from gastrula stages until the end of development. The dorsal absorptive plaque will constitute the placentome and paraplacentome during allantoplacental development. At late gastrula-early neurula stages some absorptive plaques form chorionic concavities or chorionic bags that are penetrated by a long uterine fold and seem to have a specialized histotrophic and/or metabolic role. The extraembryonic mesoderm does not ingress into the yolk sac and neither an isolated yolk mass nor a yolk cleft are formed. This derived pattern of development may be related to the drastic reduction of the egg size and obligatory placentotrophy from early developmental stages. Our results show new specialized placentotrophic structures and a novel arrangement of extraembryonic membrane morphogenesis for Squamata.