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。     

Early development of amphioxus nervous system with special reference to segmental cell organization and putative sensory cell precursors: a study based on the expression of pan-neuronal marker gene Hu/elav.

The development of the nervous system of amphioxus was investigated at the cellular level based on the expression of the pan-neuronal marker gene Hu/elav. In situ hybridization analysis showed that an amphioxus Hu/elav homolog (AmphiHu/Hel) was expressed in individual cells within the neural plate, but the cells exhibited no obvious arrangements in early embryos without distinct somites. However, in neurulae with somites, AmphiHu/Hel-positive cells were clustered along the D-V axis in close register with the boundaries of somites, resulting in reiterated cell arrangements that became evident along the neuraxis. Furthermore, AmphiHu/Hel-positive cell clusters appeared one by one along with the development of underlying somites. Double-staining in situ hybridization analysis with the islet gene revealed that the cell clusters contain presumptive motoneurons. In addition, AmphiHu/Hel expression was also observed outside the CNS, probably in the epithelial ectoderm, suggesting that amphioxus has a large number of putative sensory cell precursors as early as the early neurula stage. Taking recent gene expression studies and anatomical studies into consideration, we discuss ontogenetic and phylogenetic features of the amphioxus nervous system.     

Experimental approaches to the study of the formation of axial structures during early development of <Emphasis Type="Italic">Xenopus laevis</Emphasis>

The effect of mechanical extension on the differentiation of axial mesoderm in double explants (sandwiches) of Xenopus laevis embryonic tissues isolated during the early gastrula–late neurula developmen-tal period is studied. In explants at the early gastrula stage, artificial extension orients and stimulates isolated differentiation of the notochord and somites as well as their joint formation. Moreover, extension facilitated the formation of the normal anatomical structure of the notochord and affected expression of Chordin gene. At the late gastrula stage, the effect of artificial extension on joint somite–notochord differentiation was weaker. At the stage of late neurula, somites were sometimes formed in explants lacking a notochord anlage. Thus, at earlier stages, the formation of somites was stimulated by contacts with the notochord and joint development of both structures was mechanical dependent, while at the later stages, somites developed inde-pendently of the notochord. Thus, the role of tissue extension is primarily the establishment of normal mor-phology and expression of Chordin was located in the direction of extension.     

The retinoic acid signaling pathway regulates anterior/posterior patterning in the nerve cord and pharynx of amphioxus,a chordate lacking neural crest

Amphioxus, the closest living invertebrate relative of the vertebrates, has a notochord, segmental axial musculature, pharyngeal gill slits and dorsal hollow nerve cord, but lacks neural crest. In amphioxus, as in vertebrates, exogenous retinoic acid (RA) posteriorizes the embryo. The mouth and gill slits never form, AmphiPax1, which is normally downregulated where gill slits form, remains upregulated and AmphiHox1 expression shifts anteriorly in the nerve cord. To dissect the role of RA signaling in patterning chordate embryos, we have cloned the single retinoic acid receptor (AmphiRAR), retinoid X receptor (AmphiRXR) and an orphan receptor (AmphiTR2/4) from amphioxus. AmphiTR2/4 inhibits AmphiRAR-AmphiRXR-mediated transactivation in the presence of RA by competing for DR5 or IR7 retinoic acid response elements (RAREs). The 5' untranslated region of AmphiTR2/4 contains an IR7 element, suggesting possible auto- and RA-regulation. The patterns of AmphiTR2/4 and AmphiRAR expression during embryogenesis are largely complementary: AmphiTR2/4 is strongly expressed in the cerebral vesicle (homologous to the diencephalon plus anterior midbrain), while AmphiRAR expression is high in the equivalent of the hindbrain and spinal cord. Similarly, while AmphiTR2/4 is expressed most strongly in the anterior and posterior thirds of the endoderm, the highest AmphiRAR expression is in the middle third. Expression of AmphiRAR is upregulated by exogenous RA and completely downregulated by the RA antagonist BMS009. Moreover, BMS009 expands the pharynx posteriorly; the first three gill slit primordia are elongated and shifted posteriorly, but do not penetrate, and additional, non-penetrating gill slit primordia are induced. Thus, in an organism without neural crest, initiation and penetration of gill slits appear to be separate events mediated by distinct levels of RA signaling in the pharyngeal endoderm. Although these compounds have little effect on levels of AmphiTR2/4 expression, RA shifts pharyngeal expression of AmphiTR2/4 anteriorly, while BMS009 extends it posteriorly. Collectively, our results suggest a model for anteroposterior patterning of the amphioxus nerve cord and pharynx, which is probably applicable to vertebrates as well, in which a low anterior level of AmphiRAR (caused, at least in part, by competitive inhibition by AmphiTR2/4) is necessary for patterning the forebrain and formation of gill slits, the posterior extent of both being set by a sharp increase in the level of AmphiRAR. Supplemental data available on-line     

<Emphasis Type="Italic">AmphiFoxQ2</Emphasis>, a novel winged helix/forkhead gene,exclusively marks the anterior end of the amphioxus embryo

A full-length FoxQ-related gene (AmphiFoxQ2) was isolated from amphioxus. Expression is first detectable in the animal/anterior hemisphere at the mid blastula stage. The midpoint of this expression domain coincides with the anterior pole of the embryo and is offset dorsally by about 20 degrees from the animal pole. During the gastrula stage, expression is limited to the anterior ectoderm. By the early neurula stage, expression remains in the anterior ectoderm and also appears in the adjacent anterior mesendoderm. By the early larval stages, expression is detectable in the anteriormost ectoderm and in the rostral tip of the notochord. AmphiFoxQ2 is never expressed anywhere except at the anterior tip of amphioxus embryos and larvae. This is the first gene known that exclusively marks the anterior pole of chordate embryos. It may, therefore, play an important role in establishing and/or maintaining the anterior/posterior axis.     

Phenoloxidase, a marker enzyme for differentiation of the neural ectoderm and the epidermal ectoderm during embryonic development of amphioxus Branchiostoma belcheri tsingtaunese

The development of phenoloxidase during amphioxus embryogenesis was spectrophotometrically and histochemically studied for the first time in the present study. It was found that (1) PO activity initially appeared in the general ectoderm including the neural ectoderm and the epidermal ectoderm at the early neurula stage but not in the mesoderm or the endoderm, and (2) PO activity disappeared in the neural plate cells but remained unchanged in the epidermal cells when the neural plate was morphologically quite distinct from the rest of the ectoderm. It is apparent that PO could serve as a marker enzyme for differentiation of the neural ectoderm from the epidermal ectoderm during embryonic development of amphioxus.     

Sonic hedgehog in the pharyngeal endoderm controls arch pattern via regulation of Fgf8 in head ectoderm

Fgf8 signalling is known to play an important role during patterning of the first pharyngeal arch, setting up the oral region of the head and then defining the rostral and proximal domains of the arch. The mechanisms that regulate the restricted expression of Fgf8 in the ectoderm of the developing first arch, however, are not well understood. It has become apparent that pharyngeal endoderm plays an important role in regulating craniofacial morphogenesis. Endoderm ablation in the developing chick embryo results in a loss of Fgf8 expression in presumptive first pharyngeal arch ectoderm. Shh is locally expressed in pharyngeal endoderm, adjacent to the Fgf8-expressing ectoderm, and is thus a candidate signal regulating ectodermal Fgf8 expression. We show that in cultured explants of presumptive first pharyngeal arch, loss of Shh signalling results in loss of Fgf8 expression, both at early stages before formation of the first arch, and during arch formation. Moreover, following removal of the endoderm, Shh protein can replace this tissue and restore Fgf8 expression. Overexpression of Shh in the non-oral ectoderm leads to an expansion of Fgf8, affecting the rostral-caudal axis of the developing first arch, and resulting in the formation of ectopic cartilage. Shh from the pharyngeal endoderm thus regulates Fgf8 in the ectoderm and the role of the endoderm in pharyngeal arch patterning may thus be indirectly mediated by the ectoderm.     

Topographic changes in nascent and early mesoderm in amphioxus embryos studied by DiI labeling and by in situ hybridization for a Brachyury gene

 In amphioxus embryos, the nascent and early mesoderm (including chorda-mesoderm) was visualized by expression of a Brachyury gene (AmBra-2). A band of mesoderm is first detected encircling the earliest (vegetal plate stage) gastrula sub-equatorially. Soon thereafter, the vegetal plate invaginates, resulting in a cap-shaped gastrula with the mesoderm localized at the blastoporal lip and completely encircling the blastopore. As the gastrula stage progresses, DiI (a vital dye) labeling demonstrates that the entire mesoderm is internalized by a slight involution of the epiblast into the hypoblast all around the perimeter of the blastopore. Subsequently, during the early neurula stage, the internalized mesoderm undergoes anterior extension mid-dorsally (as notochord) and dorsolaterally (in paraxial regions where segments will later form). By the late neurula stage, AmBra-2 is no longer transcribed throughout the mesoderm as a whole; instead, expression is detectable only in the posterior mesoderm and in the notochord, but not in paraxial mesoderm where definitive somites have formed. Received: 28 November 1996 / Accepted: 2 January 1997     

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.     

Amphioxus AmphiDelta: evolution of Delta protein structure, segmentation, and neurogenesis

The amphioxus genome has a single Delta gene (AmphiDelta) encoding a protein 766 amino acids long. Comparison of Delta proteins of amphioxus and other animals indicates that AmphiDelta retains features of a basal bilaterian Delta protein--in having nine epidermal growth factor (EGF) repeats and also in having characteristic numbers of amino acids separating successive cysteines between and within EGF repeats. During development, AmphiDelta is expressed in the forming somites, in some regions of pharyngeal endoderm, and in cells (presumably differentiating neurons) scattered in both the neural plate and ectoderm. Expression is strongly associated with cells initiating movements to separate themselves from parent epithelia, either en masse by evagination (endoderm and mesoderm) or by delamination as isolated cells (ectoderm). The AmphiDelta-expressing cells delaminating from the ectoderm apparently migrate beneath it as they begin differentiating into probable sensory neurons, suggesting a scenario for the evolutionary origin of the placode-derived neurons of vertebrate cranial ganglia.