Cathepsin L involved in the freezing resistance of murine normal hatching embryos and dormant embryos

The cryopreservation of mammalian embryos is an important technology in embryo engineering. The discovery and application of the embryo's own high freezing resistance factors are the main methods to improve the utilization of mammalian embryos in cryopreservation. Cathepsin L gene expression in the frozen and thawed dormant embryos displayed a significant difference from those normal hatched ones. The aim of the present study was to dig out the potential role of Cathepsin L in anti-freezing capacity of murine blastocysts by investigating the location and expression of Cathepsin L in frozen and thawed both activated and dormant hatching blastocysts. Different concentrations of Cathepsin L recombinant protein and E-64d were then respectively added into the embryo cryoprotectant and pre-cryo culture medium. Our results found that down-regulation of Cathepsin L improves the freezing resistance of murine normal hatching embryos by reducing apoptosis. Cathepsin L inhibitors can be used to improve the efficiency of cryopreservation and recovery of blastocysts in vitro. Our study provides a theoretical basis for the further development and application of Cathepsin L.     

Characterization of the zebrafish tbx16 gene and evolution of the vertebrate T-box family

 We report on a new zebrafish T-box-containing gene, tbx16. It encodes a message that is first detected throughout the blastoderm soon after the initiation of zygotic gene expression. Following gastrulation, expression becomes restricted to paraxial mesoderm and later primarily to the developing tail bud. To gain an evolutionary prospective on the potential function of this gene, we have analyzed its phylogenetic relationships to known T-box genes from other species. Zebrafish tbx16 is likely orthologous to the chicken Tbx6L and Xenopus Xombi/Antipodean/Brat/VegT genes. Our analysis also shows that zebrafish tbx6 and mouse Tbx6 genes are paralogous to zebrafish tbx16. We present evidence which argues, that despite the same name and similar expression, zebrafish tbx6 and mouse Tbx6 genes are not orthologous to each other but instead represent relatively distant paralogs. The expression patterns of all genes are discussed in the light of their evolutionary relationships. Received: 27 November 1997 / Accepted: 27 January 1998     

Three Wnt genes expressed in a wide variety of tissues during development of the zebrafish,Danio rerio: developmental and evolutionary perspectives

 Proteins encoded by the Wnt family of genes act as signals and have been shown to play important roles in a wide variety of developmental processes. Here we describe the cloning of three Wnt family members from the zebrafish, Danio rerio, which encode proteins with homology to murine Wnt-2, -4 and -5A/B. The expression patterns of the latter two zebrafish genes, designated ZfWnt4 and ZfWnt5 show considerable similarity with their homologues in other vertebrates; ZfWnt2, however, is expressed in the developing viscera in a pattern distinct from its closest murine homologue. In the light of the similarities and differences in the patterns of expression of these genes relative to their homologues in other vertebrates, we speculate on their possible functions. Received 24 October 1995 / Accepted in revised form: 16 January 1996     

Amphibian hatching gland cells: pattern and distribution in anurans

The hatching gland (HG) is a transient organ, found in most anuran embryos and early larvae, and located on the dorsal side of the head. The enzymes secreted by hatching gland cells (HGCs) aid the embryos to escape from their enveloping coats. Analysis of HG morphology and distribution in 20 anuran species from six families using scanning electron microscopy revealed small differences in the shape and pattern of the gland particularly in the length and width of the posterior mid-dorsal extension of the gland. The four species of foam-nest making leptodactylids examined had HGs of a somewhat different shape to the others, but otherwise, there was little sign of a relationship between HG shape and taxonomic position. In the single Eleutherodactylus species examined, cells with the appearance and location of HGCs were transiently present long before the active stage of hatching. No sign of HGCs was seen on the head surface of one species, Phyllomedusa trinitatis. It seems possible that in this species, hatching is achieved by a mechanical rather than an enzymatic mechanism. The microvilli characteristic of the surfaces of HGCs were quite variable in density and length from species to species, and at different stages. HGCs remained at the surface of the embryo for some time after hatching and the possibility of a post-hatching function is briefly discussed.     

sox30: a novel zebrafish sox gene expressed in a restricted manner at the midbrain-hindbrain boundary during neurogenesis

 The Sox family of proteins is thought to act to regulate gene expression in a wide variety of developmental processes. Here we describe the cloning of sox30, a novel sox gene from the zebrafish (Danio rerio). In situ hybridization shows that sox30 is expressed in a restricted manner at the boundary between the midbrain and hindbrain during nervous system development. This expression pattern is in direct contrast to that of most other neuronally expressed Sox genes which are expressed throughout the nervous system. Received: 30 October 1998 / Accepted: 1 February 1999     

Hox gene expression reveals regionalization along the anteroposterior axis of the zebrafish notochord

 The vertebrate Hox genes have been shown to confer regional identity along the anteroposterior axis of the developing embryo, especially within the central nervous system (CNS) and the paraxial mesoderm. The notochord has been shown to play vital roles in patterning adjacent tissues along both the dorsoventral and mediolateral axes. However, the notochord’s role in imparting anteroposterior information to adjacent structures is less well understood, especially as the notochord shows no morphological distinctions along the anteroposterior axis and is not generally described as a segmental or compartmentalized structure. Here we report that four zebrafish hox genes: hoxb1, hoxb5, hoxc6 and hoxc8 are regionally expressed along the anteroposterior extent of the developing notochord. Notochord expression for each gene is transient, but maintains a definite, gene-specific anterior limit throughout its duration. The hox gene expression in the zebrafish notochord is spatially colinear with those genes lying most 3’ in the hox clusters having the most anterior limits. The expression patterns of these hox cluster genes in the zebrafish are the most direct molecular evidence for a system of anteroposterior regionalization of the notochord in any vertebrate studied to date. Received: 30 March 1998 / Accepted: 16 June 1998     

Polar effects of lithium in the heart of the zebrafish Danio rerio

 The molecular signalling mechanisms that are believed to govern the patterning of the heart early in embryonic development are not well understood. We have investigated the events which occur during patterning of the vertebrate heart by exposing gastrula stage zebrafish embryos to lithium, which is known to affect the phosphoinositol signalling pathway. Treatment of embryos at 50% epiboly (5.25 h after fertilization at 28.5°C) with 0.3 m LiCl for 5–15 min, results in embryos with defects which range from mild to severe, depending on the length of time the embryos are exposed to lithium. In the heart, defects appear progressively in the inflow tract, the sinus venosus and atrium. By using an antibody that recognizes an atrium-specific isoform of myosin, our results show that lithium treatment at gastrulation specifically affects the atrium and sinus venosus, and has little obvious effect on the ventricle. Defects induced by lithium differ from those induced by retinoic acid (RA) treatment of similarly staged embryos, and suggest that lithium and RA may affect the patterning signals important for establishment of the vertebrate heart by acting on different populations of cells or by influencing different patterning pathways. Received: 8 December 1995 / Accepted: 11 April 1996     

Analysis of axon tract formation in the zebrafish brain: the role of territories of gene expression and their boundaries

Mutant analysis in the zebrafish is revealing the genes that are expressed in the early neuroepithelium and that regulate factors responsible for the guidance of commissural axons. We review work on the developing zebrafish brain illustrating the way in which territories of regulatory gene expression influence the formation and positioning of axon pathways. Received: 20 May 1997 / Accepted: 9 June 1997     

脊尾白虾组织蛋白酶L基因的克隆及其表达分析

根据本实验室构建的脊尾白虾(Exopalaemon carinicauda)血细胞全长cDNA文库获得的EST序列,利用RACE技术克隆获得脊尾白虾组织蛋白酶L基因的cDNA全长,命名为EcCatL基因.该序列全长1136 bp,包括5'非编码区24 bp,开放阅读框960 bp和3'非编码区152 bp,开放阅读框共编码319个氨基酸,预测相对分子量为35.30×103,理论等电点为5.27.同源性分析表明,脊尾白虾组织蛋白酶LEcCatL氨基酸序列与其它甲壳动物高度保守,与变色小长臂虾(Palaemonetes varians)及北极甜虾(Pandalus borealis) CatL的同源性分别为92％和76％.系统进化分析表明,EcCatL基因氨基酸序列与变色小长臂虾的CatL聚为一支.荧光定量PCR分析结果表明,EcCatL基因在血细胞、鳃、肝胰腺、肌肉、卵巢、肠、胃及眼柄中均有表达,其中肝胰腺中的相对表达量最高.感染鳗弧菌及WSSV后6h和12h,脊尾白虾血细胞和肝胰腺中EcCatL的表达量较对照组均极显著增加(P＜0.01),且具有明显的时间差异性,表明EcCatL基因在脊尾白虾免疫反应中具有重要作用.     

Mutations affecting pigmentation and shape of the adult zebrafish

 Mutations causing a visible phenotype in the adult serve as valuable visible genetic markers in multicellular genetic model organisms such as Drosophila melanogaster, Caenorhabditis elegans and Arabidopsis thaliana. In a large scale screen for mutations affecting early development of the zebrafish, we identified a number of mutations that are homozygous viable or semiviable. Here we describe viable mutations which produce visible phenotypes in the adult fish. These predominantly affect the fins and pigmentation, but also the eyes and body length of the adult. A number of dominant mutations caused visible phenotypes in the adult fish. Mutations in three genes, long fin, another long fin and wanda affected fin formation in the adult. Four mutations were found to cause a dominant reduction of the overall body length in the adult. The adult pigment pattern was found to be changed by dominant mutations in wanda, asterix, obelix, leopard, salz and pfeffer. Among the recessive mutations producing visible phenotypes in the homozygous adult, a group of mutations that failed to produce melanin was assayed for tyrosinase activity. Mutations in sandy produced embryos that failed to express tyrosinase activity. These are potentially useful for using tyrosinase as a marker for the generation of transgenic lines of zebrafish. Received: 17 June 1996 / Accepted: 15 July 1996     

Bone morphogenetic protein-4 expression characterizes inductive boundaries in organs of developing zebrafish

 We have cloned and examined the expression pattern of zebrafish bone morphogenetic protein-4 (BMP4) as a start to evaluating signals which might participate in the fashioning of organ systems in this genetically tractable species. The predicted sequence of the mature zebrafish protein is more than 75% identical to that of other vertebrates and 66% identical to Drosophila decapentaplegic (Dpp). As in other species, BMP4 is expressed ventrally during gastrulation, but the zebrafish is unusual in having an additional dorsal domain of expression. Subsequent BMP4 expression is especially prominent in sensory organs, fin buds, and in the gut, kidney, and heart. In all these sites, it becomes particularly enriched in regions of inductive demarcations. For example, expression initially extends through the entire heart tube but then becomes limited to the boundaries between cardiac chambers (sinus venosus-atrial junction, atrio-ventricular junction, and aortic root) prior to cushion formation. In early pectoral fin development, BMP4 is at first expressed uniformly but then becomes restricted to the mesenchyme subjacent to the apical ectodermal ridge. This suggests that among its roles in development, BMP4 serves as a signal in primordial outgrowth and also as a signal demarcating the borders within organs or structures where subspecializations occur. Received: 13 January 1997 / Accepted: 3 April 1997     

The evolution of paired appendages in vertebrates: T-box genes in the zebrafish

The presence of two sets of paired appendages is one of the defining features of jawed vertebrates. We are interested in identifying genetic systems that could have been responsible for the origin of the first set of such appendages, for their subsequent duplication at a different axial level, and/or for the generation of their distinct identities. It has been hypothesized that four genes of the T-box gene family (Tbx2–Tbx5) played important roles in the course of vertebrate limb evolution. To test this idea, we characterized the orthologs of tetrapod limb-expressed T-box genes from a teleost, Danio rerio. Here we report isolation of three of these genes, tbx2, tbx4, and tbx5. We found that their expression patterns are remarkably similar to those of their tetrapod counterparts. In particular, expression of tbx5 and tbx4 is restricted to pectoral and pelvic fin buds, respectively, while tbx2 can be detected at the anterior and posterior margins of the outgrowing fin buds. This, in combination with conserved expression patterns in other tissues, suggests that the last common ancestor of teleosts and tetrapods possessed all four of these limb-expressed T-box genes (Tbx2–Tbx5), and that these genes had already acquired, and have subsequently maintained, their gene-specific functions. Furthermore, this evidence provides molecular support for the notion that teleost pectoral and pelvic fins and tetrapod fore- and hindlimbs, respectively, are homologous structures, as suggested by comparative morphological analyses. Received: 14 July 1999 / Accepted: 4 September 1999     

Expression of Brachyury-like T-box transcription factor, Xbra3 in Xenopus embryo

The Xenopus Brachyury-like Xbra3 gene is a novel T-box gene that is closely associated with Xenopus Brachyury. The expression pattern of Xbra3 during development is similar to that of Xbra. During gastrulation Xbra3 is expressed in the marginal zone, with a gradient of increasing expression from ventral to dorsal. In the early neurula stage Xbra3 is expressed in the notochord and posterior mesoderm, but by the tailbud stage its expression is restricted to the forming tailbud and the posterior portion of the notochord.     

Characterization of Brachyury genes in the dogfish S. canicula and the lamprey L. fluviatilis. Insights into gastrulation in a chondrichthyan

In order to gain insights into the evolution of gastrulation mechanisms among vertebrates, we have characterized a Brachyury-related gene in a lamprey, Lampetra fluviatilis, and in a chondrichthyan, Scyliorhinus canicula. These two genes, respectively termed LfT and ScT, share with their osteichthyan counterparts prominent expression sites in the developing notochord, the tailbud, but also a transient expression in the prechordal plate, which is likely to be ancestral among vertebrates. In addition, the lamprey LfT gene is transcribed in the endoderm of the pharyngeal arches and the epiphysis, two expression sites that have not been reported thus far in gnathostomes, and, as in the chick, in the differentiating nephrotomes. Since Brachyury expression in nascent mesoderm and endoderm is highly conserved among vertebrates as well as cephalochordates, we have used this marker to identify these cell populations during gastrulation in the dogfish. The results suggest that these cells are initially present over the whole margin of the blastoderm and are displaced during gastrulation to its posterior part, which may correspond to the site of mesoderm and endoderm internalization. These data provide the first molecular characterization of gastrulation in a chondrichthyan. They indicate that gastrulation in the dogfish and in some amniotes shares striking similarities despite the phylogenetic distance between these species. This supports the hypothesis that the extensively divergent morphologies of gastrulae among vertebrates largely result from adaptations to the presence of yolk.