Hemps, a novel EGF-like protein, plays a central role in ascidian metamorphosis

All chordates share several characteristic features including a dorsal hollow neural tube, a notochord, a pharynx and an endostyle. Unlike other chordate taxa, ascidians have a biphasic life-history with two distinct body plans. During metamorphosis, the larval nerve cord and notochord degenerate and the pharyngeal gill slits and endostyle form. While ascidians, like other marine invertebrates, metamorphose in response to specific environmental cues, it remains unclear how these cues trigger metamorphosis. We have identified a novel gene (Hemps) which encodes a protein with a putative secretion signal sequence and four epidermal growth factor (EGF)-like repeats which is a key regulator of metamorphosis in the ascidian Herdmania curvata. Expression of Hemps increases markedly when the swimming tadpole larva becomes competent to undergo metamorphosis and then during the first 24 hours of metamorphosis. The Hemps protein is localised to the larval papillae and anterior epidermis of the larva in the region known to be required for metamorphosis. When the larva contacts an inductive cue the protein is released, spreading posteriorly and into the tunic as metamorphosis progresses. Metamorphosis is blocked by incubating larvae in anti-Hemps antibodies prior to the addition of the cue. Addition of recombinant Hemps protein to competent larvae induces metamorphosis in a concentration-dependent manner. A subgroup of genes are specifically induced during this process. These results demonstrate that the Hemps protein is a key regulator of ascidian metamorphosis and is distinct from previously described inducers of this process in terrestrial arthropods and aquatic vertebrates.     

Ecological regulation of development: induction of marine invertebrate metamorphosis

In the marine environment a wide range of invertebrates have a pelagobenthic lifecycle that includes planktonic larval and benthic adult phases. Transition between these morphologically and ecologically distinct phases typically occurs when the developmentally competent larva comes into contact with a species-specific environmental cue. This cue acts as a morphogenetic signal that induces the completion of the postlarval/juvenile/adult developmental program at metamorphosis. The development of competence often occurs hours to days after the larva is morphologically mature. In the non-feeding--lecithotrophic--larvae of the ascidian Herdmania curvata and the gastropod mollusc Haliotis asinina, gene expression patterns in pre-competent and competent stages are markedly different, reflecting the different developmental states of these larval stages. For example, the expression of Hemps, an EGF-like signalling peptide required for the induction of Herdmania metamorphosis, increases in competent larvae. Induction of settlement and metamorphosis results in further changes in developmental gene expression, which apparently is necessary for the complete transformation of the larval body plan into the adult form.     

昆虫蜕皮的分子机理及应用

昆虫蜕皮是一个由PTTH启始的、激素介导的基因序列表达和相互作用的级联反应过程。阐明昆虫蜕皮的分子机理,不仅可以解释发育生物学的科学问题,为害虫控制提供新的思路,还可以从中发现新的可资生产应用的分子。作者通过蛋白质组学方法从棉铃虫Helicoverpa armigera Hubner蜕皮幼虫鉴定到30个差异表达的蛋白质。通过抑制性消减杂交技术,从棉铃虫蜕皮幼虫、变态决定幼虫和5龄取食幼虫鉴定到100个表达序列标签(EST)。证明其中的11个EST在蜕皮或变态时差异表达。通过RT-PCR方法克隆棉铃虫激素接受子3基因,研究该基因在发育中的表达模式。用该基因构建具有绿色荧光蛋白标记和多角体蛋白的基因重组病毒(AcMNPV-GFP-HHR3-Polh)。实验结果表明,AcMNPV-GFPHHR3-Polh病毒可以通过注射或口服感染棉铃虫,导致棉铃虫幼虫非正常蜕皮、生长延缓、半数存活时间下降。该研究显示昆虫蜕皮功能基因在害虫控制中有很好的应用前景。蜕皮功能基因的表达与调控、蜕皮激素介导的信号转导通路、变态过程中组织解体和重建的分子机理、激素调控基因顺序表达的分子机理、变态起始因子、JH受体等是本领域今后的主要研究方向。     

Inhibition of settlement and metamorphosis of the ascidian Herdmania curvata by non-geniculate coralline algae

The surfaces of non-geniculate coralline algae (NCA) are known to induce the settlement and metamorphosis of disparate marine taxa. In this study we investigate the responsiveness of larvae of Herdmania curvata (Ascidiacea: Stolidobranchia) to three species of NCA (Neogoniolithon brassica-florida, Hydrolithon onkodes, and Lithothamnium prolifer) that cohabit the slope and crest of Heron Reef, Great Barrier Reef. H. curvata larvae were first exposed to these NCA at or within 2 h of hatching, which is 1 to 2 h prior to attaining competence, and then cultured continuously with the NCA for 12 to 14 h. Rates of settlement and metamorphosis of H. curvata cultured in laboratory chambers in the presence of the different NCA were significantly lower than spontaneous rates in seawater. The limited settlement in treatments containing NCA were confined entirely to the chamber periphery, and settlement never occurred on the surface of the NCA. The inhibitory effect was dose-dependent and was stronger in N. brassica-florida and H. onkodes than in L. prolifer. Larvae that did not settle in treatments with NCA had rounded anterior trunks and, in extreme cases, kinked tails with rounded and dissociated tail muscle cells. In some individuals, we observed the anterior chemosensory papillae being sloughed off the larval body. Morphological analysis of trunk ectodermal and mesenchymal nuclei of larvae cultured in the presence of the NCA revealed that general necrotic cell death was occurring. Importantly, H. curvata larvae that were exposed to NCA could not subsequently be induced to metamorphose in KCl-elevated seawater, whereas larvae not exposed to NCA metamorphosed at high rates in KCl-elevated seawater.     

Neuroectodermal and endodermal expression of the ascidian Cdx gene is separated by metamorphosis

Ascidians are a group of invertebrate chordates that exhibit a biphasic life history, with chordate-specific structures developing during embryogenesis (dorsal neural tube and notochord) and metamorphosis (pharyngeal gill slits and endostyle). Here we characterize the expression of a caudal/Cdx gene homologue, Hec-Cdx, from the ascidian Herdmania curvata. Vertebrate Cdx genes are expressed at gastrulation and in the posterior of the developing neural tube and endoderm. Hec-Cdx expression is initiated at the earliest stages of gastrulation, with peaks in RNA abundance occurring first during neurulation and tailbud extension and then in 3- to 5-day-old juveniles. Hec-Cdx is expressed in a pair of cells in the anterior lip of the blastopore in the late gastrula which form the most posterior portion of the neural plate. During tailbud formation expression is maintained in and solely restricted to these cells. During metamorphosis expression is localized to the intestine of the juvenile. These data, along with data for the H. curvata Otx gene, suggest that the evolution of the novel ascidian biphasic body plan was not accompanied by a deployment of these genes into new pathways but by a temporal separation of tissue-specific expression. Received: 10 October 1999 / Accepted: 1 November 1999     

Lipid peroxidation and antioxidant defence status during larval development and metamorphosis of giant prawn,Macrobrachium rosenbergii

In the present communication we studied the involvement of reactive oxygen species and alteration in antioxidant defence status during larval development and metamorphosis of giant prawn, Macrobrachium rosenbergii. Overall results indicate that there was a decline in endogenous lipid peroxidation level during larval development. Activity of superoxide dismutase was the lowest in early larval stages (Zoea-I and II) and thereafter increased in V and VI stages, followed by a decrease in the subsequent larval stages. Catalase and glutathione peroxidase did not exhibit specific pattern of changes during development. Reduced glutathione content exhibited an incremental increase during larval progression until metamorphosis. Ascorbic acid content of the larval tissue remained unaltered during development but a sharp fall was marked in its content in the post-larvae. Hence it is concluded that early larvae face high oxidative stress as evident from the high content of thiobarbituric acid reactive substances. This may be due to direct exposure of larvae to ambient oxygen of the water as well as their low antioxidant potential. However, during development with the augmentation in antioxidant reserve of the larval tissues a diminution in the oxidative stress was recorded. Thus it is presumed that antioxidant defences play an important role in providing protection to the developing larvae from oxidative assault during larval progression and metamorphosis.     

Induction of metamorphosis decreases nitric oxide synthase gene expression in larvae of the marine mollusc Ilyanassa obsoleta (say)

Many marine organisms spend the early part of their lives as larvae suspended in the water column before metamorphosing into benthic reproductive adults. Metamorphosis does not occur until a larva has become competent to respond to appropriate stimuli and after a suitable habitat for the young juvenile has been encountered. The gaseous neurotransmitter nitric oxide is thought to be important in the regulation of metamorphosis by holding the organism in the larval state. We have investigated expression of the neuronal nitric oxide synthase (nNOS) gene in larval and metamorphosing individuals of the marine mud snail Ilyanassa obsoleta. Our results indicate that nNOS is expressed at constant levels throughout larval development. In contrast, expression of nNOS decreases markedly during the first 24 h of metamorphosis. Our observations support previous findings that demonstrate that nitric oxide is present in larvae though competence. The decrease in nNOS gene expression that occurs during metamorphosis corresponds with a previously described reduction in nNOS activity.     

Global gene expression profiling of Homarus americanus (Crustacea) larval stages during development and metamorphosis

Homarus americanus has a life history that is similar to other arthropods, including a pelagic larval phase and a benthic adult phase. The larval phase is divided into three morphologically distinct stages, followed by metamorphosis to the post-larval phase. H. americanus larval development has been studied previously, although the molecular mechanisms that regulate the consequent changes are not fully elucidated. This study is the first to use an oligonucleotide microarray to investigate global gene expression during H. americanus larval development. Stage-specific gene expression profiles of larvae and postlarvae from two-year classes were assessed. We found the expression levels of 1851 genes to be significantly different among larval stages. Functional annotations indicated that various differentially expressed genes were involved with immune function, energy regulation, and development. Ten target genes of interest were selected for expression verification using RT-qPCR. Two Phosphoenolpyruvatecarboxykinases, Argonaute 2, Ecdysone-inducible protein 75, and Procollagen-lysine 2-oxoglutarate 5-dioxygenase 3, had significantly different expression (p?相似文献   

Further characterization of genes expressed during Ciona intestinalis metamorphosis

Metamorphosis of ascidians is a dynamic event by which a nonfeeding, mobile tadpole larva is transformed into a filter-feeding, fixed juvenile. This process usually begins with the settlement of the larva and is followed by a series of coordinated morphogenetic movements that rearrange organs, tissues, and cells. To identify genes that are involved in the initiation of metamorphosis, we conducted differential screening between mRNAs of swimming larvae and those of juveniles in Ciona intestinalis. This screening permitted the isolation of cDNA clones for genes whose expression is upregulated during metamorphosis, and the characterization of four such genes (Ci-meta3, Ci-meta4, Ci-meta5 and Ci-meta6) is reported here. Ci-meta3 encodes a protein with a domain found in Sp1a and the RYanodine receptor. This gene is not expressed in early swimming larvae but is expressed in the endoderm region and part of the retractile tail region in metamorphosing juveniles. The predicted proteins encoded by Ci-meta4, Ci-meta5 and Ci-meta6 do not contain any known consensus motifs, nor do they show any similarity to known proteins. Ci-meta4 and Ci-meta5 are expressed weakly in mesenchyme cells of the early larva and strongly in the metamorphosing juvenile, while Ci-meta6 is expressed in the mesenchyme in the late larva. In addition, we characterized 53 independent cDNA clones whose expression was downregulated during the period from early swimming larvae to metamorphosing juveniles by taking advantage of the Ciona intestinalis cDNA project database and BLAST searches. The expression patterns of some of these clones were changed during the larval period.     

Chemical composition, mineral content and amino acid and lipid profiles in bones from various fish species

Insulin-like growth factor-I (IGF-I) is an important regulator of growth and development in vertebrates. Both the endocrine and paracrine actions of IGF-I are mediated through ligand-binding to a membrane-bound IGF-I receptor (IGF-IR). The characterization of this receptor and subsequent expression studies thus help elucidate the endocrine regulation of developmental processes. As other flatfish species, the Atlantic halibut (Hippoglossus hippoglossus) undergoes a dramatic larval metamorphosis. This process is largely under endocrine control, and data indicate that IGF-I could be a key regulator. IGF-I content increases up to late pre-metamorphosis and decreases during metamorphosis. The IGF-IR has, however, not been studied during flatfish metamorphosis. To examine IGF-IR gene expression, two IGF-IR mRNA were cloned and sequenced. These partial sequences share high identity (>or=95%) and similarity (>or=97%) with other fish IGF-IR and lower identity (>or=77%) and similarity (>or=83.5%) with Japanese flounder insulin receptors. The expression of mRNA for both IGF-IR was analyzed by quantitative real-time RT-PCR during six larval developmental stages from pre- to post-metamorphosis. IGF-IR1 and IGF-IR2 mRNA are differentially expressed during metamorphosis, but if this indicates an isoform-specific regulation of developmental processes by circulating and/or locally-secreted IGF-I is unclear. Both IGF-IR genes are down-regulated in halibut larvae experiencing arrested metamorphosis, suggesting the IGF-I system is critical for metamorphic success in halibut.