The zebrafish Hairy/Enhancer-of-split-related gene her6 is segmentally expressed during the early development of hindbrain and somites

Several vertebrate genes of the Hairy/Enhancer-of-split (HES) family are involved in paraxial mesoderm segmentation and intersomitic boundary establishment/maintenance. Here, we show that the zebrafish hairy-related gene, her6, highly homologous to the mammalian and chicken HES-1 genes, is expressed in the posterior part of each segmented somite and in stripes in the anterior presomitic mesoderm (PSM), and also in a dynamic, segmentally restricted pattern during hindbrain segmentation, with all rhombomeres expressing her6 at different time points and at different levels.     

<Emphasis Type="Italic">her11</Emphasis> is involved in the somitogenesis clock in zebrafish

Somitogenesis requires an intricate process of pre-patterning, which is driven by an oscillator mechanism consisting of the Delta-Notch pathway and hairy- (h) and Enhancer of split- [E(spl)] related genes. With the aim of unravelling the complex mechanism of somite pre-patterning, we have conducted an extensive search for h/E(spl)-related genes in the third release of the Danio rerio genomic sequence. We identified 14 new h/E(spl) genes and analysed them by in situ hybridisation for their potential role in the somitogenesis process. We describe here the functional analysis of one of these genes, which we have named her11. her11 is a paralogue of her1 and, similar to her1, is arranged in a head to head fashion with another her gene, namely the previously described her5. It shares an expression in the midbrain-hindbrain boundary with her5, but is in addition cyclically expressed in patterns overlapping those of her1 and her7 and complementary to those of hey1. Furthermore it is expressed in the anterior half of the most caudally formed somites. We show that Delta-Notch pathway genes and fused somites (fss) are necessary for the control of her11 expression. However, some aspects of the her11 regulation suggest that at least one additional as yet unknown gene of the Delta-Notch cascade is required to explain its expression. Morpholino-oligonucleotide-mediated knockdown of her11 shows that it is involved in the zebrafish somitogenesis clock via an interaction with her1 and her7. We have also studied the role of hey1 by morpholino injection, but could not find a direct function for this gene, suggesting that it reflects the output of the clock rather than being a core component of the mechanism.Edited by B. Herrmann     

Interaction of Wnt and caudal-related genes in zebrafish posterior body formation

Although Wnt signaling plays an important role in body patterning during early vertebrate embryogenesis, the mechanisms by which Wnts control the individual processes of body patterning are largely unknown. In zebrafish, wnt3a and wnt8 are expressed in overlapping domains in the blastoderm margin and later in the tailbud. The combined inhibition of Wnt3a and Wnt8 by antisense morpholino oligonucleotides led to anteriorization of the neuroectoderm, expansion of the dorsal organizer, and loss of the posterior body structure-a more severe phenotype than with inhibition of each Wnt alone-indicating a redundant role for Wnt3a and Wnt8. The ventrally expressed homeobox genes vox, vent, and ved mediated Wnt3a/Wnt8 signaling to restrict the organizer domain. Of posterior body-formation genes, expression of the caudal-related cdx1a and cdx4/kugelig, but not bmps or cyclops, was strongly reduced in the wnt3a/wnt8 morphant embryos. Like the wnt3a/wnt8 morphant embryos, cdx1a/cdx4 morphant embryos displayed complete loss of the tail structure, suggesting that Cdx1a and Cdx4 mediate Wnt-dependent posterior body formation. We also found that cdx1a and cdx4 expression is dependent on Fgf signaling. hoxa9a and hoxb7a expression was down-regulated in the wnt3a/wnt8 and cdx1a/cdx4 morphant embryos, and in embryos with defects in Fgf signaling. Fgf signaling was required for Cdx-mediated hoxa9a expression. Both the wnt3a/wnt8 and cdx1a/cdx4 morphant embryos failed to promote somitogenesis during mid-segmentation. These data indicate that the cdx genes mediate Wnt signaling and play essential roles in the morphogenesis of the posterior body in zebrafish.     

Fibin, a novel secreted lateral plate mesoderm signal, is essential for pectoral fin bud initiation in zebrafish

We identified a novel secreted protein, fibin, in zebrafish, mice and humans. We inhibited its function in zebrafish embryos by injecting antisense fibin morpholino oligonucleotides. A knockdown of fibin function in zebrafish resulted in no pectoral fin bud initiation and abolished the expression of tbx5, which is involved in the specification of pectoral fin identification. The lack of pectoral fins in fibin-knockdown embryos was partially rescued by injection of fibin RNA. fibin was expressed in the lateral plate mesoderm of the presumptive pectoral fin bud regions. Its expression region was adjacent to that of tbx5. fibin expression temporally preceded tbx5 expression in presumptive pectoral fin bud regions, and not abolished in tbx5-knockdown presumptive fin bud regions. In contrast, fibin expression was abolished in retinoic acid signaling-inhibited or wnt2b-knockdown presumptive fin bud regions. These results indicate that fibin is a secreted signal essential for pectoral fin bud initiation in that it potentially acts downstream of retinoic acid and wnt signaling and is essential for tbx5 expression. The present findings have revealed a novel secreted lateral plate mesoderm signal essential for fin initiation in the lateral plate mesoderm.     

Expression of five frizzleds during zebrafish craniofacial development

Wnt/Planar Cell Polarity (PCP) signaling is critical for proper animal development. While initially identified in Drosophila, this pathway is also essential for the proper development of vertebrates. Zebrafish mutants, defective in the Wnt/PCP pathway, frequently display defects in convergence and extension gastrulation movements and additional later abnormalities including problems with craniofacial cartilage morphogenesis. Although multiple Frizzled (Fzd) homologues, Wnt receptors, were identified in zebrafish, it is unknown which Fzd plays a role in shaping the early larvae head skeleton. In an effort to determine which Frizzleds are involved in this process, we analyzed the expression of five zebrafish frizzled homologues fzd2, 6, 7a, 7b, and 8a from 2–4 days post-fertilization (dpf). During the analyzed developmental time points fzd2 and fzd6 are broadly expressed throughout the head, while the expression of fzd7a, 7b and 8a is much more restricted. Closer examination revealed that fzd7b is expressed in the neural crest and the mesodermal core of the pharyngeal arches and in the chondrocytes of newly stacked craniofacial cartilage elements. However, fzd7a is only expressed in the neural crest of the pharyngeal arches and fzd8a is expressed in the pharyngeal endoderm.     

Knockdown of zebrafish crim1 results in a bent tail phenotype with defects in somite and vascular development

The Crim1 gene encodes a transmembrane protein containing six cysteine-rich repeats similar to those found in the BMP antagonist, chordin (chd). To investigate its physiological role, zebrafish crim1 was cloned and shown to be both maternally and zygotically expressed during zebrafish development in sites including the vasculature, intermediate cell mass, notochord, and otic vesicle. Bent or hooked tails with U-shaped somites were observed in 85% of morphants from 12 hpf. This was accompanied by a loss of muscle pioneer cells. While morpholino knockdown of crim1 showed some evidence of ventralisation, including expansion of the intermediate cell mass (ICM), reduction in head size bent tails and disruption to the somites and notochord, this did not mimic the classically ventralised phenotype, as assessed by the pattern of expression of the dorsal markers chordin, otx2 and the ventral markers eve1, pax2.1, tal1 and gata1 between 75% epiboly and six-somites. From 24 hpf, morphants displayed an expansion of the ventral mesoderm-derived ICM, as evidenced by expansion of tal1, lmo2 and crim1 itself. Analysis of the crim1 morphant phenotype in Tg(fli:EGFP) fish showed a clear reduction in the endothelial cells forming the intersegmental vessels and a loss of the dorsal longitudinal anastomotic vessel (DLAV). Hence, the primary role of zebrafish crim1 is likely to be the regulation of somitic and vascular development.     

Application of the systematic "DAmP" approach to create a partially defective C. albicans mutant

An understanding of gene function often relies upon creating multiple kinds of alleles. Functional analysis in Candida albicans, a major fungal pathogen, has generally included characterization of mutant strains with insertion or deletion alleles and over-expression alleles. Here we use in C. albicans another type of allele that has been employed effectively in the model yeast Saccharomyces cerevisiae, a "Decreased Abundance by mRNA Perturbation" (DAmP) allele (Yan et al., 2008). DAmP alleles are created systematically through replacement of 30 noncoding regions with nonfunctional heterologous sequences, and thus are broadly applicable. We used a DAmP allele to probe the function of Sun41, a surface protein with roles in cell wall integrity, cell-cell adherence, hyphal formation, and biofilm formation that has been suggested as a possible therapeutic target (Firon et al., 2007; Hiller et al., 2007; Norice et al., 2007). A SUN41-DAmP allele results in approximately 10-fold reduced levels of SUN41 RNA, and yields intermediate phenotypes in most assays. We report that a sun41Δ/Δ mutant is defective in biofilm formation in vivo, and that the SUN41-DAmP allele complements that defect. This finding argues that Sun41 may not be an ideal therapeutic target for biofilm inhibition, since a 90% decrease in activity has little effect on biofilm formation in vivo. We anticipate that DAmP alleles of C. albicans genes will be informative for analysis of other prospective drug targets, including essential genes.     

A model for wave control on coral breakage and species distribution in the Hawaiian Islands

The fringing reef off southern Molokai, Hawaii, is currently being studied as part of a multi-disciplinary project led by the US Geological Survey. As part of this study, modeling and field observations were utilized to help understand the physical controls on reef morphology and the distribution of different coral species. A model was developed that calculates wave-induced hydrodynamic forces on corals of a specific form and mechanical strength. From these calculations, the wave conditions under which specific species of corals would either be stable or would break due to the imposed wave-induced forces were determined. By combining this hydrodynamic force-balance model with various wave model output for different oceanographic conditions experienced in the study area, we were able to map the locations where specific coral species should be stable (not subject to frequent breakage) in the study area. The combined model output was then compared with data on coral species distribution and coral cover at 12 sites along Molokais south shore. Observations and modeling suggest that the transition from one coral species to another may occur when the ratio of the coral colonys mechanical strengths to the applied (wave-induced) forces may be as great as 5:1, and not less than 1:1 when corals would break. This implies that coral colonys mechanical strength and wave-induced forces may be important in defining gross coral community structure over large (orders of 10s of meters) spatial scales.     

R11: a cis-regulatory node of the sea urchin embryo gene network that controls early expression of SpDelta in micromeres

A gene regulatory network (GRN) controls the process by which the endomesoderm of the sea urchin embryo is specified. In this GRN, the program of gene expression unique to the skeletogenic micromere lineage is set in train by activation of the pmar1 gene. Through a double repression system, this gene is responsible for localization of expression of downstream regulatory and signaling genes to cells of this lineage. One of these genes, delta, encodes a Notch ligand, and its expression in the right place and time is crucial to the specification of the endomesoderm. Here we report a cis-regulatory element R11 that is responsible for localizing the expression of delta by means of its response to the pmar1 repression system. R11 was identified as an evolutionarily conserved genomic sequence located about 13 kb downstream of the last exon of the delta gene. We demonstrate here that this cis-regulatory element is able to drive the expression of a reporter gene in the same cells and at the same time that the endogenous delta gene is expressed, and that temporally, spatially, and quantitatively it responds to the pmar1 repression system just as predicted for the delta gene in the endomesoderm GRN. This work illustrates the application of cis-regulatory analysis to the validation of predictions of the GRN model. In addition, we introduce new methodological tools for quantitative measurement of the output of expression constructs that promise to be of general value for cis-regulatory analysis in sea urchin embryos.     

Expression patterns of lgr4 and lgr6 during zebrafish development

Leucine-rich repeat (LRR)-containing G protein-coupled receptors (LGRs) belong to the superfamily of G protein-coupled receptors, and are characterized by the presence of seven transmembrane domains and an extracellular domain that contains a series of LRR motifs. Three Lgr proteins – Lgr4, Lgr5, and Lgr6 – were identified as members of the LGR subfamily. Mouse Lgr4 has been implicated in the formation of various organs through regulation of cell proliferation during development, and Lgr5 and Lgr6 are stem cell markers in the intestine or skin. Although the expression of these three genes has already been characterized in adult mice, their expression profiles during the embryonic and larval development of the organism have not yet been defined. We cloned two zebrafish lgr genes using the zebrafish genomic database. Phylogenetic analyses showed that these two genes are orthologs of mammalian Lgr4 and Lgr6. Zebrafish lgr4 is expressed in the neural plate border, Kupffer’s vesicle, neural tube, otic vesicles, midbrain, eyes, forebrain, and brain ventricular zone by 24 h post-fertilization (hpf). From 36 to 96 hpf, lgr4 expression is detected in the midbrain–hindbrain boundary, otic vesicles, pharyngeal arches, cranial cartilages such as Meckel’s cartilages, palatoquadrates, and ceratohyals, cranial cavity, pectoral fin buds, brain ventricular zone, ciliary marginal zone, and digestive organs such as the intestine, liver, and pancreas. In contrast, zebrafish lgr6 is expressed in the notochord, Kupffer’s vesicle, the most anterior region of diencephalon, otic vesicles, and the anterior and posterior lateral line primordia by 24 hpf. From 48 to 72 hpf, lgr6 expression is confined to the anterior and posterior neuromasts, otic vesicles, pharyngeal arches, pectoral fin buds, and cranial cartilages such as Meckel’s cartilages, ceratohyals, and trabeculae. Our results provide a basis for future studies aimed at analyzing the functions of zebrafish Lgr4 and Lgr6 in cell differentiation and proliferation during organ development.     

The epaxial-hypaxial subdivision of the avian somite

In all jaw-bearing vertebrates, three-dimensional mobility relies on segregated, separately innervated epaxial and hypaxial skeletal muscles. In amniotes, these muscles form from the morphologically continuous dermomyotome and myotome, whose epaxial-hypaxial subdivision and hence the formation of distinct epaxial-hypaxial muscles is not understood. Here we show that En1 expression labels a central subdomain of the avian dermomyotome, medially abutting the expression domain of the lead-lateral or hypaxial marker Sim1. En1 expression is maintained when cells from the En1-positive dermomyotome enter the myotome and dermatome, thereby superimposing the En1-Sim1 expression boundary onto the developing musculature and dermis. En1 cells originate from the dorsomedial edge of the somite. Their development is under positive control by notochord and floor plate (Shh), dorsal neural tube (Wnt1) and surface ectoderm (Wnt1-like signalling activity) but negatively regulated by the lateral plate mesoderm (BMP4). This dependence on epaxial signals and suppression by hypaxial signals places En1 into the epaxial somitic programme. Consequently, the En1-Sim1 expression boundary marks the epaxial-hypaxial dermomyotomal or myotomal boundary. In cell aggregation assays, En1- and Sim1-expressing cells sort out, suggesting that the En1-Sim1 expression boundary may represent a true compartment boundary, foreshadowing the epaxial-hypaxial segregation of muscle.     

Galig, a novel cell death gene that encodes a mitochondrial protein promoting cytochrome c release

Galectin-3 internal gene (Galig) was recently identified as an internal gene transcribed from the second intron of the human galectin-3 gene that is implicated in cell growth, cell differentiation, and cancer development. In this study, we show that galig expression causes morphological alterations in human cells, such as cell shrinkage, cytoplasm vacuolization, nuclei condensation, and ultimately cell death. These alterations were associated with extramitochondrial release of cytochrome c, a known cell death effector. Furthermore, Bcl-xL co-transfection significantly reduced the release of cytochrome c induced by galig expression, suggesting a common pathway between the cytotoxic activity of galig and the anti-apoptotic activity of Bcl-xL. This antagonism was not observed upon co-transfection of Bcl-2 and galig. Galig encodes a mitochondrial-targeted protein named mitogaligin. Structure-activity relationship studies showed that the mitochondrial addressing of mitogaligin relies on an internal sequence that is required and sufficient for the release of cytochrome c and cell death upon cell transfection. Moreover, incubation of isolated mitochondria with peptides derived from mitogaligin induces cytochrome c release. Altogether, these results show that galig is a novel cell death gene encoding mitogaligin, a protein promoting cytochrome c release upon direct interaction with the mitochondria.     

基于nad5的西施舌漳州群体遗传分化水平分析——以蛤蜊属2个物种差异水平为参照

扩增了西施舌日照、连云港、北海、漳州4个野生群体、四角蛤蜊和中国蛤蜊各1个群体共73个样本的NAD5基因片段,测序获得了480bp核苷酸序列,分析核苷酸的多态性,旨在评估福建漳州西施舌与日照、连云港、北海西施舌之间的分化水平。结果:从73个序列中共检测到44种单倍型(Hap),其中西施舌4个群体有29种Haps,四角蛤蜊和中国蛤蜊分别有10种和5种Haps,漳州群体与北海、日照、连云港群体单倍型有明显差异;将西施舌分为北海、日照、连云港组(GP1)和漳州组(GP2)2个组,分析核苷酸差异,GP1与GP2间的T、A、G含量差异极显著(P0.01)。GP1与GP2间的遗传距离与组内(GP1、GP2)遗传距离之比为25.1—41.8,四角蛤蜊与中国蛤蜊之间的遗传距离与种内个体间遗传距离之比为24.4—36.7,GP1、GP2间的差异达到了四角蛤蜊和中国蛤蜊种间差异水平,而日照、北海群体间的遗传距离只有0.009,北海与日照群体地理位置虽远,但遗传差异则很小;AMOVA分析显示漳州西施舌发生了极显著遗传分化(FST=0.966—0.978,P0.01)。     

Innate immune gene expression in individual zebrafish after Listonella anguillarum inoculation

Zebrafish were intraperitoneally injected with 10(6)CFU (LD50) Listonella anguillarum. Three inoculated and control fish were collected at 1, 2, 4, 6 and 22h post infection (hpi) and the expression of genes related to the immune response (il1b, cebpb, tfa, mpx, tnfa, nitr9, tlr22, hsc70, cp, mrlp1, c3b and lyz) in each fish was monitored by means of real-time RT-PCR. A similar experiment was performed considering an intermediate time point at 15 hpi. Different relative levels of expression were found among genes. Also, wide interindividual variation in gene expression for most genes was detected among fish, inoculated or not. A steady increase of expression starting from the initial stages of the interaction was found for interleukin-1beta. An initial increase in levels of gene expression was found for the genes coding for the CCAAT/Enhancer Binding Protein subunit beta and the Novel Immune-Type Receptor 9, although their levels decreased later on and were indistinguishable from the controls at 22 hpi. Finally, some genes (Transferrin, Myeloid-specific Peroxidase and Tumour Necrosis Factor alpha) were upregulated at 22 hpi. Taken together, our results show an induction in gene expression of genes involved in the inflammatory and immune response upon L. anguillarum infection but also reveal the existence of a wide variation in the levels of expression of the studied genes in the zebrafish population.     

Characterization of a gene highly expressed in the Brassica napus pistil that encodes a novel proline-rich protein

Pis 30, a gene highly expressed in Brassica napus pistils and encoding a novel proline-rich protein was isolated and characterized. Sequences homologous to the Brassica Pis 30 gene were found only in Arabidopsis thaliana. The Pis 30 gene encodes a mature protein of 8.4 kDa with no previously characterized protein domains and whose function remains unknown. PIS 30 contains especially high levels of Pro (33%), but also of Leu (14%), Phe (10%) and Ser (6%). Although it is a proline-rich protein, PIS 30 shows only limited similarity to previously characterized plant proline-rich proteins. When compared to the stigma-specific activity of the B. napus SLR1 gene promoter in pistils of transgenic Arabidopsis, an 808 bp Pis 30 promoter fragment directed -glucuronidase expression primarily in the ovary, as well as in the stigma.     

Identification, cloning and tissue localization of a rainbow trout (Oncorhynchus mykiss) intelectin-like protein that binds bacteria and chitin

Intelectins are a recently identified group of animal lectins involved in innate immune surveillance. This paper describes the primary structure, expression and immunohistochemical localization of a rainbow trout plasma intelectin (RTInt). RTInt exhibited calcium-dependent binding to N-acetylglucosamine (GlcNAc) and mannose conjugated Toyopearl Amino 650 M matrices. When GlcNAc eluates from chromatography matrices were analyzed by reducing 1D PAGE and Western blots, the lectin appeared as approximately 37 kDa and approximately 72 kDa bands. Similar analysis of plasma revealed a single 72 kDa band under reducing conditions. MALDI-TOF MS demonstrated five, approximately 37 kDa isoforms (pI 5.3-6.1) separated by 2D-PAGE. A 975 bp cDNA sequence obtained by RT-PCR from liver and spleen tissue encoded a 325 amino acid secretory protein with homology to human and murine intelectins, which bind bacterial components and are induced during parasitic infections. Gene expression and immunohistochemistry detected RTInt in gill, spleen, hepatic sinusoid, renal interstitium, intestine, skin, swim bladder and within leukocytes. Direct binding assays demonstrated the ability of RTInt to bind relevant bacterial and chitinous targets. These findings suggest that RTInt plays a role in innate immune defense against bacterial and chitinous microbial organisms.