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At least 1,639 records · Page 91Linked to original sources
PubMed2005

Expression of a novel zebrafish zinc finger gene, gli2b, is affected in Hedgehog and Notch signaling related mutants during embryonic development.

Gli zinc-finger proteins are known as downstream mediators of the evolutionary conserved Hedgehog pathway. In zebrafish, gli2 functions differently from Gli2 in mammals. This difference could be due to the gli2 duplication in teleosts evolution and partial redundancy between two duplicated genes. Here, we report a novel zebrafish gli2-like cDNA. Its structure, genetic location, and distinct expression pattern in the central nervous system suggested that this gene might represent a second gli2 of teleosts, and we named it gli2b. gli2b was expressed in the neural keel, excluding the forebrain-midbrain boundary, while gli2 expression complemented this pattern. After 24 hours postfertilization, several specific domains of gli2b expression were observed in the lateral and medial hindbrain and hypothalamus. In mutants affecting the Hedgehog and Notch signaling pathways, gli2b expression was either disrupted or extended in different regions.

Amino Acid Sequence↗
PubMed2003

Evolutionary conservation of regulatory elements in vertebrate Hox gene clusters.

Comparisons of DNA sequences among evolutionarily distantly related genomes permit identification of conserved functional regions in noncoding DNA. Hox genes are highly conserved in vertebrates, occur in clusters, and are uninterrupted by other genes. We aligned (PipMaker) the nucleotide sequences of the HoxA clusters of tilapia, pufferfish, striped bass, zebrafish, horn shark, human, and mouse, which are separated by approximately 500 million years of evolution. In support of our approach, several identified putative regulatory elements known to regulate the expression of Hox genes were recovered. The majority of the newly identified putative regulatory elements contain short fragments that are almost completely conserved and are identical to known binding sites for regulatory proteins (Transfac database). The regulatory intergenic regions located between the genes that are expressed most anteriorly in the embryo are longer and apparently more evolutionarily conserved than those at the other end of Hox clusters. Different presumed regulatory sequences are retained in either the Aalpha or Abeta duplicated Hox clusters in the fish lineages. This suggests that the conserved elements are involved in different gene regulatory networks and supports the duplication-deletion-complementation model of functional divergence of duplicated genes.

Animals↗
PubMed2001

Plasticity in zebrafish hox expression in the hindbrain and cranial neural crest.

The anterior-posterior identities of cells in the hindbrain and cranial neural crest are thought to be determined by their Hox gene expression status, but how and when cells become committed to these identities remain unclear. Here we address this in zebrafish by cell transplantation, to test plasticity in hox expression in single cells. We transplanted cells alone, or in small groups, between hindbrain rhombomeres or between the neural crest primordia of pharyngeal arches. We found that transplanted cells regulated hox expression according to their new environments. The degree of plasticity, however, depended on both the timing and the size of the transplant. At later stages transplanted cells were more likely to be irreversibly committed and maintain their hox expression, demonstrating a progressive loss of responsiveness to the environmental signals that specify segmental identities. Individual transplanted cells also showed greater plasticity than those lying within the center of larger groups, suggesting that a community effect normally maintains hox expression within segments. We also raised experimental embryos to larval stages to analyze transplanted cells after differentiation and found that neural crest cells contributed to pharyngeal cartilages appropriate to the anterior-posterior level of the new cellular environment. Thus, consistent with models implicating hox expression in control of segmental identity, plasticity in hox expression correlates with plasticity in final cell fate.

Animals↗
PubMed2001

The zebrafish buttonhead-like factor Bts1 is an early regulator of pax2.1 expression during mid-hindbrain development.

Little is known about the factors that control the specification of the mid-hindbrain domain (MHD) within the vertebrate embryonic neural plate. Because the head-trunk junction of the Drosophila embryo and the MHD have patterning similarities, we have searched for vertebrate genes related to the Drosophila head gap gene buttonhead (btd), which in the fly specifies the head-trunk junction. We report here the identification of a zebrafish gene which, like btd, encodes a zinc-finger transcriptional activator of the Sp-1 family (hence its name, bts1 for btd/Sp-related-1) and shows a restricted expression in the head. During zebrafish gastrulation, bts1 is transcribed in the posterior epiblast including the presumptive MHD, and precedes in this area the expression of other MHD markers such as her5, pax2.1 and wnt1. Ectopic expression of bts1 combined to knock-down experiments demonstrate that Bts1 is both necessary and sufficient for the induction of pax2.1 within the anterior neural plate, but is not involved in regulating her5, wnt1 or fgf8 expression. Our results confirm that early MHD development involves several genetic cascades that independently lead to the induction of MHD markers, and identify Bts1 as a crucial upstream component of the pathway selectively leading to pax2.1 induction. In addition, they imply that flies and vertebrates, to control the development of a boundary embryonic region, have probably co-opted a similar strategy: the restriction to this territory of the expression of a Btd/Sp-like factor.

Amino Acid Sequence↗
PubMed2002

A zebrafish sox9 gene required for cartilage morphogenesis.

The molecular genetic mechanisms of cartilage construction are incompletely understood. Zebrafish embryos homozygous for jellyfish (jef) mutations show craniofacial defects and lack cartilage elements of the neurocranium, pharyngeal arches, and pectoral girdle similar to humans with campomelic dysplasia. We show that two alleles of jef contain mutations in sox9a, one of two zebrafish orthologs of the human transcription factor SOX9. A mutation induced by ethyl nitrosourea changed a conserved nucleotide at a splice junction and severely reduced splicing of sox9a transcript. A retrovirus insertion into sox9a disrupted its DNA-binding domain. Inhibiting splicing of the sox9a transcript in wild-type embryos with splice site-directed morpholino antisense oligonucleotides produced a phenotype like jef mutant larvae, and caused sox9a transcript to accumulate in the nucleus; this accumulation can serve as an assay for the efficacy of a morpholino independent of phenotype. RNase-protection assays showed that in morpholino-injected animals, the percent of splicing inhibition decreased from 80% at 28 hours post fertilization to 45% by 4 days. Homozygous mutant embryos had greatly reduced quantities of col2a1 message, the major collagen of cartilage. Analysis of dlx2 expression showed that neural crest specification and migration was normal in jef (sox9a) embryos. Confocal images of living embryos stained with BODIPY-ceramide revealed at single-cell resolution the formation of precartilage condensations in mutant embryos. Besides the lack of overt cartilage differentiation, pharyngeal arch condensations in jef (sox9a) mutants lacked three specific morphogenetic behaviors: the stacking of chondrocytes into orderly arrays, the individuation of pharyngeal cartilage organs and the proper shaping of individual cartilages. Despite the severe reduction of cartilages, analysis of titin expression showed normal muscle patterning in jef (sox9a) mutants. Likewise, calcein labeling revealed that early bone formation was largely unaffected in jef (sox9a) mutants. These studies show that jef (sox9a) is essential for both morphogenesis of condensations and overt cartilage differentiation.

Alleles↗
PubMed2005

Differential localization and identification of a critical aspartate suggest non-redundant proteolytic functions of the presenilin homologues SPPL2b and SPPL3.

Signal peptide peptidase (SPP) is an unusual aspartyl protease that mediates clearance of signal peptides by proteolysis within the endoplasmic reticulum (ER). Like presenilins, which provide the proteolytically active subunit of the gamma-secretase complex, SPP contains a critical GXGD motif in its C-terminal catalytic center. Although SPP is known to be an aspartyl protease of the GXGD type, several presenilin homologues/SPP-like proteins (PSHs/SPPL) of unknown function have been identified by data base searches. We now investigated the subcellular localization and a putative proteolytic activity of PSHs/SPPLs in cultured cells and in an in vivo model. We demonstrate that SPPL2b is targeted through the secretory pathway to endosomes/lysosomes, whereas SPP and SPPL3 are restricted to the ER. As suggested by the differential subcellular localization of SPPL2b compared with SPP and SPPL3, we found distinct phenotypes upon antisense gripNA-mediated knockdown in zebrafish. spp and sppl3 knockdowns in zebrafish result in cell death within the central nervous system, whereas reduction of sppl2b expression causes erythrocyte accumulation in an enlarged caudal vein. Moreover, expression of D/A mutations of the putative C-terminal active sites of spp, sppl2, and sppl3 produced phenocopies of the respective knockdown phenotypes. Thus, our data suggest that all investigated PSHs/SPPLs are members of the novel family of GXGD aspartyl proteases. Furthermore, SPPL2b is shown to be the first member of the SPP/PSH/SPPL family that is not located within the ER but in endosomal/lysosomal vesicles.

Amino Acid Motifs↗
PubMed2003

Angiogenic network formation in the developing vertebrate trunk.

We have used time-lapse multiphoton microscopy of living Tg(fli1:EGFP)y1 zebrafish embryos to examine how a patterned, functional network of angiogenic blood vessels is generated in the early vertebrate trunk. Angiogenic vascular sprouts emerge from the longitudinal trunk axial vessels (the dorsal aorta and posterior cardinal vein) in two spatially and temporally distinct steps. Dorsal aorta-derived sprouts form an initial primary network of vascular segments, followed by emergence of vein-derived secondary vascular sprouts that interact and interconnect dynamically with the primary network to initiate vascular flow. Using transgenic silent heart mutant embryos, we show that the gross anatomical patterning of this network of vessels does not require blood circulation. However, our results suggest that circulatory flow dynamics play an important role in helping to determine the pattern of interconnections between the primary network and secondary sprouts, and thus the final arterial or venous identity of the vessels in the functional network. We discuss a model to explain our results combining genetic programming of overall vascular architecture with hemodynamic determination of circulatory flow patterns.

Animals↗
PubMed2000

Regulation of gut and heart left-right asymmetry by context-dependent interactions between xenopus lefty and BMP4 signaling.

The Lefty subfamily of TGFbeta signaling molecules has been implicated in early development in mouse, zebrafish, and chick. Here, we show that Xenopus lefty (Xlefty) is expressed both bilaterally in symmetric midline domains and unilaterally in left lateral plate mesoderm and anterior dorsal endoderm. To examine the roles of Xlefty in left-right development, we created a system for scoring gut asymmetry and examined the effects of unilateral Xlefty misexpression on gut development, heart development, and Xnr-1 and XPitx2 expression. In contrast to the unilateral effects of Vg1, Activin, Nodal, or BMPs, targeted expression of Xlefty in either the left or the right side of Xenopus embryos randomized the direction of heart looping, gut coiling, and left-right positioning of the gut and downregulated the asymmetric expression of Xnr-1 and XPitx2. It is currently thought that Lefty proteins act as feedback inhibitors of Nodal signaling. However, this would not explain the effects of right-sided Xlefty misexpression. Here, we show that Xlefty interacts with the signaling pathways of other members of the TGFbeta family during left-right development. Results from coexpression of Xlefty and Vg1 indicate that Xlefty can nullify the effects of Vg1 ectopic expression and that Xlefty is downstream of left-sided Vg1 signaling. Results from coexpression of Xlefty and XBMP4 indicate that XLefty and XBMP4 interact both synergistically and antagonistically in a context-dependent manner. We propose a model in which interactions of Xlefty with multiple members of the TGFbeta family enhance the differences between the right-sided BMP/ALK2/Smad pathway and the left-sided Vg1/anti-BMP/Nodal pathway, leading to left-right morphogenesis of the gut and heart.

Amino Acid Sequence↗
PubMed2006

Insertional mutagenesis in zebrafish: genes for development, genes for disease.

In order to rapidly identify a substantial fraction of the genes with a unique and essential role in vertebrate development, the laboratory of Nancy Hopkins at MIT has performed a large insertional mutagenesis screen in zebrafish using a pseudotyped retroviral vector as the mutagen. We have recovered mutations in about one-quarter of the embryonic essential genes in this organism, and have identified the mutated genes in nearly all of these (333). As the ease of gene identification allowed us to clone the mutated genes for nearly all of the mutants rather than prioritizing based upon the initially observed phenotypes, this has provided an unbiased view of the diversity of genes required for vertebrate development as well as a large collection of mutants to be screened for more specific phenotypes. In collaboration with other labs, we have screened the insertional mutant for the development of a variety of organs and cell types, as well as phenotypes that could represent disease models, such as cystic kidney and hepatomegaly. Furthermore, while all of these mutants are embryonic lethal in their homozygous state, we are investigating the heterozygous adults for additional phenotypes, such as cancer predisposition.

Animals↗
PubMed2002

Regulation of the Lim-1 gene is mediated through conserved FAST-1/FoxH1 sites in the first intron.

The Lim-1 gene encodes a LIM-homeodomain transcription factor that is highly conserved among vertebrates and is required for successful gastrulation and head formation. The expression of this gene in the mesoderm of the gastrula is known to require an activin/nodal signal. Earlier studies have shown that the Xenopus Lim-1 (Xlim-1) gene contains an activin response element (ARE) in its first intron, which cooperates with an activin-unresponsive upstream promoter in the regulation of the gene. Here, we show that the Xlim-1 ARE contains a cluster of FAST-1/FoxH1 and Smad4 recognition sites; such sites have been shown to mediate activin/nodal responses in other genes. By using reporter constructs with mutated FAST-1/FoxH1 sites and FAST-1/FoxH1 protein chimeras, we show that the regulation of Xlim-1 by activin depends on FAST-1/FoxH1 function. Comparative studies on the zebrafish lim1 gene indicate the presence of FoxH1 sites in the first intron of this gene and provide evidence for the requirement for FoxH1 function in its regulation. These results illuminate the conserved nature of the transcriptional regulation of the Lim-1 gene in different vertebrate animals.

Activins↗
PubMed2004

A new model for olfactory placode development.

The peripheral nervous system of vertebrate animals arises primarily from the interaction of cranial neural crest and sensory placodes. Placodes are described as thickenings of ectoderm that arise through cell division during neural tube formation. The olfactory sensory system is one component of the peripheral nervous system that arises from paired sensory placodes during development. The olfactory placodes give rise to the primary sensory neurons, support cells and basal cells of the olfactory epithelium. Recent evidence from work in zebrafish and chick suggests that the olfactory and auditory placodes arise from large fields of cells that converge to form the sensory placode. The olfactory placodes arise from within the neural plate, and cell division is apparent only after the sensory placodes are morphologically distinct. As the olfactory placode is forming, its precursors must segregate from their neighboring fields which will give rise to the adenohypophyseal placode, cranial neural crest, and telencephalon. Analysis has shown that the endocrine cells thought to arise from the olfactory placode originate in the neighboring adenohypophyseal and cranial neural crest domains. The borders separating the domains are plastic, where cells sort as they move, and cell fate is dependent on the identity of neighbors once the cells have converged to form the sensory placode. Thus there is degeneracy built into the system such that cells accommodate changes in the environment until cell migrations controlling the formation of the sensory placodes are complete.

Animals↗
PubMed1996

Teleost HoxD and HoxA genes: comparison with tetrapods and functional evolution of the HOXD complex.

In tetrapods, Hox genes are essential for the proper organization and development of axial structures. Experiments involving Hox gene inactivations have revealed their particularly important functions in the establishment of morphological transitions within metameric series such as the vertebral column. Teleost fish show a much simpler range of axial (trunk or appendicular) morphologies, which prompted us to investigate the nature of the Hox system in these lower vertebrates. Here, we show that fish have a family of Hox genes, very similar in both number and general organization, to that of tetrapods. Expression studies, carried out with HoxD and HoxA genes, showed that all vertebrates use the same general scheme, involving the colinear activation of gene expression in both space and time. Comparisons between tetrapods and fish allowed us to propose a model which accounts for the primary function of this gene family. In this model, a few ancestral Hox genes were involved in the determination of polarity in the digestive tract and were further recruited in more elaborate axial structures.

Amino Acid Sequence↗
PubMed2006

Ewing's sarcoma: general insights from a rare model.

Ewing's sarcoma is characterized by the presence of fusion oncoproteins involving EWSR1 and an ETS gene, most commonly FLI1. In this issue of Cancer Cell, Smith et al. have combined RNA interference with expression profiling to study the pattern of gene expression downstream of the most common of these fusions, EWS/FLI. Using this strategy, Smith et al. have identified a homeobox gene, NKX2.2, which is both highly expressed in Ewing's sarcoma and essential for the transforming activity of EWS/FLI.

Animals↗
PubMed2002

Identification of a Wnt/Dvl/beta-Catenin --> Pitx2 pathway mediating cell-type-specific proliferation during development.

Understanding the cell type-specific molecular mechanisms by which distinct signaling pathways combinatorially control proliferation during organogenesis is a central issue in development and disease. Here, we report that the bicoid-related transcription factor Pitx2 is rapidly induced by the Wnt/Dvl/beta-catenin pathway and is required for effective cell-type-specific proliferation by directly activating specific growth-regulating genes. Regulated exchange of HDAC1/beta-catenin converts Pitx2 from repressor to activator, analogous to control of TCF/LEF1. Pitx2 then serves as a competence factor required for the temporally ordered and growth factor-dependent recruitment of a series of specific coactivator complexes that prove necessary for Cyclin D2 gene induction. The molecular strategy underlying interactions between the Wnt and growth factor-dependent signaling pathways in cardiac outflow tract and pituitary proliferation is likely to be prototypic of cell-specific proliferation strategies in other tissues.

Adaptor Proteins, Signal Transducing↗
PubMed2002

HSPG modification by the secreted enzyme Notum shapes the Wingless morphogen gradient.

The secreted signaling protein Wingless acts as a morphogen to pattern the imaginal discs of Drosophila. Here we report identification of a secreted repressor of Wingless activity, which we call Notum. Loss of Notum function leads to increased Wingless activity by altering the shape of the Wingless protein gradient. When overexpressed, Notum blocks Wingless activity. Notum encodes a member of the alpha/beta-hydrolase superfamily, with similarity to pectin acetylesterases. We present evidence that Notum influences Wingless protein distribution by modifying the heparan sulfate proteoglycans Dally-like and Dally. High levels of Wingless signaling induce Notum expression. Thus, Wingless contributes to shaping its own gradient by regulating expression of a protein that modifies its interaction with cell surface proteoglycans.

Amino Acid Sequence↗
PubMed2003

Microphthalmia-associated transcription factor in the Wnt signaling pathway.

Microphthalmia-associated transcription factor (MITF) contains a basic helix-loop-helix and leucine-zipper (bHLH-LZ) structure and consists of many isoforms with different N-termini. Melanocyte-specific MITF isoform (MITF-M) is of particular interest, because a heterozygous mutation in the MITF gene is associated with Waardenburg syndrome type 2 (WS2) that is characterized by deafness and hypopigmentation because of lack of melanocytes in the inner ear and skin. Expression of MITF-M is under the regulation of the melanocyte-specific promoter (M promoter) of the MITF gene, and transcription from the M promoter is induced by Wnt signals through a nuclear mediator, lymphoid-enhancing factor 1 (LEF-1). In addition, functional cooperation of MITF-M with LEF-1 could lead to transcriptional activation of the M promoter and the dopachrome tautomerase (DCT) gene, an early melanoblast marker. The bHLH-LZ region of MITF-M is responsible for the physical interaction with LEF-1, and beta-catenin is required for the collaboration between LEF-1 and MITF-M. Importantly, MITF-M could function as a non-DNA-binding co-factor for LEF-1. These results suggest that MITF-M may function as a self-regulator of its own expression to maintain a threshold level of MITF-M at a certain sensitive stage of melanocyte development, which could account for the dominant inheritance of WS2. MITF-M therefore plays dual roles in the Wnt signaling pathway; MITF-M represents a downstream target and a nuclear mediator of Wnt signals in melanocytes.

Animals↗