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Z Lele

Publications and source records attributed to Z Lele.

14 recordsLinked to original sources
PubMed2001

Zebrafish admp is required to restrict the size of the organizer and to promote posterior and ventral development.

Bone morphogenetic proteins (Bmps) and their roles during early dorsoventral patterning of the vertebrate embryo are well understood. The role and regulation of a more distant member of this family, the anti-dorsalizing morphogenetic protein (Admp), however, are less clear. Here, we report the isolation and characterization of zebrafish admp. Unlike other bmps, admp is exclusively expressed on the dorsal side. Expression starts at blastula stages in the region of the organizer, giving rise to anterior neuroectoderm and axial mesoderm. During the course of gastrulation, both the neuroectodermal and the mesodermal admp transcripts vanish in an anterior-posterior wave. The maintenance of admp expression is positively influenced by Nodal signaling and by Bozozok (Boz), an organizer-promoting homeodomain protein acting as a repressor of early bmp2b expression. Despite the positive effect of boz on admp expression, Boz and Admp have rather opposite effects on zebrafish patterning, as revealed in gain- and loss-of-function experiments. Upon overexpression, admp has Bmp-like activities causing a smaller organizer and enhanced ventral specification, very similar to the phenotype caused by the loss of boz function in mutant embryos. Antisense-based admp knockdown, on the other side, leads to an enlarged organizer and impaired ventral and posterior development, as observed in embryos after boz overexpression. This finding indicates that admp is required for the development of embryonic structures normally suppressed by organizer activities. The seeming discrepancy between the regulative and functional relationship of boz and admp is discussed, and models are proposed according to which Admp might be part of a negative feedback loop to pattern and confine the organizer region.

Animals↗
PubMed2001

The zebrafish glypican knypek controls cell polarity during gastrulation movements of convergent extension.

Mutations in the zebrafish knypek locus impair gastrulation movements of convergent extension that narrow embryonic body and elongate it from head to tail. We demonstrate that knypek regulates cellular movements but not cell fate specification. Convergent extension movement defects in knypek are associated with abnormal cell polarity, as mutant cells fail to elongate and align medio-laterally. Positional cloning reveals that knypek encodes a member of the glypican family of heparan sulfate proteoglycans. Double mutant and overexpression analyses show that Knypek potentiates Wnt11 signaling, mediating convergent extension. These studies provide experimental and genetic evidence that glypican Knypek acts during vertebrate gastrulation as a positive modulator of noncanonical Wnt signaling to establish polarized cell behaviors underlying convergent extension movements.

Amino Acid Sequence↗
PubMed2001

The type I serine/threonine kinase receptor Alk8/Lost-a-fin is required for Bmp2b/7 signal transduction during dorsoventral patterning of the zebrafish embryo.

Ventral specification of mesoderm and ectoderm depends on signaling by members of the bone morphogenetic protein (Bmp) family. Bmp signals are transmitted by a complex of type I and type II serine/threonine kinase transmembrane receptors. Here, we show that Alk8, a novel member of the Alk1 subgroup of type I receptors, is disrupted in zebrafish lost-a-fin (laf) mutants. Two alk8/laf null alleles are described. In laf(tm110), a conserved extracellular cysteine residue is replaced by an arginine, while in laf(m100), Alk8 is prematurely terminated directly after the transmembrane domain. The zygotic effect of both mutations leads to dorsalization of intermediate strength. A much stronger dorsalization, similar to that of bmp2b/swirl and bmp7/snailhouse mutants, however, is obtained by inhibiting both maternally and zygotically supplied alk8 gene products with morpholino antisense oligonucleotides. The phenotype of laf mutants and alk8 morphants can be rescued by injected mRNA encoding Alk8 or the Bmp-regulated transcription factor Smad5, but not by mRNA encoding Bmp2b or Bmp7. Conversely, injected mRNA encoding a constitutively active version of Alk8 can rescue the strong dorsalization of bmp2b/swirl and bmp7/snailhouse mutants, whereas smad5/somitabun mutant embryos do not respond. Altogether, the data suggest that Alk8 acts as a Bmp2b/7 receptor upstream of Smad5.

Activin Receptors↗
PubMed2000

Heat-inducible expression of a reporter gene detected by transient assay in zebrafish.

Heat-inducibility of two reporter constructs expressing lacZ gene under the control of mouse and Xenopus hsp70 promoters was tested in zebrafish (Danio rerio) embryos using a transient expression system. Cells expressing beta-galactosidase were stained blue by histochemical staining and their average number per embryo was used as an indicator of the expression level of the reporter gene. Both constructs were heat-inducible in the embryonic tissues and showed similar heat dependence (increasing expression levels from 35-36 degrees C up to 39 degrees C with an apparent decrease at 40 degrees C), resembling that of the zebrafish hsp70 genes. However, their induction kinetics were different, which might be due to differences in their 5' UTRs. Spatial expression patterns of the two hsp/lacZ constructs and an endogenous hsp70 gene were mostly similar on the RNA level. These results indicate that our approach is applicable for in vivo analysis of the heat-shock response and that exogenous heat-shock promoters may be useful for inducible expression of transgenes in fish.

Animals↗
PubMed2000

Laser-induced gene expression in specific cells of transgenic zebrafish.

Over the past few years, a number of studies have described the generation of transgenic lines of zebrafish in which expression of reporters was driven by a variety of promoters. These lines opened up the real possibility that transgenics could be used to complement the genetic analysis of zebrafish development. Transgenic lines in which the expression of genes can be regulated both in space and time would be especially useful. Therefore, we have cloned the zebrafish promoter for the inducible hsp70 gene and made stable transgenic lines of zebrafish that express the reporter green fluorescent protein gene under the control of a hsp70 promoter. At normal temperatures, green fluorescent protein is not detectable in transgenic embryos with the exception of the lens, but is robustly expressed throughout the embryo following an increase in ambient temperature. Furthermore, we have taken advantage of the accessibility and optical clarity of the embryos to express green fluorescent protein in individual cells by focussing a sublethal laser microbeam onto them. The targeted cells appear to develop normally: cells migrate normally, neurons project axons that follow normal pathways, and progenitor cells divide and give rise to normal progeny cells. By generating other transgenic lines in which the hsp70 promoter regulates genes of interest, it should be possible to examine the in vivo activity of the gene products by laser-inducing specific cells to express them in zebrafish embryos. As a first test, we laser-induced single muscle cells to make zebrafish Sema3A1, a semaphorin that is repulsive for specific growth cones, in a hsp70-sema3A1 transgenic line of zebrafish and found that extension by the motor axons was retarded by the induced muscle.

Animals↗
PubMed1999

Disruption of zebrafish somite development by pharmacologic inhibition of Hsp90.

Members of the Hsp90 family of molecular chaperones play important roles in allowing some intracellular signaling molecules and transcription factors to reach and maintain functionally active conformations. In the present study, we have utilized the specific Hsp90-binding agent, geldanamycin, to examine the requirement for Hsp90 during zebrafish development. We show that geldanamycin interacts with both the alpha and the beta-isoforms of zebrafish Hsp90 and that geldanamycin-treated embryos consistently exhibit a number of defects in tissues which express either one of these genes. Within the somites, geldanamycin treatment results in the absence of eng-2-expressing muscle pioneer cells. However, early development of adaxial cells, which give rise to muscle pioneers and which strongly express the hsp90alpha gene shortly before muscle pioneer formation, appeared unaffected. Furthermore, development of the notochord, which provides many of the signals required for proper somite patterning and which does not express detectable levels of either hsp90alpha or hsp90beta mRNA, was similarly unaffected in geldanamycin-treated embryos. The data are consistent with there being a temporal and spatial requirement for Hsp90 function within somitic cells which is necessary for the formation of eng-2-expressing muscle pioneers and possibly other striated muscle fiber types.

Animals↗
PubMed1997

A conserved element in the protein-coding sequence is required for normal expression of replication-dependent histone genes in developing Xenopus embryos.

Replication-dependent histone genes in the mouse and Xenopus share a common regulatory element within the protein-encoding sequence called the CRAS alpha element (coding region activating sequence alpha) which has been shown to mediate normal expression in vivo and to interact with nuclear factors in vitro in a cell cycle-dependent manner. Thus far, the alpha element has only been studied in rodent cells in culture, and its effect on histone gene expression during development has not been determined. Here we examine the role of the alpha element in histone gene expression during Xenopus development which features a switch in histone gene expression from a replication-independent mode in oocytes to a replication-dependent mode in embryos after midblastula stage. In vivo expression experiments involving wild-type or alpha-mutant mouse H3.2 genes show that mutation of the CRAS alpha element results in a fourfold decline of expression in embryos, but does not affect expression in oocytes. Two distinct alpha sequence-specific binding activities were detected in both oocyte and embryonic extracts. A slowly migrating DNA-binding complex was present at relatively constant levels throughout development from the earliest stages of oogenesis through larval stages. In contrast, levels of a rapidly migrating complex were high in stage I and II oocytes, declined in stage II-VI oocytes, remained low in unfertilized eggs and cleavage stage embryos, and rose dramatically after the midblastula transition. The molecular masses of the factors forming the slow and rapidly migrating complexes were estimated to be approximately 110 and 85 kDa, respectively. DNA-binding activity of the 85 kDa alpha-binding factor was affected by phosphorylation, binding with higher affinity in the dephosphorylated state. The abrupt increase in DNA-binding activity of the 85-kDa alpha-binding factor at late blastula coincides with the switch to the replication-dependent mode of histone gene expression. We propose that the conserved alpha element present in the coding sequence of mouse and Xenopus core histone genes is required for normal replication-dependent histone expression in the developing Xenopus embryo.

Animals↗
PubMed1997

hsp47 and hsp70 gene expression is differentially regulated in a stress- and tissue-specific manner in zebrafish embryos.

We have examined differences in the spatial and temporal regulation of stress-induced hsp47 and hsp70 gene expression following exposure of zebrafish embryos to heat shock or ethanol. Using Northern blot analysis, we found that levels of hsp47 and hsp70 mRNA were dramatically elevated during heat shock in 2-day-old embryos. In contrast, ethanol exposure resulted in strong upregulation of the hsp47 gene whereas hsp70 mRNA levels increased only slightly following the same treatment. Whole-mount in situ hybridization analysis revealed that hsp47 mRNA was expressed predominantly in precartilagenous cells, as well as several other connective tissue cell populations within the embryo following exposure to either stress. hsp70 mRNA displayed a very different cell-specific distribution. For example, neither stress induced hsp70 mRNA accumulation in precartilagenous cells. However, high levels of hsp70 mRNA were detectable in epithelial cells of the developing epidermis following exposure to heat shock, but not to ethanol. These cells did not express the hsp47 gene following exposure to either of these stresses. The results suggest the presence of different inducible regulatory mechanisms for these genes which operate in a cell- and stress-specific manner in zebrafish embryos.

Amino Acid Sequence↗
PubMed1997

Heat shock protein gene expression during embryonic development of the zebrafish.

Heat shock genes exhibit complex patterns of spatial and temporal regulation during embryonic development of a wide range of organisms. Our laboratory has been involved in an analysis of heat shock gene expression in the zebrafish, a model system which is now utilized extensively for the examination of early embryonic development of vertebrates. Members of the zebrafish hsp47, hsp70 and hsp90 gene families have been cloned and shown to be closely related to their counterparts in higher vertebrates. Expression of these genes has been examined using Northern blot and whole mount in situ hybridization analyses. Both the hsp47 and hsp90 genes are expressed in a highly tissue-restricted manner during normal development. The data raise a number of interesting questions regarding the function and regulation of these heat shock genes during early zebrafish development.

Animals↗
PubMed1997

Expression of genes encoding the collagen-binding heat shock protein (Hsp47) and type II collagen in developing zebrafish embryos.

Hsp47 is a heat-shock protein which interacts with newly synthesize procollagen chains in the endoplasmic reticulum (ER) of collagen-secreting cells and is thought to assist in procollagen triple helix assembly and subsequent transport to the cis-Golgi. This is supported by studies which have reported that genes encoding collagen and Hsp47 are subject to co-ordinate increases and decreases in expression in cultured cells. However, limited information is available regarding hsp47 expression in vivo, particularly during early embryonic development when a variety of collagen genes are expressed. Here we show that the zebrafish hsp47 gene is expressed in a dynamic spatiotemporal pattern in developing embryos. Strong expression of hsp47 mRNA is co-incident predominantly with expression of the type II collagen gene (col2a1) in a number of chondrogenic and non-chondrogenic tissues including the notochord, otic vesicle and developing fins. Notochordal expression of both genes is disrupted in floating head (flh) and no tail (ntl) embryos, which lack properly differentiated notochords. Surprisingly, no hsp47 mRNA is detectable in the strongly col2a1-expressing cells of the floor plate and hypochord, indicating that the two genes are not strictly co-regulated. Finally, Northern blot analysis revealed two alternative transcripts of col2a1 which are expressed in distinct temporal patterns. Appearance of the larger transcript occurs following somitogenesis, a time which coincides with the co-activation of hsp47 and col2a1 gene expression in tissues outside of the notochord.

Age Factors↗
PubMed1997

Heat shock genes and the heat shock response in zebrafish embryos.

Heat shock genes exhibit complex patterns of spatial and temporal regulation during embryonic development in a wide range of organisms. Our laboratory has initiated an analysis of heat shock protein gene expression in the zebrafish, a model system that is now utilized extensively for the examination of early embryonic development of vertebrates. We have cloned members of the zebrafish hsp47, hsp70, and hsp90 gene families and shown them to be closely related to their counterparts in higher vertebrates. Whole mount in situ hybridization and Northern blot analyses have revealed that these genes are regulated in distinct spatial, temporal, and stress-specific manners. Furthermore, the tissue-specific expression patterns of the hsp47 and hsp90 alpha genes correlate closely with the expression of genes encoding known chaperone targets of Hsp47 and Hsp90 in other systems. The data raise a number of interesting questions regarding the function and regulation of these heat shock genes in zebrafish embryos during normal development and following exposure to environmental stress.

Animals↗
PubMed1996

The zebrafish as a model system in developmental, toxicological and transgenic research.

The zebrafish has long been used as a model system in fisheries biology and toxicology. More recently, it has also become the focus of a major research effort into understanding the molecular and cellular events which dictate the development of vertebrate embryos. As well, the zebrafish has proven attractive in studies examining the factors which affect the creation of transgenic fish and the expression of transgenes. The advances which have been made in these areas have firmly established this small aquarium fish as a major model system in biological and biotechnological research.

Journal Article↗
PubMed1993

Efficient transient expression system based on square pulse electroporation and in vivo luciferase assay of fertilized fish eggs.

Electroporation mediated DNA transfer into fish eggs has been improved by using a train of square pulses. Fertilized eggs of African catfish (Clarias gariepinus), zebrafish (Brachydanio rerio) and rosy barb (Barbus conchonius) were dechorionated enzymatically followed by application of pulses. Efficiency of plasmid DNA delivery was significantly increased by applying multiple pulses on dechorionated eggs. Optimization of physical parameters such as field strength, pulse width and pulse numbers resulted in reproducible transient expression in 25-50% of embryos and larvae by using the firefly luciferase and the E. coli beta-galactosidase (lacZ) genes both driven by CMV IE1 promoter. Temporal luciferase expression was assayed using both qualitative (sheet film) and quantitative (scintillation counting) methods in developing embryos and fry in vivo. Spatial expression of lacZ was assayed by histochemical staining. A number of embryos revealed foreign gene product also localised in the vegetal pole of the embryo.

Animals↗