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J Postlethwait

Publications and source records attributed to J Postlethwait.

15 recordsLinked to original sources

Two variants of zebrafish p100 are expressed during embryogenesis and regulated by Nodal signaling.

Human p100 protein was first identified as a transcriptional coactivator of Epstein-Barr virus nuclear antigen 2, and has been shown to be a coactivator of other cellular transactivators. Its roles in development of vertebrate embryos, however, have not been reported. We have identified a zebrafish ortholog of the human p100 coactivator. The zebrafish p100 transcript is processed to two alternative variants, long and short forms, referred to as p100L and p100S, respectively. Both GFP-p100L and GFP-p100S fusion proteins are located in the cytoplasm of transfected culture cells and microinjected embryonic cells. Analysis of transcripts with Northern blots revealed the presence of p100L and lower amounts of p100S mRNAs from the one-cell stage throughout the life cycle. Whole-mount in situ hybridization shows that p100L and p100S share the same spatiotemporal expression pattern. Their zygotic expression is initially restricted to axial mesoderm precursors during gastrulation, and then spreads over other tissues during segmentation, and finally is constrained to some internal organs at day 5. We also find that Nodal signaling is essential for the zygotic expression of p100. These studies pave the way to understanding in depth the role of p100 during vertebrate embryogenesis.

Alternative Splicing↗

Roles for zebrafish focal adhesion kinase in notochord and somite morphogenesis.

We have cloned zebrafish focal adhesion kinase (Fak) and analyzed its subcellular localization. Fak protein is localized at the cortex of notochord cells and at the notochord-somite boundary. During somitogenesis, Fak protein becomes concentrated at the basal region of epithelial cells at intersomitic boundaries. Phosphorylated Fak protein is seen at both the notochord-somite boundary and intersomitic boundaries, consistent with a role for Fak in boundary formation and maintenance. The localization of Fak protein to the basal region of epithelial cells in knypek;trilobite double mutant embryos shows that polarization of Fak distribution in the somite border cells is independent of internal mesenchymal cells. In addition, we show that neither Notch signaling through Suppressor of Hairless (SuH) nor deltaD is necessary for the wild-type segmental pattern of fak mRNA expression in the anterior paraxial mesoderm. However, nonsegmental expression of fak mRNA occurs with ectopic activation of Notch signaling through SuH and also in fused somite and beamter mutant embryos, indicating that there are multiple regulators of fak mRNA expression. Our results suggest that Fak plays a central role in notochord and somite morphogenesis.

Amino Acid Sequence↗

Characterization of duplicated zebrafish cyp19 genes.

The zebrafish has recently been developed as a good genetic model system. We report here the use of zebrafish to study the regulation of estrogen biosynthesis. The CYP19 gene encodes cytochrome P450 aromatase, which catalyzes the synthesis of estrogens. Two cyp19 genes, termed cyp19a and cyp19b, have been isolated from zebrafish. Sequence comparison shows that Cyp19a and Cyp19b belong to two separate Cyp19 subfamilies. The cyp19a gene is expressed in the ovary, whereas cyp19b is expressed in the brain. The cyp19a and cyp19b genes are located on zebrafish chromosomes LG 18 and 25, respectively. Our data indicate that these gene loci arose through an ancient chromosomal duplication event. The expression of duplicated genes in distinct tissues may have evolutionary significance.

Amino Acid Sequence↗

Two sox9 genes on duplicated zebrafish chromosomes: expression of similar transcription activators in distinct sites.

Sox9 is a transcription factor required for cartilage formation and testis determination in mammals. We have cloned from zebrafish two sox9 genes, termed sox9a and sox9b. Gene phylogenies showed that both genes are orthologous to tetrapod SOX9 genes. Genetic mapping showed that these two loci reside on chromosome segments that were apparently duplicated in a large-scale genomic duplication event in ray fin fish phylogeny. Both Sox9a and Sox9b proteins bind to the HMG consensus DNA sequences in vitro. We tested different domains for transactivation potential and identified a potential activation domain located in the middle of both Sox9a and Sox9b. During embryogenesis, sox9a and sox9b expression patterns are distinct but overlap in some regions of the brain, head skeleton, and fins. Expression of sox9a/b correlates well with that of col2a1 in chondrogenic elements. In the adults, sox9a is expressed in many tissues including brain, muscle, fin, and testis, whereas sox9b expression is restricted to previtellogenic oocytes of the ovary. This expression pattern predicts that sox9a and sox9b may have unique functions in some specific tissues during development. The role of gene duplication for the evolution of developmental gene function is discussed.

Amino Acid Sequence↗

Zebrafish acetylcholinesterase is encoded by a single gene localized on linkage group 7. Gene structure and polymorphism; molecular forms and expression pattern during development.

We cloned and sequenced the acetylcholinesterase gene and cDNA of zebrafish, Danio rerio. We found a single gene (ache) located on linkage group LG7. The relative organization of ache, eng2, and shh genes is conserved between zebrafish and mammals and defines a synteny. Restriction fragment length polymorphism analysis was allowed to identify several allelic variations. We also identified two transposable elements in non-coding regions of the gene. Compared with other vertebrate acetylcholinesterase genes, ache gene contains no alternative splicing at 5' or 3' ends where only a T exon is present. The translated sequence is 60-80% identical to acetylcholinesterases of the vertebrates and exhibits an extra loop specific to teleosts. Analysis of molecular forms showed a transition, at the time of hatching, from the globular G4 form to asymmetric A12 form that becomes prominent in adults. In situ hybridization and enzymatic activity detection on whole embryos confirmed early expression of the acetylcholinesterase gene in nervous and muscular tissues. We found no butyrylcholinesterase gene or activity in Danio. These findings make zebrafish a promising model to study function of acetylcholinesterase during development and regulation of molecular forms assembly in vivo.

Acetylcholinesterase↗

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↗

Two Cyp19 (P450 aromatase) genes on duplicated zebrafish chromosomes are expressed in ovary or brain.

Cytochrome P450 aromatase (Cyp19) is an enzyme catalyzing the synthesis of estrogens, thereby controlling various physiological functions of estrogens. We isolated two cyp19 cDNAs, termed cyp19a and cyp19b, respectively, from zebrafish. These genes are located in linkage groups 18 and 25, respectively. Detailed gene mapping indicated that zebrafish linkage groups 18 and 25 may have arisen from the same ancestral chromosome by a chromosome duplication event. Cyp19a is expressed mainly in the follicular cells lining the vitellogenic oocytes in the ovary during vitellogenesis. Cyp19b is expressed abundantly in the brain, at the hypothalamus and ventral telencephalon, extending to the olfactory bulbs. The expression of duplicated cyp19 genes at two different tissues highlights the evolutionary significance of maintaining two active genes on duplicated zebrafish chromosomes for specific functions in the ovary and the brain.

Amino Acid Sequence↗

Brachyury (T) expression in embryos of a larvacean urochordate, Oikopleura dioica, and the ancestral role of T.

The Brachyury, or T, gene is required for notochord development in animals occupying all three chordate subphyla and probably also had this role in the last common ancestor of the chordate lineages. In two chordate subphyla (vertebrates and cephalochordates), T is also expressed during gastrulation in involuting endodermal and mesodermal cells, and in vertebrates at least, this expression domain is required for proper development. In the basally diverging chordate subphylum Urochordata, animals in the class Ascidiacea do not employ T during gastrulation in endodermal or nonaxial mesodermal cells, and it has been suggested that nonnotochordal roles for T were acquired in the cephalochordate-vertebrate lineage after it split with Urochordata. To test this hypothesis, we cloned T from Oikopleura dioica, a member of the urochordate class Appendicularia (or Larvacea), which diverged basally in the subphylum. Investigation of the expression pattern in developing Oikopleura embryos showed early expression in presumptive notochord precursor cells, in the notochord, and in parts of the developing gut and cells of the endodermal strand. We conclude that the ancestral role of T likely included expression in the developing gut and became necessary in chordates for construction of the notochord.

Amino Acid Sequence↗

Hhex and scl function in parallel to regulate early endothelial and blood differentiation in zebrafish.

During embryogenesis, endothelial and blood precursors are hypothesized to arise from a common progenitor, the hemangioblast. Several genes that affect the differentiation of, or are expressed early in, both the endothelial and blood lineages may in fact function at the level of the hemangioblast. For example, the zebrafish cloche mutation disrupts the differentiation of both endothelial and blood cells. The transcription factor gene scl is expressed in both endothelial and blood lineages from an early stage and can regulate their differentiation. Here we report that in zebrafish the homeobox gene hhex (previously called hex) is also expressed in endothelial and blood lineages from an early stage. We find that hhex expression in these lineages is significantly reduced in cloche mutant embryos, indicating that hhex functions downstream of cloche to regulate endothelial and blood differentiation. Ectopic expression of hhex through injection of a DNA construct leads to the premature and ectopic expression of early endothelial and blood differentiation genes such as fli1, flk1 and gata1, indicating that Hhex can positively regulate endothelial and blood differentiation. However, analysis of a hhex deficiency allele shows that hhex is not essential for early endothelial and blood differentiation, suggesting that another gene, perhaps scl, compensates for the absence of Hhex function. Furthermore, we find that hhex and scl can induce each other's expression, suggesting that these two genes cross-regulate each other during early endothelial and blood differentiation. Together, these data provide the initial framework of a pathway that can be used to further integrate the molecular events regulating hemangioblast differentiation.

Animals↗

The BMP-related protein radar: a maintenance factor for dorsal neuroectoderm cells?

We have previously cloned several members of the TGF-beta superfamily of growth factors in zebrafish, one of which, Radar, belongs to the Dpp-Vg1-related (DVR) subgroup, with highest homology to GDF6. The pattern of expression of Radar suggested a possible involvement in several induction steps during embryogenesis including in the dorsal neural tube, red blood cells, the dorsal fin and the retina. We have analyzed the pattern of expression of Radar in comparison with that of a marker of dorsal neural tube structures, msxC and show that Radar and msxC are expressed in similar and/or adjacent tissues throughout embryogenesis. In order to demonstrate a functional relationship between these two proteins, we have generated a full-length cDNA for Radar and shown that Radar overexpression by DNA injection maintains expression of msxC in tissues where it is normally expressed then turned off, in particular in the dorsal neurectoderm. Study of the phenotype of a mutant carrying a deletion of Radar shows a loss of identity and death of the cells of the dorsal neural tube. Taken together these results suggest that Radar could be involved in maintaining the identity of cells of the dorsal-most neural tube and of at least a subset of neural crest cells.

Amino Acid Sequence↗

A recessive mutation leading to vertebral ankylosis in zebrafish is associated with amino acid alterations in the homologue of the human membrane-associated guanylate kinase DLG3.

We describe the characterization of the zebrafish homologue of the human gene DLG3. The zebrafish dlg3 gene encodes a membrane-associated guanylate kinase containing a single PDZ domain. This gene was cloned using a gene-trap construct inserted in the gene's first intron. The insertion co-segregates with a viable mutation called humpback (hmp), which leads to formation of ankylotic vertebrae in adult fishes. Insertion and mutation have both been mapped to chromosome 12, in a segment which is syntenic with region p12 to q12 of human chromosome 17. The hmp mutant phenotype, however, appears to be due to two point mutations in the guanylate kinase domain rather than to the transgene insertion itself. The results of this study are discussed in the light of the possible function of the guanylate kinase domain.

Animals↗

Preservation of duplicate genes by complementary, degenerative mutations.

The origin of organismal complexity is generally thought to be tightly coupled to the evolution of new gene functions arising subsequent to gene duplication. Under the classical model for the evolution of duplicate genes, one member of the duplicated pair usually degenerates within a few million years by accumulating deleterious mutations, while the other duplicate retains the original function. This model further predicts that on rare occasions, one duplicate may acquire a new adaptive function, resulting in the preservation of both members of the pair, one with the new function and the other retaining the old. However, empirical data suggest that a much greater proportion of gene duplicates is preserved than predicted by the classical model. Here we present a new conceptual framework for understanding the evolution of duplicate genes that may help explain this conundrum. Focusing on the regulatory complexity of eukaryotic genes, we show how complementary degenerative mutations in different regulatory elements of duplicated genes can facilitate the preservation of both duplicates, thereby increasing long-term opportunities for the evolution of new gene functions. The duplication-degeneration-complementation (DDC) model predicts that (1) degenerative mutations in regulatory elements can increase rather than reduce the probability of duplicate gene preservation and (2) the usual mechanism of duplicate gene preservation is the partitioning of ancestral functions rather than the evolution of new functions. We present several examples (including analysis of a new engrailed gene in zebrafish) that appear to be consistent with the DDC model, and we suggest several analytical and experimental approaches for determining whether the complementary loss of gene subfunctions or the acquisition of novel functions are likely to be the primary mechanisms for the preservation of gene duplicates. For a newly duplicated paralog, survival depends on the outcome of the race between entropic decay and chance acquisition of an advantageous regulatory mutation. Sidow 1996(p. 717) On one hand, it may fix an advantageous allele giving it a slightly different, and selectable, function from its original copy. This initial fixation provides substantial protection against future fixation of null mutations, allowing additional mutations to accumulate that refine functional differentiation. Alternatively, a duplicate locus can instead first fix a null allele, becoming a pseudogene. Walsh 1995 (p. 426) Duplicated genes persist only if mutations create new and essential protein functions, an event that is predicted to occur rarely. Nadeau and Sankoff 1997 (p. 1259) Thus overall, with complex metazoans, the major mechanism for retention of ancient gene duplicates would appear to have been the acquisition of novel expression sites for developmental genes, with its accompanying opportunity for new gene roles underlying the progressive extension of development itself. Cooke et al. 1997 (p. 362)

Animals↗

Sex- and cell-specific regulation of yolk polypeptide genes introduced into Drosophila by P-element-mediated gene transfer.

To find whether cis-acting regulatory sequences necessary for sex- and cell-specific expression of two yolk polypeptide genes (Yps) reside near the structural genes themselves, we introduced a 5.0-kilobase genomic DNA segment containing a 3' truncated Yp1 and a complete Yp2 into five different autosomal locations by P-element-mediated gene transfer. Transcripts from the introduced Yp1 were not found in male flies but appeared on a normal developmental schedule in adult females, accumulating in their body walls and ovarian follicles but not in guts or malpighian tubules. Protein from the introduced Yp2 allele was present in female hemolymph and vitellogenic ovaries but was lacking from male hemolymph. We conclude that sequences necessary for the correct stage-, cell-, and sex-specific expression of the Yp1 and Yp2 genes are included in this genomic fragment. These results combined with published work suggest that two tissue-specific, cis-acting, bidirectional, positive regulatory elements placed on either side of a centrally located HindIII site govern expression of both Yp genes--one element specific for fat body and the other specific for ovarian follicle cells.

Animals↗