PubMed Health⌕ Search

Biomedical subjects

P W Ingham

Publications and source records attributed to P W Ingham.

At least 19 recordsLinked to original sources
PubMed1997

Notochord induction of zebrafish slow muscle mediated by Sonic hedgehog.

The patterning of vertebrate somitic muscle is regulated by signals from neighboring tissues. We examined the generation of slow and fast muscle in zebrafish embryos and show that Sonic hedgehog (Shh) secreted from the notochord can induce slow muscle from medial cells of the somite. Slow muscle derives from medial adaxial myoblasts that differentiate early, whereas fast muscle arises later from a separate myoblast pool. Mutant fish lacking shh expression fail to form slow muscle but do form fast muscle. Ectopic expression of shh, either in wild-type or mutant embryos, leads to ectopic slow muscle at the expense of fast. We suggest that Shh acts to induce myoblasts committed to slow muscle differentiation from uncommitted presomitic mesoderm.

Animals↗
PubMed1997

Axial (HNF3beta) and retinoic acid receptors are regulators of the zebrafish sonic hedgehog promoter.

The signalling molecule Sonic hedgehog is involved in a multitude of distinct patterning processes during vertebrate embryogenesis. In the nascent body axis of the zebrafish embryo, sonic hedgehog is co-expressed with axial (HNF3beta in mammals), a transcription regulator of the winged helix family. We show here that misexpression of axial leads to ectopic activation of sonic hedgehog expression in the zebrafish, suggesting that axial is a regulator of sonic hedgehog transcription. The sonic hedgehog gene was cloned from zebrafish and its promoter was characterized with respect to activation by axial. Expression of axial or rat HNF3beta in HeLa cells results in activation of co-transfected sonic hedgehog promoter-CAT fusion genes. This effect is mediated by two Axial (HNF3beta) recognition sequences. We furthermore identified a retinoic acid response element (RARE) in the sonic hedgehog upstream region which can be bound by retinoic acid receptor (RAR) and retinoid X receptor (RXR) heterodimers in vitro and confers retinoic acid inducibility to the sonic hedgehog promoter in the HeLa cell system. Our results suggest that both Axial (HNF3beta) and retinoic acid receptors are direct regulators of the sonic hedgehog gene.

Animals↗
PubMed1997

Zebrafish genetics and its implications for understanding vertebrate development.

The identification of genes via their mutant phenotypes is the most direct way of dissecting the molecular basis of developmental processes. While this approach has been extremely powerful in invertebrates such as Drosophila and Caenorhabditis elegans, it has until recently been lacking in vertebrates. Now two landmark studies by the groups of Nüsslein-Volhard and Driever have for the first time provided the basis for a comprehensive genetic dissection of vertebrate development using the teleost fish, Danio rerio. The mutations identified in these screens look set to revolutionise our understanding of vertebrate development.

Animals↗
PubMed1996

Transcriptional activation of hedgehog target genes in Drosophila is mediated directly by the cubitus interruptus protein, a member of the GLI family of zinc finger DNA-binding proteins.

Members of the Hedgehog (Hh) family of secreted proteins have been identified recently as key signaling molecules that regulate a variety of inductive interactions central to the development of both Drosophila and vertebrates. Despite their widespread importance, the way in which Hh signals are transduced inside the cell remains poorly understood. The best candidate for a transcription factor that mediates Hh signaling in Drosophila is the product of the cubitus interruptus (ci) gene, a zinc finger protein that exhibits significant homology to protein products of the vertebrate GLI gene family. Here, we show that elevated levels of Ci are sufficient to activate patched (ptc) and other hh target genes, even in the absence of hh activity. We also show that Ci can function as a transcriptional activator in yeast and demonstrate that the zinc finger domain of the protein is sufficient for its target specificity. Finally, we identify sequences in the promoter region of the ptc gene, a primary target of Hh signaling, that are identical to the consensus-binding sequence of the GLI protein and are required for reporter gene expression in response to Hh activity. Taken together, our results strongly support the role for Ci as the transcriptional activator that mediates hh signaling.

Animals↗
PubMed1996

smoothened encodes a receptor-like serpentine protein required for hedgehog signalling.

Members of the Hedgehog family of secreted proteins control a number of important inductive interactions in the development of both vertebrates and Drosophila, but little is known about the ways in which their signalling activities are transduced. In Drosophila, hedgehog is one of the segment-polarity genes, mutations of which disrupt the pattern and polarity of individual embryonic segments and their adult derivatives; several of these genes have been implicated in transduction of the hedgehog signal. Here we show that the segment-polarity gene smoothened is required for the response of cells to hedgehog signalling during the development of both the embryonic segments and imaginal discs. Sequence analysis of the smoothened transcription unit reveals a single open reading frame encoding a protein with seven putative transmembrane domains. This structure is typical of G-protein-coupled receptors, suggesting that the Smoothened protein may act as a receptor for the Hedgehog ligand.

Alleles↗
PubMed1996

Induction of a specific muscle cell type by a hedgehog-like protein in zebrafish.

The notochord plays a central role in vertebrate development, acting as a signalling source that patterns the neural tube and somites. In in vitro assays, the secreted protein Sonic hedgehog mimics the inducing effects of notochord on both presomitic mesoderm and neural plate explants of amniote embryos, suggesting that both patterning activities of the notochord may be mediated by this protein in vivo. In zebrafish, however, mutants with disrupted notochord development lack a specific muscle cell type, the muscle pioneers, although they retain the ability to induce neural differentiation, raising the possibility that neural tube and somite patterning may be mediated by distinct signals. Here we describe a new member of the hedgehog family, echidna hedgehog, that is expressed exclusively in the notochord and has the ability to rescue the differentiation of muscle pioneer cells in mutants with no notochord. Moreover, we show that a combination of ectopic echidna hedgehog and sonic hedgehog expression induces supernumary muscle pioneers in wild-type embryos, suggesting that both signals act sequentially to pattern the developing somites.

Amino Acid Sequence↗
PubMed1996

The role of segment polarity genes during early oogenesis in Drosophila.

In the Drosophila ovary, hedgehog (hh) signaling from cells near the apical tip of the germarium stimulates the proliferation and specification of somatic cells in region 2 of the germarium, 2-5 cells away from the hh-expressing cells (A. J. Forbes, H. Lin, P. Ingham and A. Spradling (1996) Development 122, 1125-1135). This report examines the role during early oogenesis of several genes that are known to function in hh-mediated signaling during embryonic and larval development (P. Ingham (1995) Current Opin. Genetics Dev. 5, 528-534). As in imaginal discs, engrailed (en) is co-expressed with hh in the germarium, while patched (ptc) and cubitus interruptus (ci) are expressed in somatic cells throughout the germarium and in developing egg chambers, with ptc expression being elevated within 10 cell diameters of the source of the hh signal. Moreover, the somatic cell overproliferation caused by ectopic hh expression is accompanied by elevated levels of ptc and is phenocopied in ptc- somatic clones. These analyses suggest that ptc and ci are components of the hh signaling pathway in the germarium. However, unlike embryos and imaginal discs, neither wingless (wg) nor decapentaplegic (dpp) appear to mediate the ovarian hh signal. wg is expressed in 'cap cells,' a subset of hh-expressing cells located adjacent to germ-line stem cells, but is unaffected by ectopic hh expression. Nor does the ectopic expression of wg or dpp mimic the effect of ectopic hh expression. We propose that Hh diffuses from apical cells, including cap cells, and regulates the proliferation of nearby ovarian somatic cells by antagonizing the negative effects of ptc on ci activity in these cells, thereby allowing the transcription of ci-dependent genes, including ptc itself.

Animals↗
PubMed1996

hedgehog is required for the proliferation and specification of ovarian somatic cells prior to egg chamber formation in Drosophila.

The hedgehog (hh) gene plays a role in regulating cell proliferation and specifying cell identity in diverse systems. We show that hh is expressed at the extreme apical end of Drosophila ovarioles in terminal filament cells and a newly identified group of associated somatic cells. Reducing or ectopically expressing hh affects somatic cells in region 2 of the germarium, 2-5 cells away from the cells in which Hh protein is detected. hh activity stimulates the proliferation of pre-follicle somatic cells, and promotes the specification of polar follicle cells. hh signaling during egg chamber assembly appears to be closely related to, or part of pathways involving the neurogenic genes.

Animals↗
PubMed1996

Spatial regulation of a zebrafish patched homologue reflects the roles of sonic hedgehog and protein kinase A in neural tube and somite patterning.

Signalling by members of the Hedgehog family of secreted proteins plays a central role in the development of vertebrate and invertebrate embryos. In Drosophila, transduction of the Hedgehog signal is intimately associated with the activity of protein kinase A and the product of the segment polarity gene patched. We have cloned a homologue of patched from the zebrafish Danio rerio and analysed the spatiotemporal regulation of its transcription during embryonic development in both wild-type and mutant animals. We find a striking correlation between the accumulation of patched1 transcripts and cells responding to sonic hedgehog activity both in the neurectoderm and mesoderm, suggesting that like its Drosophila counterpart, patched1 is regulated by sonic hedgehog activity. Consistent with this interpretation, mis-expression of sonic hedgehog results in ectopic activation of patched1 transcription. Using dominant negative and constitutively active forms of the protein kinase A subunits, we also show that expression of patched1 as well as of other sonic hedgehog targets, is regulated by protein kinase A activity. Taken together, our findings suggest that the mechanism of signalling by Hedgehog family proteins has been highly conserved during evolution.

Amino Acid Sequence↗
PubMed1996

Expression of axial and sonic hedgehog in wildtype and midline defective zebrafish embryos.

We present a description of the expression of the HNF-3 beta homolog axial (axl) in the developing zebrafish up to larva stages and compare it with that of sonic hedgehog (shh). Both genes are expressed in derivatives of all three germ layers in dynamic patterns that show substantial overlap, consistent with mutual regulatory interactions between the two genes. However, we also describe unique sites of expression of both axl and shh indicating that some aspects of their regulation are independent of one another. In no tail (ntl, zebrafish Brachyury) and floating head (flh, zebrafish Xnot1) mutants, both of which affect notochord development, early expression of axl in the organizer is unaffected, excluding a function for these genes in establishment of axl expression. At later stages, ntl and flh mutants show different effects on the expression of both axl and shh in the neuroectoderm of the trunk and tail reflecting their distinct contributions to the development of the midline mesoderm; in contrast to flh and ntl mutations whose effects are restricted to the trunk and tail, mutation of cyclops (cyc) affects axl and shh expression along the entire midline of the neuroectoderm. Endodermal expression of axl and shh is not affected by the mutations showing that development of the endoderm is under distinct control.

Animals↗
PubMed1995

Secretion of the amino-terminal fragment of the hedgehog protein is necessary and sufficient for hedgehog signalling in Drosophila.

BACKGROUND: The Drosophila segment polarity gene hedgehog encodes a member of a family of secreted proteins that are involved in a variety of patterning processes, in both vertebrates and invertebrates. Some of these processes depend upon short-range or contact-dependent interactions, whereas others seem to involve long-range signalling. Two different models have been proposed to account for the execution of these contrasting processes by the same proteins: one postulates that Hedgehog acts exclusively over short distances, its long-range influences being effected through regulation of other signalling factors; the second postulates that different aspects of Hedgehog activity are mediated by distinct forms of the protein that are generated by autoproteolysis. RESULTS: We have investigated these models by mutating the hedgehog coding region such that only the amino-terminal or carboxy-terminal half of the protein is secreted. Deletion of the carboxy-terminal portion has little effect on the signalling activity of the protein, whereas abolishing the secretion of the amino-terminal half leads to a complete loss of signalling. In addition, we find that increases in the level of expression within the normal hedgehog transcriptional domain of either the wild-type protein or the carboxy-terminal-deleted form expand the range of activity to a limited extent, but have only minor effects on cell identity. CONCLUSIONS: In Drosophila, all of the signalling activity of Hedgehog resides in the amino-terminal portion of the protein, the secretion of which is essential for its function. The range of Hedgehog is limited by the close association of the amino-terminal peptide with the cell surface but can be extended by elevating the level of its expression.

Amino Acid Sequence↗
PubMed1995

Quantitative effects of hedgehog and decapentaplegic activity on the patterning of the Drosophila wing.

BACKGROUND: Members of the hedgehog (hh) gene family encode a novel class of proteins implicated in positional signalling in both invertebrates and vertebrates. In Drosophila, the hh gene has been shown to regulate patterning of the imaginal discs, the precursors of the insect limbs. In a remarkably similar fashion, the function and expression of the sonic hedgehog (shh) gene is closely associated with the 'zone of polarizing activity' (ZPA) that controls antero-posterior patterning of the vertebrate limb. Both of these functions suggest a role for hedgehog family proteins as morphogens. An alternative possibility, however, is that hh and its homologues act to control the expression of other instructive signalling molecules. RESULTS: We have explored this issue by examining the effects on Drosophila wing patterning of ectopically expressing varying levels of hh and shh, as well as of the putative hh target gene, decapentaplegic (dpp), a member of the transforming growth factor-beta family of signalling molecules. We find that different levels of hh activity can induce graded changes in the patterning of the wing, and that zebrafish shh acts in a similar though attenuated fashion. Varying levels of ectopic hh and shh activity can differentially activate transcription of the patched and dpp genes. Furthermore, ectopic expression of dpp alone is sufficient to induce the pattern alterations caused by ectopic hh or shh activity. CONCLUSION: Thus, hh family proteins can elicit different responses in a dose-dependent manner in the imaginal disc. The principal function of hh, however, is to activate transcription of dpp at the compartment boundary, thereby establishing a source of dpp activity that is the primary determinant of antero-posterior patterning.

Animals↗
PubMed1995

Signalling by hedgehog family proteins in Drosophila and vertebrate development.

Members of the hedgehog gene family encode a novel class of secreted proteins and are expressed in embryonic cells known to possess important signalling activities in organisms as diverse as flies and chickens. Proteins of the hedgehog family act in these different developmental contexts as both permissive and instructive signals. How this signalling activity is transduced is (as yet) poorly understood, but recent studies point to the involvement of protein kinase A in both Drosophila and vertebrates.

Animals↗
PubMed1994

Groucho is required for Drosophila neurogenesis, segmentation, and sex determination and interacts directly with hairy-related bHLH proteins.

We have used the interaction trap, a yeast two-hybrid system, to identify proteins interacting with hairy, a basic-helix-loop-helix (bHLH) protein that represses transcription during Drosophila embryonic segmentation. We find that the groucho (gro) protein binds specifically to hairy and also to hairy-related bHLH proteins encoded by deadpan and the Enhancer of split complex. The C-terminal WRPW motif present in all these bHLH proteins is essential for this interaction. We demonstrate that these associations reflect in vivo maternal requirements for gro during neurogenesis, segmentation, and sex determination, three processes regulated by the above bHLH proteins, and we propose that gro is a transcriptional corepressor recruited to specific target promoters by hairy-related bHLH proteins.

Animals↗
PubMed1994

Pattern formation. Hedgehog points the way.

Recent findings indicate that the Drosophila gene hedgehog and its vertebrate homologues act in analogous ways as signalling molecules that help provide cells with positional information during development.

Animals↗
PubMed1994

The hedgehog gene family in Drosophila and vertebrate development.

The segment polarity gene hedgehog plays a central role in cell patterning during embryonic and post-embryonic development of the dipteran, Drosophila melanogaster. Recent studies have identified a family of hedgehog related genes in vertebrates; one of these, Sonic hedgehog is implicated in positional signalling processes that show interesting similarities with those controlled by its Drosophila homologue.

Amino Acid Sequence↗