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David R McClay

Publications and source records attributed to David R McClay.

At least 19 recordsLinked to original sources
PubMed2006

The genome of the sea urchin Strongylocentrotus purpuratus.

We report the sequence and analysis of the 814-megabase genome of the sea urchin Strongylocentrotus purpuratus, a model for developmental and systems biology. The sequencing strategy combined whole-genome shotgun and bacterial artificial chromosome (BAC) sequences. This use of BAC clones, aided by a pooling strategy, overcame difficulties associated with high heterozygosity of the genome. The genome encodes about 23,300 genes, including many previously thought to be vertebrate innovations or known only outside the deuterostomes. This echinoderm genome provides an evolutionary outgroup for the chordates and yields insights into the evolution of deuterostomes.

Animals↗
PubMed2006

The sea urchin kinome: a first look.

This paper reports a preliminary in silico analysis of the sea urchin kinome. The predicted protein kinases in the sea urchin genome were identified, annotated and classified, according to both function and kinase domain taxonomy. The results show that the sea urchin kinome, consisting of 353 protein kinases, is closer to the Drosophila kinome (239) than the human kinome (518) with respect to total kinase number. However, the diversity of sea urchin kinases is surprisingly similar to humans, since the urchin kinome is missing only 4 of 186 human subfamilies, while Drosophila lacks 24. Thus, the sea urchin kinome combines the simplicity of a non-duplicated genome with the diversity of function and signaling previously considered to be vertebrate-specific. More than half of the sea urchin kinases are involved with signal transduction, and approximately 88% of the signaling kinases are expressed in the developing embryo. These results support the strength of this nonchordate deuterostome as a pivotal developmental and evolutionary model organism.

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PubMed2006

Genomics and expression profiles of the Hedgehog and Notch signaling pathways in sea urchin development.

The Hedgehog (Hh) and Notch signal transduction pathways control a variety of developmental processes including cell fate choice, differentiation, proliferation, patterning and boundary formation. Because many components of these pathways are conserved, it was predicted and confirmed that pathway components are largely intact in the sea urchin genome. Spatial and temporal location of these pathways in the embryo, and their function in development offer added insight into their mechanistic contributions. Accordingly, all major components of both pathways were identified and annotated in the sea urchin Strongylocentrotus purpuratus genome and the embryonic expression of key components was explored. Relationships of the pathway components, and modifiers predicted from the annotation of S. purpuratus, were compared against cnidarians, arthropods, urochordates, and vertebrates. These analyses support the prediction that the pathways are highly conserved through metazoan evolution. Further, the location of these two pathways appears to be conserved among deuterostomes, and in the case of Notch at least, display similar capacities in endomesoderm gene regulatory networks. RNA expression profiles by quantitative PCR and RNA in situ hybridization reveal that Hedgehog is produced by the endoderm beginning just prior to invagination, and signals to the secondary mesenchyme-derived tissues at least until the pluteus larva stage. RNA in situ hybridization of Notch pathway members confirms that Notch functions sequentially in the vegetal-most secondary mesenchyme cells and later in the endoderm. Functional analyses in future studies will embed these pathways into the growing knowledge of gene regulatory networks that govern early specification and morphogenesis.

Amino Acid Sequence↗
PubMed2006

The genomic underpinnings of apoptosis in Strongylocentrotus purpuratus.

Programmed cell death through apoptosis is a pan-metazoan character involving intermolecular signaling networks that have undergone substantial lineage-specific evolution. A survey of apoptosis-related proteins encoded in the sea urchin genome provides insight into this evolution while revealing some interesting novelties, which we highlight here. First, in addition to a typical CARD-carrying Apaf-1 homologue, sea urchins have at least two novel Apaf-1-like proteins that are each linked to a death domain, suggesting that echinoderms have evolved unique apoptotic signaling pathways. Second, sea urchins have an unusually large number of caspases. While the set of effector caspases (caspases-3/7 and caspase-6) in sea urchins is similar to that found in other basal deuterostomes, signal-responsive initiator caspase subfamilies (caspases-8/10 and 9, which are respectively linked to DED and CARD adaptor domains) have undergone echinoderm-specific expansions. In addition, there are two groups of divergent caspases, one distantly related to the vertebrate interleukin converting enzyme (ICE)-like subfamily, and a large clan that does not cluster with any of the vertebrate caspases. Third, the complexity of proteins containing an anti-apoptotic BIR domain and of Bcl-2 family members approaches that of vertebrates, and is greater than that found in protostome model systems such as Drosophila or Caenorhabditis elegans. Finally, the presence of Death receptor homologues, previously known only in vertebrates, in both Strongylocentrotus purpuratus and Nematostella vectensis suggests that this family of apoptotic signaling proteins evolved early in animals and was subsequently lost in the nematode and arthropod lineage(s). Our results suggest that cell survival is contingent upon a diverse array of signals in sea urchins, more comparable in complexity to vertebrates than to arthropods or nematodes, but also with unique features that may relate to specific requirements imposed by the biphasic life cycle and/or immunological idiosyncrasies of this organism.

Amino Acid Sequence↗
PubMed2006

A genome-wide survey of the evolutionarily conserved Wnt pathways in the sea urchin Strongylocentrotus purpuratus.

The Wnt pathways are evolutionarily well-conserved signal transduction pathways that are known to play important roles in all Metazoans investigated to date. Here, we examine the Wnt pathway genes and target genes present in the genome of the echinoderm Strongylocentrotus purpuratus. Analysis of the Wnt genes revealed that eleven of the thirteen reported Wnt subfamilies are represented in sea urchin, with the intriguing identification of a Wnt-A ortholog thought to be absent in deuterostomes. A phylogenetic study of the Frizzled proteins, the Wnt receptors, performed throughout the animal kingdom showed that not all Frizzled subfamilies were present in the metazoan common ancestor, e.g. Fz3/6 emerged later during evolution. Using sequence analysis, orthologs of the vast majority of the cellular machinery involved in transducing the three types of Wnt pathways were found in the sea urchin genome. Furthermore, of about one hundred target genes identified in other organisms, more than half have clear echinoderm orthologs. Thus, these analyses produce new inputs in the evolutionary history of the Wnt genes in an animal occupying a position that offers great insights into the basal properties of deuterostomes.

Amino Acid Sequence↗
PubMed2006

Lineage-specific expansions provide genomic complexity among sea urchin GTPases.

In every organism, GTP-binding proteins control many aspects of cell signaling. Here, we examine in silico several GTPase families from the Strongylocentrotus purpuratus genome: the monomeric Ras superfamily, the heterotrimeric G proteins, the dynamin superfamily, the SRP/SR family, and the "protein biosynthesis" translational GTPases. Identified were 174 GTPases, of which over 90% are expressed in the embryo as shown by tiling array and expressed sequence tag data. Phylogenomic comparisons restricted to Drosophila, Ciona, and humans (protostomes, urochordates, and vertebrates, respectively) revealed both common and unique elements in the expected composition of these families. Galpha and dynamin families contain vertebrate expansions, consistent with whole genome duplications, whereas SRP/SR and translational GTPases are highly conserved. Unexpectedly, Ras superfamily analyses revealed several large (5+) lineage-specific expansions in the sea urchin. For Rho, Rab, Arf, and Ras subfamilies, comparing total human gene numbers to the number of sea urchin genes with vertebrate orthologs suggests reduced genomic complexity in the sea urchin. However, gene duplications in the sea urchin increase overall numbers such that total sea urchin gene numbers approximate vertebrate gene numbers for each monomeric GTPase family. These findings suggest that lineage-specific expansions may be an important component of genomic evolution in signal transduction.

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PubMed2006

The canonical Wnt pathway in embryonic axis polarity.

The canonical Wnt pathway plays crucial roles in multiple developmental processes, including in axis specification. Throughout the animal kingdom, this pathway has been reported to drive patterning of axes as different as the animal-vegetal axis in echinoderms to the dorsal-ventral axis in vertebrates. Intriguingly enough, this pathway appears structurally and functionally well conserved during evolution. However, differences between these phyla are observed that explain how a same pathway can mediate establishment of two such apparently distinct axes. This review compares the axis specification processes used in two evolutionarily distant embryos, the sea urchin and Xenopus.

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PubMed2006

p38 MAPK in development and cancer.

p38 is a MAPK that has been shown to induce a wide variety of biological effects in cell culture in response to a wide range of stimuli. These effects are dependent not only on the stimuli, but also on the cellular context, resulting in a bewildering array of possibilities. For example, p38 was shown to induce apoptosis in some cells, but prevent apoptosis in others. Similarly opposed effects had been observed with respect to cell cycle regulation. The role of p38 in inflammatory disease has been appreciated from the beginning, since it was initially identified as an cytokine inducer. More recently, p38 function has been evaluated in vivo, and through these studies p38 has emerged as an important regulator of both embryonic development and cancer progression. This review will focus on these in vivo studies in an effort to provide perspective on p38 biologically and as a pharmacological target.

Animals↗
PubMed2006

RhoA regulates initiation of invagination, but not convergent extension, during sea urchin gastrulation.

During gastrulation, the archenteron is formed using cell shape changes, cell rearrangements, filopodial extensions, and convergent extension movements to elongate and shape the nascent gut tube. How these events are coordinated remains unknown, although much has been learned from careful morphological examinations and molecular perturbations. This study reports that RhoA is necessary to trigger archenteron invagination in the sea urchin embryo. Inhibition of RhoA results in a failure to initiate invagination movements, while constitutively active RhoA induces precocious invagination of the archenteron, complete with the actin rearrangements and extracellular matrix secretions that normally accompany the onset of invagination. Although RhoA activity has been reported to control convergent extension movements in vertebrate embryos, experiments herein show that RhoA activity does not regulate convergent extension movements during sea urchin gastrulation. Instead, the results support the hypothesis that RhoA serves as a trigger to initiate invagination, and once initiation occurs, RhoA activity is no longer involved in subsequent gastrulation movements.

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PubMed2006

Frizzled5/8 is required in secondary mesenchyme cells to initiate archenteron invagination during sea urchin development.

Wnt signaling pathways play key roles in numerous developmental processes both in vertebrates and invertebrates. Their signals are transduced by Frizzled proteins, the cognate receptors of the Wnt ligands. This study focuses on the role of a member of the Frizzled family, Fz5/8, during sea urchin embryogenesis. During development, Fz5/8 displays restricted expression, beginning at the 60-cell stage in the animal domain and then from mesenchyme blastula stage, in both the animal domain and a subset of secondary mesenchyme cells (SMCs). Loss-of-function analyses in whole embryos and chimeras reveal that Fz5/8 is not involved in the specification of the main embryonic territories. Rather, it appears to be required in SMCs for primary invagination of the archenteron, maintenance of endodermal marker expression and apical localization of Notch receptors in endodermal cells. Furthermore, among the three known Wnt pathways, Fz5/8 appears to signal via the planar cell polarity pathway. Taken together, the results suggest that Fz5/8 plays a crucial role specifically in SMCs to control primary invagination during sea urchin gastrulation.

Animals↗
PubMed2006

Repression of mesodermal fate by foxa, a key endoderm regulator of the sea urchin embryo.

The foxa gene is an integral component of the endoderm specification subcircuit of the endomesoderm gene regulatory network in the Strongylocentrotus purpuratus embryo. Its transcripts become confined to veg2, then veg1 endodermal territories, and, following gastrulation, throughout the gut. It is also expressed in the stomodeal ectoderm. gatae and otx genes provide input into the pregastrular regulatory system of foxa, and Foxa represses its own transcription, resulting in an oscillatory temporal expression profile. Here, we report three separate essential functions of the foxa gene: it represses mesodermal fate in the veg2 endomesoderm; it is required in postgastrular development for the expression of gut-specific genes; and it is necessary for stomodaeum formation. If its expression is reduced by a morpholino, more endomesoderm cells become pigment and other mesenchymal cell types, less gut is specified, and the larva has no mouth. Experiments in which blastomere transplantation is combined with foxa MASO treatment demonstrate that, in the normal endoderm, a crucial role of Foxa is to repress gcm expression in response to a Notch signal, and hence to repress mesodermal fate. Chimeric recombination experiments in which veg2, veg1 or ectoderm cells contained foxa MASO show which region of foxa expression controls each of the three functions. These experiments show that the foxa gene is a component of three distinct embryonic gene regulatory networks.

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PubMed2005

p38 MAPK is essential for secondary axis specification and patterning in sea urchin embryos.

Most eggs in the animal kingdom establish a primary, animal-vegetal axis maternally, and specify the remaining two axes during development. In sea urchin embryos, the expression of Nodal on the oral (ventral) side of the embryo is the first known molecular determinant of the oral-aboral axis (the embryonic dorsoventral axis), and is crucial for specification of the oral territory. We show that p38 MAPK acts upstream of Nodal and is required for Nodal expression in the oral territory. p38 is uniformly activated early in development, but, for a short interval at late blastula stage, is asymmetrically inactivated in future aboral nuclei. Experiments show that this transient asymmetry of p38 activation corresponds temporally to both oral specification and the onset of oral Nodal expression. Uniform inhibition of p38 prevents Nodal expression and axis specification, resulting in aboralized embryos. Nodal and its target Gsc each rescue oral-aboral specification and patterning when expressed asymmetrically in p38-inhibited embryos. Thus, our results indicate that p38 is required for oral specification through its promotion of Nodal expression in the oral territory.

Amino Acid Sequence↗
PubMed2005

A Fringe-modified Notch signal affects specification of mesoderm and endoderm in the sea urchin embryo.

Fringe proteins are O-fucose-specific beta-1,3 N-acetylglucosaminyltransferases that glycosylate the extracellular EGF repeats of Notch and enable Notch to be activated by the ligand Delta. In the sea urchin, signaling between Delta and Notch is known to be necessary for specification of secondary mesenchyme cells (SMCs). The Lytechinus variegatus Fringe homologue is expressed in both the signaling and receiving cells during this first Delta-Notch signal. Perturbation of Fringe expression through morpholino antisense oligonucleotide (MO) injection results in fewer SMCs but also causes decreased and delayed archenteron invagination. Partial endoderm specification occurs but expression of some endoderm genes is compromised. The data are consistent with a Fringe-requiring Notch signal as one upstream component of archenteron morphogenesis. Finally, Fringe perturbations result in more severe phenotypes than those previously reported for Notch dominant-negative (LvN(neg)) injections or reported here for Notch MO (NMO) injections. Injecting a combination of LvN(neg) and NMO results in a more severe phenotype than either treatment alone, and this combination phenocopies the fringe MO embryos. Taken together, the results show that Fringe is necessary both for maternal and zygotic Notch signals, and these Notch signals affect specification of mesoderm and endoderm.

Animals↗
PubMed2005

LvGroucho and nuclear beta-catenin functionally compete for Tcf binding to influence activation of the endomesoderm gene regulatory network in the sea urchin embryo.

In the sea urchin embryo, specification of the endomesoderm is accomplished by the activity of a network of regulatory genes in the vegetal hemisphere, called the endomesoderm gene regulatory network (GRN). The activation of this network is mediated primarily through the activity of the Wnt pathway, though details of pathway activation remain unclear. To gain further insight into control of endomesoderm GRN activation, we have identified a sea urchin homologue of the co-repressor Groucho (LvGroucho) that has been shown to antagonize beta-catenin/Tcf activation complexes during Wnt signaling in other systems. Groucho functions by recruiting the histone deacetylase Rpd3 to the DNA template via interaction with site-specific transcription factors, resulting in localized chromatin condensation and transcriptional silencing. Our results show that the LvGroucho protein localizes to all nuclei throughout embryonic development. Interaction assays demonstrate that LvGroucho interacts with Tcf via both the Q and the WD domains of the protein. LvGroucho interacts with Tcf to antagonize the expression of key endomesoderm regulatory genes. Assays demonstrate that LvGroucho and n beta-catenin functionally compete for binding to Tcf as a major mechanism by which the Tcf-control switch is regulated. Functional analysis of the N-terminal AES197 domain of LvGroucho shows that it is sufficient to recapitulate the function of full-length LvGroucho. This finding strongly supports the conclusion that the effects of LvGro overexpression are due primarily to its interactions with Tcf and not other Groucho interacting partners, since Tcf is the only protein present in the sea urchin known to interact with AES197. Because the Q domain is unable to bind Rpd3, it was expected to behave as a dominant negative LvGroucho. Unexpectedly, overexpression of the Q domain gave functional results similar to LvGroucho and the AES197 domain. This is the first evidence for an inherent repressive function for the Q domain alone. Together, our results indicate that LvGroucho functionally competes with beta-catenin for Tcf binding, and this competitive mechanism regulates one of the earliest steps in the initiation of the sea urchin endomesoderm GRN.

Amino Acid Sequence↗
PubMed2004

SpHnf6, a transcription factor that executes multiple functions in sea urchin embryogenesis.

The Strongylocentrotus purpuratus hnf6 (Sphnf6) gene encodes a new member of the ONECUT family of transcription factors. The expression of hnf6 in the developing embryo is triphasic, and loss-of-function analysis shows that the Hnf6 protein is a transcription factor that has multiple distinct roles in sea urchin development. hnf6 is expressed maternally, and before gastrulation its transcripts are distributed globally. Early in development, its expression is required for the activation of PMC differentiation genes such as sm50, pm27, and msp130, but not for the activation of any known PMC regulatory genes, for example, alx, ets1, pmar1, or tbrain. Micromere transplantation experiments show that the gene is not involved in early micromere signaling. Early hnf6 expression is also required for expression of the mesodermal regulator gatac. The second known role of hnf6 is its participation after gastrulation in the oral ectoderm gene regulatory network (GRN), in which its expression is essential for the maintenance of the state of oral ectoderm specification. The third role is in the neurogenic ciliated band, which is foreshadowed exactly by a trapezoidal band of hnf6 expression at the border of the oral ectoderm and where it continues to be expressed through the end of embryogenesis. Neither oral ectoderm regulatory functions nor ciliated band formation occur normally in the absence of hnf6 expression.

Amino Acid Sequence↗
PubMed2004

Nuclear beta-catenin-dependent Wnt8 signaling in vegetal cells of the early sea urchin embryo regulates gastrulation and differentiation of endoderm and mesodermal cell lineages.

The entry of beta-catenin into vegetal cell nuclei beginning at the 16-cell stage is one of the earliest known molecular asymmetries seen along the animal-vegetal axis in the sea urchin embryo. Nuclear beta-catenin activates a vegetal signaling cascade that mediates micromere specification and specification of the endomesoderm in the remaining cells of the vegetal half of the embryo. Only a few potential target genes of nuclear beta-catenin have been functionally analyzed in the sea urchin embryo. Here, we show that SpWnt8, a Wnt8 homolog from Strongylocentrotus purpuratus, is zygotically activated specifically in 16-cell-stage micromeres in a nuclear beta-catenin-dependent manner, and its expression remains restricted to the micromeres until the 60-cell stage. At the late 60-cell stage nuclear beta-catenin-dependent SpWnt8 expression expands to the veg2 cell tier. SpWnt8 is the only signaling molecule thus far identified with expression localized to the 16-60-cell stage micromeres and the veg2 tier. Overexpression of SpWnt8 by mRNA microinjection produced embryos with multiple invagination sites and showed that, consistent with its localization, SpWnt8 is a strong inducer of endoderm. Blocking SpWnt8 function using SpWnt8 morpholino antisense oligonucleotides produced embryos that formed micromeres that could transmit the early endomesoderm-inducing signal, but these cells failed to differentiate as primary mesenchyme cells. SpWnt8-morpholino embryos also did not form endoderm, or secondary mesenchyme-derived pigment and muscle cells, indicating a role for SpWnt8 in gastrulation and in the differentiation of endomesodermal lineages. These results establish SpWnt8 as a critical component of the endomesoderm regulatory network in the sea urchin embryo.

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