Biomedical subjects

Eric H Davidson

Publications and source records attributed to Eric H Davidson.

At least 19 recordsLinked to original sources

PubMed2006

The C2H2 zinc finger genes of Strongylocentrotus purpuratus and their expression in embryonic development.

The C2H2 zinc finger is one of the most abundant protein domains and is thought to have been extensively replicated in diverse animal clades. Some well-studied proteins that contain this domain are transcriptional regulators. As part of an attempt to delineate all transcription factors encoded in the Strongylocentrotus purpuratus genome, we identified the C2H2 zinc finger genes indicated in the sequence, and examined their involvement in embryonic development. We found 377 zinc finger genes in the sea urchin genome, about half the number found in mice or humans. Their expression was measured by quantitative PCR. Up to the end of gastrulation less than a third of these genes is expressed, and about 75% of the expressed genes are maternal; both parameters distinguish these from all other classes of regulatory genes as measured in other studies. Spatial expression pattern was determined by whole mount in situ hybridization for 43 genes transcribed at a sufficient level, and localized expression was observed in diverse embryonic tissues. These genes may execute important regulatory functions in development. However, the functional meaning of the majority of this large gene family remains undefined.

Animals↗

PubMed2006

Phosphatized polar lobe-forming embryos from the Precambrian of southwest China.

In developing embryos of some extant spiralian animals, polar lobe formation is one of the symmetry-breaking mechanisms for segregation of maternal cytoplasmic substances to certain blastomeres and not others. Polar lobe formation leads to unique early cleavage morphologies that include trilobed, J-shaped, and five-lobed structures. Fossil embryos similar to modern lobeforming embryos are recognized from the Precambrian Doushantuo Formation phosphates, Weng'an, Guizhou Province, China. These embryos are abundant and form a developmental sequence comparable to different developing stages observed in lobe-forming embryos of extant spiralians. These data imply that lobe formation is an evolutionarily ancient process of embryonic specification.

Animals↗

PubMed2006

Confocal quantification of cis-regulatory reporter gene expression in living sea urchin.

Quantification of GFP reporter gene expression at single cell level in living sea urchin embryos can now be accomplished by a new method of confocal laser scanning microscopy (CLSM). Eggs injected with a tissue-specific GFP reporter DNA construct were grown to gastrula stage and their fluorescence recorded as a series of contiguous Z-section slices that spanned the entire embryo. To measure the depth-dependent signal decay seen in the successive slices of an image stack, the eggs were coinjected with a freely diffusible internal fluorescent standard, rhodamine dextran. The measured rhodamine fluorescence was used to generate a computational correction for the depth-dependent loss of GFP fluorescence per slice. The intensity of GFP fluorescence was converted to the number of GFP molecules using a conversion constant derived from CLSM imaging of eggs injected with a measured quantity of GFP protein. The outcome is a validated method for accurately counting GFP molecules in given cells in reporter gene transfer experiments, as we demonstrate by use of an expression construct expressed exclusively in skeletogenic cells.

Animals↗

PubMed2006

cis-regulatory processing of Notch signaling input to the sea urchin glial cells missing gene during mesoderm specification.

The glial cells missing regulatory gene of Strongylocentrotus purpuratus (spgcm) was proposed earlier to be the genomic target of Delta/Notch (D/N) signaling required for specification of the mesodermal precursors of pigment cells. Here, we show that microinjection of a spgcm antisense morpholino oligonucleotide results in larvae without pigment cells. Microinjection of an mRNA encoding a dominant negative form of Suppressor of Hairless (dn-Su(H)) results in reduced levels of spgcm mRNA, disruption of mesodermal founder cell specification and failure to produce pigment cells. These results confirm that this gene is required for pigment cell specification. Three cis-regulatory modules of the spgcm gene were identified, which when incorporated in a GFP expression construct recapitulate the early expression pattern of this gene. Spatial expression of this GFP expression construct is severely disrupted by co-expression of dn-Su(H) mRNA, confirming that spgcm is a direct target of canonical N signaling mediated through Su(H) inputs. cis-perturbation analysis by mutation of consensus Su(H) sites identified a conserved motif paired-site and a lone site in the middle module that function both to drive expression in SMC precursors which receive the Delta signal and to repress expression in ectopic locations which lack this signal. While these Su(H) target sites provide the cis-regulatory architecture with the core of an N signaling transcriptional response switch, both the on and off outputs from this module require additional inputs.

Animals↗

PubMed2006

Expression and function of blimp1/krox, an alternatively transcribed regulatory gene of the sea urchin endomesoderm network.

The blimp1/krox gene of Strongylocentrotus purpuratus, formerly krox1, encodes zinc finger transcription factors which play a central role in both early and late endomesoderm specification. Here we show that there are two alternative splice forms transcribed under the control of different regulatory regions. The blimp/krox1b form was previously unknown, and is the form expressed during cleavage, beginning 6-9 h postfertilization. This form is required for the early events of endomesoderm specification. A different splice variant, blimp1/krox1a, is expressed only from gastrula stage onward. During cleavage stages the blimp1/krox gene is expressed in the large micromeres and veg2 descendents. Soon after, it is expressed in the ring of specified mesoderm cells at the vegetal pole of the blastula. Its expression is later restricted to the blastopore region and the posterior of the invaginating archenteron, and finally to the midgut and hindgut of the pluteus larva. The expression of blimp1/krox is dynamic, and involves several distinct spatial territories. A GFP recombinant BAC was created by substituting the GFP coding sequence for that of the second exon (1b), in order to distinguish the expression pattern of the early form from that of the late form. This construct closely mimics blimp1/krox1b expression during early stages of sea urchin development. To expand our knowledge of the downstream linkages of this gene, additional experiments were carried out using antisense morpholino oligos (MASO). We confirmed previously published data that blimp1/krox autoregulates its own expression, but discovered, surprisingly, that this gene represses rather than activates itself. This negative autoregulation is restricted to the mesodermal and probably skeletogenic territories during the blastula stage, as shown by in situ hybridization analysis of MASO injected embryos. The MASO perturbation analysis also revealed blimp1/krox inputs into other genes of the endomesoderm regulatory network.

Alternative Splicing↗

PubMed2006

Regulation of spblimp1/krox1a, an alternatively transcribed isoform expressed in midgut and hindgut of the sea urchin gastrula.

The sea urchin regulatory gene Spblimp1/krox produces alternatively transcribed and spliced isoforms, 1a and 1b, which have different temporal and spatial patterns of expression. Here we describe a cis-regulatory module that controls the expression of the 1a splice form in the midgut and hindgut at the beginning of gastrulation. Conserved sequence patches revealed by a comparison of the blimp1/krox locus in Strongylocentrotus purpuratus and Lytechinus variegatus genomes were tested by gene transfer, in association with GFP or CAT reporter genes. An expression construct containing a conserved sequence patch immediately 5' of exon 1a included the transcription initiation site for blimp1/krox1a. This construct displays specific mid and hindgut expression, indicating that these are the locations of endogenous blimp1/krox1a transcription during the gastrula stage. Its sequence contains binding sites for Brn1/2/4, Otx, and Blimp1/Krox itself, as predicted in a prior regulatory network analysis.

Alternative Splicing↗

PubMed2006

cis-Regulatory control of cyclophilin, a member of the ETS-DRI skeletogenic gene battery in the sea urchin embryo.

The Strongylocentrotus purpuratus cyclophilin1 gene (Sp-cyp1) is expressed exclusively in skeletogenic mesenchyme cells of the embryo, beginning in the micromere lineage of the early blastula stage and continuing after gastrulation during the syncytial deposition of the skeleton. This gene encodes a protein which is a member of the peptidyl-prolyl cis-trans isomerase (PPIase) family. Sp-cyp1 is among the differentiation genes activated in the skeletogenic territory as a terminal function of the endomesodermal gene regulatory network. Network perturbation analysis had predicted the skeletogenic regulators Ets1 and Deadringer (Dri) to be its driver inputs. Here, we show that a 218-bp cis-regulatory DNA fragment recapitulates skeletogenic Sp-cyp1 expression; that elimination of either Ets1 or Dri inputs severely depresses the activity of expression constructs containing this DNA fragment; and that Ets1 and Dri target sites within the 218 bp fragment are required for normal expression. This indicates that the predicted inputs are direct. Other studies indicate that the same inputs are evidently necessary for expression of several other skeletogenic differentiation genes, and these genes probably constitute a skeletogenic gene battery, defined by its Ets plus Dri regulatory inputs.

Amino Acid Sequence↗

PubMed2006

Gene regulatory networks and the evolution of animal body plans.

Development of the animal body plan is controlled by large gene regulatory networks (GRNs), and hence evolution of body plans must depend upon change in the architecture of developmental GRNs. However, these networks are composed of diverse components that evolve at different rates and in different ways. Because of the hierarchical organization of developmental GRNs, some kinds of change affect terminal properties of the body plan such as occur in speciation, whereas others affect major aspects of body plan morphology. A notable feature of the paleontological record of animal evolution is the establishment by the Early "Cambrian of virtually all phylum-level body plans. We identify a class of GRN component, the kernels" of the network, which, because of their developmental role and their particular internal structure, are most impervious to change. Conservation of phyletic body plans may have been due to the retention since pre-Cambrian time of GRN kernels, which underlie development of major body parts.

Adaptation, Biological↗

PubMed2006

Unusual gene order and organization of the sea urchin hox cluster.

While the highly consistent gene order and axial colinear patterns of expression seem to be a feature of vertebrate hox gene clusters, this pattern may be less well conserved across the rest of the bilaterians. We report the first deuterostome instance of an intact hox cluster with a unique gene order where the paralog groups are not expressed in a sequential manner. The finished sequence from BAC clones from the genome of the sea urchin, Strongylocentrotus purpuratus, reveals a gene order wherein the anterior genes (Hox1, Hox2 and Hox3) lie nearest the posterior genes in the cluster such that the most 3' gene is Hox5. (The gene order is 5'-Hox1, 2, 3, 11/13c, 11/13b, 11/13a, 9/10, 8, 7, 6, 5-3'.) The finished sequence result is corroborated by restriction mapping evidence and BAC-end scaffold analyses. Comparisons with a putative ancestral deuterostome Hox gene cluster suggest that the rearrangements leading to the sea urchin gene order were many and complex.

Animals↗

PubMed2006

Hindgut specification and cell-adhesion functions of Sphox11/13b in the endoderm of the sea urchin embryo.

Sphox11/13b is one of the two hox genes of Strongylocentrotus purpuratus expressed in the embryo. Its dynamic pattern of expression begins during gastrulation, when the transcripts are transiently located in a ring of cells at the edge of the blastopore. After gastrulation, expression is restricted to the anus-hindgut region at the boundary between the ectoderm and the endoderm. The phenotype that results when translation of Sphox11/13b mRNA is knocked down by treatment with morpholino antisense oligonucleotides (MASO) suggests that this gene may be indirectly involved in cell adhesion functions as well as in the proper differentiation of the midgut-hindgut and midgut-foregut sphincters. The MASO experiments also reveal that Sphox11/13b negatively regulates several downstream endomesoderm genes. For some of these genes, Sphox11/13b function is required to restrict expression to the midgut by preventing ectopic expression in the hindgut. The evolutionary conservation of these functions indicates the general roles of posterior Hox genes in regulating cell-adhesion, as well as in spatial control of gene regulatory network subcircuits in the regionalizing gut.

Animals↗

PubMed2005

cis-Regulatory inputs of the wnt8 gene in the sea urchin endomesoderm network.

Expression of the wnt8 gene is the key transcriptional motivator of an intercellular signaling loop which drives endomesoderm specification forward early in sea urchin embryogenesis. This gene was predicted by network perturbation analysis to be activated by inputs from the blimp1/krox gene, itself expressed zygotically in the endomesoderm during cleavage; and by a Tcf1/beta-catenin input. The implication is that zygotic expression of wnt8 is stimulated in neighboring cells by its own gene product, since reception of the Wnt8 ligand causes beta-catenin nuclearization. Here, the modular cis-regulatory system of the wnt8 gene of Strongylocentrotus purpuratus was characterized functionally, and shown to respond to blockade of both Blimp1/Krox and Tcf1/beta-catenin inputs just as does the endogenous gene. The genomic target sites for these factors were demonstrated by mutation in one of the cis-regulatory modules. The Tcf1/beta-catenin and Blimp1/Krox inputs are both necessary for normal endomesodermal expression mediated by this cis-regulatory module; thus, the genomic regulatory code underlying the predicted signaling loop thus resides in the wnt8 cis-regulatory sequence. In a second regulatory region, which initiates expression in micromere and macromere descendant cells early in cleavage, Tcf1 sites act to repress ectopic transcription in prospective ectoderm cells.

Animals↗

PubMed2005

An evolutionary constraint: strongly disfavored class of change in DNA sequence during divergence of cis-regulatory modules.

The DNA of functional cis-regulatory modules displays extensive sequence conservation in comparisons of genomes from modestly distant species. Patches of sequence that are several hundred base pairs in length within these modules are often seen to be 80-95% identical, although the flanking sequence cannot even be aligned. However, it is unlikely that base pairs located between the transcription factor target sites of cis-regulatory modules have sequence-dependent function, and the mechanism that constrains evolutionary change within cis-regulatory modules is incompletely understood. We chose five functionally characterized cis-regulatory modules from the Strongylocentrotus purpuratus (sea urchin) genome and obtained orthologous regulatory and flanking sequences from a bacterial artificial chromosome genome library of a congener, Strongylocentrotus franciscanus. As expected, single-nucleotide substitutions and small indels occur freely at many positions within the regulatory modules of these two species, as they do outside the regulatory modules. However, large indels (>20 bp) are statistically almost absent within the regulatory modules, although they are common in flanking intergenic or intronic sequence. The result helps to explain the patterns of evolutionary sequence divergence characteristic of cis-regulatory DNA.

Animals↗

PubMed2005

Computational representation of developmental genetic regulatory networks.

Developmental genetic regulatory networks (GRNs) have unique architectural characteristics. They are typically large-scale, multi-layered, and organized in a nested, modular hierarchy of regulatory network kernels, function-specific building blocks, and structural gene batteries. They are also inherently multicellular and involve changing topological relationships among a growing number of cells. Reconstruction of developmental GRNs requires unique computational tools that support the above representational requirements. In addition, we argue that DNA-centered network modeling, separate descriptions of network organization and network behavior, and support for network documentation and annotation are essential requirements for computational modeling of developmental GRNs. Based on these observations, we have developed a freely available, platform-independent, open source software package (BioTapestry) which supports both the process of model construction and also model visualization, analysis, documentation, and dissemination. We provide an overview of the main features of BioTapestry. The BioTapestry software and additional documents are available from http://www.biotapestry.org. We recommend BioTapestry as the substrate for further co-development for and by the developmental biology community.

Computer Simulation↗

PubMed2005

Brn1/2/4, the predicted midgut regulator of the endo16 gene of the sea urchin embryo.

A specific prediction of our detailed cis-regulatory analysis of the Strongylocentrotus purpuratus (Sp) endo16 gene was that the later expression of this gene would be driven by a midgut-specific transcriptional regulator. We have now identified this factor and determined some of its functions. The cDNA sequence reveals it to be a POU domain factor related closely to the mammalian factors Brain-1, -2, and -4. The factor was termed SpBrn1/2/4 (henceforth Brn1/2/4). Quantitative measurements of transcript prevalence show that the gene is first activated in the 20-h blastula, but there remain only about 100 molecules of brn1/2/4 mRNA per embryo (only a few per endoderm cell) until an abrupt 10-fold increase occurs as gastrulation begins. Measured in the same embryos, the late rise in prevalence of endo16 transcripts follows that of brn1/2/4 transcripts. As predicted by the endo16 model, brn1/2/4 expression is confined perfectly to the midgut, coincident with the domain of endo16 expression. The kinetics of accumulation of these transcripts indicates that the switch into the late phase of endo16 expression occurs when the brn1/2/4 transcript level nears its plateau (2000 molecules mRNA per embryo), after which each endo16 gene produces about 1 mRNA every 2 min (about 380 molecules mRNA per min in the whole embryo). Arrest of Brn1/2/4 translation by MASO treatment blocks the late phase of endo16 expression and specifically abolishes expression of cis-regulatory Module B of endo16, while not affecting Module A, also as predicted. The brn1/2/4 gene lies downstream of the regulatory genes executing post-gastrular specification of the midgut, as shown by further gene expression perturbation experiments which provide an initial glimpse of the underlying network architecture.

Amino Acid Sequence↗

PubMed2005

Paircomp, FamilyRelationsII and Cartwheel: tools for interspecific sequence comparison.

BACKGROUND: Comparative sequence analysis is an effective and increasingly common way to identify cis-regulatory regions in animal genomes. RESULTS: We describe three tools for comparative analysis of pairs of BAC-sized genomic regions. Paircomp is a tool that does windowed (ungapped) comparisons of two sequences and reports all matches above a set threshold. FamilyRelationsII is a graphical viewer for comparisons that enables interactive exploration of several different kinds of comparisons. Cartwheel is a Web site and compute-cluster management system used to execute and store comparisons for display by FamilyRelationsII. These tools are specialized for the discovery of cis-regulatory regions in animal genomes. All tools and their source code are freely available at http://family.caltech.edu/. CONCLUSION: These tools have been shown to effectively identify regulatory regions in echinoderms, mammals, and nematodes.

Algorithms↗

PubMed2005

Gene regulatory networks for development.

The genomic program for development operates primarily by the regulated expression of genes encoding transcription factors and components of cell signaling pathways. This program is executed by cis-regulatory DNAs (e.g., enhancers and silencers) that control gene expression. The regulatory inputs and functional outputs of developmental control genes constitute network-like architectures. In this PNAS Special Feature are assembled papers on developmental gene regulatory networks governing the formation of various tissues and organs in nematodes, flies, sea urchins, frogs, and mammals. Here, we survey salient points of these networks, by using as reference those governing specification of the endomesoderm in sea urchin embryos and dorsal-ventral patterning in the Drosophila embryo.

Animals↗

PubMed2005

Logic functions of the genomic cis-regulatory code.

cis-Regulatory modules that control developmental gene expression process the regulatory inputs provided by the transcription factors for which they contain specific target sites. A prominent class of cis-regulatory processing functions can be modeled as logic operations. Many of these are combinatorial because they are mediated by multiple sites, although others are unitary. In this work, we illustrate the repertoire of cis-regulatory logic operations, as an approach toward a functional interpretation of the genomic regulatory code.

Animals↗

PubMed2005

Expression of AmHNF6, a sea star orthologue of a transcription factor with multiple distinct roles in sea urchin development.

The sea urchin transcription factor SpHNF6 is an early activator of differentiation genes in skeletogenic lineages and regulatory genes in the oral ectoderm. We report here the cloning and the expression of an orthologue of this gene, AmHNF6, from the sea star Asterina miniata. The vertebrate and the echinoderm hnf6 and onecut genes belong to the novel ONECUT homeo domain class of transcription factors. In blastula stage sea star embryos, AmHNF6 is expressed everywhere except around the vegetal pole. As is observed in sea urchin, by the end of gastrulation, the expression of AmHNF6 is distinctly localized to the ciliary bands. This terminal phase of expression has remained unchanged since the divergence of these two taxa half a billion years ago.

Amino Acid Sequence↗