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Published June 1, 2002 | public
Journal Article

A Provisional Regulatory Gene Network for Specification of Endomesoderm in the Sea Urchin Embryo


We present the current form of a provisional DNA sequence-based regulatory gene network that explains in outline how endomesodermal specification in the sea urchin embryo is controlled. The model of the network is in a continuous process of revision and growth as new genes are added and new experimental results become available; see http://www.its.caltech.edu/~mirsky/endomeso.htm (End-mes Gene Network Update) for the latest version. The network contains over 40 genes at present, many newly uncovered in the course of this work, and most encoding DNA-binding transcriptional regulatory factors. The architecture of the network was approached initially by construction of a logic model that integrated the extensive experimental evidence now available on endomesoderm specification. The internal linkages between genes in the network have been determined functionally, by measurement of the effects of regulatory perturbations on the expression of all relevant genes in the network. Five kinds of perturbation have been applied: (1) use of morpholino antisense oligonucleotides targeted to many of the key regulatory genes in the network; (2) transformation of other regulatory factors into dominant repressors by construction of Engrailed repressor domain fusions; (3) ectopic expression of given regulatory factors, from genetic expression constructs and from injected mRNAs; (4) blockade of the β-catenin/Tcf pathway by introduction of mRNA encoding the intracellular domain of cadherin; and (5) blockade of the Notch signaling pathway by introduction of mRNA encoding the extracellular domain of the Notch receptor. The network model predicts the cis-regulatory inputs that link each gene into the network. Therefore, its architecture is testable by cis-regulatory analysis. Strongylocentrotus purpuratus and Lytechinus variegatus genomic BAC recombinants that include a large number of the genes in the network have been sequenced and annotated. Tests of the cis-regulatory predictions of the model are greatly facilitated by interspecific computational sequence comparison, which affords a rapid identification of likely cis-regulatory elements in advance of experimental analysis. The network specifies genomically encoded regulatory processes between early cleavage and gastrula stages. These control the specification of the micromere lineage and of the initial veg_2 endomesodermal domain; the blastula-stage separation of the central veg_2 mesodermal domain (i.e., the secondary mesenchyme progenitor field) from the peripheral veg_2 endodermal domain; the stabilization of specification state within these domains; and activation of some downstream differentiation genes. Each of the temporal–spatial phases of specification is represented in a subelement of the network model, that treats regulatory events within the relevant embryonic nuclei at particular stages.

Additional Information

© 2002 Elsevier Science (USA). Received for publication January 3, 2002. Revised February 20, 2002. Accepted February 20, 2002. The work summarized in this paper is the product of many minds, many kinds of technology, many different experiments, and several different laboratory groups and institutions, as indicated in the list of authors. Much of the computational work and model assembly was done with Hamid Bolouri and his group at the University of Hertfordshire, the remainder with C. Titus Brown of Caltech (see Bolouri and Davidson, 2002; Brown et al., 2002). Most of the experimental molecular biology directly underlying the model was carried out by the first author's group at Caltech (individual contributions that are otherwise yet unpublished are indicated in the QPCR data Web site, and in papers elsewhere in this issue, viz Ransick et al., 2002; Rast et al., 2002; Oliveri et al., 2002; Yuh et al., 2002). Much of the experimental embryology on which the model rests comes from the laboratory of David R. McClay of Duke University. Most of the BAC sequences were obtained at Leroy Hood's sequencing center at the Institute for Systems Biology, Seattle, Washington. We thank Scott Bloom at ISB for his technical assistance. We are also extremely grateful to Elbert Branscombe of JGI who jump-started this whole project by offering to arrange for JGI to sequence S. purpuratus BACs containing endomesodermal genes, and in the event provided about a third of the BAC sequences listed in Table 2. The work described in this paper required an intense expenditure of technical effort, and would never have been possible without the contributions of Jane Wyllie, Ping Dong, Niñon Le, Miki Jun, Jina Jun, and Patrick Leahy, all superbly able, experienced technical assistants. We are grateful to Maria Rosa Ponce and José Luis Micol, who participated in the early phases of the genomics underlying the network analysis when we were first feeling our way forward. The project also depended heavily on the Arraying Facility at the Beckman Institute, directed by R. Andrew Cameron and staffed by Julie Hahn, Arnufo Lorico, and Ted Biondi; and supported by a grant from NCRR (RR-15044). The project has intensively utilized the computational and technological facilities in the Transcription Factor Research Center of the Beckman Institute, headed by Chiou-Hwa Yuh. The embryological and molecular biology aspects of the network analysis were supported by grants from NIH (HD-37105 and RR-06591); the computational aspects by a grant from NIGMS (GM-61005); the comparative aspects by a grant from NASA's Fundamental Space Biology program (NAG2-1368); and other support was provided by the Stowers Institute for Medical Research, the Beckman Institute, and the Lucille P. Markey Trust. The first author would particularly like to acknowledge the continuing support and encouragement of Dr. Richard Tasca of NICHHD. Many other scientists have contributed in very important ways to this project. In addition to those whose published contributions are referred to in text, we are very grateful to Koji Akasaka of Hiroshima University, William Klein of MD Anderson Hospital; and Athula Wikramanayake of the University of Hawaii for making available for reference their unpublished data. Prof. Ellen Rothenberg of Caltech provided a perspicacious and valuable critical review of the manuscript, for which it benefited greatly. Finally, one should perhaps acknowledge the unusual properties of sea urchin embryos, which seem preordained for developmental gene network analysis, and many millions, if not billions, of which contributed directly to each of the diagrams in this paper.

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