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

Abstract

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 beta-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.

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

“…To obtain the tracts of sequence from the genomic regions surrounding the relevant cis-regulatory modules in S. franciscanus, primers that lie outside the highly conserved protein coding regions were required. For each gene, alignments between the S. purpuratus and L. variegatus BAC inserts had been previously performed in the course of the cis-regulatory analyses (20,27,28,30,31). To find suitable conserved regions for primer design, we also used BLASTN (32) and additional FAMILY RELATIONS (FR) analyses (20).…”

Molecular basis of lithium action: integration of lithium-responsive signaling and gene expression networks

“…Recently, a regulatory gene network that directs specific developmental events has been identified in developing sea urchin embryo. 114,115 This provides a heuristic model for constructing a lithium-responsive gene network as a means to identify signature genes that direct the therapeutic or nontherapeutic action of lithium. While chromosome immunoprecipitation could start with antibodies against several known lithiumresponsive transcription factors such as AP-1, CREB, NF-kB, LEF/TCF, these are general transcription Figure 3 Model for a gene network for lithium-responsive genes: defining the therapeutic effect of chronic lithium.…”

Regulatory gene networks and the properties of the developmental process

“…Furthermore, it is easy to inject sea urchin eggs with regulatory DNA expression constructs and with micro-or macromolecules that efficiently perturb expression of given genes or sets of genes. A GRN for specification of endomesoderm in the sea urchin embryo has been published (9)(10)(11). The GRN covers the period from the time of the initial divisions that divide the eggs up into the first cells (cleavage), through the several hundred-cell stage when the embryo is a hollow cellular sphere (blastula), to the point when invagination of the gut is about to begin (gastrulation).…”

“…The major method of perturbation used here is blockade of mRNA translation by injection of morpholino-substituted antisense oligonucleotides or alternatively, the normal mRNA is made to be translated ectopically. In addition, some transcription factors have been converted into obligate repressors by fusing their DNAbinding domains with a sequence encoding the Engrailed repressor domain (9)(10)(11)19). Other more general perturbations that result in specific, complete arrest of all endomesoderm specification (except skeletogenic), or arrest of all mesoderm but not endoderm specification, were also applied (refs.…”