Biomedical subjects

R Andrew Cameron

Publications and source records attributed to R Andrew Cameron.

22 records · Page 2Linked to original sources

PubMed2002

New computational approaches for analysis of cis-regulatory networks.

The investigation and modeling of gene regulatory networks requires computational tools specific to the task. We present several locally developed software tools that have been used in support of our ongoing research into the embryogenesis of the sea urchin. These tools are especially well suited to iterative refinement of models through experimental and computational investigation. They include: BioArray, a macroarray spot processing program; SUGAR, a system to display and correlate large-BAC sequence analyses; SeqComp and FamilyRelations, programs for comparative sequence analysis; and NetBuilder, an environment for creating and analyzing models of gene networks. We also present an overview of the process used to build our model of the Strongylocentrotus purpuratus endomesoderm gene network. Several of the tools discussed in this paper are still in active development and some are available as open source.

Chromosomes, Artificial, Bacterial↗

PubMed2002

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 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.

Animals↗

PubMed2002

brachyury Target genes in the early sea urchin embryo isolated by differential macroarray screening.

Brachyury is a transcription factor that functions in gastrulation and endoderm development throughout the Bilateria. Here, we identify genes that are expressed downstream of brachyury during gastrulation of the sea urchin embryo. Screens with two different complex probes generated by subtractive hybridization were carried out on high-density arrays of embryonic cDNA libraries. An mRNA sequence population from embryos expressing brachyury at its peak stage of expression was subtracted with message sequence from embryos in which Brachyury function had been "knocked-out" by injection of a morpholine-substituted antisense oligonucleotide to generate a differential probe for brachyury target genes. Another probe was made by using an mRNA population from embryos that mis-express brachyury at a stage just prior to the normal onset of expression, subtracted with message sequence taken from normal embryos at this stage. Screens carried out with these probes target overlapping but distinct sets of downstream genes. After partial sequence characterization, promising genes were independently analyzed by quantitative real-time PCR and by in situ hybridization. Two major classes of genes emerge in this study: genes expressed in the subset of the secondary mesenchyme cells (SMC) that will become pigment cells, and genes that are expressed in portions of the endoderm coincident with brachyury expression. The latter genes are candidates for direct transcriptional targets of Brachyury. Some of the endodermal genes that respond to Brachyury are cytoskeletal modulators that may play a role in gut morphogenesis. This finding is consistent with the block in gastrulation induced by interfering with Brachyury function in sea urchins, and with known or suggested Brachyury function in other species. Other endodermal target genes are expressed in the archenteron and might be terminal differentiation enzymes of the gut. Brachyury expression patterns for Strongylocentrotus purpuratus reported in this paper are entirely consistent with data from other echinoderm species. Brachyury expression in the vegetal plate is confined to the presumptive endodermal cells. Therefore, the SMC genes are likely to be indirect targets of Brachyury-induced signaling from the surrounding endoderm to the central mesoderm, or the effects on these genes may be indirect consequences of gross disruption of the vegetal plate. These results and other data suggest that the brachyury gene transduces information about the state of endodermal specification to genes that modulate morphogenesis and genes that perform terminal functions in the gut.

Animals↗

PubMed2002

A genomic regulatory network for development.

Development of the body plan is controlled by large networks of regulatory genes. A gene regulatory network that controls the specification of endoderm and mesoderm in the sea urchin embryo is summarized here. The network was derived from large-scale perturbation analyses, in combination with computational methodologies, genomic data, cis-regulatory analysis, and molecular embryology. The network contains over 40 genes at present, and each node can be directly verified at the DNA sequence level by cis-regulatory analysis. Its architecture reveals specific and general aspects of development, such as how given cells generate their ordained fates in the embryo and why the process moves inexorably forward in developmental time.

Animals↗