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

Abstract

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.

Gene regulatory network subcircuit controlling a dynamic spatial pattern of signaling in the sea urchin embryo

“…As reviewed previously, this is a feedback subcircuit composed of the wnt8 and blimp1 genes, expression of which progresses concentrically outward from the SM to the NSM and then to the surrounding endoderm. At each stage, after some hours of transcription, the blimp1 gene represses itself, causing loss of wnt8 expression as well (16,18), and also loss of expression of downstream regulatory genes (20) in what now becomes the silenced center of a torus of gene expression. Meanwhile, Wnt8 diffusion causes expansion of the subcircuit expression torus to the adjacent cells of the next domain.…”

“…We showed that the regulatory circuitry underlying this phenomenon is a double-feedback loop linking the wnt8 and the blimp1 regulatory genes in a causal embrace (18). cis-Regulatory studies show that the wnt8 gene requires inputs from both ␤-catenin/Tcf (i.e., from the same signal transduction system that it activates) and from Blimp1 factor for expression, and conversely, the blimp1 gene requires inputs from the same Wnt8/Tcf signaling system as well as from another then ubiquitous factor, Otx (16,18). However, the blimp1 gene also contains autorepression sites which, after some hours when the Blimp1 factor attains a high concentration, shut down its own expression, and therefore that of wnt8 as well (18,19).…”

A missing link in the sea urchin embryo gene regulatory network: hesC and the double-negative specification of micromeres

“…Antibody incubation was carried out containing a 1,000-fold dilution of anti-DIG antibody (Fab fragments; Roche Diagnostics, Indianapolis, IN) for DIG-labeled probes or anti-DNP antibody-alkaline phosphatase (Mirus, Madison, WI) for DNP-labeled probes. Double-WMISH protocol (from Sagar Damle and E.H.D., unpublished data) was based on the above protocol for WMISH and the double-WMISH protocol described earlier (34). Steps before the hybridization reaction were as described above for WMISH.…”

Global regulatory logic for specification of an embryonic cell lineage

“…By late fourth-cleavage stage the micromeres begin to transcribe the wnt8 gene, and both the network perturbation analysis and a direct cis-regulatory study (15,37) demonstrate that the inputs required for its expression in the micromere lineage are Blimp1 and ␤-catenin/Tcf. To initiate wnt8 expression, the micromeres initially rely on the maternal ␤-catenin localization system, which is activated pre- cociously in these cells, as discussed above.…”