C. elegans embryos exhibit an invariant lineage comprised primarily of a stepwise binary diversification of anterior-posterior (A-P) blastomere identities. This binary cell fate specification requires input from both the Wnt and MAP kinase signaling pathways. The nuclear level of the TCF protein POP-1 is lowered in all posterior cells. We show here that the ␤-catenin SYS-1 also exhibits reiterated asymmetry throughout multiple A-P divisions and that this asymmetry is reciprocal to that of POP-1. Furthermore, we show that SYS-1 functions as a coactivator for POP-1, and that the SYS-1-to-POP-1 ratio appears critical for both the anterior and posterior cell fates. A high ratio drives posterior cell fates, whereas a low ratio drives anterior cell fates. We show that the SYS-1 and POP-1 asymmetries are regulated independently, each by a subset of genes in the Wnt/MAP kinase pathways. We propose that two genetic pathways, one increasing SYS-1 and the other decreasing POP-1 levels, robustly elevate the SYS-1-to-POP-1 ratio in the posterior cell, thereby driving A-P differential cell fates.KEY WORDS: C. elegans, TCF/POP-1, ␤-catenin/SYS-1, Cell fate specification Development 134, 2685Development 134, -2695Development 134, (2007 POP-1 (Kidd et al., 2005). Recently, SYS-1 has been implicated in endoderm precursor specification (Phillips et al., 2007). Whereas animals homozygous for a reduction-of-function mutation are sterile, sys-1(RNAi) resulted in a very low penetrance gutless phenotype.We show here that SYS-1 is a limiting coactivator for POP-1 in the activation of Wnt/MAPK-responsive genes in the E blastomere. SYS-1 exhibits a reiterated asymmetry that is reciprocal to the reiterated asymmetry of nuclear POP-1 through all A-P divisions examined. We show that the SYS-1-to-POP-1 ratio appears critical for both anterior and posterior cell fates at multiple divisions: a high ratio drives the posterior cell fate, whereas a low ratio drives the anterior cell fate. SYS-1 and POP-1 levels are regulated in opposite directions by two pathways known to regulate endoderm specification: SYS-1 levels are increased primarily by the MOM-2/MOM-5/APR-1 pathway, whereas nuclear POP-1 levels are decreased primarily by the MOM-4/LIT-1/WRM-1 pathway. Together, these two pathways efficiently increase the SYS-1-to-POP-1 ratio in the posterior cell, promoting asymmetric cell fates. MATERIALS AND METHODS StrainsN2 was used as the wild-type strain. Genetic markers:LGI, pop-1(zu189), dpy-5(e61), mom-5(or57), mom-4(ne19), sys-1(q544), fog-3(q520), teIs3 (P med-1 gfp::pop-1), hT1(I:V), szT1(I:X);LGII, rol-1(e91), mom-3(or78), mnC1; LGIII, unc-119(ed3), lit-1(t1512), lit-1(t1534), unc32(e189);LGIV, teIs46(P end-1 gfp::H2B); LGV, mom-2(or42), DnT1(IV;V), teIs18 (P sdz-23 gfp::H2B); LGX,. TX796 [teEx321(P med-1 gfp::sys-1)]. gfp::H2B)(V)]. TX691 [teIs46(P end-1 gfp::H2B)(IV)] (Shetty et al., 2005). TX932 [sys-1(q544)/fog-3(q470)(I); teIs3(P med-1 gfp::pop-1)(V)] (Miskowski et al., 2001). TX964 [teIs98(P pie-1 gfp::sys-1)]. JM139 [P pho-1 ...

Canonical Wnt signaling converts the TCF/LEF transcription factor from repressor to activator by increasing nuclear levels of its coactivator, beta-catenin. A striking exception had been reported for Wnt-induced endoderm formation during C. elegans embryogenesis. It has long been believed that transcriptional activation of Wnt target genes in the endoderm precursor occurred due to a lowering of nuclear levels of the worm TCF/LEF protein, POP-1, effectively alleviating POP-1 repressive activity. Contrary to this model, we demonstrate here that POP-1 directly activates Wnt target genes in the endoderm precursor. Wnt converts POP-1 from a repressor to an activator, and this conversion requires that POP-1 nuclear levels be lowered in the endoderm precursor. We propose that the balance between TCF/LEF and coactivator(s), achieved by elevating coactivator levels (the canonical pathway) and/or reducing TCF/LEF levels (worm endoderm), determines Wnt signal strength.

During Caenorhabditis elegans embryogenesis, a maternally supplied transcription factor, SKN-1, is required for the specification of the mesendodermal precursor, EMS, in the 4-cell stage embryo. When EMS divides, it gives rise to a mesoderm-restricted precursor, MS, and an endoderm-restricted precursor, E. To systematically identify genes that function as key regulators of MS and/or E-derived tissues, we identified, by microarray analyses, genes that are newly transcribed within a short developmental window (approximately 30 min) encompassing the generation and fate specification of the MS and E blastomeres. By comparing total cDNAs generated from individual, carefully staged embryos, we identified 275 genes up-regulated in 12-cell embryos compared to 4-cell embryos. Fifty of these 275 genes are down-regulated in 12-cell skn-1 mutant embryos and are designated skn-1-dependent zygotic (sdz) genes. The spatial and temporal expression patterns in C. elegans embryos of 10 randomly selected sdz genes were analyzed by a nuclear GFP reporter driven by the endogenous 5' regulatory sequence of each gene. GFP expression, although absent at the 4-cell stage, was detected at the 12- to 16-cell stage for all 10 genes and was restricted to EMS-derived lineages for 7 of the 10. Among the seven lineage-specific genes, three genes are expressed equally in both MS and E lineages, two are expressed exclusively or predominantly in the MS lineage, and two are expressed exclusively in the E lineage. Depletion of skn-1 by RNAi abolishes the expression of all seven reporter transgenes in vivo, confirming that these genes are indeed skn-1 dependent. These results demonstrate the successful combination of single-staged embryo cDNAs, genetic mutants, and whole transcriptome microarray analysis to identify stage- and lineage-specific transcripts in early C. elegans embryos.

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To study the physiological regulation and function of cell-cell gap junction communication in vivo, we developed a bioconjugate of caged dye, named dextran-CANPE-HCC, for imaging cell coupling in small model organisms. In vitro, the compound was photolyzed efficiently with robust fluorescence enhancement. Dextran-CANPE-HCC delivered into Caenorhabditis elegans oocytes was retained in cells throughout development. Using local uncaging, we photolyzed dextran-CANPE-HCC to release the small HCC dye and imaged the dynamics of intercellular dye transfer through gap junction channels, a technique we named Trojan-local activation of molecular fluorescent probes (LAMP). Early during embryonic development, the pattern of cell coupling undergoes dramatic remodeling and imaging revealed that the germ cell precursors, P2, P3 and P4, were isolated from the somatic cell communication compartment. As dextran-CANPE-HCC is chemically and metabolically stable, Labeled worms showed very bright signal upon photoactivation after hatching, which allowed us to examine cell coupling in living worms noninvasively.