The N- or C-terminal domains of DSH-2 can activate the C. elegans Wnt/beta-catenin asymmetry pathway.

Dishevelleds are modular proteins that lie at the crossroads of divergent Wnt signaling pathways. The DIX domain of dishevelleds modulates a beta-catenin destruction complex, and thereby mediates cell fate decisions through differential activation of Tcf transcription factors. The DEP domain of dishevelleds mediates planar polarity of cells within a sheet through regulation of actin modulators. In Caenorhabditis elegans asymmetric cell fate decisions are regulated by asymmetric localization of signaling components in a pathway termed the Wnt/beta-catenin asymmetry pathway. Which domain(s) of Disheveled regulate this pathway is unknown. We show that C. elegans embryos from dsh-2(or302) mutant mothers fail to successfully undergo morphogenesis, but transgenes containing either the DIX or the DEP domain of DSH-2 are sufficient to rescue the mutant phenotype. Embryos lacking zygotic function of SYS-1/beta-catenin, WRM-1/beta-catenin, or POP-1/Tcf show defects similar to dsh-2 mutants, including a loss of asymmetry in some cell fate decisions. Removal of two dishevelleds (dsh-2 and mig-5) leads to a global loss of POP-1 asymmetry, which can be rescued by addition of transgenes containing either the DIX or DEP domain of DSH-2. These results indicate that either the DIX or DEP domain of DSH-2 is capable of activating the Wnt/beta-catenin asymmetry pathway and regulating anterior-posterior fate decisions required for proper morphogenesis.

Creating precise GFP fusions in plasmids using yeast homologous recombination.

Using a combination of primer amplification, homologous recombination, and yeast genetics, we established a method for creating precise promoter and protein fusions in genes originating from organisms other than yeast. One major advantage of this new method is its versatility. Fusions can be produced within a target gene without constraints regarding the site of insertion. Thus, fusions can be generated within a target sequence exactly at the site desired, and all sequences upstream and downstream of the insertion site were preserved. To illustrate the general applicability of this technique, we fused the gene encoding GFP to a Caenorhabditis elegans homologue of the dishevelled gene, dsh-2. This approach is not restricted to GFP fusions but can be utilized to create fusions between almost any two sequences regardless of the source. Therefore, this method provides a flexible alternative to other PCR-mediated techniques.

C. elegans HAM-1 positions the cleavage plane and regulates apoptosis in asymmetric neuroblast divisions.

Asymmetric cell division occurs when a mother cell divides to generate two distinct daughter cells, a process that promotes the generation of cellular diversity in metazoans. During Caenorhabditis elegans development, the asymmetric divisions of neural progenitors generate neurons, neural support cells and apoptotic cells. C. elegans HAM-1 is an asymmetrically distributed cortical protein that regulates several of these asymmetric neuroblast divisions. Here, we show that HAM-1 is a novel protein and define residues important for HAM-1 function and distribution to the cell cortex. Our phenotypic analysis of ham-1 mutant embryos suggests that HAM-1 controls only neuroblast divisions that produce apoptotic cells. Moreover, ham-1 mutant embryos contain many unusually large cell-death corpses. An investigation of this corpse phenotype revealed that it results from a reversal of neuroblast polarity. A misplacement of the neuroblast cleavage plane generates daughter cells of abnormal size, with the apoptotic daughters larger than normal. Thus, HAM-1 regulates the position of the cleavage plane, apoptosis and mitotic potential in C. elegans asymmetric cell divisions.

MOM-5 frizzled regulates the distribution of DSH-2 to control C. elegans asymmetric neuroblast divisions.

Asymmetric cell divisions produce all 302 neurons of the C. elegans hermaphrodite. Here, we describe a role for a C. elegans Dishevelled homolog, DSH-2, in an asymmetric neuroblast division. In dsh-2 mutants, neurons normally descended from the anterior neuroblast daughter of the ABpl/rpppa blast cell were frequently duplicated, while non-neuronal cells produced by the posterior daughter cell were often missing. These observations indicate that in the absence of dsh-2 function, the posterior daughter cell was transformed into a second anterior-like cell. Loss of mom-5, a C. elegans frizzled homolog, produced a similar phenotype. We also show that the DSH-2 protein localized to the cell cortex in most cells of the embryo. In the absence of MOM-5/Fz, DSH-2 was localized to the cytoplasm, suggesting that MOM-5 regulates asymmetric cell division by controlling the localization of DSH-2. Although all neurons in C. elegans are produced by an invariant pattern of cell divisions, our results indicate that cell signaling may contribute to asymmetric neuroblast division during embryogenesis.