Use of an Activated Beta-Catenin to Identify Wnt Pathway Target Genes inCaenorhabditis elegans, Including a Subset of Collagen Genes Expressed in Late Larval Development

Abstract

The Wnt signaling pathway plays a fundamental role during metazoan development, where it regulates diverse processes, including cell fate specification, cell migration, and stem cell renewal. Activation of the beta-catenin−dependent/canonical Wnt pathway up-regulates expression of Wnt target genes to mediate a cellular response. In the nematode Caenorhabditis elegans, a canonical Wnt signaling pathway regulates several processes during larval development; however, few target genes of this pathway have been identified. To address this deficit, we used a novel approach of conditionally activated Wnt signaling during a defined stage of larval life by overexpressing an activated beta-catenin protein, then used microarray analysis to identify genes showing altered expression compared with control animals. We identified 166 differentially expressed genes, of which 104 were up-regulated. A subset of the up-regulated genes was shown to have altered expression in mutants with decreased or increased Wnt signaling; we consider these genes to be bona fide C. elegans Wnt pathway targets. Among these was a group of six genes, including the cuticular collagen genes, bli-1col-38, col-49, and col-71. These genes show a peak of expression in the mid L4 stage during normal development, suggesting a role in adult cuticle formation. Consistent with this finding, reduction of function for several of the genes causes phenotypes suggestive of defects in cuticle function or integrity. Therefore, this work has identified a large number of putative Wnt pathway target genes during larval life, including a small subset of Wnt-regulated collagen genes that may function in synthesis of the adult cuticle.

Multiple cis elements and GATA factors regulate a cuticle collagen gene in Caenorhabditis elegans

“…Temporally, however, COL-41 appears to be a larval-specific cuticle collagen, with expression absent from embryos and adults. This result is consistent with RNAseq analyses that demonstrate a peak of col-41 expression in L2 (Gerstein et al, 2010; Jackson et al, 2014). Expression of the fluorescent marker may be visible later than the peak in mRNA expression due to the time lag for protein synthesis and maturation, and/or perdurance of the mCherry protein.…”

“…Collagen is secreted from the apical surface of the epidermal cells to the exterior of the body. Cuticle collagen genes show highly specific temporal expression patterns that may be stage-specific or cycle in phase with the cuticle molts (Jackson et al, 2014; Johnstone and Barry, 1996; Park and Kramer, 1994). Additionally, levels and timing of cuticle collagen gene expression respond to cell signaling pathways, such as the TGFβ and Wnt pathways (Jackson et al, 2014; Liang et al, 2007; Roberts et al, 2010).…”

Regulation of C. elegans L4 cuticle collagen genes by the heterochronic protein LIN‐29

“…First, we showed that the L4 col genes col-38, col-49, and col-71 are regulated by the Wnt pathway transcription factor BAR-1 (beta-catenin) (Gorrepati et al, 2015;Jackson et al, 2014;Van Der Bent et al, 2015), which binds to its target genes via interaction with the TCF transcription factor POP-1 (Jackson & Eisenmann, 2012). First, we showed that the L4 col genes col-38, col-49, and col-71 are regulated by the Wnt pathway transcription factor BAR-1 (beta-catenin) (Gorrepati et al, 2015;Jackson et al, 2014;Van Der Bent et al, 2015), which binds to its target genes via interaction with the TCF transcription factor POP-1 (Jackson & Eisenmann, 2012).…”

“…*p 0.01 and **p < 0.0001 (Fisher's exact test) compared to WT (Jackson et al, 2014). Animals were scored as either positive or negative for YFP expression, since animals at the L4 stage all showed similar YFP intensity.…”

“…Previous work has identified four transcription factors regulating col gene expression. First, we showed that the L4 col genes col-38, col-49, and col-71 are regulated by the Wnt pathway transcription factor BAR-1 (beta-catenin) (Gorrepati et al, 2015;Jackson et al, 2014;Van Der Bent et al, 2015), which binds to its target genes via interaction with the TCF transcription factor POP-1 (Jackson & Eisenmann, 2012).…”