The roles of EGF and Wnt signaling during patterning of the C. elegans Bgamma/delta Equivalence Group

Abstract

Background: During development, different signaling pathways interact to specify cell fate by regulating transcription factors necessary for fate specification and morphogenesis. In Caenorhabditis elegans, the EGF-Ras and Wnt signaling pathways have been shown to interact to specify cell fate in three equivalence groups: the vulval precursor cells (VPCs), the hook competence group (HCG) and P11/12. In the VPCs, HCG and P11/12 pair, EGF and Wnt signaling positively regulate different Hox genes, each of which also functions during fate specification. In the male, EGF-Ras signaling is required to specify the Bgamma fate within the Bgamma/delta equivalence pair, while Notch signaling is required for Bdelta fate specification. In addition, TGF-beta signaling by dbl-1/dpp controls ceh-13/labial/Hox1 expression in Bgamma.

Results: We show that EGF-Ras signaling is required for Bgamma expression of ceh-13/labial/Hox1. The transcription factors lin-1/ETS and lin-31/Forkhead, functioning downstream of the EGF pathway, as well as sur-2/MED23 (a component of the Mediator complex) also control ceh-13 expression in Bgamma. In addition, our results indicate that lin-44/Wnt, mom-2/Wnt and lin-17/Fz are necessary to maintain the division of Bgamma along a longitudinal axis. We also show that dbl-1/dpp acts either in parallel or downstream of EGF pathway to regulate ceh-13/Hox1 expression in Bgamma. Lastly, we find that a dbl-1/dpp null mutation did not cause any vulval or P12 defects and did not enhance vulval and P12 defects of reduction-of-function mutations of components of the EGF pathway.

Conclusions: ceh-13/labial/Hox1 expression in Bgamma is regulated by the EGF pathway and downstream factors lin-1/ETS lin-31/Forkhead and sur-2/MED23. Wnt signaling is required for proper Bgamma division, perhaps to orient the Bgamma mitotic spindle. Our results suggest that dbl-1/dpp is not required for VPC and P12 specification, highlighting another difference among these EGF-dependent equivalence groups.

Figure 1

The Bγ/δ equivalence group during development. (A) Arrangement of the B.a progeny during the mid-L3 stage, adapted from Chamberlin and Sternberg (1993). Left lateral view and cross section. (B) Cell division patterns of Bγ and Bδ, adapted from Sulston et al. (1980). Circled crosses indicate pairs of cell in which the left or right cell dies.

Figure 2

EGF signaling is necessary and sufficient for ceh-13::GFP expression in the Bγ/δ pair (A-B) Mid-L3. Wild-type ceh-13::GFP expression was only observed in Bγ. (C-D) Mid-L3 let-23(rf) males. ceh-13::GFP was not expressed in Bγ. Similar observations were made in sem-5(rf) and let-60(rf) mutants. (E-F) Mid-L3. Increased EGF signaling in heat-shocked HS::EGF males caused ectopic ceh-13::GFP expression in Bδ, in addition to wild-type Bγ expression. Similar observations were made in lin-15(lf) and let-60(gf) mutants. Left lateral views. Scale bar in B, 20 μm for A-F.

Figure 3

Wnt signaling is required to maintain the division axis of Bγ. (A-B) Mid-L3. Bγ divides along the longitudinal axis in wild-type males. (C-D) Mid-L3 lin-17(n698rf) male. Bγ divides in a transverse manner. Only Bγ.a can be seen in this plane and the posterior daughter of Bγ is out of focus in this picture. Left lateral views. Scale bar in B, 20 μm for A-D.

Figure 4

Patterning of equivalence groups in C. elegans (A) Model for EGF, Wnt and TGF-β signaling during Bγ/δ specification. The EGF and TGF-β pathways specify Bγ fate by regulating the transcription of target genes such as ceh-13/hox1. Wnt controls the axis of division of Bγ, possibly by orienting the mitotic spindle. POPTOP expression suggests Wnt may play a role in Bγ fate specification. (B) A comparison of the HCG, VPCs, P11/12 and Bγ/δ groups. EGF and Wnt signaling have different requirements relative to each other during the patterning of each equivalence group. This difference may account for the specificity of fate by both pathways induced in each group. In addition, Wnt signaling is required for Bγ division along the correct axis. Such a role for Wnt signaling has not been observed in the other equivalence groups. Another factor that may contribute to fate specification in each equivalence group is the use of a third pathway during patterning. TGF-β signaling by dbl-1/dpp is required to specify Bγ fate and does not appear to act during VPC and P12 specification, equivalence groups in which EGF signaling is the major inductive signal. Finally, downstream of the EGF and Wnt pathways, a different Hox gene is expressed in each equivalence group and required to specify fate within that group. One exception is ceh-13/Hox1 for which a functional role in Bγ fate specification has not been identified.