The AC/VU decision is a simple example of lateral specification that occurs during the development of the somatic gonad. Two initially equivalent cells, Z1.ppp and Z4.aaa, communicate with each other via LIN-12, a receptor of the LIN-12/Notch family, and LAG-2, a ligand of the DSL family. As a result, one of the two cells becomes the anchor cell (AC) while the other becomes a ventral uterine precursor cell (VU). Although one of these cells always becomes the AC, which cell does so is random. Loss of activity of either
lag-2 or
lin-12 results in both cells taking on AC fate. Conversely, too much activity of
lag-2 or
lin-12 results in both cells taking on VU fate. We are interested in examining other genes that may play a role in this decision. Our investigation has focused on
hlh-2, which encodes a bHLH transcription factor that is homologous to Drosophila Daughterless and the mammalian E proteins. We have shown that a difference in HLH-2 expression is the first known indication that Z1.ppp and Z4.aaa are becoming different. RNAi studies indicate that
hlh-2 plays two different roles with respect to the AC/VU decision. First, we find that
hlh-2 activity is required in early L1 for the AC fate, as animals lacking
hlh-2 activity at this time do not form an AC. However,
hlh-2 is also required in early L2 in order to restrict the number of ACs formed from 2 to 1, as animals lacking
hlh-2 activity at this time exhibit a 2AC phenotype. Several observations prompted us to ask whether the 2AC phenotype could indicate a role for HLH-2 in directly activating
lag-2 transcription. 1) HLH-2 is likely a transcriptional activator and is expressed in the same cell as
lag-2 during the AC/VU decision (the presumptive AC). 2) The 2AC phenotype is similar to the phenotype observed when
lag-2 activity is removed. 3) The
lag-2 promoter contains several potential binding sites for HLH-2. 4) In Drosophila, Daughterless and its dimerization partners are known to directly activate transcription of Delta, a homologue of
lag-2. RNAi experiments and mutation of potential HLH-2 binding sites in the
lag-2 promoter do suggest that HLH-2 is necessary for
lag-2 transcription in Z1.ppp and Z4.aaa during the AC/VU decision. Interestingly,
hlh-2 also appears necessary for
lag-2 expression in the mature AC. This is indicated by loss of
lag-2::lacZ expression in the mature AC following
hlh-2(RNAi), as well as a phenotype indicative of loss of
lag-2 activity in the AC, failure of pi-cell induction. In summary,
hlh-2 activity is necessary for two apparently separate roles in specification and function of the AC. The first is a requirement for AC fate, which our available evidence thus far suggests is a
lag-2-independent role. The second is to activate
lag-2 transcription both during the AC/VU decision and in the mature AC.