Figure 4. Post-transcriptional regulation of HLH-2 during the AC/VU decision. (A) Schematic depiction of the
hlh-2::lacZ transcriptional reporter and the HA-HLH-2 translational reporter. Coding regions are shown as boxes, with noncoding regions as lines. Red indicates that the region will be translated, whereas gray coding regions should not be translated, as they follow a stop codon. The reporters include the region from the end of the upstream-most gene (left white box) to the beginning of the downstream most gene (right white box). (B,C) Confocal photomicrographs, small yellow arrows indicate Z1.ppp and Z4.aaa. Larger white arrows indicate HLH-2 expression in the presumptive AC. Distal tip cells are marked with an asterisk. An
hlh-2::lacZ transcriptional reporter consistently shows expression in both Z1.ppp and Z4.aaa and their sisters (LacZ-positive cells on either side of Z1.ppp and Z4.aaa) in two independent transgenic lines carrying complex arrays (full genotypes:
smg-1(
r861)
unc-54(
r293);
pha-1 (
e2123); arEx445 and
smg-1(
r861)
unc-54(
r293);
pha-1 (
e2123); arEx467). All animals (and only animals) with both distal tip cells expressing
hlh-2::lacZ were counted; 39/40 arEx445 animals and 27/30 arEx467 animals had detectable LacZ expression in both Z1.ppp and Z4.aaa. The level of LacZ appeared to be the same in Z1.ppp, Z4.aaa, and their sisters. Post-transcriptional regulation is inferred because the patterns of LacZ and endogenous HLH-2 accumulation are not coincident. Of animals with detectable LacZ expression in both Z1.ppp and Z4.aaa, 39/39 arEx445 animals and 27/27 arEx467 animals showed HLH-2 accumulation in neither B or only one C of the two cells (B is a dorsolateral view and C is a ventral view). In addition, HLH-2 was not observed in the sisters of Z1.ppp and Z4.aaa, although
hlh-2::lacZ is expressed there. (D,E) To examine HLH-2 accumulation under conditions that are directly comparable with the
hlh-2::lacZ transcriptional reporter, we generated complex arrays using the identical regulatory region (A), in which HLH-2 was tagged with HA. In lines carrying these arrays [
smg-1(
r861)
unc-54(
r293);
pha-1 (
e2123); arEx431 (shown),
smg-1(
r861)
unc-54(
r293);
pha-1 (
e2123); arEx425], HA-HLH-2, detected with an anti-HA antibody, displayed the same pattern of accumulation as endogenous HLH-2; when we could be confident of scoring Z1.ppp and Z4.aaa, HA-HLH-2 was absent from both Z1.ppp or Z4.aaa, 15/28 arEx425 animals and 18/45 arEx431 animals (D), or present in only one of the two cells, 13/28 arEx425 animals and 27/45 arEx431 animals (E). We also generated lines in which HA-HLH-2 and
hlh-2::lacZ were present in the same transgenic array; however, we were unable to find staining conditions in which both antigens could be visualized simultaneously (data not shown).