C. elegans sym-1 is a downstream target of the hunchback-like-1 developmental timing transcription factor

Abstract

In the nematode Caenorhabditis elegans, the let-7 microRNA (miRNA) and its family members control the timing of key developmental events in part by directly regulating expression of hunchback-like-1 (hbl-1). C. elegans hbl-1 mutants display multiple developmental timing deficiencies, including cell cycle defects during larval development. While hbl-1 is predicted to encode a transcriptional regulator, downstream targets of HBL-1 have not been fully elucidated. Here we report using microarray analysis to uncover genes downstream of HBL-1. We established a transgenic strain that overexpresses hbl-1 under the control of a heat shock promoter. Heat shock-induced hbl-1 overexpression led to retarded hypodermal structures at the adult stage, opposite to the effect seen in loss of function (lf) hbl-1 mutants. The microarray screen identified numerous potential genes that are upregulated or downregulated by HBL-1, including sym-1, which encodes a leucine-rich repeat protein with a signal sequence. We found an increase in sym-1 transcription in the heat shock-induced hbl-1 overexpression strain, while loss of hbl-1 function caused a decrease in sym-1 expression levels. Furthermore, we found that sym-1(lf) modified the hypodermal abnormalities in hbl-1 mutants. Given that SYM-1 is a protein secreted from hypodermal cells to the surrounding cuticle, we propose that the adult-specific cuticular structures may be under the temporal control of HBL-1 through regulation of sym-1 transcription.

Figure 1

Heat shock inducible hbl-1 expression causes a developmental abnormality. (A) RT-PCR analysis to compare hbl-1 gene levels in two independent hsp::hbl-1 integrated lines. Synchronized L1 animals of Is[hsp::hbl-1 myo-2::gfp] were cultured with food (bacterial strain OP50) at 25°C and harvested 36 hours later. The animals, which were L4 larvae, were heat shocked at 30°C for 6 hours, then recovered at 25°C one hour after heat shock. (B and C) Typical adults bearing the hsp::hbl-1 transgene showing a broken alae phenotype and a molting defect. eft-2 was used as an internal control. (B) hsp::hbl-1 animal expressed a mix of larval and adult fates in seam cells, indicated by the break in the alae (arrow). (C) hsp::hbl-1 animal exhibiting the molting defect. Unshed cuticle was observed at the L4 molt (arrowheads).

Figure 2

Common sequence motifs found in upstream regions of candidate HBL-1 targets. The MEME program was applied to upstream sequences taken from the start codon of each target gene up to the neighboring gene, or up to 1 kb. One and two common 8-bp motifs were extracted from 23 upregulated and 53 downregulated genes, respectively, in the hbl-1 overexpression L4 animals listed in Table 2. The consensus binding motif of Drosophila HB is derived from ref. The motif sequence logo was created using the WebLogo3 program. Also see Materials and Methods.