The Drosophila gap gene hunchback (hb) is required during embryogenesis for the specification of thorax segments and posterior head parts. Using BLAST search, we identified a predicted C. elegans open reading frame on LGX that contains a region with significant homology to the N-terminal zinc-finger domain of hb. We have cloned the C. elegans
hbl-1 (hunchback-like) cDNA from the Okkema embryonic library and have undertaken studies to determine the function of
hbl-1 during embryogenesis. Partial sequence analysis indicates that the
hbl-1 gene structure differs significantly from the predictions of Genefinder: exon 7 encodes an additional 25 amino acids to include a complete C-terminal zinc-finger domain, while an eighth exon predicted by Genefinder was found to be part of the 1.4-kb 3'UTR of
hbl-1. Also, the translation initiation site of
hbl-1 appears to be located approximately 1,200 bp upstream of the predicted start site, and several additional exons were identified in this new N-terminal domain. Our findings suggest that
hbl-1 encodes a protein of 982 amino acids which is most similar to the leech hb homologue Lzf2. The N-terminal zinc finger domain of
hbl-1 shows a 68% identity with Drosophila melanogaster hb and 78% identity with H. Triserialis Lzf2 in this region. In addition, a BLAST search of
hbl-1 against the entire GENBANK database identifies only putative hb homologues in the top 12 matches. Northern blot analyses indicate that
hbl-1 is expressed at high levels during mid-to late embryogenesis and at lower but detectable levels in L1 larvae. In contrast,
hbl-1 does not appear to be expressed maternally or at appreciable levels in later larval stages or adults. To further analyze the nature of
hbl-1 expression, multiple transgenic lines were generated (containing both integrated and extrachromosomal transgenes) that carry an
hbl-1-GFP gene fusion (at
hbl-1 amino acid position 195) and include a 6.3 kb region upstream of the predicted
hbl-1 translation start site. GFP staining reveals that expression begins before the 200 cell stage of embryogenesis in several anterior cells. Strong hypodermal expression appears transiently at the lima bean stage, while extensive neuronal expression is seen during later embryonic stages. Staining of the amphid neurons is detectable in pretzel-stage embryos and in L1 larvae. Details await further characterization of the GFP lines as well as generation of an HBL-1-specific antibody. Antisense injection experiments were carried out in an attempt to identify potential
hbl-1 loss-of-function phenotypes. While low levels of embryonic lethality were observed following injection of
hbl-1 antisense RNAs, the incidence of inviable embryos did not appear to be substantially greater than those levels observed in control injections. This apparent lack of effect may simply reflect the general finding that zygotic gene functions are less sensitive to antisense RNA inhibition than those of maternally required genes. To obtain mutations in
hbl-1, we are collaborating with members of the Culotti lab who are screening their Tc1 library for insertions within the gene. Finally, we are testing candidate genes that map near
hbl-1 on LGX for rescue with
hbl-1 sequences. Our initial search for a C. elegans homologue of hunchback was prompted by an interest in identifying genes that, based on findings in Drosophila, might interact with
pal-1. Experiments are planned to determine whether hypodermal expression of
hbl-1 is dependent on
pal-1 function. Interestingly, the 3'UTR of
hbl-1 contains a number of motifs similar to Drosophila nanos Response Elements (NREs) which are known to be required for the repression of hb by pumilio during embryogenesis. We have generated lines containing an
hbl-1-GFP fusion that includes the
hbl-1 3'UTR. Preliminary analysis indicates that the expression pattern of this transgene is identical to that of our original
hbl-1 fusion construct. This reporter could prove useful for identifying genes that may regulate
hbl-1 via its 3'UTR.