Worm Breeder's Gazette 15(1): 46 (October 1, 1997)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
MCD Biology, University of Colorado Boulder, Colorado 80309-0347
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.