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.