Worm Breeder's Gazette 11(5): 48

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.

A Putative Nuclear Hormone Receptor Gene is Expressed During Early Embryogenesis

Ann Sluder, Kim McCall and Gary Ruvkun

We have previously described the cloning of crf-2, a gene with 
homology to nuclear hormone receptors.  In situ hybridization 
experiments and developmental Northern blot analysis had indicated 
that crf-2 RNA is present primarily, if not exclusively, during the 
first half of embryogenesis (WBG, Vol.  11, #3).  Subsequent Northern 
blot analysis has shown that the crf-2 message is present in N2 adults 
and in fem-2(b245ts) adults raised at restrictive temperature, but not 
in glp-1(e2141ts) adults raised at restrictive temperature.  Thus crf-
2 is expressed maternally in the germ line.  Whether or not it is also 
transcribed zygotically remains to be determined.
In order to further define the crf-2 expression pattern a 6 kb 
fragment from the 5' end of the crf-2 gene has been fused to the lacZ 
gene in a derivative of one of Andy Fire's transformation vectors (see 
Burglin et al., this issue).  Worms were transformed with this fusion 
gene in combination with a dominant rol-6 allele as a marker.  
Immunostaining of transgenic animals reveals expression of the crf-
2/lacZ fusion protein in nuclei of early embryos.  Staining is first 
observed in 16-20 cell embryos, in most but probably not all cells.  
The crf-2/lacZ protein disappears in mitotic cells, and reappears 
after mitosis in a progressively restricted pattern as embryogenesis 
advances: fusion protein expression first appears to become 
concentrated primarily in the dorsal region of the embryo, but is 
ultimately found in only two to four cells near or in the cluster of 
neuroblasts in the presumptive head region before disappearing for 
good just as morphogenesis is beginning.  Attempts to identify the 
exact cells which do or do not produce the fusion protein are 
currently underway.  While the fusion gene expression pattern is 
consistent with the Northern blot and in situ hybridization analyses, 
we are also attempting to isolate a crf-2 cDNA, and hope eventually to 
generate antibodies against the actual crf-2 protein in order to 
compare its expression pattern with that of the fusion gene.
These expression studies suggest that crf-2 is involved in some 
aspect of embryogenesis which is at least partially, if not entirely, 
under maternal control.  Thus we expect (hope) that a crf-2 mutation 
will have a maternal-effect embryonic lethal or perhaps a zygotic 
lethal phenotype.  The physical map location of crf-2 places it 
between dpy-5 and dpy-14 on Chromosome I, a region which the Ann Rose 
lab has saturated for lethal mutations.  In order to take advantage of 
the wealth of information which the Rose group has collected, we have 
mapped crf-2 relative to the duplications which they have used in 
mapping their collection of lethals.  A Bristol-Bergerac polymorphism 
has been detected two cosmids to the left of the crf-2 cosmid; the 
restriction patterns of the polymorphism (mgP29) are consistent with 
it being a Tc1 insertion.  The Rose lab generously provided strains 
bearing duplications (Bristol-derived) with breakpoints at various 
sites between dpy-5 and dpy-14 as well as a strain (KR408) in which 
the dpy-5-unc-29 interval is derived from Bergerac (see Starr et aL, 
1989, Genome 32: 365-372).  The duplications were moved into the KR408 
background.  Since KR408 has the Bergerac allele of mgP29 while the 
duplications, if they cover the site of the polymorphism, have the 
Bristol allele, we were then able to map mgP29 relative to the 
duplications by genomic Southern blot analysis.  This placed mgP29 
outside of hDp15, hin hDp12 and 
hDp16.  Additional probings of the blots with cosmids between mgP29 
and crf-2 detected no evidence of a duplication breakpoint between 
these two markers, so crf-2 should also lie between the hDp37 and 
hDp12 breakpoints.  We now plan to examine the terminal phenotypes and 
attempt transformation rescue of lethal genes from this region in 
efforts to correlate crf-2 with a genetically defined locus.  Our 
initial candidate genes will include 8-10 loci from the Rose lethal 
collection which have been mapped relative to at least some of the 
duplications used above, though crf-2 could have been missed in their 
screens if maternal expression is required or sufficient since their 
screening strategy was not designed to detect maternal-effect genes.  
We also plan to look at some emb and zyg genes from the Stock Center 
collection which are less precisely mapped, but some of which do have 
maternal effects.