Worm Breeder's Gazette 10(3): 21

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.

Analysis of tra-1 Mosaic Intestines by in situ Hybridization with a vit-5 Probe

Craig P. Hunter and W.B. Wood

We have previously reported results consistent with a cell-
autonomous requirement for tra-1 function in development of 
hermaphrodite structures (WBG Nov.  1987; May 1988).  The intersexual 
phenotypes of several tra-1 mosaics suggested that tra-1 function may 
also be required in descendants of E, the founder cell of the 
intestine, for production of the hermaphrodite specific gene product 
vitellogenin.  These intersexes were either hermaphrodite in all 
respects but failed to express vitellogenins or were hermaphrodite for 
all AB- and P2-derived structures but male for all EMS-derived 
structures.  We have further tested this suggestion by analyzing tra-1 
mosaic intestines, using in situ hybridization with a vit-5 probe to 
detect presence or absence of vitellogenin mRNA production in 
individual cells of dissected intestines as described previously (e.g. 
Schedin and Wood, WBG Nov.  1985).  The free duplication ctDp2, which 
carries tra-1(+), is lost approximately once every 200 cell divisions. 
The E cell and its descendants undergo 19 divisions in generating the 
20 cells of the adult intestine.  Therefore, on average, 1 in 10 
animals of a strain homozygous for a tra-1(lf) mutation and carrying 
ctDp2 should have genetically mosaic intestines.  If tra-1 function is 
required cell-autonomously for intestinal vitellogenin synthesis, then 
tra-1(+) cells should express the vit genes and tra-1(+) cells should 
not.  If the duplication is lost only once in the E lineage, then the 
cells not expressing the vit genes will be clonally related.  Specific 
patterns of nonexpressing cells are predicted, because some cells in 
the lineage exchange places during development, giving rise to 
noncontiguous sister cells in the adult intestine (Sulston et al., Dev.
Biol, 100:64, 1983).  To assay vit gene expression, [35S]-labelled 
vit-5 probe was hybridized in situ to partially dissected, fixed 
intestines (3:1 ethanol:acetic acid) from animals of the appropriate 
strain.  On average, about half the intestine was available for 
hybridization.  From 329 dissected hermaphrodites, 17 mosaic 
intestines were identified.  As expected, most of these showed only a 
single cell or pair of cells not hybridizing to the vit-5 probe.  
However, one mosaic intestine contained 5 nonhybridizing cells, and 
another contained 10.  In these mosaics, some of the nonhybridizing 
cells were not adjacent, but interspersed with hybridizing cells.  
However, in each mosaic, the nonhybridizing cells could be identified 
and shown to represent an exclusive clone, consistent with a single 
loss of the duplication during intestinal development and a cell-
autonomous requirement for tra-1 function in vitellogenin synthesis.