Worm Breeder's Gazette 11(5): 79

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Positional Information and mab-5 Expression in the Embryo

Deborah Cowing and Cynthia Kenyon

We are interested in understanding the positional information in C.  
elegans which we believe directs the region-specific expression of mab-
5.  To approach this question, we are using a strain with a 
chromosomally integrated mab-5 promoter:  -gal fusion (Salser and 
Kenyon, this issue).  We have found that in this strain (and also in 
several transformants carrying the same fusion extra-chromosomally),  -
gal is first expressed in the embryo, at about 260 minutes after the 
first cleavage.  At this point elongation has not yet begun.  Most of 
the cells that will later be affected by mab-5 mutations have been 
born and are in the posterior region of the embryo.  We haven't yet 
determined exactly which cells stain for  -gal, but  -gal staining is 
in the posterior, consistent with the position of the cells that are 
known to be affected by mab-5 mutations.  This embryonic staining was 
surprising, since the known phenotype of mab-5 mutations is almost 
exclusively postembryonic.  Nevertheless, it suggests that positional 
information that directs the expression of mab-5 exists in the embryo. 
The mab-5 promoter:  -gal fusion thus serves as a marker to allow us 
to study anterior-posterior pattern formation in C.  elegans embryos.
We have begun a series of experiments to try to learn how this 
positional information might be set up in the embryo.  One can imagine 
a number of possibilities.  'Posterior' cytoplasmic determinants might 
be segregated through the first several cell divisions, like P 
granules and intestinal determinants, to a blastomere which gives rise 
to a cell or cells which are later positioned in the posterior.  This 
cell or cells would then induce the expression of mab-5 in the correct 
region.  If this were true, one might expect the pattern of mab-5 
expression to be perturbed in par mutants, since par mutations affect 
the known segregation events in the first several cell divisions.  In 
addition, it should be possible to identify, by embryonic ablations, 
the blastomeres to which the 'posterior determinants' were segregated. 
On the other hand, the posterior-specific expression of mab-5 could 
be directed by a par-independent mechanism.  One possibility for such 
a mechanism would be a gradient of a molecule such as retinoic acid, 
which would be independent of cell boundaries.
So far, we have tested the effect of one par mutation, par-2(e2030ts)
, on the expression of the mab-5: -gal fusion.  1- to 4-cell par-2(
e2030ts) embryos carrying the mab-5: -gal fusion were dissected from 
mothers that had been at 25 C (the non-permissive temperature) 
overnight, and incubated 4-5 hours longer at 25 C.  This was long 
enough for wild-type embryos to develop to 1 1/2 fold, and to stain 
for  -gal.  50% of the e2030 embryos had abnormal morphogenesis and no 
gut granules (the other 50% were less severely affected.) In these 
embryos, nevertheless,  -gal staining was still localized to the 
posterior.  It is not yet clear how to interpret this, since e2030 is 
a weak allele in which segregation of P granules is relatively normal 
in the first several cleavages, and only fails in later cell divisions 
(Kemphues et al., Cell 52, 311.).  We are in the midst of constructing 
strains with other par alleles.
We have also asked whether glp-1 affects mab-5 expression.  glp-1 is 
known to be involved in signalling pathways, and in the embryo is 
required in inductive interactions that specify the normal development 
of at least some AB-derived cells (Priess et al.  Cell 51, 601).  glp-
1(q231ts) embryos carrying the fusion were shifted from 15 C to 25 C 
at the 1-cell or 2-cell stage, and incubated long enough for wild-type 
embryos to express  -gal.  Despite the fact that q231ts embryos 
shifted to 25 C at the 2-cell stage develop abnormally and do not 
hatch (see Austin and Kimble, Cell 51, 589), the pattern of  -gal 
staining in these embryos was similar to wild-type.  Thus, although 
glp-1 could affect mab-5 expression in some cells, it must not play a 
major part in setting up the pattern of mab-5 expression.
We think it is unlikely that mab-5 expression is directed solely by 
lineage rather than by positional information (the other kind of 
hypothetically possible model) since the cells affected by mab-5 
mutations are not related by lineage.  However, looking at the pattern 
of  -gal expression in cleavage-blocked embryos carrying the mab-5: -
gal fusion should help to distinguish between this type of model, and 
a positional information model.  At the four cell stage all four 
blastomeres give rise to cells which will later express the mab-5: -
gal fusion (unpublished observations.) If this were due to lineage-
specific segregation of mab-5 determinants, one might expect all four 
blastomeres to express  -gal in a cleavage blocked embryo.  Instead, 
in three out of six embryos which were blocked from further cleavage 
at the four cell stage using cytochalasin D, P2 alone expressed  -gal.  (
Two out of six had no  -gal expression, and the sixth stained in a 
position that was most consistent with P2, but the orientation of the 
embryo was such that it was impossible to be sure.)  Thus, there 
appears to be some mechanism by which only this one blastomere has the 
potential to express the mab-5: -gal fusion.  This result is more 
consistent with a positional information model than with lineage-
specific segregation of mab-5 determinants.