Many years ago (Beh et al., 1991), we described an acid phosphatase activity (
pho-1) that was expressed along the intestinal brush border in every gut cell except
int-1 and
int-2, beginning at the three fold stage of embryogenesis. Thus, compared to early gut differentiation markers such as the
ges-1 esterase,
pho-1 should allow us to investigate three different polarities within the gut: anterior-posterior, apical-basal, and temporal. The
pho-1 gene was cloned and its expression pattern is now being analyzed. First of all, fusions of the
pho-1 promoter to GFP reporters reflect the endogenous expression pattern and do not express in
int-1 and
int-2; in other words, the anterior-posterior patterning is at the level of the
pho-1 promoter. We have previously shown that A/P patterned expression of a modified
ges-1 reporter depends on zygotic
pop-1 activity (Schroeder and McGhee, 1998). This patterning within the E-lineage is gut-autonomous and occurs after the Wnt-dependent P2-EMS contact of the four cell stage. We are now investigating whether
pho-1 responds to the same A/P patterning cues but in an opposite manner, i.e. we would expect that high level of
pop-1 within the gut cell nuclei would repress
pho-1 expression, whereas high level of
pop-1 in gut nuclei activates the modified
ges-1 reporter. An experiment to test this model is to inject
lit-1 doublestranded RNA into a
pop-1(
zu189) mother. The gut is still formed (
zu189 results in low maternal
pop-1 and hence a double E cell) but loss of
lit-1 activity should prevent downregulation of zygotic
pop-1 within the gut. Our prediction is that
pho-1 should be repressed throughout the gut because of high
pop-1 and (in preliminary experiments) this is indeed what we see. The availability of a temperature sensitive allele of
lit-1 should allow us to investigate the gut patterning events in much finer detail. To investigate apical-basal asymmetry, we are attempting to find a GFP construct that will reflect the endogenous
pho-1 distribution, in order to allow for genetic screens of polarity defects. At the moment, all we know is that mutations in
lin-2,
lin-7 and
lin-10 (shown by Stuart Kim's lab to be involved in epithelial polarity) have no apparent effect on the normal distribution of
pho-1 activity. Finally, we are investigating why
pho-1 is expressed 2-3 E-cell divisions later than
ges-1. There are GATA sites in the promoter, that might be a target for
elt-2. However, ectopic expression of either
elt-2 or
end-1 does not activate ectopic expression of either endogenous
pho-1 gene or
pho-1 reporter gene, suggesting other factors or events may be required. We are analyzing the
pho-1 promoter to identify the site of action of these timing factors or timing events.