The MS and E blastomeres are born at the 7-cell stage of development in C. elegans. E generates the gut, while MS generates cells that are primarily mesodermal, including GLP-1-independent pharynx, body muscles, and four embryonically-derived coelomocytes. We have previously reported that
tbx-35, a target of the MED-1,2 factors, is important for MS specification. In
tbx-35(-) embryos, MS-derived pharynx is specifically absent, and embryos appear to have PAL-1-dependent muscle and hypodermis, characteristic of C. However,
tbx-35(-) embryos still make coelomocytes from the MS cell. This suggests that it is possible for MS to adopt a mixed fate, making tissues characteristic of both MS and C, and that additional factors contribute to MS fate in the absence of TBX-35. One of these factors may be
pal-1, as
pal-1(RNAi);
tbx-35(-) embryos make coelomocytes less frequently. Our next studies with MS and E address the role of POP-1 in MS. Using a bona fide
end-1 end-3 double mutant, we find that it lacks all gut as expected, but shows a surprising ability to gastrulate normally and even hatch. Using laser ablation analysis with various GFP reporters [both early and late markers of the MS lineage], it has become clear that
pop-1(-);
end-1,3(-) triple mutant embryos still make GLP-1-independent pharynx tissue from MS, and also sometimes from E. To our surprise, the
end-1,3 doubles and
pop-1(-) alone also show activation of early MS lineage reporters in the E lineage, suggesting that both POP-1 and END-1,3 act to repress aspects of MS specification in the E cell. Finally, we have been characterizing a target of TBX-35 in the early MS lineage, provisionally named
dlx-1, which encodes a homeoprotein. Overexpression of DLX-1 causes ectopic muscle to be generated, as assayed by
unc-120 expression and in situ detection of ectopic
myo-3 transcripts. A
dlx-1::GFP reporter is expressed in the early MS lineage, and ectopically in hs-
tbx-35 embryos, though it is still expressed in the
tbx-35(-) mutant. A shorter promoter (-187bp) drives apparently normal early MS lineage expression but becomes completely
tbx-35-dependent, suggesting that
dlx-1 is a target of TBX-35 and at least one other factor in the MS lineage. Using the TBX-35 binding site identified by DNaseI footprinting of the
dlx-1 promoter, we have identified one other target of TBX-35, an apparent RING finger protein. We will present the results of ongoing work on these and other projects on
end-1,3 and
tbx-35 regulation.