To characterize the M lineage expression pattern of
mls-2, double-labeling experiments were performed using anti-MLS-2 antibodies and the M lineage-specific
hlh-8::gfp or
hlh-8::lacZ markers.
mls-2 expression in the M lineage was first detectable in the M mesoblast, and was retained during the first three rounds of cell divisions, such that
mls-2 expression was still detectable in eight M descendants (designated 8-M stage, n>200). However, after one more round of cell division (at the 16-M stage), no MLS-2 signal was detected either by anti-MLS-2 antibodies or by the gfp::
mls-2 fusion construct. Although it is possible that a low level of MLS-2 protein is present after the 8-M stage, the loss of MLS-2 signal at the 16-M stage appears to be due to the instability of the MLS-2 protein, because the
mls-2 promoter is still active in M lineage descendants after the fourth round of cell division, as detected by a transcriptional
mls-2p::gfp::
mls-2 3' UTR construct. Neither the
mls-2 promoter activity nor the MLS-2 protein was detected in the SM lineage or the differentiated BWMs and CCs. Thus
mls-2 is expressed in the proliferating cells of the early M lineage.