The expression of genes encoding powerful developmental regulators is exquisitely controlled, often at multiple levels. Here, we investigate developmental expression of three conserved genes, Caenorhabditis elegans mpk-1, lag-1, and lag-3/sel-8, which encode homologs of ERK/MAPK and core components of the Notch-dependent transcription complex, respectively. We use single-molecule FISH (smFISH) and MATLAB to visualize and quantify nuclear nascent transcripts and cytoplasmic mRNAs as a function of position along the germline developmental axis. Using differentially labeled probes, one spanning an exceptionally long first intron and the other spanning exons, we identify two classes of active transcription sites (ATS). The iATS class, for “incomplete” ATS, harbors only partial nascent transcripts; the cATS class, for “complete” ATS, harbors full-length nascent transcripts. Remarkably, the frequencies of iATS and cATS are patterned along the germline axis. For example, most mpk-1 ATS are iATS in hermaphrodite germline stem cells, but most are cATS in differentiating stem cell daughters. Thus, mpk-1 ATS class frequencies switch in a graded manner as stem cell daughters begin differentiation. Importantly, the patterns of ATS class frequency are gene-, stage-, and sex-specific, and cATS frequency strongly correlates with transcriptional output. Although the molecular mechanism underlying ATS classes is not understood, their primary difference is the extent of transcriptional progression. To generate only partial nascent transcripts in iATS, progression must be slowed, paused, or aborted midway through the gene. We propose that regulation of ATS class can be a critical mode of developmental gene regulation.

编码强大的发育调节因子的基因的表达通常在多个水平上受到精确控制。在这里，我们调查了三个保守基因，秀丽隐杆线虫mpk-1，lag-1和lag-3 / sel-8的发育表达，它们分别编码ERK / MAPK的同源物和Notch依赖性转录复合物的核心成分。我们使用单分子FISH（smFISH）和MATLAB可视化和量化核新生转录本和胞质mRNA作为沿种系发育轴位置的函数。使用差异标记的探针，一个跨越一个非常长的第一内含子，另一个跨越外显子，我们确定了两类活性转录位点（ATS）。对于“不完整”的ATS，iATS类仅包含部分新生的笔录；对于“完整的” ATS，cATS类包含完整的新生成绩单。值得注意的是，iATS和cATS的频率沿种系轴进行了模式化。例如，大多数mpk-1ATS是雌雄同体种系干细胞中的iATS，但大多数是分化中的干细胞子代中的cATS。因此，随着干细胞子代开始分化，mpk-1 ATS类频率以分级方式切换。重要的是，ATS类频率的模式是基因，阶段和性别特异性的，而cATS频率与转录输出密切相关。尽管尚不清楚ATS类的潜在分子机制，但它们的主要区别是转录进展的程度。要在iATS中仅生成部分新生的转录本，必须在该基因的中途减慢，暂停或中止进展。我们建议，ATS类的调控可以是发育基因调控的关键模式。