Cellular dataset for: The anterior Hox gene ceh-13 and elt-1/GATA activate the posterior Hox genes nob-1 and php-3 to specify posterior lineages in the C. elegans embryo

Hox transcription factors play a conserved role in specifying positional identity during animal development, with posterior Hox genes typically repressing the expression of more anterior Hox genes. Here, we dissect the regulation of the posterior Hox genes nob-1 and php-3 in the nematode C. elegans. We show that nob-1 and php-3 are co-expressed in gastrulation-stage embryos in cells that previously expressed the anterior Hox gene ceh-13. This expression is controlled by several partially redundant transcriptional enhancers. Surprisingly, these enhancers require ceh-13 for expression, providing an example of an anterior Hox gene positively regulating a posterior Hox gene. Several other regulators including elt-1/GATA, ceh-20/ceh-40/Pbx, unc-62/Meis, pop-1/TCF, ceh-36/Otx and unc-30/Pitx also act positively through nob-1/php-3 enhancers. We identified defects in both cell position and cell division patterns in ceh-13 and nob-1;php-3 mutants, suggesting that these factors regulate lineage identity in addition to positional identity. Taken together, our results highlight the complexity and remarkable flexibility of Hox gene regulation and function.

Methods
We acquired confocal images with a Leica TCS SP5, Stellaris or Nikon A1RSi resonance scanning confocal microscope (67 z planes at 0.5 μm spacing and 1.5 minute time spacing, with laser power increasing by 4-fold through the embryo depth to account for attenuation of signal with depth). Embryos from self-fertilized hermaphrodites were mounted in egg buffer/methyl cellulose with 20μm beads as spacers (Bao and Murray, 2011) and imaged at 22°C using a stage temperature controller (Brook Industries, Lake Villa, IL). We used StarryNite software to automatically annotate nuclei and trace lineages, and AceTree software to identify and fix any errors from the automated analysis, and quantified reporter expression in each nucleus relative to local background (using the “blot” background correction technique) as previously described (Bao et al., 2006; Boyle et al., 2006; Murray et al., 2006, 2008; Santella et al., 2014).