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.

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). 

For each cell (named according to C. elegans convention (Sulston, 1983)), for each timepoint collected, there is an expression level (blot column) and x, y, z position values. Cells continue until the time at which they divide or the dataset (or cell) was no longer curated. Additional information about the strains and experimental conditions for each embryo are found in the related manuscript.

Data is presented as comma separated value files (.CSV) which can easily be opened in Excel, other spreadsheet program, or with a text editor. Each file contains the following columns: cellTime (unique identifier based on concatanation of cell name and timepoint), cell (name as based on curated assignment according to Sulston 1983), time (order of z-stacks collected with 1.5 minute time spacing), none (uncorrected reporter intensity), global (corrected reporter intensity with "global" methodology), local (corrected reporter intensity with "local" methodology), blot (orrected reporter intensity with "blot" methodology, USE THIS ONE), cross (corrected reporter intensity with "cross" methodology), z (z plane, uncorrected for embryo orientation), x (x position in microns, uncorrected for embryo orientation), y (y position in microns, uncorrected for embryo orientation), size (cell diameter in pixels), gweight (intensity of lineage channel).

n.b. In file names, ceh-13 refers to JIM118, ceh-13_GFP to JIM356, nob-1 to RW10896 and nob-1_GFP to JIM284. The CRISPR GFP reporter strains are labeled with their strain numbers. Furthermore, the filenames refer to -3.4 kb enhancer as e76 or enh76, the -4.3kb enhancer as e85 or enh85, and -8.3 kb enhancers as e92 or enh92. RNAi against a gene of interest is indicated by a letter "i" tag following the name of the targeted gene, which comes after the reporter name. Additional strain information can be found in Supplemental Table 1 of the related manuscript.