DEV030668 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Reporter gene constructs identify the location of hlh-1 enh-2. The genomic regions, as indicated by nucleotide positions listed at the ends of each line, were cloned into basal promoter plasmids and tested for bodywall muscle enhancer activity in transgenic lines during embryogenesis and postembryonic development. A set of overlapping oligonucleotides that were also tested is shown below the line. Red lines indicate fragments that were positive for muscle enhancer activity; a broken line indicates weak or variable expression. MS+D+C indicates expression in the MS, D and C founder blastomere embryonic muscle lineages. BWM indicates expression in mature bodywall muscle cells of larvae and adults. A dash indicates no expression detected.

• Supplemental Figure S2 -

  Fig. S2. Reporter gene constructs identify the location of enh-4 within the first intron of hlh-1. The genomic regions, as indicated by nucleotide positions listed at the ends of each line, were cloned and assayed for expression as described above.

• Supplemental Figure S3 -

  Fig. S3. Both N- and C-terminal, 6×His-tagged PAL-1 proteins retain biological activity. The ability of 6×His-tagged PAL-1 fusion proteins to convert cells to a bodywall muscle-like fate was assayed in transgenic embryos. The fusions proteins were induced by heat-shock treatment of early embryos (see text) and cell fates assayed by staining terminally arrested embryos with antibodies against either muscle myosin heavy chain A (MHCA) or the hypodermal transcription factor LIN-26. In the absence of PAL-1 fusion protein induction, depletion of POP-1 by RNAi results in embryos arresting with a posterior mass of muscle and scattered hypodermal cells. As previously shown for wild type PAL-1 (Fukushige and Krause, 2005), heat shock induction of both 6×His-tagged PAL-1 fusion proteins efficiently convert most blastomeres to muscle when POP-1 levels are depleted by RNAi. DAPI staining of DNA reveals nuclei size and number for each embryo. Where possible to determine, embryos are oriented with anterior to the left. Scale bar: 10 µm.

• Supplemental Figure S4 -

  Fig. S4. Time course of PAL-1 protein accumulation after heat shock induction. Transgenic mixed population embryos harboring a heat shock promoter driven, C-terminal 6xHis-tagged PAL-1 encoding cDNA were checked by western blot analysis using an anti-6xHis antibody prior to (no), at (0) or at 1-hour intervals after heat-shock treatment. Peak levels of PAL-1 protein were detected (red arrow) at 3 hours post-heat shock treatment. Wild-type (N2) animals with and without heat-shock treatment were used as a negative control. An unidentified, ∼42 kDa cross-reacting protein serves as a loading control in all lanes.

• Supplemental Figure S5 -

  Fig. S5. An hlh-1 enh-1 P1 site containing reporter gene requires PAL-1 for activity in vivo. Transgenic strain PD4743, containing four tandem copies of the oligonucleotide JKL26 that includes the evolutionarily conserved P1 site, drives expression in D+C muscle lineages as shown at top. Each series of images is from a single embryo at mid-embryogenesis with GFP imaging, staining for myosin heavy chain A (MHCA), DAPI staining of nuclei. GFP expression from this reporter gene is lost following pal-1 RNAi (lower panels) and development is disrupted. All embryos are oriented with anterior towards the left and dorsal towards the top. Scale bar: 10 µm.

• Supplemental Figure S6 -

  Fig. S6. RNAi phenotypes of maternally expressed hmg-related genes. Genes encoding HMG-box containing proteins that are: (1) related to POP-1 and (2) likely to be expressed maternally or in early embryos (based on information in WormBase WS197 and NextDB databases) were knocked down by RNAi. Gravid adults were injected with double-stranded RNA corresponding to coding regions of each of five genes and the resulting progeny scored for phenotypes. Although hmg-12 RNAi resulted in no obvious phenotype, knock down of hmg-1.2, hmg-3, hmg-4 and sem-2 resulted in phenotypes as shown and quantitated in the figure. Immunological assays following sem-2 RNAi demonstrated that at least some bodywall muscles likely derived from D and C founder cells (based on position) were born and differentiated. Images are of various magnifications and size is indicated by the developmental stage shown for each panel.

• Supplemental Table S1
• Supplemental Table S2