DEV02507 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. HSNL axon and cell body defects. HSNL was visualized with the kyIs179 transgene. The cell bodies of HSNL/R are located just posterior to the vulva, and their axons project first ventrally and then anteriorly in the ipsilateral fascicle of the VNC in the wild type. Only HSNL was scored, as the axon of HSNR was obscured by other axons in the right VNC that express the reporter transgene, kyIs179. With the exception of the posteriorly directed HSNL axon, which is almost certainly a developmental defects, we have not assessed whether the axonal midline defects are developmental or maintenance defects. The cell body position defect of HSNL is due to failure of maintenance, as it is initially wild type in 98% (n=51) of ky188;kyIs179 young L1 larvae. This result indicates that dig-1 functions also to maintain the cell body position of neurons that are located outside of ganglia and lie isolated along the body wall of the worm.

• Supplemental Figure 2 -

  Fig. S2. Reporter gene constructs. Three reporter gene constructs were generated, two transcriptional gfp reporter fusions that include 3.3 and 4.9 kb of upstream regulatory regions and one translational fusion that comprises 0.3 kb of upstream regulatory region, the beginning of the genomic coding region of dig-1 up to the fourth exon and gfp. The translational fusion includes all the upstream region of an isoform of dig-1, which is deposited in GenBank (www.wormbase.org; EMBL accession AY117398). Because the first exon of dig-1 contained within the translational construct contains a signal sequence, the transmembrane domain from PAT-3 (pPD122.39) was introduced in the translational reporter construct to prevent diffusion of the reporter protein. All reporter fusions were generated by PCR fusion (Hobert, 2002) and injected at 20 ng/μl with pRF4 rol-6(su1006d) (100 ng/μl). Primer sequences are as follows: dig-1^prom4.9kb::gfp: A* primer (nomenclature according to Hobert, 2002)=oCB62=5′-cgttgagtagaagaagagcacc; B primer=oCB63=5′-agtcgacctgcaggcatgcaagctcgtgctacagttcttcgctgg; dig-1^prom3.3kb::gfp: A* primer=oCB67=5′-gcttttcctggcggcaagag; B primer=oCB63=5′-agtcgacctgcaggcatgcaagctcgtgctacagttcttcgctgg; dig-1^{translational}::gfp: A* primer=oCB68=5′-ccctcttgtgaggttctttcc; B primer=oCB66=5′-agtcgacctgcaggcatgcaagcttggcggcggaatcggagg. All transcriptional reporter gene lines show similar expression patterns in multiple independent transgenic lines (see main text for gfp images). The sites of expression of the translational reporter construct in embryos, larvae and adults, including all the mesodermal cells, are the same as for the transcriptional fusions, but the expression level is much weaker. Possibly, cis-regulatory elements driving strong mesodermal expression of dig-1 are absent in this translational fusion or the protein-coding region included in this construct destabilizes the reporter. In addition, expression of the translational reporter is detected in hypodermal cells along the entire body, including the head and tail, hyp 7 and the seam cells. As with the transcriptional reporter lines, no expression is detected in neurons.