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Supplementary Material

DEV042051 Supplementary Material

Files in this Data Supplement:

  • Supplemental Figure S1 -

    Fig. S1. Sequence alignment of SMED-CHD4 with other CHD4 homologs. The SMED-CHD4 protein sequence was deduced from the full-length cDNA sequence. The protein structure of SMED-CHD4 is depicted at the top of the figure. Alignments with the human, mouse, X. laevis, zebrafish, C. elegans and D. melanogaster homologs are shown. The plant homeodomains, the chromodomains 1 and 2, the DEAD-like helicase superfamily domain (DEXDc) and the helicase superfamily c-terminal domain (HELICc) are indicated. Smed-CHD4 is 43.8% identical to mouse CHD4. The NCBI accession number for Smed-CHD4 is GU980571.

  • Supplemental Figure S2 -

    Fig. S2. Phylogenetic analysis of CHD genes. The represented tree is a maximum-likelihood tree. The SMED-CHD4 protein sequence was aligned with other CHD family members using ClustalX. Neighbor-joining phylogenetic analysis was carried out in ClustalX correcting for multiple substitutions and 1000-fold bootstrapped. Ce, Caenorhabditis elegans; Ci, Ciona intestinalis; Dm, Drosophila melanogaster; Dr, Dario renio; Hs, Homo sapiens; Mm, Mus musculus; Sc, Saccaromyces cerevisiae.

  • Supplemental Figure S3 -

    Fig. S3. CHD4(RNAi) animals cannot regenerate 5 days following the first dsRNA feeding.CHD4(RNAi) worms cannot regenerate after 7 days and 13 days following amputation (20/20 worms showed this phenotype). Worms have been RNAi-fed at days 0 and 4 and amputated 5 days after the first RNAi feeding. White dotted line indicates the blastema boundary. Anterior is to the left. Scale bars: 0.1 mm.

  • Supplemental Figure S4 -

    Fig. S4. CHD4 is expressed at wound sites following amputation. (A) RNAi of CHD4 greatly reduced detectable CHD4 message. The animals were fixed 14 days after first RNAi feeding (5/5 worms displayed similar results). (B) Irradiation (6000 rads) does not affect the expression of non-neoblast genes. H1.3B labels a subset of subepidermal adhesive cells. Anterior is to the left. (C) Relative quantitative PCR for CHD4 in wild-type animals (WT), lethally irradiated animals (6000 rads; IRR), purified X1 cells (X1) and purified X2 cells (X2). UDP has been used as an internal control. Data is normalized to the WT. Mean ± s.d. is shown. (D) CHD4 is expressed at the wound site 3 days following amputation (>20 regenerating pieces displayed similar results). Following irradiation (6000 rads), the CHD4 expression near the wound site was eliminated (>15 regenerating pieces displayed similar results). Regenerating head, trunk and tail pieces are shown. Arrows indicate the site of amputation. Anterior is up. Scale bars: 0.1 mm.

  • Supplemental Figure S5 -

    Fig. S5. CHD4(RNAi) animals have normal numbers of neoblasts. Fluorescence in situ hybridization of animals fixed 10 days after three dsRNA feedings using Smed-H2B and smedwi-1 riboprobes (>12 worms per riboprobe showed similar results). Anterior is to the left. Scale bars: 0.1 mm.

  • Supplemental Figure S6 -

    Fig. S6. CHD4(RNAi) animals have reduced numbers of Smed-AGAT-1-expressing cells. Wholemount in situ hybridizations using Category 2 probe Smed-NB.21.11e (upper two rows) and Category 3 probe Smed-AGAT-1 (lower two rows) of control and CHD4(RNAi) animals at several time points following the first dsRNA feeding (>4 worms per time point, per riboprobe, displayed similar results). Anterior is to the left. Scale bars: 0.1 mm.

  • Supplemental Figure S7 -

    Fig. S7. CHD4 is required for the formation of Smed-AGAT-1-expressing cells. Percentage of double-labeled BrdU-Smed-AGAT-1-expressing cells in the total number of Smed-AGAT-1-expressing cells in control and CHD4(RNAi) animals (left graph), and percentage of double-labeled BrdU-Smed-AGAT-1-expressing cells normalized by the area of the head region (mm2; right graph). Data are represented as means ± s.e.m. (n=4 worms per group). Animals were fed at days 0 and 4 and BrdU was injected at day 6 following initial RNAi feeding and fixed at day 10. Fluorescence in situ hybridizations using a Smed-AGAT-1 riboprobe (green) and antibody staining against BrdU (red) of control and CHD4(RNAi) animals have been performed. An optical section of the head region is shown. Insets show a higher magnification of the double-labeled cells found within the white box. pr, photoreceptors. Anterior is to the upper left. Scale bars: 0.1 mm.

  • Supplemental Figure S8 -

    Fig. S8. Summary time-course of the progression of the CHD4(RNAi) phenotype.CHD4(RNAi) animals could not regenerate after 5 days following the initial dsRNA feeding, although they maintained normal numbers of neoblasts. However, neoblasts had a reduced proliferative response to wounding. As early as 6 days following the initial dsRNA feeding, CHD4(RNAi) animals had reduced numbers of neoblast progeny cells. Later in the progression of the phenotype, the CHD4(RNAi) animals displayed head regression, lesions and curling, indicating that they could not maintain tissue turnover, as well as decreased neoblast numbers.