Fig 2: Sall4 binds to collagen promoters and regulates transcription. (a–c) Chromatin immunoprecipitation with anti-Sall4 followed by qPCR revealed enrichment of collagen I and XII from 14-day injured skin (shown are means of fold change over pull down with IgG). Unpaired t-test. Error bars are s.d. **=P<0.01. Replicas=2, with each replicate pooling tissue from three animals. (d, e) A luciferase assay was performed after transfecting HEK293T cells with the pGL3 Enhancer vector containing either (d) axolotl collagen I Intron 1 or (e) axolotl collagen XII intron 1 together with either a Sall4 over expression construct or empty vector control. After 48 h the cells were lysed and luciferase activity was measured relative to β-galactosidase activity. An unpaired two-tailed t-test was used to determine significance. Error bars are s.d. ***=P<0.0001.

Fig 3: Knockdown of SALL4 leads to excessive collagen deposition during wound healing. Acid fuchsin/ Orange G stain on (a) mismatch control morpholino or (b) Sall4 morpholino-injected animals at 21 days after injury. Yellow arrows indicate the re-formation of the basal lamina. Representative images shown for two replicates of four animals each. (c) Expression of Sall4 relative to 18S at 7 days post injury in tissue that was treated with either mismatch morpholino or Sall4 morpholino. Representative graph of three repeats. Significance determined by an unpaired t-test. Error bars are standard deviation (s.d.) ***=P<0.0001. (d) Analysis of the number of cells in a 100 μm2 area below the basal lamina at 21 days post injury after Sall4 knockdown. Replicas=2 with 10 animals total in each condition. Error bars are s.d. ***=P<0.001. Scale bar = 20 μm.

Fig 4: Sall4 expression during wound healing. (a) qRT-PCR of Sall4 expression levels during wound healing versus control uninjured tissue. Significance determined by a one-way ANOVA with Dunnett’s multiple comparisons test. N=3. Error bars are s.e.m. ***=P<0.001. NS=not significant. (b–e″) Co-Immunofluorescence analysis of SALL4 and TP63 (b–b″), Keratin 15 (c–c″), Vimentin (d–d″) and smooth muscle actin (SMA) (e–e″) at 28 days post injury. Examples of double positive cells are indicated with yellow arrows. Nuclei are stained with DAPI. Dotted lines represent separation between the epidermis and the dermis. Representative images shown for three replicates of each co-stain. Scale bars = 50 μm. (f) The percentage of Sall4+ keratinocytes, dermal fibroblasts (vimentin) and myofibroblasts (smooth muscle actin) are shown within the wound bed at 14 and 28 days post injury. An unpaired two-tailed t-test was used to determine significance. Error bars are standard deviation. **=P<0.01.

Fig 5: Discovery of a Novel SALL4 DNA-Binding Motif(A–C) DNA sequence motifs bound by WT SALL4A (A), a mutant lacking the 2nd zinc finger cluster (AΔZFC2) (B), or a mutant lacking the 4th ZFC (AΔZFC4) (C), discovered in universal PBM assays. The color bars above the position weighted matrices indicate the linear structure of SALL4, and the ZFCs are denoted by black ovals.(D) EMSA showing SALL4A shifts the AT-rich motif-containing oligos (lanes 1–3) but not when the motif is scrambled (lanes 4–6, gel cut for clarity); UC, unlabeled competitor probes.(E) SALL4-DNA complex is super-shifted by a SALL4 monoclonal antibody (lane 4) but not by mouse IgG isotype control (lane 5); mAb, SALL4 mouse antibody (Santa Cruz EE-30).(F) EMSA showing that SALL4-DNA complex was reduced or super-shifted in the presence of FLAG or SALL4 antibodies; rAb-1, SALL4 rabbit antibody (Cell Signaling D16H12); rAb-2, SALL4 rabbit antibody (Abcam ab57577). Lanes 8–10 show that the AΔZFC3 mutant binds to the same WT sequence, whereas binding by AΔZFC2 and AΔZFC4 mutants is abrogated. All EMSA reactions contain poly dI:dC competitor to reduce background binding.(G) Isothermal titration calorimetry (ITC) experiments showing purified SALL4 ZFC4 (amino acids 864–929) binds DNA oligos containing the WT WTATB motif (top) and not when the motif was mutated (bottom). All EMSA and ITC oligo sequences can be found in the Key Resources Table.