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. 2016 Dec 8;1:16016. doi: 10.1038/npjregenmed.2016.16

Figure 6.

Figure 6

MiR-219 regulates Sall4. (a) Log transformed qRT-PCR of Sall4 and miR-219 transcript abundance during wound healing normalised as a per cent of 18S or SnoRD25, respectively. miR-219 levels decrease at 2 days post injury, when SALL4 level increase and at 21 days, miR-219 levels increase, at which time SALL4 levels decrease. Average of samples collected from three different animals. Error bars represent s.d.. Unpaired t-test was used to determine significance (**=P<0.01, *=P<0.05) (b) qRT-PCR confirmed that injection of miR-219 mimic into the injury site increased levels of miR-219 transcript and decreased levels of Sall4 message within tissue collected 1 day post injury. N=2. Unpaired t-test was used to determine significance (***=P<0.001) Errors bars=s.d. across four samples, sample are generated from tissue pooled from three animals. (c–f) Immunofluorescence of SALL4 protein 1 and 7 days post injury in skin samples injected with a non-targeting mimic control or miR-219 mimic. SALL4 protein levels decrease when mature miR-219 mimic is injected into the dermal cells (d, f). Dashed grey line indicates epidermal/ dermal boarder (Scale bars = 50 μm). (g, h) Acid fuchsin/ Orange G stain on mimic control or miR-219 mimic-injected animals 7 days after injury. Yellow arrows indicate the injury site. Representative images are from two replicates with 7 or 9 animals total in mimic control or miR-219-injected animals, respectively. Scale bars: c–f = 50 μm, g, h = 20 μm and inset=10 μm. (i) Model illustrating the normal process that leads to scar-free regeneration in axolotls. Deregulation of SALL4 early in regeneration causes a loss of normal repression of collagen due to SALL4 binding to collagen I and 12. This result in early aberrant collagen deposition that does not get remodelled later, ultimately resulting is imperfect skin regeneration. (j). A model illustrating the mechanism of SALL4 regulation. In steady state conditions, miR-219 binds to the 3′ UTR of SALL4 and represses its expression. On injury miR-219 repression of SALL4 is released, SALL4 protein then binds to collagen 1 and collagen XII and represses there expression during the early phases of regeneration.