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Fig. 3. Setd7 is required for specification of pancreatic progenitor cells. (A-F) Whole-mount ISH analysis of pdx1 (A,B), ptf1a (C,D) and hhex (E,F) in
uninjected (ctrl) and setd7-MO-injected (5 ng) Xenopus embryos. Arrowheads indicate dorsal (dp) and ventral pancreatic (vp) buds. Brackets indicate ptf1a
expression in the eye and hindbrain. Scale bars: 1 mm. (G) RT-qPCR analysis of indicated genes in Xenopus AE explants injected with increasing doses of setd7-
MO. n=10. (H) RT-qPCR analysis shows that co-injection of mouse Setd7 mRNA (1 ng) together with setd7-MO in AE restores the expression of indicated
endodermal and pancreatic genes; n=4. Data were normalized to that of odc and represented as fold changes compared with AE samples (set to 1). Error bars
represent ±s.e.m. *P<0.05, **P<0.01, ***P<0.001. |
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Fig. 4. Setd7-H297A histone methyltransferase mutant lacks pancreatic
fate-inducing activity. (A) Whole-mount ISH of pdx1, ptf1a and hhex in
uninjected (ctrl) and setd7-H297A-injected (1 ng) Xenopus embryos.
Arrowheads indicate dorsal (dp) and ventral pancreatic (vp) buds. Brackets
indicate ptf1a expression in the eye and hindbrain. Scale bar: 1 mm. lv, liver.
(B) RT-qPCR analysis of setd7-H297A-injected AE explants for indicated
markers. Data were normalized to that of odc and represented as fold changes
compared with uninjectedAEsample (set to 1). n=8; error bars represent ±s.e.m.
*P<0.05, **P<0.01, ***P<0.001. |
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Figure S5. Activity of Setd7-H297A mutant in Xenopus embryos. (A) Schematic of Setd7
protein structure and aminoacid allignment of the highly conserved region in the SET domain
(288-306), harboring the histonemethyltransferase activity of SET proteins (Nishioka et al., 2002).
The SET7 domain is highly conserved across vertebrate species, with 81% identity between the
human and Xenopus laevis homologues. In Red, the key conserved histidine residue within the
SET domain; its single amino acid substitution (H297A) results in the loss of methyltransferase
activity (Nishioka et al., 2002). Xenopus setd7-H297A was generated by site-directed
mutagenesis PCR. (B-C) Xenopus embryos injected with setd7-H297A mRNA recapitulate the
same pancreatic phenotype observed in setd7-MO-injected embryos. Transverse sections of
control (ctrl) and setd7-H297A-injected Xenopus embryos processed by whole mount ISH for
hhex (B) and ptf1a (C). Hhex+ and ptf1a+ expression areas were quantified on transverse
sections. Error bars represent ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. Abbreviations, dp, dorsal
pancreas; vp, ventral pancreas; lv, liver. |
