Figure 1.
Raldh1 expression in mouse and human embryonic pancreas.
(A) In situ hybridization of an e13.5, e14.5 and e15.5 mouse pancreas using a Raldh1 probe. (B) qRT-PCR using cDNA prepared from human fetal pancreas (n = 3 for week 8, 9, 10, 11, 13, 14 and 18, n = 2 for week 12, 15, 17 and 21, n = 1 for week 19 and 20) with RALDH1 and INSULIN specific primers. (C) In situ hybridizations of week 12, week 14 and week 17 human fetal pancreas using a RALDH1 probe. (D) The stomach-duodenal region from a wild-type and an Ipf1/RAR403 transgenic neonatal mouse.
Figure 2.
RA stimulates β-cell differentiation in vitro.
(A) Representative sections of control and RA-treated e10.5 dorsal pancreatic explants cultured for 6 days and analyzed for insulin (green) and glucagon (red) expression. (B) Quantification of insulin and glucagon positive cells in control pancreatic explants and pancreatic explants exposed to RA for 6 days (n = 6 for both) displayed as % positive cells/total number of cells. (C) Representative sections of control and retinol-treated e10.5 pancreatic explants cultured for 6 days and stained with antibodies against insulin (green). (D) Quantification of insulin expression in control and retinol-treated pancreatic buds (n = 2 for both). Scale bar = 40 µM in (A) and 30 µM in (C). Data show mean±S.E.M, *p<0.05.
Figure 3.
RA induces Ngn3 expression in vitro.
(A) Representative sections of a control and RA-treated e10.5 dorsal pancreatic explants cultured for 2 days and stained with antibodies specific for Ngn3. (B) Quantification of Ngn3+ cells in control and RA-treated explants cultured for 2 or 4 days (n = 4 for each condition) displayed as % Ngn3 expressing cells/total number of cells. (C) qRT-PCR using Bhlhb2, Hes1 and NeuroD specific primers on cDNA from control and RA treated e10.5 pancreatic explants cultured for 2 (n = 13) or 4 days (n = 9). Data represent the mean value±S.E.M. *p<0.05, **p<0.01, for RA treated explants versus controls.
Figure 4.
Temporal changes in the expression of Bhlh and homeobox genes in response to RA.
(A) Quantification of Ins+ cells in control pancreatic explants (n = 6) and pancreatic explants exposed to 25 nM RA for the first 2 (n = 3) or 4 days (n = 4) of the 6 day in vitro culture period displayed as % positive cells/total number of cells. Data show mean±S.E.M, *p<0.05 **p<0.01. (B) qRT-PCR using Pax4 specific primers on cDNA from control and RA treated e10.5 pancreatic explants cultured for two (n = 13) or four days (n = 9). Data represent the mean value±S.E.M. *p<0.05, **p<0.01. (C) 6d RA treated explants double-stained for insulin (green) and Ipf1/Pdx1, Nkx6.1, Hb9 or C-peptide (all red). (D) Schematic model summarizing the multiple proposed roles of RA during pancreatic development. Our data provides evidence that RA sequentially specifies: i) proendocrine cells; ii) committed pre-β-cells; and finally iii) differentiated β-cells. Scale bar = 30 µM in (C) (7,5 µM for the enlarged inserts).