DEV015859 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Nkx2.2 regulation of Phox2b. (A) Early expression of Phox2b. Phox2b mRNA levels were determined by RT-PCR of RNA isolated from the pancreas and stomach of Nkx2.2^+/+ and Nkx2.2^−/− E10.5 embryos (upper gel), and compared with β-actin mRNA levels (lower gel). mRNA isolated from E12.5 Nkx2.2^+/+ embryos was used as a positive control. (B-D) Co-expression of Phox2b and Sox10 in the pancreas. E12.5 embryonic mouse pancreas was harvested, stained by immunofluorescence for Phox2b (Alexa 633, blue in B,D), Sox10 (FITC, green in C,D), and Nkx2.2 (Alexa 568, red in C,D), and imaged by confocal microscopy. (E-H) Ghrelin-positive cells do not express Phox2b and do not originate in the neural crest. Pancreases from Nkx2.2^+/+ E12.5 (E), Nkx2.2^−/− E15.5 (G) and Nkx2.2^−/− E17.5 (H) embryos were co-stained with immunofluorescence for Phox2b (Cy3, red) and Ghrelin (FITC, green). In F, pancreases from E17.5 Wnt1-cre/R26R embryos were co-stained for the neural crest lineage marker β-gal (Cy3, red) and ghrelin (FITC, green). Scale bars: 50 µm.

• Supplemental Figure S2 -

  Fig. S2. Neural and glial marker expression in neural-crest-derived cells. (A-E) Lineage tracing of differentiated neurons in the pancreas and stomach. Pancreas and stomach from Wnt1-cre/R26R embryos (A,B) and pancreas from wild-type embryos (C-E) at E17.5 were co-stained by immunofluorescence for the neural marker HuC/D (FITC, green in A-C,E) and either the neural crest lineage marker β-gal (Cy3, red in A,B) or the neural marker Pgp9.5 (Cy3, red in D,E). (F-I) Pancreases from Nkx2.2^+/+ (F), Nkx2.2^+/− (H) and Nkx2.2^−/− (G,I) embryos at E17.5 (F,G) and E18.5 (H,I) were co-stained by immunofluorescence for Fabp7 (FITC, green) and HuC/D (Cy3, red, F,G) or Pdx1 (Cy3, red, H,I). Scale bars: 50 µm.

• Supplemental Figure S3 -

  Fig. S3. Loss of Nkx2.2 does not affect pancreatic acinar and ductal structures. Pancreases from Phox2b^+/+ (A) and Phox2b^−/− (B) embryos at E17.5 were stained by immunofluorescence for the acinar marker amylase (FITC, green), and with a duct cell stain, biotinylated lectin DBA (Alexa 546, red). Scale bars: 50 µm.