Figure 3. Nkx6.1 functions as an endocrine fate determinant.

(A,B) A tamoxifen-inducible form of Cre recombinase (CreER™) expressed under the control of the Pdx1 promoter removes a floxed Bgeo (lacZ) cassette to permanently activate expression of green fluorescent protein (GFP) from the Z/EG transgene (A) or Nkx6.1 and GFP from the Nkx6.1^OE transgene (B). Immunofluorescence staining of pancreas sections from e10.5 (C-J), e12.5 (L-W) and e15.5 (Y-H′) Pdx1-CreER™;Z/EG and Pdx1-CreER™;Nkx6.1^OE embryos injected with one dose of tamoxifen at e8.5. Quantification of GFP⁺/marker⁺ cells relative to the total number of GFP⁺ cells at e10.5 (K), e12.5 (X), or e15.5 (I′). At e10.5, similar numbers of GFP⁺ cells have initiated expression of the endocrine markers Ngn3, Pax6, and hormones in Pdx1-CreER™;Nkx6.1^OE and Pdx1-CreER™;Z/EG control embryos (K). Similar analysis at e12.5 reveals a preference of GFP⁺ cells in Pdx1-CreER™;Nkx6.1^OE embryos to express Ngn3, Pax6, and hormones, while showing a decreased propensity to express Ptf1a and CPA (X). Expression of the proliferation marker Ki67 does not show a difference between the two groups (X). At e15.5, GFP⁺ cells in Pdx1-CreER™;Z/EG embryos express endocrine hormones (Y) as well as the acinar marker amylase (Z) (arrowheads in inset, Y,Z). By contrast, in Pdx1-CreER™;Nkx6.1^OE embryos, GFP⁺/amylase⁺ cells are rarely found (E′), while GFP⁺/hormone⁺ cells are abundantly detected (arrowheads in inset, D′). GFP⁺ cells from Pdx1-CreER™;Nkx6.1^OE and Pdx1-CreER™;Z/EG embryos have a similar potential to differentiate into DBA⁺ ductal cells (arrowheads in inset show GFP⁺/DBA⁺ cells, C′,H′). (J′) Model of cell fate shifts induced by heritable Nkx6.1 misexpression in multipotent pancreatic progenitors. Scale bar=50 μm; triangles represent loxP sites; filled ovals, IRES; Amy, amylase; Horm, hormone (insulin, glucagon, somatostatin, pancreatic polypeptide); Endo, endocrine; Ac, acinar.