akreas is a hypomorphic allele of ptf1a. (A,B) Three-dimensional rendering of Tg(gutGFP) (green) wild-type (A) and akreas mutant (B) embryos at 56 hpf stained for Isl1 (red) and Insulin (Ins; blue) showing a smaller ventral pancreas (arrows) in akreas mutants. (C,D) Tg(elastase:GFP); Tg(lfabp:dsRed) wild-type (C) and akreas mutant (D) at 100 hpf stained for pan-Cadherin showing the absence of Tg(elastase:GFP) expression (green) in akreas mutants. Asterisks mark the EPD. (E) Genomic DNA sequence of wild-type and akreas mutant ptf1a showing the molecular lesion in the akreas ptf1a gene. (F) Schematic of the Ptf1a bHLH domain and corresponding amino acid sequences of wild-type and akreas mutant. (G) Luciferase assay to evaluate I142N substitution on Ptf1a transcriptional activity. Wild-type Ptf1a + E47 results in >100-fold induction of the reporter, while I142N Ptf1a results in a modest 40-fold induction. (H–K) Retina of 52 hpf Tg(ptf1a:eGFP); akreas mutant (H; green channel shown in I) and akreas mutant injected with ptf1aMO1 (J; green channel shown in K) stained for Isl1 (red) showing that Tg(ptf1a:eGFP)-expressing cells are restricted to the horizontal (white arrowheads) and amacrine (white arrows) layers in akreas mutants (H,I), but are scattered in ptf1aMO1-injected akreas mutants (J,K) indicating a more severe retina phenotype in the latter. Yellow arrows point to the photoreceptor cell layer. Blue arrows point to Tg(ptf1a:eGFP)/Isl1-coexpressing cells.

Ptf1a represses endocrine development. (A–H,K,L) Tg(ptf1a:eGFP) (green) expression in wild-type (A,C,D,I) and akreas mutant (B,E–H,J) embryos stained for Isl1 (red) and pan-Cadherin (blue) show mutually exclusive expression of Tg(ptf1a:eGFP) and Isl1 in wild-type (except in a single cell; arrow) but not in akreas mutants at 50 hpf (A,B) and 80 hpf (C–H). (D) Tg(ptf1a:eGFP) intensity of C showing the single cell (arrow) with relatively low Tg(ptf1a:eGFP) expression. Note that most Tg(ptf1a:eGFP)/Isl1-coexpressing cells in akreas mutants are found at the distal EPD (E–H; arrows) where the Tg(ptf1a:eGFP) intensity is relatively high (H, GFP intensity of E) (shown at higher magnification in Supplemental Fig. S3A–F). (I,J) Tg(ptf1a:eGFP) (green) expression in 80 hpf wild type (I) and akreas mutants (J) stained for Isl1 (red) and Insulin (blue) show mutually exclusive expression of Tg(ptf1a:eGFP) and Insulin in wild-type and coexpression in akreas mutants (arrows). (K,L) At 140 hpf, Isl1 expression is no longer detectable in the principal endocrine cluster in wild-type (K), suggesting that Isl1 expression is lost from more mature endocrine cells. However, in akreas mutants (L), Isl1-positive cells, some of which coexpress Tg(ptf1a:eGFP) (arrows), are present, suggesting that endocrine neogenesis continues in akreas mutants. Asterisks mark the EPD.

Ventral pancreas specification and outgrowth in akreas mutants. (A–H) Three-dimensional rendering of Tg(ptf1a:eGFP) (green) wild-type (A,B,E,F) and akreas mutant (C,D,G,H) embryos at 38 hpf (A–D) and 46 hpf (E–H) stained for Prox1 (red) and Nkx6.1 (blue) showing Tg(ptf1a:eGFP)-expressing pancreatic cells coexpressing Prox1 and Nkx6.1 (arrow) in wild-type but not akreas mutants at 38 hpf. Arrowheads point to Prox1 and Nkx6.1 expression in the dorsal pancreas. At 46 hpf, wild type and akreas mutants show Tg(ptf1a:eGFP) coexpression with Prox1 and Nkx6.1 (arrows) (E–H). Only red and blue channels are shown in B, D, F, and H (shown at higher magnification in Supplemental Fig. S2). (I,J) Three-dimensional rendering of 42 hpf Tg(gutGFP) (green) wild-type (I) and akreas mutant (J) embryos stained for Isl1 (red) showing a ventral pancreatic outgrowth in wild-type but not akreas mutants (arrows). (K,L) Three-dimensional rendering of 44 hpf Tg(gutGFP) (green) wild-type (K) and akreas mutant (L) embryos stained for Isl1 (red) and Ins (blue) showing the appearance of a small ventral pancreatic bud in akreas mutants (arrow). (M,N) Three-dimensional rendering of 80 hpf Tg(gutGFP) (green) wild-type (M) and akreas mutant (N) stained for Isl1 (red) and Ins (blue) showing that the ventral pancreas in akreas mutants can fuse (arrow) with the dorsal pancreas. (O,P) Wild-type (O) and akreas mutant (P) at 80 hpf stained for Nkx6.1 (red) and pan-Cadherin (green) showing Nkx6.1-positive cells marking the EPD (arrows), which does form in akreas mutants. L, liver.

Low levels of Ptf1a expression promote, whereas high levels repress, endocrine fate. (A–L) Tg(ptf1a:eGFP) expression (green) in control wild type (A–C) and ptf1aMO1 injected wild type (D–L) stained for Isl1 (red) and pan-Cadherin (blue). (A) Three-dimensional rendering of a 56 hpf control. (B,C) Z-focal plane of A at 2× magnification revealing mostly mutually exclusive expression of Tg(ptf1a:eGFP) and Isl1 in control with the exception of a single cell (arrows; red and blue channels in C). (D) Three-dimensional rendering of a 2 ng ptf1aMO1 injected wild type at 56 hpf. (E,F) Magnification (2×) of D shows ectopic Isl1-expressing cells all along the EPD (arrows; red and blue channels of E shown in F). (G,H) Two different Z-focal planes of E revealing extensive coexpression of Tg(ptf1a:eGFP) and Isl1 along the entire EPD (arrows; red and blue channels of H shown in I). (J) Three-dimensional rendering of a wild-type embryo injected with 0.5 ng of ptf1aMO1 at 76 hpf. (K) Z-focal plane of J showing a cluster of high level Tg(ptf1a:eGFP)-expressing cells that do not express Isl1 (arrowheads). (L) Tg(ptf1a:eGFP) intensity of K showing relatively high GFP expression in a cluster of cells that do not express Isl1 (K). (M,N) Three-dimensional rendering of ptf1aMO2 [which knocks down both Ptf1a and Tg(ptf1a:eGFP) expression] injected wild type at 76 hpf (M; green channel shown in N). (O–R) Z-focal plane through M showing that cells with higher levels of Tg(ptf1a:eGFP) expression (arrowheads) do not express Isl1. (P) Shows red and blue and (Q) shows green channels of O. Cells with relatively low levels of Tg(ptf1a:eGFP) expression (arrows) coexpress Isl1 (shown at higher magnification in Supplemental Fig. S3G–L). (R) Tg(ptf1a:eGFP) intensity of O.