ZFIN ID: ZDB-FIG-160316-22
Xu et al., 2016 - Four and a Half LIM Domains 1b (Fhl1b) Is Essential for Regulating the Liver versus Pancreas Fate Decision and for β-Cell Regeneration. PLoS Genetics   12:e1005831 Full text @ PLoS Genet.
ADDITIONAL FIGURES
EXPRESSION / LABELING:
Genes:
Antibody:
Fish:
Knockdown Reagents:
Anatomical Terms:
Stage Range: Prim-15 to Long-pec
PHENOTYPE:
Fish:
Knockdown Reagents:
Observed In:
Stage Range: Prim-15 to Long-pec

Fig. 2

Loss of Fhl1b activity enhances induction of pancreatic cells and compromises liver specification.

(A-D) Whole-mount in situ hybridization showing the expression of hhex (A and B) and pdx1 (C and D), comparing control embryos (A and C) and fhl1b morphants (B and D) at 30 hpf. hhex is expressed in the liver (black arrows) and the dorsal pancreatic bud (white dotted circles). pdx1 is expressed in the developing pancreas including the dorsal pancreatic bud (white dotted circles) and intestine (black brackets), but not in the liver. hhex expression was reduced in the liver of fhl1b morphants, while expanded in the dorsal pancreatic bud (B). pdx1 expression in the dorsal pancreatic bud was also expanded, while its expression in the intestinal bulb primordium appeared to be reduced in fhl1b morphants (D) compared to that of control embryos (C). (E–F′) Confocal images of control embryos (E and E′) and fhl1b morphants (F and F′) at 30 hpf, stained for Pdx1 (red; expression in the dorsal pancreatic bud is outlined by white dotted circles) and Prox1 (blue). The somites are also Pdx1 positive. Compared to that of control embryos (E and E′), in fhl1b morphants (F and F′), the Pdx1 expression domain in the dorsal pancreatic bud was expanded, while the Prox1 expression domain was reduced. Note that the Pdx1 expression domain in the intestinal primordium (yellow brackets) appeared to be decreased in morphants (F′). (G-H′) Confocal images of Tg(ins:GFP)zf5 control embryos (G and G′) and fhl1b morphants (H and H′) at 36 hpf, stained for Islet (red; expression in the dorsal pancreatic bud is outlined by white dotted circles) and Prox1 (blue in G and H; grey in G′ and H′). In fhl1b morphants (H and H′), the Prox1 expression domain was greatly reduced, whereas the number of Tg(ins:GFP)zf5- and Islet-expressing cells was increased. (I and J) Confocal images of Tg(sox17:GFP)s870 control embryos (I) and fhl1b morphants (J) at 55 hpf, stained for Insulin (red; outlined by white dotted circles) and Prox1 (blue). fhl1b morphants (J) continuously exhibited an enlarged Insulin-expressing β-cell population with a reduced number of Prox1-positive cells in the liver. (K) Quantification of the number (mean±SD) of Insulin-positive cells in the pancreas (red) and Prox1-positive cells in the liver (blue) at 55 hpf. 33.9±2.1 cells were Insulin-positive in control embryos, whereas 57.8±3.6 cells were Insulin-positive in fhl1b morphants. 148.0±15.2 cells expressed Prox1 in fhl1b morphants, while 262.7±14.0 cells were Prox1-positive in control embryos. Cells in 20 planes of confocal images from 5 individual embryos were counted. Asterisks indicate statistical significance: ***, P < 0.001. A-D, dorsal views, anterior to the left. E-J, confocal projection images, ventral views, anterior to the top. Scale bars, 20 µm.

Gene Expression Details
Gene Antibody Fish Conditions Stage Qualifier Anatomy Assay
GFP s870Tg control Long-pec endoderm IHC
s870Tg + MO1-fhl1b + MO2-fhl1b standard conditions Long-pec endoderm IHC
zf5Tg control Prim-25 insulin secreting cell IHC
zf5Tg + MO1-fhl1b + MO2-fhl1b standard conditions Prim-25 insulin secreting cell IHC
hhex WT control Prim-15 liver ISH
Prim-15 posterior pancreatic bud ISH
WT + MO1-fhl1b + MO2-fhl1b standard conditions Prim-15 liver ISH
Prim-15 posterior pancreatic bud ISH
pdx1 WT control Prim-15 intestine ISH
Prim-15 Not Detected liver ISH
Prim-15 pancreatic bud ISH
Prim-15 posterior pancreatic bud ISH
WT + MO1-fhl1b + MO2-fhl1b standard conditions Prim-15 intestine ISH
Prim-15 posterior pancreatic bud ISH
Antibody Labeling Details
Antibody Assay Fish Conditions Stage Anatomy
Ab2-isl IHC s870Tg control Long-pec insulin secreting cell
IHC s870Tg + MO1-fhl1b + MO2-fhl1b standard conditions Long-pec insulin secreting cell
IHC zf5Tg control Prim-25 posterior pancreatic bud
IHC Prim-25 primary islet
IHC zf5Tg + MO1-fhl1b + MO2-fhl1b standard conditions Prim-25 posterior pancreatic bud
IHC Prim-25 primary islet
Phenotype Details
Fish Conditions Stage Phenotype
WT + MO1-fhl1b + MO2-fhl1b standard conditions Prim-15 intestine pdx1 expression decreased distribution, abnormal
Prim-15 liver hhex expression decreased distribution, abnormal
Prim-15 liver development decreased occurrence, abnormal
Prim-15 pancreas induction increased occurrence, abnormal
Prim-15 posterior pancreatic bud hhex expression increased distribution, abnormal
Prim-15 posterior pancreatic bud pdx1 expression increased distribution, abnormal
s870Tg + MO1-fhl1b + MO2-fhl1b standard conditions Long-pec insulin secreting cell ab2-isl labeling increased distribution, abnormal
Long-pec insulin secreting cell increased amount, abnormal
Long-pec liver development decreased occurrence, abnormal
Long-pec pancreas induction increased occurrence, abnormal
zf5Tg + MO1-fhl1b + MO2-fhl1b standard conditions Prim-25 insulin secreting cell GFP expression increased distribution, abnormal
Prim-25 insulin secreting cell increased amount, abnormal
Prim-25 liver development decreased occurrence, abnormal
Prim-25 pancreas induction increased occurrence, abnormal
Prim-25 posterior pancreatic bud ab2-isl labeling increased distribution, abnormal
Prim-25 primary islet ab2-isl labeling increased distribution, abnormal
Acknowledgments:
ZFIN wishes to thank the journal PLoS Genetics for permission to reproduce figures from this article. Please note that this material may be protected by copyright. Full text @ PLoS Genet.