fhl1b is a target of the Bmp2b pathway

In order to uncover novel Bmp2b target genes essential for regulating the fate of bipotential hepatopancreatic progenitors, we performed the transcriptome analysis on endodermal tissues exposed to either increased or decreased levels of Bmp2b signaling (S1A Fig). Total RNA from FACS-sorted Tg(sox17:GFP)^s870-expressing endodermal cells [33] was used in gene expression profiling analysis. Known genes showing at least a 2-fold (in the case of increased Bmp2b signaling) or a 2.75-fold (in the case of decreased Bmp2b signaling) changes with p ≤ 0.05 were clustered by biological processes, which were derived from Gene Ontology analysis using PANTHER (http://www.pantherdb.org/) (S1B Fig). A total of 998 and 1261 genes showed changes in increased (S1 Table) and decreased (S2 Table) Bmp2b signaling, respectively. Among the genes that exhibited at least a 2-fold change in expression in both conditions (S3 Table), four and a half LIM domains 1b (fhl1b), which encodes a LIM domain only protein (S2B Fig), had a prominent change in expression (S1C Fig). The microarray results were confirmed by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) analysis (Fig 1A). The transcription of fhl1b, which was detected by RT-qPCR analysis of whole embryos, starts at the 12-somite stage after the endogenous bmp2b expression is initiated in the lateral plate mesoderm (LPM) (Fig 1B; [1]). Double antibody and in situ hybridization staining in Tg(sox17:GFP)^s870 embryos revealed that fhl1b is primarily expressed in the anterior part of the endoderm, which corresponds to the prospective liver anlage, at 22–24 hours-post-fertilization (hpf) (Fig 1C, 1D, 1G and 1G”, black arrows; early-forming dorsal pancreatic bud, which gives rise exclusively to the principal islet, a cluster of endocrine cells, is marked by white and black dotted circles in G-G”). Additionally, fhl1b is expressed in the pronephric duct (Fig 1C and 1E, blue arrowheads) and heart (Fig 1C, 1E and 1F, black arrowheads) from 24 hpf onwards. Double antibody and in situ hybridization staining in TgBAC(neurod:EGFP)^nl1 [34] embryos revealed that fhl1b continues to be highly expressed in the liver when the liver has started budding from the medially migrated endodermal rod [35] at 30 hpf (Fig 1E, 1F and 1H, black arrows) and 78 hpf (Fig 1I, black arrow). At 78 hpf, levels of fhl1b are also high in patches of cells in the distal intestine (Fig 1I), low in the HPD system (Fig 1I, black bracket), and absent in most of the pancreatic cells except for a few cells in the periphery of the principal islet (Fig 1I, yellow arrow; magnified images of fhl1b expression in the principal islet in Fig 1I’ and 1I”). To confirm that Bmp2b signaling regulates fhl1b, we examined fhl1b expression in the excess or absence of Bmp2b signaling. Compared to control embryos (Fig 1J, black bracket, and Fig 1M), in embryos where bmp2b expression was induced at the 8-somite stage, which is before the initiation of endogenous bmp2b expression, fhl1b expression showed a significant anterior-posterior (A-P) and M-L expansion in the liver (Fig 1K, black bracket, and Fig 1M). Furthermore, suppression of Bmp signaling with DMH1 (a highly selective inhibitor of the BMP type I receptors Alk3 (Bmpr1a) and Alk8 (Acvr1/Alk2)) [36] led to a marked reduction of fhl1b expression in the liver at 30 hpf (Fig 1L, black bracket, and Fig 1M). These data indicate that fhl1b is a target of Bmp2b signaling and it is primarily expressed in the prospective liver anlage.

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Fig 1. fhl1b is a target of the Bmp2b pathway.

(A) Quantitative real-time PCR analysis of fhl1b in bmp2b-overexpressing or DMH1-treated embryos at 20 hours-post-fertilization (hpf). Gene expression was normalized to that of β-actin and presented as fold changes (mean±SD) against control expression. Asterisks indicate statistical significance: ***, P < 0.001. (B) fhl1b full-length transcript, which was detected by RT-qPCR analysis of whole embryos, starts to be expressed from the 12-somite stage onwards. (C-F) Whole-mount in situ hybridization showing the expression of fhl1b at 24 (C-D) and 30 (E-F) hpf. At 24 hpf (C-D), fhl1b is expressed in the anterior part of the endoderm, which corresponds to the prospective liver anlage (black arrows). Additionally, fhl1b is expressed in the pronephric duct (blue arrowhead) and heart (black arrowhead). At 30 hpf (E-F), fhl1b continues to be highly expressed in the liver (black arrows) and remains to be expressed in the pronephric duct (blue arrowhead) and heart (black arrowheads). (G-G”) Double antibody and in situ hybridization staining of fhl1b at 24 hpf in Tg(sox17:GFP)^s870 embryos. fhl1b is primarily expressed in the anterior part of the endoderm, which corresponds to the prospective liver anlage (black arrows). White and black dotted circles locate the dorsal pancreatic bud, which gives rise exclusively to the principal islet, a cluster of endocrine cells. A merged view of G’ and G” is shown in G. (H-I”) Double antibody and in situ hybridization staining of fhl1b in TgBAC(neurod:EGFP)^nl1 embryos at 30 (H) and 78 (I-I”) hpf. TgBAC(neurod:EGFP)^nl1 expression marks the posteriorly located pancreatic endocrine cells. At 30 hpf (H), fhl1b is highly expressed in the liver (black arrow). At 78 hpf (I-I”), the level of fhl1b expression is continuously high in the liver (black arrow) and in patches of cells in the distal intestine, low in the HPD system (black bracket), and absent in most of the pancreatic cells except for a few cells in the periphery of the principal islet (yellow arrow). In the principal islet, fhl1b expression is confined to the peripheral boundary and does not significantly overlap with the TgBAC(neurod:EGFP)^nl1 expression. Magnified images of fhl1b and TgBAC(neurod:EGFP)^nl1expression in the principal islet are shown in I’ and I”. (J-L) Double antibody and in situ hybridization staining showing the endogenous expression of fhl1b in the liver (black brackets), comparing control (J), bmp2b-overexpressing (K), and DMH1-treated (L) TgBAC(neurod:EGFP)^nl1 embryos at 30 hpf. fhl1b expression was greatly expanded when bmp2b expression was induced at the 8-somite stage (K), but was reduced in DMH1-treated embryos (L). (M) Quantification of the fhl1b-positive in situ hybridization signal at 30 hpf. The areas of fhl1b-positive signal were selected and measured using Image J with normalization to control. 3 individual embryos were analyzed for each condition. Asterisks indicate statistical significance: ***, P < 0.001. G-L, confocal single-plane in situ hybridization images combined with the projection images of Tg(sox17:GFP)^s870 (G-G”) and TgBAC(neurod:EGFP)^nl1 (H-L) expression, ventral views, anterior to the top. C and E, lateral views, anterior to the left. D and F, dorsal views, anterior to the left. n = 10 per each time point and condition. Scale bars, 20 μm.

https://doi.org/10.1371/journal.pgen.1005831.g001

Based on the phylogenetic tree of zebrafish Fhl1b and the related proteins in mammals, Fhl1 was selected as the mouse ortholog of zebrafish Fhl1b (S2A Fig). Fhl1 ablation exacerbated the cardiomyopathy in hypertrophic cardiomyopathy (HCM) mice [37]. Fhl1 shares 61% amino acid identity with Fhl1b (S2B Fig). We examined mRNA and protein expression of Fhl1 in developing mouse embryos. At embryonic day 8.5 (E8.5)- E9.5, Fhl1 is detected in the foregut endoderm where the liver and the pancreas are derived [3] (S2C Fig). From E10.5 onwards, Fhl1 is expressed in the liver (S2C Fig). At E14.5, Fhl1 proteins are highly expressed in the Prospero homeobox protein 1 (Prox1)-positive liver cells (S2D–S2D”‘ Fig), whereas their expression is weakly detected in the Prox1-positive pancreatic cells [38,39] (S2E–S2E”‘ Fig). These findings suggest that similar to zebrafish Fhl1b, Fhl1 is expressed in the developing liver. Taken together, these results indicate that the hepatopancreatic expression of Fhl1b and its mouse ortholog Fhl1 is evolutionarily conserved.

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

To elucidate the role of Fhl1b in regulating the fate choice of endodermal progenitors, we disrupted the function of fhl1b with morpholino oligonucleotides (MOs) either against the splice acceptor site of the second exon, which includes the start codon (MO 1), or against the splice donor site of the third exon (MO 2) (S3A Fig). At 30 hpf, either single MO 1- or MO 2- as well as a mixture of MO 1- and 2-injected embryos (morphants) showed a decrease of the hhex [40] expression domain in the liver (Fig 2A and 2B, black arrows), whereas its expression appeared to be expanded in the early-forming dorsal pancreatic bud (Fig 2A and 2B, white dotted circles). The pdx1 expression domain in morphants was also expanded in the dorsal pancreatic bud (Fig 2C and 2D, white dotted circles), whereas its expression in the intestinal bulb primordium appeared to be reduced (Fig 2C and 2D, black brackets). Immunostaining with the antibodies recognizing Pdx1 and the early liver marker Prospero homeobox protein 1 (Prox1; [35]) in Tg(sox17:GFP)^s870 embryos [33] (Figs 2E–2F’ and S4A–S4B’) as well as Islet and Prox1 in Tg(ins:GFP)^zf5 embryos ([41], Fig 2G–2H’), respectively, showed an evident reduction of the Prox1 expression domain in the liver (Fig 2E–2H’), an increase in the number of Tg(ins:GFP)^zf5-expressing and Islet-positive pancreatic endocrine cells (Fig 2G–2H’, white dotted circles), and an expansion of the Pdx1-expressing cell population in the dorsal pancreatic bud in morphants at 30–36 hpf (Figs 2E–2F’ and S4A, S4B, S4C and S4D, white dotted circles; 78.3±3.2 cells in controls vs. 101.6±4.1 cells in morphants; n = 5 per condition; P = 0.0009). The Pdx1 expression domain in the intestinal primordium appeared to be decreased in morphants (Fig 2E–2F’, yellow brackets). At 55 hpf, morphants continuously exhibited an enlarged Insulin-expressing β-cell population (Figs 2I–2K and S3C, S8I; 33.9±2.1 cells in controls vs. 57.8±3.6 cells in morphants; n = 5 per condition; P = 0.00003) with a reduced number of Prox1-positive cells in the liver (Figs 2I–2K and S3C and S8I; 262.7±14.0 cells in controls vs. 148.0±15.2 cells in morphants; n = 5 per condition; P = 0.00003). No TUNEL-positive liver cells were observed in fhl1b morphants at 48 hpf, suggesting that the small liver observed in morphants was not caused by enhanced cell death (S5A and S5B Fig). In addition to the endodermal phenotypes, morphants displayed pericardial edema and a reduced heart rate from 30 hpf onwards (S3H and S3I Fig, black arrowheads).

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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.

https://doi.org/10.1371/journal.pgen.1005831.g002

To validate the specificities of fhl1b MOs, reverse transcription polymerase chain reaction (RT-PCR) was performed. MO 1 and 2 each blocked the endogenous splice sites of fhl1b and, as a result, either a deletion of exon 2 (S3B Fig, MO 1, white asterisk) or a formation of a cryptic splice form of exon 3 (S3B Fig, MO 2, white asterisk) occurred. A mixture of both MO 1 and 2 led to the deletion of both exon 2 and 3 (S3B Fig, MO 1& 2, white asterisk). MO-mediated knockdown can often induce apoptosis via aberrant p53 activation. Hence, we performed simultaneous knockdown of tp53 and fhl1b to ameliorate apoptosis induced by MO off-targeting [42]. Single fhl1b and double fhl1b/tp53 MO-injected embryos and larvae showed no difference in the phenotypes of small liver (S3E and S3F, white circles, and S3K, S3M and S3N Fig), an increased Insulin-expressing β-cell population (S3E, S3F and S3H–S3J Fig), and pericardial edema (S3H–S3I, black arrowheads) at 55 hpf and 5 days-post-fertilization (dpf). These data indicate that fhl1b knockdown phenotypes in the endoderm and heart are independent of the p53 pathway. Throughout this study, fhl1b MOs were used as a mixture of MO 1 and 2 (total 4 ng) as each MO caused essentially the same phenotype (S3C Fig), and standard control MO was used as a negative control. Furthermore, co-injection of fhl1b mRNA with a mixture of MO 1 and 2 partially rescued the effect of fhl1b MO knockdown (S6A–S6D Fig).

To complement fhl1b MO knockdown studies, knockout of fhl1b was performed by applying the CRISPR/Cas9 nuclease targeting system [43], which has been shown to lead to highly efficient biallelic conversion in somatic cells in zebrafish [44]. We microinjected cas9 mRNA and two guide RNAs (gRNAs), which were both designed to target overlapping regions in the exon 2 of fhl1b (S7A Fig), into one-cell stage embryos. We found that 11.62% of Cas9/gRNA-treated embryos (38 out of 327 embryos) showed an enlarged Insulin-expressing β-cell population (31.6±3.51 cells in controls vs. 45.3±6.0 cells in Cas9/gRNA-treated embryos; n = 5 per condition; P = 0.02) with a reduced number of Prox1-positive cells in the liver (265±18.6 cells in controls vs. 164±16.5 cells in Cas9/gRNA-treated embryos; n = 5 per condition; P = 0.002) as in fhl1b MO knockdown embryos at 55 hpf (S7D–S7F Fig). We randomly selected 4 embryos with these phenotypes and confirmed to contain insertions/deletions (indels) with the T7 endonuclease I (T7EI) assay and Sanger sequencing. T7EI assay revealed that the percent gene modification in the 4 tested embryos was between 21.75% and 31.58% (S7B Fig). Sanger sequencing of these 4 embryos (20–30 PCR amplicons were sequenced for each embryo) confirmed site-specific insertions/deletions (indels) including 2–17 bp deletions or 2–11 bp insertions (S7C Fig). Consistent with the report that Cas9 cuts the target DNA at six base pairs upstream of the protospacer adjacent motif (PAM) [45], all mutations occurred at the 3′ end of the target sequence, further validating the sequence specificity of this targeting process. Taken together, these comparable MO knockdown and CRISPR/Cas9 knockout results suggest that Fhl1b is required for restraining endodermal progenitors from specifying to pancreatic endocrine cells and for the proper induction of the liver.

Decreased Fhl1b activity augments induction of pancreatic endocrine cells

To further analyze which pancreatic cell types are induced in fhl1b morphants, we first examined the expression of Tg(P0-pax6b:GFP)^ulg515, a pan-endocrine progenitor reporter [46]. The number of Tg(P0-pax6b:GFP)^ulg515-expressing cells increased from 82.6±4.5 in controls to 103.2±2.0 in morphants at 30 hpf (Figs 3A, 3B and 3I and S8G; n = 5 per condition; P = 0.0009). Next, we investigated which endocrine subpopulation was expanded in the morphants. The number of Insulin-expressing β-cells was increased from 30.6±1.5 in controls to 44.6±2.0 in morphants at 30 hpf (Figs 3C–3D and 3I and S8G; n = 5 per condition; P = 0.0004). While the number of Somatostatin-expressing δ-cells was also increased in morphants (Figs 3E, 3F and 3I and S8G; 20.7±0.8 cells in controls vs. 26.7±2.0 cells in morphants; n = 5 per condition; P = 0.0033), the number of Glucagon-expressing α-cells appeared unaffected in morphants (Figs 3G, 3H and 3I and S8G; 26.5±0.7 cells in controls vs. 24.6±1.5 cells in morphants; n = 5 per condition; P = 0.2). As recently reported [47], Insulin and Glucagon, but not Insulin and Somatostatin, are co-expressed in both control embryos and fhl1b morphants at 30 hpf (S8G Fig). The number of these dual-hormone expressing cells was slightly increased in fhl1b morphants at 30 hpf (S8G Fig; 8.0±1.0 cells in controls vs. 10.6±1.5 cells in morphants; n = 5 per condition; P = 0.03).

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Fig 3. Decreased Fhl1b activity augments induction of pancreatic endocrine cells.

(A-H) Confocal images showing Tg(P0-pax6b:GFP)^ulg515 (A and B, green), Insulin (C and D, red), Somatostatin (E and F, grey), and Glucagon (G and H, blue) expression at 30 hpf, comparing control embryos (A, C, E, and G) and fhl1b morphants (B, D, F, and H). The number of Tg(P0-pax6b:GFP)^ulg515 (B)- and Insulin (D)-expressing cells was significantly increased in fhl1b morphants compared to that of control embryos (A and C, respectively). The number of Somatostatin-expressing cells was also increased (F), while that of Glucagon-expressing cells appeared unaffected (H) in fhl1b morphants compared to that of control embryos (E and G, respectively). (I) Quantification of the number (mean±SD) of total and individual pancreatic endocrine hormone-expressing cells, comparing that of control embryos and fhl1b morphants at 30 hpf. The absolute number of Tg(P0-pax6b:GFP)^ulg515-, Insulin-, and Somatostatin-expressing cells was increased from 82.6±4.5, 30.6±1.5, and 20.7±0.8, respectively, in control embryos, to 103±2.0, 44.6±2.0, and 26.7±2.0, respectively, in fhl1b morphants, while that of Glucagon-expressing cells appeared unaffected (26.5±0.7 cells in controls vs. 24.6±1.5 cells in fhl1b morphants). Cells in 20 planes of confocal images from 5 individual embryos were counted. Asterisks indicate statistical significance: **, P < 0.01; ***, P < 0.001. (J-K’) Confocal images of Tg(ins:GFP)^zf5;Tg(ins:dsRed)^m1018 control embryos (J and J’) and fhl1b morphants (K and K’) at 48 hpf. Compared with the control embryos (J), fhl1b morphants showed an increased number of GFP-only-positive β-cells (K, white arrows). (L) Quantification of the number (mean±SD) of GFP- and dsRed-double positive (yellow) and GFP-only-positive (green) β-cells, comparing that of control embryos and fhl1b morphants at 48 hpf. In control embryos, 5.0±0.7 β-cells were GFP-only-positive, while 12.2±2.3 β-cells were GFP-only-positive in fhl1b morphants. Cells in 20 planes of confocal images from 5 individual embryos were counted. Asterisks indicate statistical significance: ***, P < 0.001. A-H and J-K’, confocal projection images, ventral views, anterior to the top. Scale bars, 20 μm.

https://doi.org/10.1371/journal.pgen.1005831.g003

A previous report showed that cell-autonomous suppression of Bmp signaling is critical for the induction of endocrine cells derived not only from the early-forming dorsal bud but also from the late-forming ventral bud [7]. In zebrafish, the late-forming ventral pancreas, which mostly generates pancreatic exocrine cells (acinar and duct cells) and endocrine cells, subsequently encapsulates the early-forming, pdx1-positive, dorsal pancreas, which gives rise exclusively to the endocrine cells, thus establishing the mature pancreatic structure [14]. To test the role of Fhl1b in the induction of endocrine cells from the ventral bud specifically, we examined the numbers of the newly differentiated ventral bud-derived endocrine cells in Tg(ins:GFP)^zf5;Tg(ins:dsRed)^m1018 double transgenic embryos. As dsRed takes 18–22 hours longer than GFP to mature, we can distinguish GFP only (ventral bud-derived) from GFP/dsRed double-positive (dorsal bud-derived) β-cells until at least 60 hpf [48]. At 48 hpf, the number of GFP-only-positive β-cells increased in morphants compared to that of control embryos (Figs 3J–3L and S8H; 5.0±0.7 cells in controls vs. 12.2±2.3 cells in morphants; n = 5 per condition; P = 0.0002), suggesting an augmented induction of β-cells from the ventral bud. We further quantified total and subpopulations of pancreatic endocrine cells at 72 hpf. The number of Tg(P0-pax6b:GFP)^ulg515-, Insulin-, and Somatostatin-expressing cells was increased from 98.6±3.0, 32.3±2.0, and 28.7±1.4, respectively, in control embryos, to 132.0±5.2, 54.8±3.5, and 40.0±2.1, respectively, in morphants (S8A–S8G Fig; n = 5 per condition; P = 0.0003, P = 0.0003, P = 0.006, respectively), while the number of Glucagon-expressing cells appeared unaffected (S8C, S8F and S8G Fig; 28.6±1.1 cells in controls vs. 27.3±1.5 cells in morphants; n = 5 per condition; P = 0.2). Altogether, these data suggest that Fhl1b is required for restricting the induction of pancreatic endocrine cells, specifically Insulin- and Somatostatin-expressing cells, from endodermal progenitors.

Increased Fhl1b activity suppresses specification of pancreatic cells and induces liver

In a converse experiment, we assessed the effects of ectopic expression of fhl1b on liver and pancreas induction. We overexpressed fhl1b using a heat-inducible transgene, Tg(hsp:fhl1b; hsp:GFP)^gt3. In response to heat shock, robust ectopic expression of GFP was observed in a variety of tissues throughout the embryos without any discernible body phenotype. Concurrent expression of fhl1b all over the embryos was confirmed with whole-mount in situ hybridization. When fhl1b expression was induced at the 8-somite stage, the initial time point of pdx1 expression in the pancreatic exocrine and intestinal progenitors and before the beginning of endogenous fhl1b expression, hhex expression domain was greatly expanded in the liver at 45 hpf (Fig 4A and 4B, black arrows). In these embryos, pdx1 expression was significantly reduced in the intestinal bulb primordium and ventral pancreas, which gives rise mainly to the pancreatic exocrine cells, intestine cells, and a few endocrine cells (Fig 4C and 4D, black brackets). pdx1 expression in the dorsal pancreatic bud appeared unaffected (Fig 4C and 4D, white dotted circles), consistent with the previous data that the lineage of this bud is specified primarily during the gastrulation stage [1]. To determine whether specification of pancreatic exocrine cells is affected in fhl1b-overexpressing embryos, we examined the expression of Tg(ptf1a:GFP)^jh1 [49], which is largely restricted to the developing exocrine pancreas [50], along with Prox1, which is highly expressed in the liver and developing exocrine pancreas at 50 hpf. Compared to control embryos, we found that in the embryos where fhl1b expression was induced at the 8-somite stage, Tg(ptf1a:GFP)^jh1expression was almost completely eliminated whereas the Prox1 expression domain was markedly expanded, suggesting that virtually all Prox1-expressing cells are liver cells (Figs 4E–4F’ and S9A). Quantification showed that while 235.5±7.3 cells were Prox1-positive in control embryos, 306.5±12.6 cells expressed Prox1 in fhl1b-overexpressing embryos (Figs 4G and S9B; n = 5 per condition; P = 0.000067). In contrast, the number of Tg(ptf1a:GFP)^jh1-expressing cells was decreased from 82.2±6.4 in controls to 16.0±5.2 in fhl1b-overexpressing embryos (Figs 4H and S9B; n = 5 per condition; P = 0.000004). These results suggest that Fhl1b is sufficient to inhibit specification of pancreatic exocrine cells and induce the liver.

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Fig 4. Increased Fhl1b activity suppresses specification of pancreatic cells and induces liver.

(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-overexpressing embryos (B and D, heat shock applied at the 8-somite stage) at 45 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. When fhl1b expression was induced at the 8-somite stage, hhex expression was greatly expanded in the liver (B, black arrow), while pdx1 expression in the developing gut was reduced (D, black bracket). hhex and pdx1 expression in the dorsal pancreatic bud in fhl1b-overexpressing embryos was comparable to that of control embryos. (E-F’) Confocal images showing Islet (red), Prox1 (blue in E and F; grey in E’ and F’), and Tg(ptf1a:GFP)^jh1 (green) expression at 50 hpf, comparing control embryos (E and E’) and fhl1b-overexpressing embryos (F and F’, heat shock applied at the 8-somite stage). When fhl1b expression was induced at the 8-somite stage (F and F’), the Prox1 expression domain was expanded, whereas Tg(ptf1a:GFP)^jh1 expression was drastically reduced. (G) Quantification of the number (mean±SD) of Prox1-positive cells in the liver at 50 hpf. 235.5±7.3 cells were Prox1-positive in control embryos, while 306.5±12.6 cells were Prox1-positive in fhl1b-overexpressing embryos (heat shock applied at the 8-somite stage). Cells in 20 planes of confocal images from 5 individual embryos were counted. Asterisks indicate statistical significance: ***, P < 0.001. (H) Quantification of the number (mean±SD) of Tg(ptf1a:GFP)^jh1-expressing cells in the exocrine pancreas at 50 hpf. The number of Tg(ptf1a:GFP)^jh1-expressing cells decreased from 82.2±6.4 in control embryos to 16.0±5.2 in fhl1b-overexpressing embryos (heat shock applied at the 8-somite stage). 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 and F, confocal projection images; E’ and F’, confocal single-plane images, ventral views, anterior to the top. Scale bars, 20 μm.

https://doi.org/10.1371/journal.pgen.1005831.g004

Fhl1b regulates the patterning and subsequent fate of the medial and lateral endodermal progenitors

To determine the role of Fhl1b in the M-L patterning of endodermal progenitors, which is essential for the fate decision of liver versus pancreas [1], we first examined the pdx1 gradient in the endodermal sheet of fhl1b-depleted embryos. From the 14-somite stage onwards, morphants showed a dramatic lateral expansion of the pdx1 expression domain (Fig 5A and 5B). The expression domain of neurod, which marks pancreatic endocrine progenitor cells that express high levels of pdx1 (corresponding to the cells with white asterisks in Fig 5A and 5B; [1,51]), was markedly expanded (Fig 5C and 5D). Furthermore, multiple TgBAC(neurod:EGFP)^nl1-expressing cells were found even in the lateral part of the endodermal sheet, which normally gives rise to the liver, exocrine pancreas, and intestine (Fig 5E and 5F, white arrows) [1]. Next, we performed single-cell lineage tracing experiments to examine possible cell fate changes caused by modulation of Fhl1b activity. Tg(sox17:GFP)^s870 embryos were injected at the one-cell stage with the photoactivatable lineage tracer CMNB-caged fluorescein dextran conjugate, and single endodermal cells at 3 different M-L positions (medial, lateral 1, and lateral 2) at the level of somite 2 were uncaged using a 405nm laser at the 6–8 somite stage. In consistent with earlier data [1], in control embryos, lateral 2 cells at the level of somite 2 predominantly gave rise to the exocrine pancreas, intestine, and liver, but rarely to the endocrine pancreas (Figs 5G and S10C–S10E and S10F (as L2) and S10H; in 1 out of 10 control embryos lateral 2 cells gave rise to the endocrine pancreas). In every fhl1b-depleted embryo, lateral 2 cells contributed to the pancreatic endocrine cells (Figs 5H and S10D and S10F (as fhl1b MO L2) and S10H; n = 10). Assessment of exocrine pancreas development and differentiation by analyzing the expression of Tg(ptf1a:GFP)^jh1, which labels developing exocrine pancreatic cells, as well as that of Tg(fabp10a:DsRed;ela3l:EGFP)^gz15 [52], which marks differentiated hepatocytes and pancreatic acinar cells, showed a reduced number of pancreatic exocrine cells at 72 and 96 hpf (S11A–S11D Fig). These data suggest that depletion of Fhl1b function results in the conversion from no/low to high pdx1-expressing cells, leading to a significant increase in the number of pancreatic endocrine cells along with a concomitant compromise of the development of liver and pancreatic exocrine cells, which are derivatives of no and low pdx1-expressing cells [1].

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Fig 5. Fhl1b regulates the patterning and subsequent fate of the medial and lateral endodermal progenitors.

(A-D) Whole-mount in situ hybridization showing the expression of pdx1 (A and B) and neurod (C and D), comparing that of control embryos (A and C) and fhl1b morphants (B and D) at the 14-somite stage (A and B) and 18 hpf (C and D). pdx1 is expressed at high levels in the most medial cells (white asterisks) and at low levels in the lateral cells (gray arrows). neurod is expressed in the high-level pdx1-expressing cells. In fhl1b morphants, high levels of pdx1 (white asterisks) and neurod expression were expanded laterally (B and D). (E-F) Ventral confocal images showing TgBAC(neurod:EGFP)^nl1, β-catenin (white), and Topro (blue) at 18 hpf (the notochord is outlined by yellow dashed lines). Somites are numbered from anterior to posterior (S1-S4). (E) In control embryos, TgBAC(neurod:EGFP)^nl1-expressing cells are located close to the notochord. (F) Ectopic TgBAC(neurod:EGFP)^nl1-expressing cells were found in lateral endodermal regions in fhl1b morphants (white arrows). (G and H) Confocal images of Tg(sox17:GFP)^s870 embryos at 55 hpf, stained for uncaged-Fluorescein (red) and Islet (blue). In control embryos (G), lateral 2 (L2) cells gave rise to the liver (white rhombi), intestine (white arrowhead), and exocrine pancreas (white double arrows), but rarely gave rise to the endocrine pancreas. In fhl1b morphants (H), L2 cells contributed to the Islet-positive pancreatic endocrine cells (white arrows), but not to the liver or exocrine pancreas. (I-L) Whole-mount in situ hybridization showing the expression of pdx1 (I and J) and neurod (K and L) at 18 hpf, comparing control embryos (I and K) and fhl1b-overexpressing embryos (J and L, heat shock applied at the 8-somite stage). In embryos induced to overexpress fhl1b at the 8-somite stage (J and L), neurod and high levels of pdx1 expression (white asterisks in J) were maintained, while low levels of pdx1 expression (gray arrows) were reduced. (M-N’) Confocal images of Tg(sox17:GFP)^s870 embryos at 50 hpf, stained for uncaged-Fluorescein (red) and Prox1 (blue in M and N; grey in M’ and N’). In control embryos (M and M’), lateral 1 (L1) cells gave rise to the exocrine pancreas (white double arrows) and the intestine (white arrowheads), but not to the liver. In embryos induced to overexpress fhl1b at the 8-somite stage (N and N’), L1 cells mostly contributed to the Prox1-positive liver cells (white rhombi). A-D and I-L, dorsal views, anterior to the top. G-H and M-N, confocal projection images; E-F, M’, and N’, confocal single-plane images, ventral views, anterior to the top. Scale bars, 20 μm.

https://doi.org/10.1371/journal.pgen.1005831.g005

Conversely, we examined the pdx1 gradient in fhl1b-overexpressing embryos. In Tg(hsp:fhl1b; hsp:GFP)^gt3 embryos in which fhl1b expression was induced at the 8-somite stage, medial cells, as their counterpart in control embryos, exhibited high levels of pdx1 (Fig 5I–5J, white asterisks). Consistently, neurod expression appeared unaffected (Fig 5K and 5L). In contrast, lateral cells exhibited greatly reduced levels of pdx1 compared to that of control embryos (Fig 5I–5J, gray arrows), demonstrating that fhl1b overexpression during the post-gastrulation stage led to a decrease of pdx1 expression in the pancreatic exocrine and intestinal progenitors. Next, a single lateral 1 cell in Tg(hsp:fhl1b; hsp:GFP)^gt3 embryos was heat- shocked and uncaged at the 6–8 somite stage. In every embryo where fhl1b expression was induced at the 6–8 somite stage, lateral 1 cells contributed to the liver (Figs 5N and S10D and S10G (as HS @ 8s L1) and S10H; n = 11). However, in most control embryos lateral 1 cells only gave rise to the pancreas and intestine, but not to the liver (Figs 5M and S10B and S10D and S10E and S10G (as L1) and S10H; 1 out of 10 control embryos showed contribution of lateral 1 cells to the liver). These results indicate that augmentation of Fhl1b activity decreases pdx1 expression levels in pancreatic exocrine and intestinal progenitor cells, leading them to become liver cells.

As previously reported [1], the medial cells at the 6–8 somite stage, which express high levels of pdx1, give rise mostly to pancreatic endocrine cells (S10A and S10D and S10E and S10H Fig; n = 11), indicating an early fate restriction of these cells primarily during the gastrulation stage. Intriguingly, forced induction of fhl1b during the gastrulation stage led to a significant reduction in the number of high and low pdx1-expressing cells resulting in a decrease in the number of Insulin-expressing cells and pancreatic exocrine cells (S12A–S12G Fig). Taken together, these results suggest that Fhl1b plays an essential role in determining the precise patterning of medial and lateral endodermal progenitors by directly or indirectly modulating the levels of pdx1 expression for proper fate choice of liver versus pancreas.

Relationship between Bmp2b, Fhl1b, and Id2a in fate choice of liver versus pancreas

Our data indicate that Fhl1b is a novel physiological effector of Bmp2b signaling that regulates the adequate fate choice for liver and pancreas. To investigate the epistatic relationship between Bmp2b signaling and Fhl1b, we induced bmp2b expression at the 8-somite stage in the presence or absence of fhl1b. As previously reported [1], in bmp2b-overexpressing embryos, the Prox1 expression domain in the liver was significantly expanded (S13B Fig), whereas the number of Islet-positive pancreatic endocrine cells appeared unaffected (S13B Fig; red; dorsal pancreatic bud is outlined by white dotted circle). We found that the majority of bmp2b-overexpressing fhl1b morphants exhibited an enlarged Islet-positive pancreatic endocrine cell population (S13D Fig, 80%; red; dorsal pancreatic bud is outlined by white dotted circle) with a reduced number of Prox1-positive cells in the liver (S13D Fig, 80%) as in fhl1b morphants (S13C Fig), whereas a small portion of bmp2b-overexpressing fhl1b morphants restored the developmental defects of the liver and pancreatic endocrine formation (S13D Fig, 20%). These results suggest that Fhl1b is a critical mediator of Bmp2b signaling in governing the liver versus pancreas fate decision. Furthermore, these data raise the possibility of other effector(s) of Bmp2b signaling that may act in concert with Fhl1b in this process. Hence, we analyzed the function of Id2, which has been shown to suppress the function of Neurod [19] and an immediate target of Bmp signaling [18]. Zebrafish have two id2 genes: id2a and id2b [53]. Only id2a is expressed in the liver from 30 hpf onwards [53]. We conducted loss-of-function analyses using published id2a MO [54]. While id2a morphants showed a decrease of the hhex expression domain in the liver (S13F Fig, black arrow), its expression in the dorsal pancreas appeared unaffected at 30 hpf (S13F Fig, white dotted circle). Consistently, the pdx1 expression domain in id2a morphants was comparable to that of control embryos (S13J Fig). Double id2a/fhl1b morphants exhibited synergistically more severe defects in liver formation (S13H Fig, black arrow) than that of single id2a (S13F Fig, black arrow) or single fhl1b morphants (S13G Fig, black arrow), whereas the dorsal pancreas of double morphants (S13H and S13L Fig, white dotted circles) phenocopied that of single fhl1b morphants (S13G and S13K Fig, white dotted circles). The expression of id2a in the liver biliary epithelial cells of morphants was comparable to that of control embryos at 72 hpf (S13M and S13N Fig). These data suggest that Id2a is required for hepatic outgrowth, not for the fate decision of liver versus pancreas. Given our results showing incomplete penetrance of phenotype in fhl1b morphants (S3C Fig) and restoration of the liver and pancreatic endocrine formation defects in a small portion of bmp2b-overexpressing fhl1b morphants (S13D Fig, 20%), these data indicate that other effector(s) of Bmp2b signaling may also function to regulate the liver versus pancreas fate decision at least in part (S13O Fig), while Fhl1b plays a major role to govern this process.

Modulation of Fhl1b activity regulates the capacity of β-cell regeneration

Given the critical role of Fhl1b in restricting the induction of pancreatic endocrine cells, we investigated whether altering Fhl1b activity changes β-cell regeneration efficiency. Using Tg(ins:CFP-Eco.NfsB)^s892 (abbreviated as Tg(ins:CFP-NTR)^s892) [55] together with Tg(ins:Kaede)^jh6 [56], we compared the β-cell regeneration efficiency in control vs. fhl1b MO-injected larvae. We first converted the fluorescence of the Kaede protein from green to red by exposing the larvae to UV light. This conversion permanently marked all β-cells that were present before the ablation step. We then treated the larvae from 84−108 hpf with metronidazole (MTZ) to ablate the β-cells. In this set-up, newly formed β-cells express green-fluorescent Kaede only, whereas β-cells that survive the ablation co-express red- and green-fluorescent Kaede. We observed that a greater number of green-only β-cells regenerated in fhl1b MO-injected recovering larvae than in control recovering larvae (Figs 6A–6C and S14A; 3.8±1.3 cells per islet in controls vs. 9.6±1.4 cells per islet in fhl1b MO-injected larvae; n = 10 per condition; P = 0.00000005). Conversely, we overexpressed fhl1b using Tg(hsp:fhl1b; hsp:GFP)^gt3 in conjunction with Tg(ins:CFP-NTR)^s892 and Tg(ins:Kaede)^jh6 to measure the regenerative efficiency of β-cells in control vs. fhl1b-overexpressing larvae. We found that the number of regenerated β-cells significantly decreased when fhl1b was induced at 50 hpf (S14A Fig; 3.8±1.3 cells per islet in controls vs. 1.5±0.5 cells per islet in fhl1b-overexpressing larvae; n = 10 per each condition; P = 0.0008). We further examined the underlying mechanism of how Fhl1b modulates the efficiency of β-cell regeneration. At 72 hpf, the number of Islet-positive cells in or adjacent to the HPD system dramatically decreased after inducing fhl1b at 50 hpf even in the presence of Fgf receptor inhibitor SU5402, which induces ectopic Islet1-positive cells in the HPD system [7] (S15A–S15C’ Fig). Conversely, at 72 hpf, fhl1b morphants showed a dramatic increase of pdx1 and neurod expression in the principal islet (Fig 6D–6G, white dotted circles) and in the HPD system (Fig 6D–6G, white brackets). In line with these expression data, in recovering fhl1b MO-injected larvae, multiple regenerating β-cells were found at the junction between the pancreas and the HPD system marked with 2F11 [57] (S14C–S14C” Fig, white arrows). Intriguingly, fhl1b and pdx1 exhibit a reciprocal expression pattern in control embryos at 3 dpf. The level of fhl1b expression is high in the liver (Fig 6I, black arrow) and in patches of cells in the distal intestine (Fig 6I, white dotted lines), low in the HPD system (Fig 6I, white bracket), and absent in most pancreatic cells except for a few cells in the principal islet (Fig 6I, yellow arrow). Double antibody and in situ hybridization staining in Tg(ins:GFP)^zf5 embryos at 3 dpf showed that in the principal islet, fhl1b expression is confined to the peripheral boundary and does not overlap with the centrally located β-cells (S14D Fig, yellow arrow) nor does with the δ-cells (S14E Fig, black arrowheads) but partially with a small number of α-cells (S14F Fig, black arrowheads). The pdx1 level of expression is high in the proximal intestine (Fig 6H, white dotted lines) and in most pancreatic cells, moderate in the HPD system (Fig 6H, white bracket), and absent in the liver (Fig 6H). These results indicate that the antagonistic interplay between fhl1b and pdx1 may affect β-cell regeneration by directly or indirectly modulating pdx1 and neurod expression in the HPD system.

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Fig 6. Reduction of Fhl1b activity enhances the capacity of β-cell regeneration.

(A and B) Confocal images of [Tg(ins:CFP-NTR)^s892; Tg(ins:Kaede)^jh6] control larvae (A) and fhl1b MO-injected (B) larvae at 36 hours-post-ablation (hpa) stained with Topro (blue). A greater number of β-cells regenerated in recovering fhl1b-MO injected larvae (B) compared to that of control larvae (A). (C) Quantification of the number (mean±SD) of regenerated and survived β-cells. After photoconversion and ablation, the survived β-cells are red and green (yielding a combined color of yellow), whereas the newly formed β-cells are green only. 3.8±1.3 β-cells were green-only-positive in control recovering larvae, while 9.6±1.4 β-cells expressed as green-only in fhl1b-MO injected recovering larvae. Almost no β-cells survived the ablation in both the control and fhl1b-MO injected recovering larvae. Cells in 20 planes of confocal images from 10 individual larvae were counted. Asterisks indicate statistical significance: ***, P < 0.001. (D-G) Whole-mount in situ hybridization showing the expression of pdx1 (D and E) and neurod (F and G) at 72 hpf, comparing control embryos (D and F) and fhl1b morphants (E and G). pdx1 is expressed in the pancreas including the principal islet (white dotted circles), the HPD system (white brackets), and the proximal intestine, but not in the liver. neurod is expressed mainly in the principal islet (white dotted circles) with slight expression in the HPD system (white brackets). pdx1 (E) and neurod (G) expression in the principal islet and the HPD system was greatly increased in fhl1b morphants. (H-I) Whole-mount in situ hybridization showing the expression of pdx1 (H) and fhl1b (I) in wild-type embryos at 3 dpf. (H) pdx1 is expressed in the pancreas including the principal islet, the HPD system (white bracket), and the proximal intestine (white dotted line), but not in the liver. (I) fhl1b is expressed at high levels in the liver cells (black arrow), which never express pdx1, whereas the HPD system (white bracket) expresses low levels of fhl1b. Most pancreatic cells except for a few cells in the principal islet (yellow arrow) do not express fhl1b. The distal intestine also expresses fhl1b (white dotted lines). A-B, confocal projection images, ventral views, anterior to the top. D-I, dorsal views, anterior to the left. Scale bars, 20 µm.

https://doi.org/10.1371/journal.pgen.1005831.g006

Previous studies showed that glucose is crucial for β-cell differentiation and regeneration [47,58] and acts as a potent β-cell mitogen [59–61]. To test the possibility of whether Fhl1b regulates β-cell regeneration by affecting liver-derived glucose production, we measured free glucose levels. At 3 dpf, prior to MTZ treatment, there was no significant difference in free glucose levels between control/WT, fhl1b-MO injected, and fhl1b-overexpressing larvae (S14G Fig). Free glucose levels were dramatically elevated after β-cell ablation, but were recovered to a great extent from 5–7 dpf in MTZ-treated, MTZ/fhl1b MO-injected, and MTZ/fhl1b-overexpressing larvae (S14G Fig). Importantly, normal levels of free glucose were recovered significantly faster in the MTZ/fhl1b MO-injected larvae (S14G Fig, green line) than in the MTZ-treated (S14G Fig, red line) or MTZ/fhl1b-overexpressing larvae (S14G Fig, purple line). Furthermore, MTZ/fhl1b-overexpressing larvae still had increased levels of free glucose at 7 dpf (S14G Fig, purple line) compared to MTZ-treated (S14G Fig, red line) or MTZ/fhl1b MO-injected larvae (S14G Fig, green line). Taken together, these data suggest that the activity of Fhl1b on the HPD system, rather than the liver-derived glucose production, can modulate the efficiency in restoration of functional β-cells.

Ethics statement

This study was approved by the Institutional Animal Care and Use Committee at Georgia Institute of Technology (A13075). All animal work was performed according to procedures approved by the Institutional Animal Care and Use Committee at Georgia Institute of Technology.

Zebrafish strains

This study was approved by the Institutional Animal Care and Use Committee at Georgia Institute of Technology (A13075). All animal work was performed according to procedures approved by the Institutional Animal Care and Use Committee at Georgia Institute of Technology. Adult fish and embryos were raised and maintained under standard laboratory conditions [68]. We used the following published zebrafish transgenic lines: Tg(P0-pax6b:GFP)^ulg515 [46], Tg(ins:GFP)^zf5 [41], Tg(ins:dsRed)^m1018 (from W. Driever, Freiburg), TgBAC(neurod:EGFP)^nl1 [34], Tg(sox17:GFP)^s870 [33], Tg(hsp70l:bmp2b)^f13 [69], Tg(fabp10:dsRed, ela3l:GFP)^gz12 [52], Tg(ptf1a:GFP)^jh1 [49], Tg(ins:Kaede)^jh6 [56], Tg(ins:CFP-NTR)^s892 [55], and Tg(fabp10a:CFP-Eco.NfsB)^gt1 [70]. To generate the Tg(hsp:fhl1b; hsp:GFP)^gt3, fhl1b coding sequence was amplified (forward: 5’-CCGGAATTCATGGCAAGCCGGTCCAACTG-3’, reverse: 5’-CCGGAATTCTTACAGTTTCTTGGAGCAGTCG-3’) and cloned into a vector containing a multimerized minimal heat shock promoter, which drives gfp and fhl1b transcription bi-directionally in response to a heat shock [71]. Tol2-mediated transgenesis was achieved as described [72].

Microarray and phylogenetic analysis

Tg(sox17:GFP)^s870 embryos were either crossed with Tg(hsp70l:bmp2b)^f13 to induce overexpression of bmp2b at the 8-somite stage or treated with 0.3 μM DMH1. For each condition, 100 embryos were used. At 20 hpf, sox17:GFP-positive endodermal cells from dissected zebrafish trunks containing the organ-forming area were isolated by FACS and subjected to transcriptome profiling using the Zebrafish 44K gene expression microarray (Agilent Technologies). Data with an average fold change of 2 (bmp2b overexpressing) or 2.75 (DMH1-treated) at p ≤ 0.05 were considered for GO analysis using PANTHER (http://www.pantherdb.org/). The phylogenetic tree of zebrafish Fhl1b (NM_199217) was constructed using Phylogeny.fr [73] with mammalian homologous proteins sorted by performing alignment on UniProtKB/Swiss-Prot database.

Reverse transcription quantitative real-time polymerase chain reaction

Total RNA was extracted using the Trizol Reagent (Invitrogen). cDNA synthesis was performed using Superscript III First-strand Synthesis System (Invitrogen). PCR was conducted using iTaq Universal SYBR Green Supermix in triplicate (Bio-Rad). Optimized primers targeting each gene were designed using Primer3 [74]. The StepONE Plus PCR System (Applied Biosystems) was used to obtain the Ct value. The relative gene expression of each sample was determined using the comparative Ct method with β-actin as an internal control [75]. The following primers were used: fhl1b: forward 5’-GTGAGGAAAGACGAGAAACAAG-3’, reverse 5’-GGCACATCGGAAACAATCAG-3’; β-actin: forward 5’-CGAGCTGTCTTCCCATCCA-3’, reverse 5’-TCACCAACGTAGCTGTCTTTCTG-3’; mouse Fhl1: forward 5’- ATAAGGTGGGCACCATGTCGG-3’, reverse 5’- GTGATTCCTCCAGATGTGATGG-3’.

Embryo microinjection

Knockdown of fhl1b was performed via injection of individual fhl1b MO 1 (2 ng; 5’-CCCGCGAAAAGCTGTGAGAAATAAT-3’) or MO 2 (2 ng; 5’-ATAAATATCTGTCCCCTCACCTGGC-3’) or a combination of MO 1 and 2 (4 ng; Gene Tools, LLC). A standard control MO (4 ng; 5’-CCTCTTACCTCAGTTACAATTTATA-3’) targeting a human beta-globin intron mutation was used as a negative control (Gene Tools, LLC). id2a MO (5’ -GCCTTCATGTTGACAGCAGGATTTC-3’) [54] and tp53 MO (5’- GACCTCCTCTCCACTAAACTACGAT-3’) [42]were purchased from Gene Tools, LLC. 4 ng of id2a MO or 2 ng of tp53 MO was used. The primers annealing to the first (5’- GCAAAACACTTTGCTGTGGC-3’) and the sixth (5’- GCCAGGTTGAGGGAGCATTT-3’) coding exons were used to confirm the specificity of fhl1b MOs. Sense-strand-capped fhl1b-P2A-mCherry mRNA was synthesized with mMESSAGE mMACHINE kit (Ambion). For rescue experiments, embryos were injected with 200 pg of fhl1b-P2A-mCherry mRNA with a mixture of fhl1b MO 1 and MO 2.

In situ hybridization and immunohistochemistry

Whole-mount in situ hybridization was performed as previously described [76], using the following probes: pdx1 [77], neurod [78], hhex [79], and fhl1b (template for antisense RNA probe was amplified from embryonic cDNA with the following primers: forward: 5’-CCGCTCGAGATGGCAAGCCGGTCCAACTG-3’, reverse: 5’-ACGGCTGGTCCTGGTAATTC-3’). Immunohistochemistry on whole-mount zebrafish embryos was performed as previously described [12] using the following antibodies: mouse anti-Glucagon (1:100; Sigma), mouse anti-2F11 (1:200; Abcam), mouse anti-β-catenin (1:100; BD Transduction Laboratories), chicken anti-GFP (1:1000; Aves Labs), rabbit anti-Somatostatin (1:100; MP Biomedicals), mouse anti-Islet1/2 (1:10; Developmental Studies Hybridoma Bank (DSHB), clone 39.4D5), guinea pig anti-Insulin (1:100; Sigma), rabbit anti-Prox1 (1:100; Millipore), guinea pig anti-Pdx1 (1:200; gift from C. Wright), rabbit anti-pan-Cadherin (1:1000; Sigma), goat anti-Fluorescein (1:100; Molecular Probes), rabbit anti-Carboxypeptidase (1:100; Rockland), and fluorescently conjugated Alexa antibodies (1:200; Molecular Probes). Nuclei were visualized with TOPRO (1:10000; Molecular Probes). For the TUNEL assay, embryos were fixed in 3% formaldehyde, preincubated in PBST, and then labeled with the TUNEL kit (Roche) for 1 hour at 37°C. For coimmunostaining with Prox1, sections were first incubated with primary antibodies, then with TUNEL solutions, and finally with secondary antibodies. For the detection of mouse Fhl1 protein, the entire gut, including the liver and pancreas of E14.5 mice, was isolated and fixed in 4% paraformaldehyde, then embedded in Tissue-Tek OCT compound (Sakara Finetek). 8 μm cryostat sections were obtained by using a cryostat microtome (Leica CM1520). Immunohistochemistry was performed using the following antibodies: rabbit anti-FHL1 (1:200; Abcam), goat anti-Prox1 (1:20; R&D Systems), and fluorescently conjugated Alexa antibodies (1:200; Molecular Probes). Embryos and sections were mounted in Vectashield (Vector Laboratories) and imaged on a Zeiss LSM 510 VIS confocal microscope.

fhl1b gene disruption with the CRISPR/Cas9 system

The guide RNA (gRNA) targeting sites, which are downstream of the start codon (gRNA 1: 5’- CTGTCGTGAGGACCTCAG-3’, gRNA 2: 5’- AGTGGAAAGAAGTTCGTG-3’), were selected using the online application available at crispr.mit.edu. Complementary oligonucleotides corresponding to the target sequences were annealed as previously described [44]. Annealed oligonucleotides (1 μl) were mixed with 500 ng of the gRNA cloning vector pDR274, 0.5 μl of BsaI-HF, 0.5 μl of T4 DNA ligase, 1 μl of 10× NEB buffer 2, 1 μl of 10× T4 ligase buffer, and water for a total of 10 μl. Digestion and ligation were performed in a single step as previously described [44]. The gRNAs were transcribed using HindIII-digested expression vectors as templates and the MEGAshortscript T7 kit (Life Technologies). The cas9 mRNA was transcribed using NotI-digested Cas9 expression vector and the mMESSAGE mMACHINE kit (Ambion). The mixture of 1 nl of cas9 mRNA (300–450 ng/μl) and an individual or a combination of gRNA 1 and 2 (final concentration 12.5 ng/μl) were injected into one-cell stage embryos.

T7 Endonuclease I (T7EI) assay

The genomic region flanking the target sites was amplified using PCR (forward: 5’-ACTTACACATGAGGGGCTGTG-3’, reverse: 5’- ATAGTCCTTAATGGAAAACATGCTG-3’). A total of 200 ng of the purified PCR products was denatured and re-annealed, as previously described [44] to facilitate heteroduplex formation. The re-annealed products were digested with 10 units of T7 endonuclease I (New England Biolabs) at 37°C for 30 min. The reaction was stopped by adding 1 μl of 0.5 M EDTA. Samples were analyzed by 2% agarose gel. Band intensity was quantified using ImageJ software (National Institutes of Health). Gene modification levels were estimated based on the following equation [80]:

DNA sequencing

For sequencing the target region in injected embryos, the PCR products were cloned into pGEM T-easy vector (Promega). Plasmid DNA was isolated from individual transformants and sequenced.

Chemical treatment and heat-shock experiment

Embryos were treated with 0.3 μM DMH1 (EMD Chemicals) from 12 hpf to 20 hpf or 3 μM SU 5402 (Tocris Bioscience) from 50 hpf to 72 hpf in egg water. To ablate β-cells, Tg(ins:CFP-NTR)^s892 embryos were treated with freshly prepared 5 mM metronidazole (MTZ) (Sigma) from 84 hpf to 108 hpf in the dark, followed by 24–48 hours recovery. Before ablation, Tg(ins:Kaede)^jh6-expressing β-cells were converted from green to red by exposing them to UV light. Control embryos from the same batch were treated with DMSO in egg water. Tg(hsp:fhl1b; hsp:GFP)^gt3 and Tg(hsp70l:bmp2b)^f13 embryos were heat shocked at various stages by transferring them into egg water pre-warmed at 40°C and 37°C, respectively. After a 30-minute heat shock, embryos were returned into a 28°C incubator and harvested at various stages.

Synthesis and photochemical properties of caged fluorescein dextran

Caged fluorescein dextran was synthesized by coupling a dextran-spermine conjugate to 5-carboxymethoxy-2-nitrobenzyl (CMNB)-caged carboxyfluorescein succinimidyl ester. Dextran-spermine conjugate was produced from dextran (MW 10 kDa, Molecular Probes). In the final product, 20% glucose units were bonded with spermine (estimated by ¹H NMR spectroscopy). A mixture of dextran-spermine conjugate (8 mg) and CMNB-caged carboxyfluorescein succinimidyl ester (1 mg, Molecular Probes) in 1 mL of borate buffer (0.1 M, pH 8.5) was added into a tinted tube and reacted on a vortexer at room temperature for 24 hours, protected from light. After the reaction, the solution was poured into a dialysis membrane (14000 cutoff cellulose membrane) and dialyzed with deionized water at 4°C for 1 day. The dialysate was gravimetrically filtered to remove insoluble parts and lyophilized to dryness. A total of 4.6 mg were obtained (yield 57% w/w). The average loading of CMNB-caged carboxyfluorescein on dextran was 3.2 dye molecules per dextran chain (estimated from UV-Visible spectra). Light exposure at 360 nm removed the CMNB cage and rendered the free fluorescein modified dextran. Uncaging was followed in solution by the increase in absorbance in the region of 350–550 nm and the increase in fluorescence emission between 460–700 nm. Apparent quantum yield of uncaging is calculated to be 0.0051 from fluorescence spectra.

Lineage tracing

Tg(sox17:GFP)^s870 embryos were injected with 2 nl of 0.5% caged fluorescein dextran and allowed to develop until the 6-somite stage (corresponding to 12 hours-post-fertilization (hpf)). After manual dechorionation, embryos were mounted ventrally in a mold filled with egg water. Using a Nikon Eclipse Ti confocal microscope, we visualized the endodermal sheet in live embryos at the 6–8 somite stage, and the A-P position of endodermal cells was determined by counting somites. Caged-fluorescein was activated in a single endodermal cell in each embryo with a 405 nm laser focused through a 40X objective lens. The uncaged embryos were fixed at various time points and stained with antibodies against GFP, uncaged-Fluorescein, and Islet1 or Prox1.

Glucose measurements

Glucose measurements were performed 3 times on 10 zebrafish larvae per condition using a fluorescence-based enzymatic detection kit (Biovision Inc.) [26]. The larvae were collected in 1.5 ml microcentrifuge tubes. Excess medium was removed and embryos were frozen on crushed dry ice. After thawing, 200 μl PBS was added and the larvae were homogenized using a hand-held mechanical homogenizer. Reactions were assembled on ice in black, flat bottom 96-well plates (Costar). Standard curves were generated using glucose standard solution (according to instructions) and were included in each assay. To measure glucose in embryo extracts, 15 μl of sample were used. Control reactions without sample lysate were included in each row. Reactions were incubated for 30 minutes at 37°C in the dark. Fluorescence (excitation 535 nm; emission, 590 nm) was measured using a Safire II plate reader equipped with XFLUOR4 software (v 4.51). Fluorescence values were corrected by subtracting measurements from control reactions without sample. Glucose levels were interpolated from standard curves. Each sample was measured in triplicate and each experiment repeated three times.

Statistical analysis

The p-values were calculated using an unpaired two-tailed Student t-test with Excel (Microsoft, Redmond, WA).