Screening resources, design, and controls.

(A) Transgenic line used for the primary screen, Tg(ins:PhiYFP-2a-nsfB, sst2:tagRFP)lmc01 (β/δ reporter; Walker et al., 2012), the insulin promoter drives YFP-expression in β cells (yellow), the somatostatin 2 promoter drives RFP expression in neighboring δ cells (red). Photomicrograph of the anterior region of a 7 dpf larva shows YFP and RFP labeling of the principal islet (arrow). (B) Confocal z-projection of the principal islet in a β/δ-reporter fish (scale bar: 10 µM), YFP labeling β cells (yellow) and RFP labeling δ cells (red)-note, apparent ‘orange’ co-labeling is an artifact of z-projection in 2D format. (C) Illustration of two potential mechanisms by which drug exposures could lead to increased β-cell mass: (1) enhanced endocrine differentiation, indicated by secondary (2°) islet formation (left path) and (2) increased β-cell proliferation, indicated by supernumerary β cell numbers in the principal islet (right path) in the absence of effects on endocrine differentiation-that is, no effect on 2° islet formation. (D) Schematic of the ARQiv-HTS screening process: Day 0, mass breeding produced 5000-10,000 eggs per day; Day 2 (evening), JHDL compounds were serially diluted into drug plates; Day 3, the COPAS-XL (Union Biometrica) was used to dispense individual 3 dpf larvae into single wells of drug plates, and plates were then maintained under standard conditions for 4 days; Day 7, larvae were anesthetized and reporters quantified by automated reporter quantification in vivo (ARQiv). (E) β/δ-reporter larvae were exposed to 0.1% DMSO (negative control) or the γ-secretase/Notch inhibitor DAPT (positive control) at six different concentrations from 3 to 7 dpf. ARQiv was then used to measure fluorescent signals from β cells (yellow line, left y-axis) and δ cells (red line, right y-axis). The DAPT to DMSO ratio (DAPT/DMSO) was used to indicate signal strength for each fluorophore independently, as per the primary screen. The β-cell data show a non-monotonic dose response (yellow dashed line, polynomial curve fit), with maximal signal observed at 25-50 µM DAPT. The δ-cell data show a similar trend (red dashed line, polynomial curve fit), but with approximately fourfold lower signal strength due to higher autofluorescent background in the RFP emission range.

Observation of 2o islet formation in live β/δ-reporter larvae after drug treatment.

(A, B) Representative in vivo confocal images - brightfield and fluorescence images merged-of pancreata in β/δ-reporter larvae following treatment with 0.1% DMSO (A) or a representative Hit I drug (B, Beta-estradiol) from 3 to 7 dpf. White arrows indicate 2° islets in the tail of the pancreas. Scale bar = 25 µm. (C) Percentages of larvae having 2° islets following treatment from 3 to 7 dpf with the indicated control of Hit I compounds at optimal concentrations. n > 20. negative control: 0.1% DMSO. Positive control: RO2949097 (5 µM).

Validation of endocrine differentiation induction: precocious 2° islet assay.

(A, B) Representative confocal images - brightfield and fluorescence images merged - of dissected pancreata (dashed lines) from neurod:EGFP transgenic larvae treated from 3 to 5 dpf with 0.1% DMSO (A) or a Hit I drug (B, example shown is parthenolide). Early endocrine cells are labeled with GFP (green) allowing precocious formation of 2^° islets (white arrows) to be visualized following drug exposures. (C) The number of precocious 2° islets was quantified following treatment with the indicated Hit I compounds from 3 to 5 dpf. Results obtained with the optimal concentration were plotted relative to negative (0.1% DMSO) and positive controls (RO2949097, 5 µM). Of 20 Hit I compounds tested, 11 were confirmed as Lead I drugs for inducing endocrine differentiation (optimal concentrations for validated leads are shown in parentheses). Arrows indicate drugs that inhibit NF-κB signaling. Scale bar, 25 µm. Error bars, standard error. All p-values were calculated using Dunnett′s test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. n = 5–10 larvae per condition, experiment was repeated 3 times per compound.

Validation of endocrine differentiation induction: precocious 2° islet assay (neurod reporter).

Precocious 2° islet assays were performed as per Figure 3. (A-J) Representative confocal images - brightfield and fluorescence images merged - of dissected pancreata (dashed lines) from neurod:EGFP transgenic larvae treated with indicated Lead I compounds (at optimal concentrations) from 3 to 5 dpf: (A) Thioctic acid (5 µM); (B) N-acetylmuramic acid (12.5 µM); (C) Maprotiline (5 µM); (D) β-estradiol (12.5 µM); (E) Biperiden HCl (12.5 µM); (F) Benzamidine (20 µM); (G) Dexetimide (1 µM); (H) Oxacillin (5 µM); (I) Dimethindene (S, +) maleate (1 µM); (J) Methylthiouracil (12.5 µM). Secondary islets are indicated by arrows. Scale bar, 25 µm.

Validation of endocrine differentiation induction: precocious 2° islet assay (pax6b reporter).

Precocious 2° islet assays were performed as per Figure 3. (A-L) Representative confocal images - brightfield and fluorescence images merged - of dissected pancreata (dashed lines) from pax6b:EGFP transgenic larvae treated with indicated Lead I compounds (at optimal concentrations) from 3 to 5 dpf: (A) Thioctic acid (5 µM); (B) N-acetylmuramic acid (12.5 µM); (C) Maprotiline (5 µM); (D) β-estradiol (12.5 µM); (E) Biperiden HCl (12.5 µM); (F) Benzamidine (20 µM); (G) Dexetimide (1 µM); (H) Oxacillin (5 µM); (I) Dimethindene (S, +) maleate (1 µM); (J) Parthenolide (5 µM); (K) Methylthiouracil (12.5 µM). Secondary islets are indicated by arrows. (M) The number of precocious 2° islets was quantified following treatment with the indicated Hit I compounds from 3 to 5 dpf. Results obtained with the optimal concentration were plotted relative to negative (0.1% DMSO) and positive controls (RO2949097, 5 µM). The same 11 Lead I compounds validated with the neurod:GFP reporter line (Figure 3, Figure 3 - figure supplement 1, Table 1) showed significant results - albeit producing fewer numbers of 2° islets than the neurod:EGFP line. Scale bar, 25 µm. All p-values were calculated using Dunnett′s test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. n = 5-10 larvae per condition, experiment was repeated 3 times per compound.

Validation of increased β-cell proliferation: cell counts.

(A) Quantification of β-cell numbers following incubation of ins:hmgb1-EGFP transgenic larvae from 3 to 5 dpf in one of 30 Hit compounds, 0.1% DMSO, or the Notch inhibitor RO4929097 (5 µM). 15 compounds were confirmed as Lead II drugs for increasing β-cell numbers. Arrows indicate drugs that enhance serotonin signaling. (B, C) ARQiv screen data for paroxetine: box plots of β cells (B) and δ cells (C) suggest a β cell-specific effect - that is, a dose-response in YFP but not RFP signal (dashed line, single polynomial curve fit). (D) Numbers of δ cells (red bars) and β cells (green bars) were quantified following treatment with paroxetine, 0.1% DMSO, or RO4929097. Increased β-cell numbers were seen following paroxetine and RO4929097 treatments. However, only RO4929097 increased both β and δ cells. (E) Ratio of the number of β cells to δ cells, which confirms that the number of β cells increases following paroxetine treatment relative to δ cells, suggesting cell-type selective effects. Error bars, standard error. n = 5-10 larvae per condition, experiment was repeated 2-3 times per compound. (F-H) Representative z-projection confocal images of the principal islets in dissected pancreata (post-paraformaldehyde fixation) fromTg(ins:hmgb1-EGFP; β/δ-reporter ) triple transgenic lines treated with DMSO (F), paroxetine (G), or RO4929097 (H). Shown are EGFP⁺ β-cell nuclei (green) and TagRFP⁺ δ cells (red); note, PhiYFP in the β/δ-reporter line does not withstand fixation, allowing ‘clean’ labeling of β-cell nuclei with EGFP. In addition, apparent overlap between the β-cell and δ-cell markers (i.e., occasional ‘yellow’ cells) is an artifact of z-projection images shown in 2D format. For clarity, the inset panels show a single z-slice image of partial islet showing no co-localization of cell type specific reporters. All p-values were calculated using Dunnett′s test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. N.S., non-significant. Scale bar, 10 µm.

NF-κB pathway inhibition induces endocrine differentiation.

(A, B) Representative confocal images - brightfield and fluorescence images merged - of dissected pancreata (dashed lines) from neurod:EGFP transgenic larvae treated from 3 to 5 dpf with NF-κB signaling inhibitor II (A) or III (B). Both inhibitors induced precocious secondary islet formation (white arrows). (C) Secondary islet numbers were quantified and plotted relative to vehicle control (0.1% DMSO). n = 5-10 larvae per condition, experiment was repeated 3 times. Error bar, standard error. All p-values were calculated using Dunnett′s test. ****p < 0.0001. (D, D′) Representative in vivo confocal z-projection of pancreas (dashed lines) in 6xNFκB:EGFP ;Tp1:hmgb1:mCherry double transgenic larvae at 5 dpf showing co-labeling of the NF-κB reporter (green) and Notch reporter (red) in endocrine progenitor cells (arrows in D′), suggesting endocrine progenitors respond to both Notch and NF-κB signaling. Scale bars, 25 µm (D), 10 µm (D′).

Thioctic acid and parthenolide inhibit NF-κB signaling.

(A-E) Confocal images of 5 dpf pancreata (dashed lines) from 6xNFκB:EGFP/Tp1:hmgb1-mCherry larvae treated with indicated compounds or DMSO control from 3dpf to 5dpf. The NF-κB reporter showed reduced fluorescence levels in the pancreas following exposure to all tested NF-κB inhibitors (A-D) compared with DMSO (E). Scale bar, 25 µm. (F) ARQiv scans were performed on individually tracked 6xNFκB:EGFP/Tp1:hmgb1-mCherry larvae prior to (3 dpf) and after compound exposures (5 dpf). All compounds induced a significant reduction of NF-κB reporter activity relative to 0.1% DMSO controls. GFP reporter expression levels were normalized to pre-treatment levels - that is, plotted as percent change in fluorescence over time (as per Walker et al., 2012) - and showed significant signal loss for all tested compounds. Error bar = standard deviation. NFκBi-II: NF-κB inhibitor II, NFκBi-III: NF-κB inhibitor III. All p-values were calculated using Dunnett′s test. ***p < 0.001, ****p < 0.0001.

Serotonin signaling stimulates β-cell proliferation in a cell type-specific manner.

(A) β-cell quantification following 25 µM serotonin or 25 µM fluoxetine treatment of ins:hmgb1-eGFP transgenic larvae from 3 to 5 dpf indicates enhanced serotonin signaling increases β-cell numbers in zebrafish larvae. (B) β-cell quantification in the principal islet (All) and the number of EdU-labeled β cells (EdU⁺) are plotted following treatments with EdU and either DMSO, 1 µM paroxetine, or 5 µM RO4929097. More β cells overall, and more EdU⁺ β cells, are observed with 1 µM paroxetine and 5 µM RO4929097 treatments, suggesting effects on β-cell proliferation. (C) Plot of EdU⁺ β cells as a percentage all β cells shows that paroxetine treatment stimulates β-cell proliferation, whereas Notch inhibition does not. Error bars, standard error. (D-F) Single-plane confocal fluorescence images of ins:hmgb1-eGFP islets (dashed lines) treated with EdU and either DMSO (D), 1 µM paroxetine (E), or 5 µM RO4929097 (F) - β cell nuclei (green); EdU⁺ cells (red); double-labled EdU⁺ β cells (yellow). Scale bar, 10 µm. n = 5-10 larvae per condition, experiment was repeated 3 times. (G, G2) Confocal images of immunostained adult zebrafish pancreas indicate that serotonin signaling is active in islets (white arrows, islets indicated by dashed lines). aTub: acetylated tubulin (red); 5HT (5-hydoxytryptamine): serotonin (green); insulin (magenta). Scale bar, 10 µm. (H, I) Confocal images of adult zebrafish pancreas following injections with EdU and either DMSO (H) or 1 mM paroxetine (I), and immunostained as indicated. (J) Plot of EdU⁺ β cells as a percentage all β cells shows that paroxetine treatment stimulates β-cell proliferation in adult zebrafish. Error bars, standard deviation. n = 3-5 adult fish per condition, experiment was repeated 3 times. All p-values were calculated using Dunnett′s test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.