EMBO/soda-nel huggingface dataset:
The kinetochore module Okp1CENP-Q/Ame1CENP-U is a reader for N-terminal modifications on the centromeric histone Cse4CENP-A (2018)
Figure 2

Figure 2: Mutations in OKP1 suppress growth defects of mutation of Cse4-R37.

A Overview of the amino-acid sequence of Cse4. R37Me and K49Ac sites are part of the essential N-terminal domain (END, aa 28 - 60, yellow) and are indicated in red. The localization of α-helices in the histone fold domain are shown in grey. Amino acid residues that are relevant for this study are indicated with numbers.

B okp1-R164C suppressed the temperature-sensitive growth defect of cbf1∆ cse4-R37A. Serial dilutions of strains with the indicated genotypes were spotted on full medium and grown for three days at 30ºC or 37ºC. The original okp1-R164C isolate from the suppressor screen is indicated as "UV mutagenesis".

C okp1-R164C suppressed the growth defect of cse4-R37A with chl4∆. Representation as in (B).

D okp1-R164C was unable to suppress the lethality of cse4-R37A with ame1-4. Tetrad dissection of a genetic cross between a cse4-R37A okp1-R164C and an ame1-4 strain is shown. The four spores from individual asci are aligned in vertical rows.

E okp1-R164C suppressed the maintenance defect of cse4-R37A for plasmids lacking the CDEI sequence of CEN6 (CEN ∆CDEI, at 37ºC). Error bars give SD of at least three independent transformants. * p = 0.03.

F The okp1 mutations I45T, S94T and E208V suppressed the temperature-sensitive growth defect of cbf1∆ cse4-R37A. Strains with the indicated okp1 alleles on a plasmid (derivatives of AEY5584) are shown as in (B).
The kinetochore module Okp1CENP-Q/Ame1CENP-U is a reader for N-terminal modifications on the centromeric histone Cse4CENP-A (2018)
Figure 3

Figure 3: okp1-R164C restored binding of kinetochore components to centromeric sequences.

A okp1-R164C restored binding of Okp1 to centromeric sequences. ChIP of 9xmyc-tagged Okp1 was performed in strains with the indicated genotypes that were grown at 37ºC for 4 hours before ChIP. Enrichment of CEN4 and POL1 (as a control) relative to input is given. Below, Western blot analysis of the amounts of 9xmyc-Okp1 and histone H2B (loading control) in whole cell extracts.

B okp1-R164C restored binding of Ame1 to centromeric sequences. ChIP of 9xmyc-tagged Ame1 was performed as in (A). Below, Western blot analysis of the amounts of 9xmyc-Ame1 and H2B as in (A).

C okp1-R164C restored binding of Mtw1 to centromeric sequences. ChIP of 9xmyc-Mtw1 and Western blots (right) are presented as in (A) and (B).

D okp1-R164C improved in vivo association of Okp1 with Cse4-R37A. Co-immunoprecipitation of Cse4-R37A and Okp1 was carried out in cells carrying cse4-R37A with (+) or without (-) 3xHA-tag and OKP1 or okp1-R164C with (+) or without (-) 9xmyc-tag (AEY5040, AEY5972, AEY5973, AEY6589, AEY6592). Cells were grown at 23ºC or were shifted to 37ºC for 5 hours prior to harvesting. Cse4-R37A was immunoprecipitated using an α-HA antibody, and the presence of Cse4-R37A and Okp1 or Okp1-R164C in the immunoprecipitate was tested by Western blotting with α-HA and α-myc antibody, respectively. Left, inputs (the two bands indicate Okp1 and a shorter degradation product); right, α-HA immunoprecipitates.

Data information: (A-C) Mean ± SD, n = 4 (p-values determined by Student's t-test).
The kinetochore module Okp1CENP-Q/Ame1CENP-U is a reader for N-terminal modifications on the centromeric histone Cse4CENP-A (2018)
Figure 4

Figure 4: The N-terminus of Cse4 interacts in vitro with Okp1/ Ame1.

A Cse4(21-129, Cse4N) and Okp1/ Ame1 were expressed and purified separately from bacteria and co-incubated before separation by analytical gel filtration. Top, representative size-exclusion chromatography (SEC) chromatogram with absorbance measurements at 280 nanometers (nm) is shown. Below, image of Coomassie Blue-stained SDS-PAGE gels with fractions from principal SEC peaks of runs with Okp1/ Ame1 alone (top), Cse4N alone (middle) and Okp1/ Ame1 pre-incubated with Cse4N before SEC (bottom). Molecular weight standards are shown in kilodalton (kDa).

B Intersubunit and self-link map for the Cse4N/ Okp1/ Ame1 complex formed in vitro shows direct contacts of Cse4N with both Okp1 and Ame1. Self-links and intersubunit cross-links are coloured in purple and green, respectively.

C The ame1 mutations S59P, N159H, S275N and a C-terminal truncation after aa 273 (ame1-273*) suppressed the growth defect of cbf1∆ cse4-R37A. cbf1∆ cse4-R37A ame1∆ strains carrying the indicated plasmid-borne ame1 alleles are shown as in Fig. 2B.
The kinetochore module Okp1CENP-Q/Ame1CENP-U is a reader for N-terminal modifications on the centromeric histone Cse4CENP-A (2018)
Figure 5

Figure 5: Acetylation of Cse4 K49 counteracts mutation of Cse4-R37.

A Cse4-K49Ac and -R37Me are increased in S-phase of the cell cycle. Cells carrying 3xHA-tagged Cse4 were used to immunoprecipitate Cse4 with an α-Cse4-K49Ac (left, top) and an α-HA antibody (left, bottom). To detect Cse4-R37 methylation, 3xHA-tagged Cse4 was immunoprecipitated with an α-Cse4-R37Me2a (right, top) (Samel et al., 2012) and an α-HA antibody (right, bottom). Western blots were probed for the presence of Cse4 with the α-HA antibody. G1, treatment with α-factor; S, arrest with hydroxyurea; G2/ M, arrest in nocodazole; asynch, asynchronous culture.

B Mimicking the unacetylated state of Cse4-K49 (cse4-K49R) partially suppressed the temperature-sensitivity of cbf1∆ cse4-R37A. cbf1∆ cse4∆ strains carrying the indicated cse4 alleles on a plasmid were spotted on full medium and grown as in Fig. 2B.

C cse4-K49R suppressed the lethality of cse4-R37A with ctf19∆. Tetrad dissection of a ctf19∆ strain crossed with cse4-R37A (left) and cse4-R37A K49R (right). Representation as in Fig. 2D.

D Acetylation of Cse4-K49 was reduced in the absence of Gcn5 and the SAGA complex, but not in the absence of the HAT Sas2. Cse4 was precipitated as in (A) from strains wild-type (wt) or gcn5∆ (top) or sas2∆ strains (bottom) carrying HA-tagged Cse4. w/o, control immunoprecipitation without antibody.

E Cse4-K49Ac depended on the SAGA components Ada2 and Spt7. Cse4-K49Ac was determined as in (A, left) with cells carrying ada2∆ or spt7∆.
The kinetochore module Okp1CENP-Q/Ame1CENP-U is a reader for N-terminal modifications on the centromeric histone Cse4CENP-A (2018)
Figure 6

Figure 6: Methylation of Cse4-R37 and acetylation of Cse4-K49 inhibit binding of Cse4 to Okp1/ Ame1.

A A synthetic 78-aa Cse4 peptide (aa 33 - 110) binds to Okp1/ Ame1 in vitro. Microscale thermophoresis (MST) measurement of binding of fluorescently labeled Cse4 (33 - 110) to Okp1/ Ame1 is shown. Normalized fluorescence, from which the fluorescence of unbound Okp1/ Ame1 was subtracted (∆F norm), is plotted against Cse4(33-110) concentration (logarithmic scale). Mean values ± SD (n = 3) are shown.

B Plot of MST measuring the competition of fluorescently labelled Cse4 (33 - 110) bound to Okp1/ Ame1 (black) (or Okp1-R164C/ Ame1, red) with unlabelled Cse4 (33 - 87) that is unmodified or monomethylated on R37 (R37Me). Normalized fluorescence from which the fluorescence of unbound Cse4 (33 - 110) was subtracted (∆F norm) is plotted against peptide concentration. Mean values ± standard deviation (n = 3) are given. Control, normalized fluorescence of Cse4 (33 - 110) alone at increasing concentrations.

Tables

Table 1: Overview of the suppression of cse4-R37A phenotypes by okp1-R164C and ame1-273*

Suppression by

okp1-R164C

Suppression by

by ame1-273*

n.d., not determined

Table 2: Cse4-R37 methylation and K49 acetylation inhibit the in vitro binding of Cse4 to Okp1/ Ame1, and binding is restored by Okp1-R164C. Mean ± SD values (n = 3) of measurements by microscale thermophoresis as performed in Fig. 6 are given.
Synergistic prostaglandin E synthesis by myeloid and endothelial cells promotes fetal
hematopoietic stem cell expansion in vertebrates (2022)
Figure 1

Figure 1. The PGE2 synthesis pathway in zebrafish CHT.

(A) Experimental outline of qPCR analysis after dissection of CHT (caudal region) at 48 hpf from kdrl:GFP, ikaros:GFP, mpeg1:GFP, mpx:GFP transgenic animals, and FACS-sorted GFP-positive cells to purify ECs, HSPCs, macrophages and neutrophils.

(B-C) The phospholipases pla2g4aa and pla2g4ab are highly expressed in neutrophils.

(D-E-F) The expression of cyclooxygenases cox1, cox2a and cox2b is enriched in macrophages.

(G-H) The prostaglandin synthases ptges3a and ptges3b are only expressed by ECs.

(I-O) The prostaglandin receptors ptger1a, ptger1b, ptger2a, ptger3, ptger4a are mostly expressed in HSPCs.

(P-Q) The prostaglandin transporters slco2b1 and abcc4 are specifically expressed in ECs.

Data information: For each panel, data represents biological triplicates plated in technical duplicates. Statistical analysis was completed using one-way ANOVA, multiple comparison test. * P<.05; **P<.01; ***P<.001; ****P<.0001. Center values denote the mean, and error bars denote s.e.m.
Synergistic prostaglandin E synthesis by myeloid and endothelial cells promotes fetal
hematopoietic stem cell expansion in vertebrates (2022)
Figure 2

Figure 2. The loss of HSCs after myeloid ablation can be rescued by PGE2 or PGH2 treatments.

(A) Schematic indicating the imaging area in the tail at 72hpf, as indicated by the black box; fluorescence imaging of the CHT in double transgenic cd45:CFP-NTR/runx1:mcherry embryos in DMSO and after treatment with MTZ and PGH2 or PGE2.

(B) Quantification of runx1:mcherry positive-cells in double transgenic cd45:CFP-NTR/runx1:mcherry embryos in DMSO and after treatment with MTZ and PGH2 or PGE2.

(C) Quantification of cd45:CFP positive-cells in double transgenic cd45:CFP-NTR/runx1:mcherry embryos in DMSO and after treatment with MTZ and PGH2 or PGE2.

Data information: Statistical analysis was completed using one-way ANOVA, multiple comparison test. * P<.05; **P<.01; ***P<.001 ****P<.0001. Scale bar 200μm (A).
Synergistic prostaglandin E synthesis by myeloid and endothelial cells promotes fetal
hematopoietic stem cell expansion in vertebrates (2022)
Figure 3

Figure 3. The deficiency of slco2b1 induces a decrease of HSPC in the CHT.

(A) WISH for cmyb expression at 60hpf in wild type and slco2b1-/- embryos.

(B) Quantification of cmyb-expressing cells. Each n represents the number of cmyb-expressing cells for each embryo (biological replicates). Each experiment has been repeated three independent times.

(C) Schematic indicating the imaging area in the tail at 60hpf, as indicated by the black box; fluorescence imaging in the CHT of kdrl:mCherry;cmyb:GFP embryos injected with control- and slco2b1-Mos.

(D) Quantification of HSPCs associated to ECs. Each n represents the number of yellow spots for each embryo (biological replicates). Each experiment has been repeated three independent times.

(E) Experimental outline and quantification of time-lapse live imaging in controls and slco2b1-morphants cmyb:GFP+ cells, using Imaris software.

Data information: Centre values denote the mean, and error values denote s.e.m. The statistical analysis was completed using an unpaired two tailed t test. ***P < .001 Scale bar 500μm (A); 200μm (C)
Synergistic prostaglandin E synthesis by myeloid and endothelial cells promotes fetal
hematopoietic stem cell expansion in vertebrates (2022)
Figure 4

Figure 4. The defect in HSPCs proliferation in slco2b1-deficient embryos can be rescued by PGE2 treatment.

(A) Anti-GFP and pH3 immunostainings of either controls (Ctrl-Mo) or slco2b1-morphants (slco2b1-MO) cmyb:GFP embryos.

(B) Quantification of the number of the pH3+ HSCs in controls or slco2b1-morphants. Centre values denote the mean, and error values denote s.e.m, statistical analysis was completed using an unpaired two tailed t test. ***P < .001.

(C) Experimental outline to measure PGE2 level by ELISA-kit in control and slco2b1-MO at 60hpf.

(D) Quantification of PGE2 concentration in control and slco2b1 morphants. The statistical analysis was completed using an unpaired two tailed t test *P < .01. Centre values denote the mean, and error bars denote s.e.m.

(E) Fluorescence imaging in the CHT of cmyb:GFP embryos injected with control- and slco2b1-MOs and treated with PGE2.

(F) Quantification of GFP positive cells. Statistical analysis: one-way ANOVA, multiple comparison, *P < .01; ****P < .0001. Centre values denote the mean, and error bars denote s.e.m. Data information: Scale bar 50μm (A); 200μm (E).
Synergistic prostaglandin E synthesis by myeloid and endothelial cells promotes fetal
hematopoietic stem cell expansion in vertebrates (2022)
Figure 5

Figure 5. Slco2b1 is necessary to import PGH2 into ECs.

(A) Fluorescence imaging of the CHT of double transgenic cd45:CFP-NTR/runx1:mcherry embryos injected with control- or slco2b1-morpholinos, after treatment with DMSO.

(B) Fluorescence imaging of the CHT of double transgenic cd45:CFP-NTR/runx1:mcherry embryos injected with control- or slco2b1-morpholinos, after treatment with DMSO+MTZ.

(C) Fluorescence imaging of the CHT of double transgenic cd45:CFP-NTR/runx1:mcherry embryos injected with control- or slco2b1-morpholinos, after treatment with DMSO+MTZ+PGH2.

(D) Quantification of runx1:mcherry positive-cells. Each dot represents the number of red cells for each embryo (biological replicates). Each experiment has been repeated three independent times.

(E) Quantification of cd45:CFP positive-cells. Each dot represents the number of blue cells for each embryo (biological replicates). Each experiment has been repeated three independent times.Data information for (D) and (E): Statistical analysis was completed using one-way ANOVA, multiple comparison test. ****P<.0001. Centre values denote the mean, and error bars denote s.e.m

Scale bar 200μm (A-C).
Synergistic prostaglandin E synthesis by myeloid and endothelial cells promotes fetal
hematopoietic stem cell expansion in vertebrates (2022)
Figure 6

Figure 6. Slco2b1-overexpression in ECs can rescue the loss of HSPCs in slco2b1- morphants.

(A) WISH for cmyb at 60hpf in kdrl:Gal4+ embryos injected with control- and slco2b1- morpholinos and/or co-injected with the Tol2-UAS:slco2b1 vector and tol2 mRNA. The region indicated by the dashed bas been magnified and shows in (B). (C) Quantification and statistical analysis were completed using one-way ANOVA, multiple comparison test. ****P< .0001. Centre values denote the mean, and error bars denote s.e.m. Each dot represents the number of cmyb-expressing cells for each embryo (biological replicates). Each experiment has been repeat three independent times.
Synergistic prostaglandin E synthesis by myeloid and endothelial cells promotes fetal
hematopoietic stem cell expansion in vertebrates (2022)
Figure 8

Figure 8. Myeloid and endothelial cells are very close to HSPCs in the mouse fetal liver.

(A) Immunohistochemistry on paraffin sections for cKit (HSPCs marker) and CD31 (endothelial cell marker) at E13 and E16 stages of mouse fetal liver.

(B) Distance distribution between cKithigh cells and endothelial cells (n=79 from 3 E13 fetal livers; n=92 from 3 E16 fetal livers), binned into 20μm intervals. Statistical analysis was completed using t-test. *P<.01; **P<.001; ***P<.0001. Centre values denote the mean, and error values denote s.e.m

(C) Immunohistochemistry on paraffin sections for cKit (HSPCs marker) and F4/80 (macrophage cell marker) at E13 and E16 stages of mouse fetal liver.

(D) Distance distribution between cKithigh cells and macrophages (n=54 from 3 E13 fetal livers; n=64 from 3 E16 fetal livers), binned into 5μm intervals. Statistical analysis was completed using t-test.

(E) Immunohistochemistry on paraffin sections for cKit (HSPCs marker) and MBP (neutrophil cell marker) at E13 and E16 stages of mouse fetal liver.

(F) Distance distribution between cKithigh cells and neutrophils (n=45 from 3 E13 fetal livers; n=53 from 3 E16 fetal livers), binned into 20μm intervals. Statistical analysis was completed using t-test ***P<.001.

All nuclei marked with DAPI. Scale bar 20μm (a-c-e).
IGFBP1 Increases β-cell Regeneration by Promoting α- to β-cell Transdifferentiation (2016)
Figure 1

Figure 1. Genetic screening in zebrafish identifies igfbp1a as a promoter of β-cell regeneration.

(A) Schema of the analysis of gene expression in islets isolated from control larvae and larvae subjected to β-cell ablation. β cells were ablated by exposing nitroreductase (NTR)-expressing transgenic larvae to metronidazole (MTZ) from 3-4 dpf. Islets were then isolated, and their RNA extracted and analyzed by microarray. Out of the 470 genes that were upregulated more than 50%, 33 genes encoded proteins that harbored a signal peptide for secretion (according to the algorithm of SignalP). Excluding genes that encode enzymes or proteins with a transmembrane (TM) domain, we selected 11 genes for overexpression studies in zebrafish larvae (C-E). (B) Microarray heat map showing the upregulation in expression of the 11 candidate genes in β cell-ablated versus control islets. Igfbp1a and b were the genes whose expression increased the most after β-cell ablation. (C) Schema of the construct used for overexpression of the candidate genes (under the control of the beta-actin promoter), and expression of GFP in the heart (as an internal control for genomic integration). (D-E) Representative images at 6 dpf of Tg(ins:kaede);Tg(ins:CFP-NTR) transgenic larvae that had been injected at the 1-2-cell stage with transposase mRNA (control) or transposase mRNA + bactin:igfbp1a (bactin:igfbp1a), subjected to β-cell ablation by metronidazole (MTZ) during 3-4 dpf, and subsequently allowed to regenerate for 2 days. The GFP+ heart (arrowhead) visualizes successful integration of the construct. Islets are indicated by white arrows. Scale bars: 100 μm. (F) Quantification of β-cell regeneration at 6 dpf in control (n=23), bactin:igfbp1a-overexpressing (n=13), and bactin:igfbp1b-overexpressing (n=8) Tg(ins:kaede);Tg(ins:CFP-NTR) larvae; ***P=0.0002, ns= nonsignificant (P=0.3106). (G) Immunohistochemistry showing Igfbp1 protein expression in 6 dpf Tg(ins:GFP) following β-cell ablation between 3-4 dpf. Scale bar: 50 μm. (H) Both liver-specific (lfabp promoter; n=46) and widespread (bactin promoter; n=18) overexpression of igfbp1a increase β-cell regeneration when compared to control (n=77). (I-M) Quantification of β cells with or without β-cell ablation and igfbp1a overexpression by confocal microscopy, which detects even weakly insulin-expressing β cells. Scale bars: 15 μm. See also Figure EV1.
IGFBP1 Increases β-cell Regeneration by Promoting α- to β-cell Transdifferentiation (2016)
Figure 3

Figure 3. Cellular mechanisms of Igfbp1a’s effect on β-cell regeneration.

(A-C) Tg(ins:GFP);Tg(tp1:H2B-mCherry);Tg(ins:Flag-NTR) transgenics, with or without Tg(bactin:igfbp1a), were treated with MTZ from 3-4 dpf to ablate the β cells, and were then allowed to regenerate from 4-6 dpf. Representative confocal images at 6 dpf of control (A) and Tg(bactin:igfbp1a) (B) larvae showing a modest number of ins+ tp1+ co-expressing cells, indicated by arrows, after 2 days of regeneration. Scale bars: 15 μm. (C) Quantification of the total number of β cells (green bars) at 6 dpf, and of β cells expressing Tg(tp1:H2B-mCherry), i.e., of ductal origin (yellow bars). ****P<0.0001, ns= nonsignificant. n=23 larvae in the control group, n=17 larvae in the Tg(bactin:igfbp1a) group.

(D-F) Tg(ins:Venus-zGeminin) was examined in control and Tg(bactin:igfbp1a) larvae at 6 dpf, after β-cell ablation 3-4 dpf by using Tg(ins:Flag-NTR). (D-E) Representative confocal images at 6 dpf of control and Tg(bactin:igfbp1a) larvae showing cell cycle activation of β cells in green. Scale bars: 15 μm. (F) Quantification of the total number of β cells with activated cell cycle at 6 dpf, ***P<0.001, n=32 larvae in the control group, n=41 larvae in the Tg(bactin:igfbp1a) group. The number of β cells with activated cell cycle are displayed together with the total number of β cells in experiments with the same setup, ****P<0.0001. n=39 larvae in the control group, n=33 larvae in the Tg(bactin:igfbp1a) group.

(G-I) EdU was used as a marker for cell cycle progression. Tg(ins:H2B-GFP);Tg(ins:Flag-NTR) transgenics, with or without Tg(bactin:igfbp1a), were treated with MTZ from 3-4 dpf to ablate their β cells, and subsequently incubated with EdU during regeneration from 4-6 dpf. (G-H) Representative confocal images at 6 dpf of control and Tg(bactin:igfbp1a) larvae showing β cells in green and the β cells that had incorporated EdU in yellow (green and red overlap; arrowheads). Scale bars: 20 μm. (I) Quantification of the total number of β cells (green bars) at 6 dpf, and of β cells that incorporated EdU (yellow bars) during β-cell regeneration from 4-6 dpf. **P<0.01, ns= nonsignificant. n=16 in both the control and the Tg(bactin:igfbp1a) group.

(J-L) Tg(ins:kaede);Tg(ins:CFP-NTR) transgenics, with or without Tg(bactin:igfbp1a), were injected with a control morpholino or a morpholino that knocked down arx, which is necessary for α-cell differentiation and thus glucagon expression (J-K). Scale bars: 20 μm. (L) Quantification of the total number of regenerating β cells at 4 dpf (after β-cell ablation at 2-3 dpf). ****P<0.0001, *P<0.05, ns=nonsignificant. n=42, 20, 48 and 20, respectively.
IGFBP1 Increases β-cell Regeneration by Promoting α- to β-cell Transdifferentiation (2016)
Figure 4

Figure 4. Igfbp1a increases β-cell regeneration by potentiating α- to β-cell transdifferentiation.

(A-E) Control and Tg(bactin:igfbp1a)-overexpressing larvae were treated with MTZ from 3-4 dpf to ablate β cells, and analyzed at 6 dpf, after 2 days of regeneration. (A-B) Representative confocal images of control and Tg(bactin:igfbp1a)-overexpressing Tg(ins:dsRed);Tg(gcg:GFP);Tg(ins:Flag-NTR) larvae. A bihormonal glucagon- and insulin-expressing cell (gcg+;ins+) is indicated by an arrow. Scale bars: 20 μm. (C-D) Representative confocal images of control and Tg(bactin:igfbp1a)-overexpressing Tg(gcg:GFP);Tg(ins:Flag-NTR) larvae stained for Pdx1. Pdx1- and glucagon-expressing cells (pdx1+;gcg+) are indicated by arrows. Scale bars: 20 μm. (E) Quantification of gcg+;ins+ and pdx1+;gcg+ cells in the control and Tg(bactin:igfbp1a) group; P<0.001, P=0.0012; n=18 and 18 in the control groups, n=15 and 10 in the Tg(bactin:igfbp1a) groups, for the gcg+;ins+ and pdx1+;gcg+ quantification, respectively. (F-I) Lineage-tracing evidence supports α- to β-cell transdifferentiation. (F) Schema for temporal and spatial lineage tracing. loxP-mediated excision of the STOP-cassette permits heat-inducible expression of the stable fusion-protein H2B-GFP. Control and bactin:igfbp1a-overexpressing Tg(hsp:loxP-mCherry-STOP-loxP-H2B-GFP);Tg(gcg:CRE);Tg(ins:Flag-NTR) larvae were first heat-shocked at 3 dpf to label the glucagon-expressing cells with the stable fluorescent protein H2B-GFP, thereafter treated with MTZ from 3-4 dpf to ablate the β cells, and analyzed at 6 dpf after 2 days of regeneration. Note that the temporal control of the lineage tracing is mediated by a heat-shock. (G) Quantification of insulin-expressing cells that originate from α-cells. Control larvae, n=13; bactin:igfbp1a larvae, n=16. P=0.0051. (H-I) Representative confocal images at 6 dpf (after 2 days of regeneration) of control and bactin:igfbp1a-overexpressing larvae stained for insulin, glucagon, to-pro, and H2B-GFP. ins+;H2B-GFP+ cells are indicated by the arrowheads, and H’ and I’ show staining against only insulin and H2B-GFP. Moreover, most insulin+;H2B-GFP+ cells no longer express glucagon. Scale bars: 15 μm.