Negative cell cycle regulation by calcineurin is necessary for proper beta cell regeneration in zebrafish

  1. Laura Massoz  Is a corresponding author
  2. David Bergemann
  3. Arnaud Lavergne
  4. Célia Reynders
  5. Caroline Désiront
  6. Chiara Goossens
  7. Lydie Flasse
  8. Bernard Peers
  9. Marianne M Voz
  10. Isabelle Manfroid
  1. Zebrafish Development and Disease Models Laboratory, GIGA-Stem Cells, University of Liège, Belgium
  2. GIGA-Genomics Core Facility, GIGA, University of Lièg, Belgium
7 figures, 1 table and 3 additional files

Figures

Figure 1 with 1 supplement
Transcriptomic profiling of ductal cells during beta cell regeneration and validation in larvae.

(A, B) Enrichment ratio of selected non-redundant signatures of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways overrepresented in ductal cells after beta cells ablation (UP – A and DOWN – …

Figure 1—figure supplement 1
Transcriptomic profiling of ductal cells during beta cell regeneration and validation in larvae.

(1) Design of the experimental pipeline for transcriptomic experiment. (2) Expression of ppp3cca; ppp3ccb; nfatc3a; nfatc3b in acinar, alpha, beta, delta, or ductal cells population from the …

Figure 2 with 1 supplement
Calcineurin inhibition with Cyclosporin A (CsA) increases the ductal regenerative response.

(A) Experimental design for regeneration test in larvae with CsA treatment. Briefly, after nifurpirinol treatment from 3 to 4 dpf, larvae were treated with CsA from 1 to 3 dpt and fixed and analyzed …

Figure 2—figure supplement 1
Calcineurin inhibition with Cyclosporin A (CsA) increases the ductal regenerative response.

(1) Whole mount fluorescent immunohistochemistry (mCherry) of the pancreas of Tg(ins:NTR-P2A-mCherry) larvae at 4–7 and 14 dpt. 3D projection (stack) of ablated larvae treated with DMSO or CsA …

Transgenic mediated overexpression of calcineurin abolish the ductal regenerative response.

(A) Experimental design for regeneration test in larvae with heat-shocks. Briefly, after nifurpirinol treatment from 3 to 4 dpf, four heat-shock were performed from 1 to 3 dpt and larvae were fixed …

Figure 4 with 1 supplement
Calcineurin (CaN) repression potentializes the effect of Notch inhibition on beta cell formation.

(A) Experimental design for Notch inhibition test in non-ablated condition. Larvae were treated concomitantly with LY411575 (Notch inhibitor) and Cyclosporin A (CsA) from 3 to 4 dpf and were fixed …

Figure 4—figure supplement 1
Calcineurin (CaN) repression potentializes the effect of Notch inhibition on beta cell formation.

(1) Experimental design for Notch inhibition test in non-ablated condition. Larvae were treated concomitantly with LY411575 (Notch inhibitor) and FK506 from 3 to 4 dpf and were fixed and analyzed at …

Figure 5 with 1 supplement
Calcineurin (CaN) repression increases the proportion of ductal proliferating cells.

(A) Experimental design for 5‐ethynyl‐2′‐deoxyuridine (EdU) assay in Notch test. Larvae were treated concomitantly with LY411575 (Notch inhibitor) and Cyclosporin A (CsA) from 3 to 4 dpf and then …

Figure 5—figure supplement 1
Calcineurin (CaN) repression increases the proportion of duct proliferating cells.

(1–3) Barplot representing the number of GFP+ ductal cells which in pancreatic tail of Tg(ins:NTR-P2A-mCherry); Tg(nkx6.1:GFP) larvae at 4 dpf for the Notch test. (1) Mild Notch inhibition with …

Figure 6 with 1 supplement
Calcineurin (CaN) regulation is important in juveniles/adults and necessary for correct glycemia recovery.

(A) Whole mount fluorescent immunohistochemistry (GFP and mCherry) of the pancreas of Tg(ins:NTR-P2A-mCherry); Tg(nkx6.1:GFP) 2-month-old zebrafish at 7 dpt. 3D projection (stack) of non-ablated and …

Figure 6—figure supplement 1
Calcineurin (CaN) regulation is important in juveniles/adults and necessary for correct glycemia recovery.

(1) Barplot representing the glycemia (mg/dl) of Tg(ins:NTR-P2A-mCherry); adult zebrafish at 14 dpt. The pink triangles represent the ablated condition; the inverted green triangles ablated + …

Model of calcineurin (CaN) action on ductal progenitors to regenerate beta cells.

Under physiological conditions, the behavior of the ductal progenitors is determined by Notch signaling. CaN is active in these progenitors and enable a proper control between proliferation and …

Tables

Key resources table
Reagent type (species) or resourceDesignationSource or
reference
IdentifiersAdditional
information
Genetic reagent (Danio rerio)TgBAC(nkx6.
1:eGFP)ulg004
PMID:26329351ZFIN:
ZDB-ALT-160205-1
Genetic reagent (Danio rerio)Tg(ins:NTR-P2A-mCherry)ulg034PMID:29663654ZFIN:
ZDB-ALT-171122-9
Genetic reagent (Danio rerio)Tg(cftr:gal4)PMID:25592226ZFIN:
ZDB-FISH-150901-25442
Genetic reagent (Danio rerio)Tg(tp1:
VenusPest)
PMID:22492351ZFIN:
ZDB-FISH-150901-8023
Genetic reagent (Danio rerio)Tg(hsp70:eGFP-
P2A-ppp3ccaCA) ulg068
This paperSee Zebrafish
husbandry
and generation
of the Tg(hsp70:eGF
P-P2A-ppp3ccaCA)
zebrafish line
Genetic reagent (Danio rerio)Tg(UAS:eGFP-P2A-ppp3ccaCA) ulg069This paperSee Zebrafish
husbandry
and generation
of the Tg(UAS:eGFP-
P2A-ppp3ccaCA)
zebrafish line
AntibodyAnti-GFP (chicken
polyclonal)
Aves LabsGFP-10201:1000
AntibodyAnti-mCherry/ds
Red
(rabbit polyclonal)
Clontech6324961:500
AntibodyAnti-glucagon
(mouse polyclonal)
SigmaG26541:300
AntibodyGoat polyclonal
anti-Chicken IgY
(H+L), Alexa Fluor 488
InvitrogenA-110391:750
AntibodyGoat polyclonal
anti-dsred 568
Invitrogen1:750
AntibodyGoat polyclonal anti-
Mouse IgG (H+L)
Cross-Adsorbed
Secondary
Antibody, Alexa
Fluor 633
Invitrogen1:750
Chemical compoundNifurpirinol
(NFP)
Sigma-Aldrich32439
Chemical compoundMetronidazole
(MTZ)
Sigma-AldrichM1547
Chemical compoundCyclosporine A
(CsA)
SelleckchemS2286
Chemical compoundLY411575Sigma-AldrichSML0506
Chemical compoundCHIR990211SellekchemCT99021
Commercial assay or kitGateway LR
Clonase II
Enzyme mix
Invitrogen11791020
Commercial assay
or kit
Gateway BP
Clonase
II Enzyme
mix
Invitrogen11789020
Sequence-based
reagent
IM369This paperPCR primergaagaaaaccccggtcctatgtcgacgaaagagccgaaag
Sequence-based reagentIM380This paperPCR primerccttacacattcccgtcagtgc
Sequence-based
reagent
IM371This paperPCR primerCGGCTCTTTCGTCGACATAGGACCGGGGTTTTCTTCCACG
Sequence-based reagentO226This paperPCR primerGCCACCATGGTGAGCAAGGGCGAGGA
Sequence-based reagentIM370This paperPCR primerttattagatcttatttctgatcacctcctt
Sequence-based reagentIM459This paperPCR primercacacgaattcgccgccaccATGGTGAGCAAGGGCGAG
Sequence-based reagentIM460This paperPCR primerggatcggtcgagatccttacGATCTTATTTCTGATCACCTCCTTACG
Sequence-based reagentIM457This paperPCR primerGTAAGGATCTCGACCGATCCTG
Sequence-based reagentIM458This paperPCR primerGGTGGCGGCGAATTCGTG
Commercial assay
or kit
Nextera XT DNA
Library kit
IlluminaFC-131–1024
Commercial assay
or kit
Click-iT EdU
Cell Proliferation
Kit for
Imaging,
Alexa
Fluor 647 dye
InvitrogenC10340
Software, algorithmImarisBitplane
(http://www.bitplane.com/imaris/imaris)
RRID:SCR_007370Version 9.5
Software, algorithmGraphPad
Prism
GraphPad
Prism
(https://graphpad.com)
RRID:SCR_015807Version 8
Software, algorithmDESeq2DESeq2
(https://bioconductor.org/packages/release/bioc/html/DESeq2.html)
RRID:SCR_015687
Software, algorithmWebGestaltWebGestalt
(http://www.webgestalt.org/)
RRID:SCR_006786

Additional files

Supplementary file 1

Table of DE (differentially expressed) genes od ductal cells 3 days after beta cell ablation compared to non-ablated controls.

https://cdn.elifesciences.org/articles/88813/elife-88813-supp1-v1.xlsx
Supplementary file 2

Table of ORA analysis results with data from Supplementary file 1.

https://cdn.elifesciences.org/articles/88813/elife-88813-supp2-v1.xlsx
MDAR checklist
https://cdn.elifesciences.org/articles/88813/elife-88813-mdarchecklist1-v1.pdf

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