Reduced synchroneity of intra-islet Ca2+ oscillations in vivo in Robo-deficient β cells
- Department of Cell and Regenerative Biology, University of Wisconsin-Madison, United States
- Department of Bioengineering, University of Colorado Denver, Anschutz Medical Campus, United States
- Herman B Wells Center for Pediatric Research and Center for Diabetes and Metabolic Diseases, Indiana University School of Medicine, United States
- Department of Medicine, Division of Endocrinology, Diabetes, and Metabolism, University of Wisconsin-Madison, United States
- Barbara Davis Center for Diabetes, University of Colorado Anschutz Medical Campus, United States
- Kovler Diabetes Center and the Department of Medicine, University of Chicago, United States
Figures
Figure 1 with 1 supplement
Robo βKO islets have a decreased ratio of homotypic nearest neighbors than controls.
(A) Immunofluorescence images (left and middle panels) and cell connectivity maps generated by nearest-neighbor analysis (right panels) of control and Robo βKO islets. β cells (red), α cells …
Figure 1—figure supplement 1
Robo βKO islets retain β cell differentiation and maturity, and Ca2+ regulatory genes.
Volcano plot of differential gene expression from bulk RNA sequencing on lineage-traced FACS-sorted β cells from Robo βKO and control mice showing no significant differential gene expression of …
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Figure 1—figure supplement 1—source data 1
RNA sequensing source data.
- https://cdn.elifesciences.org/articles/61308/elife-61308-fig1-figsupp1-data1-v2.xlsx
Figure 2
Dissociated Robo βKO β cells show no difference in glucose-stimulated Ca2+ oscillations.
(A) Representative [Ca2+]i trace (Fura2) of a dispersed β cell from a control islet. 10G line marks the addition of 10 mM glucose. (B) Representative [Ca2+]i trace (Fura2) of a dispersed β cell from …
Figure 3
Control islets show highly synchronized whole-islet [Ca2+]i oscillations.
(A) Control islet in vivo in an AAV8-RIP-GCaMP6s-injected mouse showing GCaMP6s in green, nuclear mCherry β cell lineage tracing in red, and collagen (second harmonic) in blue. (B) Stills from a …
Figure 4 with 3 supplements
Robo βKO islets show decreased synchronization of whole-islet [Ca2+]i oscillations.
(A) Robo βKO islet in vivo in an AAV8-RIP-GCaMP6s-injected mouse showing GCaMP6s in green, nuclear mCherry β cell lineage tracing in red, and collagen (second harmonic) in blue. (B) Stills from a …
Figure 4—figure supplement 1
Robo βKO islets show uncoordinated whole-islet [Ca2+]i oscillations.
(A) Robo βKO islet in vivo in an AAV8-RIP-GCaMP6s-injected mouse showing GCaMP6s in green, nuclear mCherry β cell lineage tracing in red, and collagen (second harmonic) in blue. Scale bar is 100 μm. …
Figure 4—figure supplement 2
Robo βKO islets show uncoordinated whole-islet [Ca2+]i oscillations regardless of imaging speed or islet size while a subset display wildtype-like wave properties.
(A) Largest fraction of area in islet exhibiting coordinated [Ca2+]i oscillations for control and Robo βKO islets imaged at ~1 Hz. Control n = 3 islets collectively from two mice, Robo βKO n = 4 …
Figure 4—figure supplement 3
A subset of Robo βKO islets show coordinated whole-islet [Ca2+]i oscillations.
(A) Robo βKO islet in vivo in an AAV8-RIP-GCaMP6s-injected mouse showing GCaMP6s in green, nuclear mCherry β cell lineage tracing in red, and collagen (second harmonic) in blue. Scale bar is 100 µm. …
Figure 5
Network dynamics differ in Robo βKO islets compared to controls.
(A) Connectivity maps of control (left panel top and bottom), asynchronous Robo βKO (middle panel top and bottom), and synchronous Robo βKO (right panel top and bottom) islets. Nodes are connected …
Figure 6 with 1 supplement
Amount and perfusion of vascularization remains unchanged in Robo βKO islets.
(A) Representative immunofluorescent staining of collagen IV marking vasculature showing similar amounts in Robo βKO and control islets. (B) Representative immunofluorescent staining of laminin …
Figure 6—figure supplement 1
Robo βKO shows normal innervation within the islet.
(A) Immunofluorescent images of acetylated-tubulin3 (Tubb3) marking nerves in white, insulin in red, and DAPI in blue in islets from Robo βKO and control pancreatic tissue sections. (B) …
Figure 7
Amount of Cx36 gap junctions remains unchanged in Robo βKO.
(A) Immunofluorescent images showing DAPI in blue, histone H2B-mCherry β cell lineage trace in red, F-actin (phalloidin) in green, and Cx36 in gray demonstrating normal localization of Cx36 to …
Author response image 1
Videos
Video 1
Control islets show highly synchronized [Ca2+]i oscillations.
Intravital time-course video of an islet within the in vivo pancreas of a control β cell lineage-traced mouse infected with AAV8-Ins1-GcaMP6s. Lineage-traced β cells are marked by mCherry in red and …
Video 2
Control islets show highly synchronized [Ca2+]i oscillations.
Intravital time-course video of an islet within the in vivo pancreas of a control β cell lineage-traced mouse infected with AAV8-ins1-GCaMP6s. Lineage-traced β cells are marked by mCherry in red and …
Video 3
Robo βKO islets show unsynchronized [Ca2+]i oscillations.
Intravital time-course video of an islet within the in vivo pancreas of a Robo βKO β cell lineage-traced mouse infected with AAV8-ins1-GCaMP6s and retroorbitally injected with rhodamine-dextran to …
Video 4
Robo βKO islets show unsynchronized [Ca2+]i oscillations.
Intravital time-course video of an islet within the in vivo pancreas of a Robo βKO β cell lineage-traced mouse infected with AAV8-ins1-GCaMP6s. Lineage-traced β cells are marked by mCherry in red …
Video 5
Robo βKO islets show unsynchronized [Ca2+]i oscillations.
Intravital time-course video of an islet within the in vivo pancreas of a Robo βKO β cell lineage-traced mouse infected with AAV8-ins1-GCaMP6s. Lineage-traced β cells are marked by mCherry in red …
Video 6
A subset of Robo βKO islets retain synchronized [Ca2+]i oscillations.
Intravital time-course video of an islet within the in vivo pancreas of a Robo βKO β cell lineage-traced mouse infected with AAV8-ins1-GCaMP6s. Lineage-traced β cells are marked by mCherry in red …
Video 7
A subset of Robo βKO islets retain synchronized [Ca2+]i oscillations.
Intravital time-course video of an islet within the in vivo pancreas of a Robo βKO β cell lineage-traced mouse infected with AAV8-ins1-GCaMP6s. Lineage-traced β cells are marked by mCherry in red …
Video 8
Control islet perfused with the fluorescent glucose analog 2-NBDG.
Intravital time-course video of an islet within the in vivo pancreas of a control mouse at 1 Hz imaging speed. Mouse was injected through tail vein IV with the fluorescent glucose analog 2-NBDG …
Video 9
Robo βKO islet perfused with the fluorescent glucose analog 2-NBDG.
Intravital time-course video of an islet within the in vivo pancreas of a Robo βKO mouse at 1 Hz imaging speed. Mouse was injected through tail vein IV with the fluorescent glucose analog 2-NBDG …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (Mus musculus) | Robo1tm1Matl ;Robo2tm1Rilm | PMID:26743624 | RRID:MGI:3043177 RRID:MGI:5705332 | Linked alleles provided by Xin Sun |
| Genetic reagent (M. musculus) | Ins1tm1.1(cre)Thor | Jackson Laboratory | RRID:IMSR_JAX:026801 | |
| Genetic reagent (M. musculus) | B6.FVB(Cg)-Tg(Ucn3-cre)KF43Gsat/Mmucd | MMRRC | RRID:MMRRC_037417-UCD | |
| Genetic reagent (M. musculus) | R26H2BCherry | PMID:25233132 | R26H2BCherry | Generated as described in source |
| Genetic reagent (AAV8) | VB171020-1118pud | PMID:31186447 Generated by VectorBuilder | AAV8-INS-GcAMP6s | Sequence available in Supplementary materials of source |
| Antibody | Anti-insulin (guinea pig polyclonal) | Agilent | Cat#:IR002 RRID:AB_2800361 | (1:6) |
| Antibody | Anti-glucagon (mouse monoclonal) | Sigma | Cat#:G2654 RRID:AB_259852 | (1:500) |
| Antibody | Anti-glucagon (rabbit polyclonal) | Cell Signaling | Cat#:2760S RRID:AB_659831 | (1:200) |
| Antibody | Anti-somatostatin (rabbit polyclonal) | Phoenix | Cat#:G-060-03 RRID:AB_2890901 | (1:1000) |
| Antibody | Anti-Cx36 (rabbit polyclonal) | Invitrogen | Cat#:36-4600 RRID:AB_2314259 | (1:80) |
| Antibody | Anti-Col IV (rabbit polyclonal) | Abcam | Cat#:Ab6586 RRID:AB_305584 | (1:300) |
| Antibody | Anti-laminin β1 (rat monoclonal) | Invitrogen | Cat#:MA5-14657 RRID:AB_10981503 | (1:500) |
| Antibody | Anti-Tubb3 (rabbit polyclonal) | BioLegend | Cat#:poly18020 RRID:AB_2564645 | (1:4000) |
| Antibody | Anti-guinea pig 594 (donkey polyclonal) | Jackson | Cat#:706-585-148 RRID:AB_2340474 | (1:500) |
| Antibody | Anti-guinea pig 647 (donkey polyclonal) | Jackson | Cat#:706-605-148 RRID:AB_2340476 | (1:500) |
| Antibody | Anti-rabbit 594 (donkey polyclonal) | Invitrogen | Cat#:A21207 RRID:AB_141637 | (1:500) |
| Antibody | Anti-goat 647 (donkey polyclonal) | Invitrogen | Cat#:A21447 RRID:AB_141844 | (1:500) |
| Antibody | Anti-rat 488 (donkey polyclonal) | Invitrogen | Cat#:A21208 RRID:AB_141709 | 1:500 |
| Antibody | Anti-rabbit 488 (donkey polyclonal) | Invitrogen | Cat#:A21206 RRID:AB_2535792 | (1:500) |
| Antibody | Anti-mouse 488 (donkey polyclonal) | Jackson | Cat#:715-546-151 RRID:AB_2340850 | (1:500) |
| Antibody | Anti-biotin 488 (mouse polyclonal) | Jackson | Cat#:200-542-211 RRID:AB_2339040 | (1:500) |
| Chemical compound, drug | Phalloidin-647 | Invitrogen | A22247 | (1:400) |
| Chemical compound, drug | 2-NBDG | Invitrogen | N13195 | (5 mg/mL) |
| Chemical compound, drug | Fura2-AM | Thermo Fischer | F1201 | (5 μm) |
| Peptide, recombinant protein | Accutase | Thermo Fischer | A1110501 | |
| Peptide, recombinant protein | Geltrex | Thermo Fischer | A1413302 | |
| Software, algorithm | Prism | GraphPad | RRID:SCR_005375 | |
| Software, algorithm | MATLAB | MathWorks | RRID:SCR_001622 |
Additional files
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Supplementary file 1
Differentially expressed transcripts linked to [beta] cell maturity and differeantion.
- https://cdn.elifesciences.org/articles/61308/elife-61308-supp1-v2.csv
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Supplementary file 2
Robo βKO and control oscillation patterns.
- https://cdn.elifesciences.org/articles/61308/elife-61308-supp2-v2.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/61308/elife-61308-transrepform-v2.docx
