A conserved bacterial protein induces pancreatic beta cell expansion during zebrafish development
- University of Oregon, United States
- Harvard T. H. Chan School of Public Health, United States
- The Broad Institute, United States
- Canadian Institute for Advanced Research, Canada
Figures
Figure 1
The microbiota are required for normal expansion of the larval β cell mass.
(A) Total number of β cells per larva in GF (white box plots) and CV (grey box plots) fish at 3, 4, 5 and 6 dpf. In this, and in all subsequent figures, CV data are shown in grey box plots, and GF …
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Figure 1—source data 1
Quantifications and statistical analysis of larval β cells corresponding to Figure 1A.
Exact values of N, mean, median, SD, and SEM are reported in the source data file and are highlighted in yellow, magenta, blue, green, and cyan, respectively.
- https://doi.org/10.7554/eLife.20145.004
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Figure 1—source data 2
Quantifications and statistical analysis of 6 dpf larval free glucose levels corresponding to Figure 1C.
Exact values of N, mean, median, SD, and SEM are reported in the source data file and are highlighted in yellow, magenta, blue, green, and cyan, respectively.
- https://doi.org/10.7554/eLife.20145.005
Figure 2
Specific bacterial members of the zebrafish microbiota are sufficient to rescue normal expansion of the GF β cell mass.
(A) Experimental timeline for all subsequent zebrafish experiments, unless stated otherwise.Experimental manipulations are denoted by red text. Important zebrafish developmental events are denoted …
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Figure 2—source data 1
Quantifications and statistical analysis of larval β cells corresponding to Figure 2B.
Exact values of N, mean, median, SD, and SEM are reported in the source data file and are highlighted in yellow, magenta, blue, green, and cyan, respectively.
- https://doi.org/10.7554/eLife.20145.007
Figure 3 with 1 supplement
Aeromonas secretes a factor that rescues normal expansion of the GF β cell mass.
(A) Total β cell numbers in GF, CV and GF fish treated at 4 dpf with different cell free supernatant (CFS) samples. 'Aero.' refers to bacteria of the genus Aeromonas, with each number (1, 2, 3) …
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Figure 3—source data 1
Quantifications and statistical analysis of larval β cells corresponding to Figures 3A.
Exact values of N, mean, median, SD, and SEM are reported in the source data file and are highlighted in yellow, magenta, blue, green, and cyan, respectively.
- https://doi.org/10.7554/eLife.20145.009
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Figure 3—source data 2
Quantifications and statistical analysis of larval β cells corresponding to Figure 3B.
- https://doi.org/10.7554/eLife.20145.010
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Figure 3—source data 3
Quantifications and statistical analysis of larval β cells corresponding to Figure 3C.
- https://doi.org/10.7554/eLife.20145.011
Figure 3—figure supplement 1
10165 (BefA) protein purification.
SDS-page gel image showing subsequent steps in the purification of BefA (black arrowhead) from E. coli cell lysate; lane 1: ladder, lane 2: cell lysate after IPTG induction, lane 3: supernatant from …
Figure 4 with 1 supplement
BefA is required for Aeromonas to induce GF β cell expansion.
(A) Quantification of the colony forming units (CFUs) per gut in GF fish mono-associated (MA) with either wild type (WT) or mutant (△befA) A. veronii strains for 48 hr. Dashed line denotes the limit …
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Figure 4—source data 1
Quantifications and statistical analysis of larval β cells corresponding to Figure 4B.
Exact values of N, mean, median, SD, and SEM are reported in the source data file and are highlighted in yellow, magenta, blue, green, and cyan, respectively.
- https://doi.org/10.7554/eLife.20145.014
Figure 4—figure supplement 1
BefA confers a colonization advantage in the larval zebrafish gut.
(A) Growth rates of A. veroniiWT (black trace) and A. veroniiΔbefA (grey trace) in vitro. Density measurements (OD600) were taken every half hour for 25 hours on three replicate cultures grown in …
Figure 5 with 1 supplement
BefA facilitates β cell mass expansion through proliferation.
(A, D & E) Representative 2D slices from confocal scans through the primary islets of GF, CV and BefA (10165) protein treated 6 dpf larvae. Scale bars = 40 μM. (A) Insulin promoter expressing β …
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Figure 5—source data 1
Quantifications and statistical analysis of proliferation of larval β cells corresponding to Figure 5B.
Exact values of N, mean, median, SD, and SEM are reported in the source data file and are highlighted in yellow, magenta, blue, green, and cyan, respectively.
- https://doi.org/10.7554/eLife.20145.017
Figure 5—figure supplement 1
The microbiota increase β cell neogenesis from the EPD.
Quantification of EDP localized insulin expressing cells per animal in 6 dpf CV and GF larvae.Error bars represent the standard deviation. **t520=3.28, p=0.0011.
Figure 6 with 1 supplement
Homologs of BefA encoded in the human microbiome have conserved function in zebrafish.
(A) Close homologs of BefA across microbial species. Each species is represented by its closest BefA homolog, with a minimum allowed amino acid sequence identity of 50% (relative to the query …
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Figure 6—source data 1
Quantifications and statistical analysis of larval β cells corresponding to Figure 6D.
Exact values of N, mean, median, SD, and SEM are reported in the source data file and are highlighted in yellow, magenta, blue, green, and cyan, respectively.
- https://doi.org/10.7554/eLife.20145.020
Figure 6—figure supplement 1
Amino acid sequence alignment of BefA and functionally conserved homologs.
Amino acid sequence alignment by MUSCLE.Egal = Enterococcus gallinarum homolog sequence, Eaero = Enterobacter aerogenes homolog sequence, and BefA = original Aeromonas veronii HM21 BefA sequence. …
Additional files
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Supplementary file 1
Mass spectrometry: Aeromonas CFS inventory.
Relative abundance of protein species (spectral count) within the 60–80% ammonium sulfate fraction of the A. veroniiΔT2SS CFS. BefA is listed in bold and highlighted in green (uncharacterized protein).
- https://doi.org/10.7554/eLife.20145.022
