Genomic knockout of alms1 in zebrafish recapitulates Alström syndrome and provides insight into metabolic phenotypes

Abstract

Alström syndrome (OMIM #203800) is an autosomal recessive obesity ciliopathy caused by loss-of-function mutations in the ALMS1 gene. In addition to multi-organ dysfunction, such as cardiomyopathy, retinal degeneration and renal dysfunction, the disorder is characterized by high rates of obesity, insulin resistance and early-onset type 2 diabetes mellitus (T2DM). To investigate the underlying mechanisms of T2DM phenotypes, we generated a loss-of-function deletion of alms1 in the zebrafish. We demonstrate conservation of hallmark clinical characteristics alongside metabolic syndrome phenotypes, including a propensity for obesity and fatty livers, hyperinsulinemia and glucose response defects. Gene expression changes in β-cells isolated from alms1-/- mutants revealed changes consistent with insulin hypersecretion and glucose sensing failure, which were corroborated in cultured murine β-cells lacking Alms1. We also found evidence of defects in peripheral glucose uptake and concomitant hyperinsulinemia in the alms1-/- animals. We propose a model in which hyperinsulinemia is the primary and causative defect underlying generation of T2DM associated with alms1 deficiency. These observations support the alms1 loss-of-function zebrafish mutant as a monogenic model for mechanistic interrogation of T2DM phenotypes.

Figure 1

Generation of zebrafish alms1^−/− line. (A) Schematic of alms1 genomic region with and without CRISPR/Cas9-induced deletion resulting in premature stop codon (orange). Sequence shown indicates region in exon 4 with sgRNA target (green) and deletion (red). (B) Ratios of identified mutants in heterozygous in-cross progeny indicating reduced rate of homozygous mutants relative to expected Mendelian ratios. (C) Representative images of alms1^+/+ (n = 82) and alms1^−/− (n = 70) larvae at 5 dpf. Numbers of alms1^−/− larvae having ciliopathy body dysmorphogenesis shown. Scale bar, 1 mm. (D)alms1^−/− larvae have reduced RNA expression levels compared to alms1^+/+ at 5 dpf (n = 60–90 larvae). Statistics, Student's t-test, ^****P < 0.0001. Dots, replicates; error bars, 95% confidence interval (CI). (E)alms1^−/− larvae have reduced protein levels compared to alms1^+/+ at 5 dpf.

Figure 2

alms1^−/− zebrafish display multiple defective organ systems. (A) Representative images of alms1^+/+ and alms1^−/− larvae showing severe cardiac edema at 48 h post-fertilization. Scale bar, 1 mm. Quantification of cardiac edema rates in alms1^+/+ (n = 71) and alms1^−/− (n = 44) larvae. Significance, chi-squared, ^****P < 0.0001. (B) H&E sections from alms1^+/+ and alms1^−/− zebrafish at 6 months showing gross structure in the heart. Scale bar, 500 μm. A, atrium; V, ventricle; M, muscle; IN, intestine. (C) H&E sections from alms1^+/+ and alms1^−/− zebrafish showing high magnification of ventricular wall. Quantification of thickness of ventricular wall in alms1^+/+ and alms1^−/− animals (n = 4 animals). Bracket, ventricular wall. Scale bar, 25 μm. A, atrium; V, ventricle. Dots, individual measurements; error bars, 95% CI. (D) Representative images of H&E staining of retinal layers from alms1^+/+ and alms1^−/− zebrafish at 6 months showing degradation of multiple retinal layers in alms1^−/− animals. Retina directionality indicated by photoreceptor and ganglion cell sides. Scale bar, 50 μm. (E) Quantification of scotopic b-wave amplitudes in response to light stimulation from alms1^+/+ (n = 4) and alms^−/− (n = 3) zebrafish at 9 months of age. Error bars, 95% CI. (F) Representative H&E images of kidneys in alms1^+/+ and alms1^−/− animals at demonstrating the abnormal shape and size of kidney tubules in alms1^−/− animals. Distal tubule, yellow arrow; proximal tubule, red arrow. Scale bar, 100 μm. (G) Lumen width along short axis in kidneys from alms1^+/+ and alms1^−/− animals (n = 4 animals). Error bars, 95% CI. Where indicated, ^*P < 0.05, ^**P < 0.01.

Figure 3

alms1^−/− zebrafish exhibit increased weight gain and systemic metabolic defects. (A)alms1^+/+ and alms1^−/− zebrafish adults at 3 months were fed daily with set weights of either maintenance diet (control) or overfeeding with HF diet for 8 weeks (n = 4–6 animals per condition). Statistics, two-way ANOVA compared to alms1^+/+ control diet. (B) Representative images of alms1^+/+ and alms1^−/− zebrafish after 8 weeks of either control diet or HF diet. (C) Representative images of Oil Red O staining in livers of alms1^+/+ (n = 27) and alms1^−/− (n = 23) larvae at 6 dpf. Scale bar, 500 μm. Quantification of Oil Red O positive livers. Significance, chi-squared. (D) Representative regions of sectioned H&E liver tissue from alms1^+/+ and alms1^−/− zebrafish at 6 months. Scale bar, 25 μm. BV, blood vessel.

Figure 4

alms1^−/− zebrafish islets have fewer β-cells and reduced glucose responsiveness. (A) Aldehyde fuchsin staining of sectioned zebrafish at 6 months showing aberrant islet structure (`ins+', dark purple regions) and tissue degradation (arrowheads) in alms1^−/− as compared to alms1^+/+. BV, blood vessel; D, secretory duct. Scale bar, 50 μm. (B)β-cell imaging and quantification (count method in bottom panel) of 5 dpf larvae in alms1^+/+ (n = 31) and alms1^−/− (n = 22) larvae via ins:mCherry reporter expression. Scale bar, 25 μm. Dots, individual larvae. Statistics, Student's t-test with Welch's Correction. (C) Representative images of β-cells with and without exposure to 40 mm glucose in alms1^+/+ and alms1^−/− larvae. Scale bar, 25 μm. (D) Quantification of β-cells at 5 dpf from alms1^+/+ (NT = 22, Glu = 31) and alms1^−/− (NT = 25, Glu = 18) larvae via ins:mCherry reporter expression. Statistics, one-way ANOVA. Dots, individual larvae. Error bars, 95% CI. Where indicated, ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001.

Figure 5

Transcriptomic analysis indicates dysregulation of insulin secretion and glucose sensing in zebrafish alms1^−/−β-cells. (A) Schematic of experimental design for comparative gene expression in β-cell-enriched populations from age-matched control and alms1^−/− larvae. (B) Volcano plot of significantly differentially expressed genes from GRZ10 between control and alms1^−/− larvae. (C) Selected tissue specific genes identified in isolated alms1^+/+ cells indicating high expression of pancreatic markers. Error bars, standard deviation. ND, not identified. (D) Subset of significantly up-regulated pathway nodes, identified by ConsensusPath DB, in alms1^−/−β-cells. Genes within intersections are listed in table alongside the fold increase and significance compared to alms1^+/+β-cells.

Figure 6

Hyperinsulinemia accompanied by defective glucose sensing with loss of Alms1. (A) Expression of glucose response genes in si-Alms1β-cells under basal (2.5 mm) and high-glucose (16.7 mm) conditions (n = 4). Cells were collected after 10 min after glucose stimulation. (B) Relative insulin by ELISA-based detection in culture media from β-cells after 30 min of exposure to 2.5 mm and 16.7 mm glucose (n = 3), normalized to basal si-control, shows failure of si-Alms1 cells to alter insulin secretion. (C and D) 2-NBDG at indicated concentrations was provided to (C) alms1^+/+ and (D) alms1^−/− larvae for 6 h. The kidney and retinal fluorescence intensity were quantified at each dose. Dots, individual larvae. (E) Relative insulin by ELISA-based detection under the indicated conditions in alms1^+/+ (n = 30–50 per group) and alms1^−/− (n = 30–50 per group) larvae at 5 dpf. All statistics, two-way ANOVA. Dots, replicates; error bars, 95% CI. Where indicated, ^*P < 0.05, ^**P < 0.01.