Progression from the initial vascular response upon hyperglycemia to a proliferative stage with neovacularizations is the hallmark of proliferative diabetic retinopathy. Here, we report on the novel diabetic pdx1−/− zebrafish mutant as a model for diabetic retinopathy that lacks the transcription factor pdx1 through CRISPR-Cas9–mediated gene knockout leading to disturbed pancreatic development and hyperglycemia. Larval pdx1−/− mutants prominently show vasodilation of blood vessels through increased vascular thickness in the hyaloid network as direct developmental precursor of the adult retinal vasculature in zebrafish. In adult pdx1−/− mutants, impaired glucose homeostasis induces increased hyperbranching and hypersprouting with new vessel formation in the retina and aggravation of the vascular alterations from the larval to the adult stage. Both vascular aspects respond to antiangiogenic and antihyperglycemic pharmacological interventions in the larval stage and are accompanied by alterations in the nitric oxide metabolism. Thus, the pdx1−/− mutant represents a novel model to study mechanisms of hyperglycemia-induced retinopathy wherein extensive proangiogenic alterations in blood vessel morphology and metabolic alterations underlie the vascular phenotype.

Novel and better strategies to prevent or reverse diabetic retinopathy (DR) as the leading cause of visual impairment in the middle-aged Western population are needed (1). Research depends on animal studies, but only selected disease aspects are represented in single models. Hyperglycemia leads to DR through initial induction of microangiopathy, impaired autoregulation, and increased vascular permeability. Progressively, this leads to pericyte dropout and microaneurysm formation in the human retina, followed by capillary occlusion and downstream formation of acellular nonperfused capillaries. Retinal ischemia is induced and provides the trigger for subsequent development of pathological neoangiogenesis driven by vascular endothelial growth factor (VEGF) and a switch to proliferative DR (PDR) with neovascularizations (2,3).

This progression, however, is often missing in the commonly used animal models, like streptozotocin (STZ)-induced murine diabetes, and, as such, may lead to mechanisms of disease progression that remain undiscovered (4,5). Some larger mammalian species show selected characteristics of late-stage PDR, but neovascularization as seen in humans is not represented by any model (3). Dogs have been studied in long-term experiments leading to a retinal phenotype with vascular lesions, including microaneurysms coming closer to human DR, but ethical concerns in larger mammals are great disadvantages and remain controversial (3,5). Most studies favor rodents, but DR phenotypes in these animals do not reach similar translational value for proliferative aspects. Nonhyperglycemic rodents are therefore recognized as alternative tools to study neovascularization in the retina. The oxygen-induced retinopathy of prematurity model is commonly used to study retinal neoangiogenesis and leads to overexpression of VEGF, erythropoietin, and angiopoietin 2 in the retina but should be interpreted with care as a primarily hypoxic model (3).

Zebrafish provide easy access to visualization of the vasculature, conserved vascular physiology, and a vertebrate anatomy with high translational value, making them a strong model for vascular diseases (611). Similar to the murine retinopathy of prematurity model, zebrafish respond to hypoxia with new vessel formation close to the inner optic circle (IOC) (12), highlighting the capability of zebrafish for the study of retinal neoangiogenesis. Responsiveness of zebrafish to hyperglycemic stimuli was shown by short-term incubation in external high-glucose solutions leading to thickening of the retinal vasculature, although under unphysiological glucose concentrations (13,14). Despite this, the influence of a permanent genetic diabetic model in a long-term setting, relating closer to the genuine clinical situation, has not been established in zebrafish.

Glucose homeostasis in matured zebrafish shares physiological responses to human recombinant insulin, and zebrafish develop insulin resistance when under chronic exposure to higher doses (1517). Chemical β-cell ablation using STZ is commonly used in rodents to disrupt the glucose homeostasis; however, in zebrafish, the STZ model faces off against the regenerative capacity in long-term settings because β-cell mass will be restored (18). Genetic models avoid direct activation of regenerative programs; therefore, they could reflect the development of diabetic microvascular complications more accurately.

Pancreatic and duodenal homeobox 1 (Pdx1) is needed for β-cell differentiation and insulin gene transcription and is one of the earliest pancreatic transcription factors (15). Pdx1 shows conserved function and relevance for islet function across species (19). Heterozygous mutations induce modest onset of diabetes in the young in humans and are recognized as a risk factor for type 2 diabetes (20). Homozygous mutations induce a permanent neonatal diabetes caused by pancreatic agenesis (2123), resulting in severe insulin deficiency. Kimmel et al. (24) first reported characterization of a homozygous pdx1 mutant in zebrafish as a model for diabetes and characterized the disturbed islet development and glucose homeostasis leading to hyperglycemia.

In this study, we have generated a pdx1−/− zebrafish mutant as a new monogenetic animal model for hyperglycemia-mediated retinal blood vessel alterations and investigated the influence on the larval hyaloid and corresponding adult retinal vasculature. We also highlight the pharmacological response and pathophysiological similarity to the human disease with regard to proliferative capacity.

Zebrafish Husbandry and Zebrafish Lines

Embryos of AB wild-type, Tg(hb9:GFP) (25), Tg(fli1:EGFP) (6), Tg(nflk:EGFP) (26), and Tg(gata1a:DsRed) (27) lines were raised in egg water at 28.5°C with 0.003% 1-phenyl-2-thiourea to suppress pigmentation. After 72 h postfertilization (hpf), zebrafish are referred to as larvae as previously described (28). Zebrafish larvae were transferred to adult boxes after 6 days postfertilization (dpf) and raised under a 13-h light/11-h dark cycle. Fish are referred to as adults after 90 dpf. Adult zebrafish were fed freshly hatched Artemia salina and fish flake food daily.

Antibodies

Antibodies used included rabbit anti-Pdx1 (TA345380; OriGene Technologies), goat anti-actin (I-19) (sc-1616; Santa Cruz Biotechnology), and rabbit anti-goat horseradish peroxidase–conjugated (Dako) and rabbit anti-rat horseradish peroxidase–conjugated antibody (Dako).

Morpholinos

Morpholinos included SB-pdx1-Mo: 5′-GATAGTAATGCTCTTCCCGATTCAT-3′ and control-Mo: 5′-CCTCTTACCTCAGTTACAATTTATA-3′ and were used as recently described (29).

Mutant Generation

For mutant generation through the CRISPR/Cas9 technique, one guide RNA (gRNA) targeting exon 1 of pdx1 was designed using the ZiFiT Targeter 4.2 and cloned into a T7-driven promotor expression vector (pT7-gRNA; Addgene) (Pdx1-CRISPR-for: 5′-TAGGAGACTCTCTGGACCTCTG-3′, pdx1-CRISPR-rev: 5′-AAACCAGAGG TCCAGAGAGTCTCC-3′). The pT3TS-nCas9n vector (Addgene) was used for in vitro transcription to attain Cas9 mRNA (30). Synthesis of mRNA for injection was achieved by use of the mMESSAGE mMACHINE T3 Transcription Kit for Cas9 mRNA and MEGAshortscript T7 Kit for gRNA while following the protocol of the manufacturer (Invitrogen). One nanoliter of 0.1 mol/L KCl solution containing the gRNA (250 pg/nL) and Cas9 mRNA (250 pg/nL) were injected at one-cell stage (30). F0 mosaic fish were analyzed for germline transmission and selectively bred. Genotyping was performed through Sanger sequencing of PCR products (Pdx1-genotyping-for: 5′-TTTCCCCGGTCTATGGCAAT-3′, Pdx1-genotyping-rev: 5′-TGGCCAAAGTACGA GTTACCT-3′). Mutations were analyzed by evaluation of the chromatograms and use of Yost tools (31).

Western Blot Analysis

For analysis of pdx1 expression, 50 zebrafish larvae per group were collected at 5 dpf, deyolked in egg water, and prepared for Western blot analysis as previously described (32).

Glucose Determination in Zebrafish Larvae

Zebrafish larvae were collected at 120 hpf and snap frozen. Approximately 20 (CBA086) or 50 (GAHK-20) larvae per clutch were deyolked in PBS, and glucose content was determined according to manufacturer’s instructions for Glucose Assay Kits GAHK-20 and CBA086 (Sigma-Aldrich).

Blood Glucose Measurement

Adult zebrafish were transferred to single boxes the day before and fasted overnight. After 16–18 h, zebrafish were either directly euthanized for measurements under fasting conditions or fed 0.5 g fish flake made available for 1 h followed by an additional hour in fresh tank water for postprandial conditions. Zebrafish were euthanized in 250 mg/L tricaine, and blood glucose was measured according to Zang et al. (33).

In Vivo Fluorescence Microscopy in Tg(hb9:GFP) and Tg(fli1:EGFP) Larvae

Tg(hb9:GFP) embryos and Tg(fli1:EGFP) larvae were anesthetized with 0.003% tricaine in egg water, and Tg(hb9:GFP) were embedded in 1% low melting point agarose dissolved in egg water for visualization on an inverted microscope (DMI 6000 B; Leica) with a camera (DFC420 C; Leica) and the Leica Application Suite 3.8 software. The endocrine islet was imaged at 10× and quantified using ImageJ software. For quantification of altered trunk vessels, the first 5 intersegmental vessel and dorsal longitudinal anastomotic vessel pairs of each zebrafish larva were skipped, and in the following 17 pairs, alterations were counted at 4 and 8 dpf.

Preparation, Microscopy, and Quantification of Larval Hyaloid Vasculature

Tg(fli1:EGFP), Tg(nflk:EGFP), and Tg(gata1a:DsRed) zebrafish larvae were euthanized either at 5 dpf or at 6 dpf in 250 mg/L tricaine and fixed in 4% paraformaldehyde/PBS overnight at 4°C. Fixed larvae were washed three times for 20 min in double distilled water and incubated for 80 min at 37°C in 0.25% trypsin/EDTA solution (25200-056; Gibco) buffered with 0.1 mol/L Tris (Nr. 4855.3; Roth) and adjusted to pH 7.8 with 1 mol/L HCl solution. Larval hyaloid vasculature was dissected under a stereoscope and mounted in PBS for visualization as adapted from Jung et al. (14). Confocal images for phenotype evaluation were acquired using a confocal microscope (DM6000 B) with a scanner (TCS SP5 DS; Leica) using a 10 × 0.3 objective, 1,024 × 1,024 pixels, 0.5 μm Z-steps, and 2.0 zoom factor. Vascular diameters were measured at four different positions along the circumference of the hyaloid network and averaged per sample.

Preparation, Microscopy, and Quantification of Adult Retinal Vasculature

Wholemounts were prepared as previously described by our laboratory (34). Confocal images were acquired using a confocal microscope (DM6000 B) with a scanner (TCS SP5 DS) in a tile scan, depending on retinal size (e.g., 4 × 5, 5 × 5 tiles). Images were taken with a 20 × 0.3 water immersion objective, 400 Hz bidirectional, 1,024 × 1,024 pixels (retina vessels), and Z-stack steps of 1.5-μm thickness. Max projections of the acquired Z-stacks were used to assess the vascular morphology on squared cutouts of 350 × 350 μm near the IOC generated using GIMP2 software (www.gimp.org), and results are reported per retinal square. Branching points are defined as positions wherever a singular vascular lumen originating from the central retinal artery separated into more than one lumen. IOC diameter was measured at three equally spaced positions and averaged. Intervascular distance is reported as the mean of all single measurements on a parallel line with 175 μm distance to the IOC. Retinal area was divided into four quarters and sorted into three vascularity groups (25% low/50% middle/25% high) on the basis of an uneven pattern of intervascular distance along the circumference of the retinal wholemounts with highest and lowest vascularity opposite of each other in zebrafish. Data were adjusted to keep a ratio of 1:2:1 (low:middle:high) to avoid bias for retinal averages.

Pharmacological Treatment of Zebrafish Embryos

Zebrafish embryos were raised until 48 hpf under standard conditions before incubation with additional substances. Pharmacologically active drugs were then added in the respective concentration to the egg water, and embryos were raised until 120 hpf before euthanasia. Substances were as follows: metformin 10 μmol/L (Nr. 317240; Sigma-Aldrich), PK11195 10 μmol/L (C0424; Sigma-Aldrich), vatalanib/PTK787 0.1 and 0.2 μmol/L (S1101; Selleck Chemicals), and l-NG-nitro-l-arginine methyl ester (l-NAME) 10 μmol/L (N5751-1G; Sigma-Aldrich).

Metabolite Analysis by Ultra-Performance Liquid Chromatography Fluorescence

Detection was done in cooperation with the Metabolomics Core Technology Platform from the Centre of Organismal Studies Heidelberg. At 120 hpf, ∼50 zebrafish larvae per measurement were anesthetized with 0.003% tricaine and snap frozen. Adenosine compounds and free amino acids were measured as previously described (11).

Statistics

Results are expressed as mean ± SD. Statistical significance between different groups was analyzed using two-sided unpaired Student t test or one-way or two-way ANOVA followed by appropriate multiple comparison tests using GraphPad Prism software. P < 0.05 was considered significant.

Study Approval

All experimental procedures on animals were approved by the local government authority Regierungspräsidium Karlsruhe (License no. G-98/15 and G-57/19) and by Medical Faculty Mannheim (License no. I-19/02) and carried out in accordance with the approved guidelines.

Data and Resource Availability

The data sets generated and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Establishment of a Diabetic pdx1−/− Zebrafish Line With Impaired Glucose Homeostasis Using CRISPR-Cas9–Mediated Gene Editing

To investigate the effects of chronic hyperglycemia on the retinal vasculature in zebrafish, we created a homozygous knockout for pdx1 using CRISPR/Cas9 to disturb β-cell development, leading to reduced insulin production. We designed a gRNA targeting exon 1 of the pdx1 gene using ZiFiT Targeter 4.2 and injected the pdx1-gRNA together with Cas9 mRNA at the one-cell stage. F0-positive founders were outcrossed, and offspring were screened through genotyping for frameshift mutations near the target site (Fig. 1A). Two frameshift mutations, a 2-base pair deletion (Δ2bpD) and 1-base pair insertion (Δ1bpI), in exon 1 (Fig. 1A and B) were selected for establishment of a stable F2 generation through selective breeding. Gross morphology of 3-dpf-old pdx1−/− zebrafish did not show outer developmental phenotypes compared with wild-type siblings (Fig. 1C). Remaining functional protein expression was analyzed through Western blot and showed a total loss of pdx1 protein (Fig. 1D).

Homozygous pdx1sa280 zebrafish mutants show decreased β-cell count and insulin production, leading to elevated glucose levels in larvae (24). To verify this, we established a pdx1−/− line carrying the Tg(hb9:GFP) reporter to assess functional β-cell mass of the primary endocrine islet. After 20 hpf, pancreatic expression of Hb9 (Mnx1) is confined only to insulin expressing β-cells (35). The pdx1−/− embryos have less cellular mass at 3 dpf, indicating the expected defect in insulin expressing β-cell development (Fig. 2A and B), quantified by significant reduction in respective primary islets size and fluorescence intensity (Fig. 2C and D). Heterozygous pdx1+/− littermates similarly exhibit a reduction in pancreatic size to a lesser degree, indicative of a gene dose effect for pdx1 (Fig. 2C and D). Whole-body glucose in pdx1−/− larvae at 5 dpf, before external feeding behavior, was monitored for alterations in basal glucose metabolism and showed a more than twofold increase in glucose levels (Fig. 2E). This demonstrates successful establishment of a novel pdx1−/− knockout line and highlights induction of a diabetic condition with impaired glucose homeostasis in zebrafish larvae.

pdx1−/− Zebrafish Larvae Are Susceptible to Microvascular Damage Exhibiting Organ-Specific Changes in the Larval Hyaloid Vasculature

To study short-term influence, we analyzed the retinal hyaloid network (Fig. 3A and B) as an early precursor of the mature retinal vasculature. In contrast to the mammalian retina, where the retinal vasculature is formed by angiogenesis and the hyaloid vasculature regresses, the hyaloid vasculature does not regress in zebrafish (36). Retinal vessel dilation and reduced constrictive autoregulation is recognized as an early sign of DR (37). The pdx1−/− larvae showed a significant increase in the hyaloid vascular diameters of both offbranching vessels and the inner annular vessel (Fig. 3C and D) after 6 dpf. Also, significantly more vascular interconnections were formed, quantified by a higher number of branches and number of free-ending sprouts (Fig. 3E and F). Morpholino-mediated pdx1 knockdown led to higher numbers of endothelial nuclei as a sign of increased vascular proliferation in Tg(nflk:EGFP) larvae (Fig. 3G and H) and increased endothelial extravasation of fluorescent dye after injection, but not of erythrocytes, in the hyaloid network (Supplementary Figs. 1 and 2). Therefore, on the basis of pdx1 inactivation, a proangiogenic retinal phenotype with hypersprouting, hyperbranching, increased vessel diameters, and permeability can be induced in diabetic zebrafish larvae. This effect seems to be organ specific because the trunk vasculature in pdx1−/− mutants did not show any increase in sprouting behavior or vessel alterations (Supplementary Fig. 3).

Early Vascular Phenotype Induced Through the Hyperglycemic Condition in Zebrafish Larvae Continues Into Adulthood With Aggravated Vascular Alterations in the Mature Retinal pdx1−/− Vasculature

To investigate aggravation through prolonged exposure to the hyperglycemic condition, we raised pdx1−/− fish and wild-type siblings into adulthood. Genotyping in the heterozygous in-cross breedings showed a homozygous animal frequency greatly below expected Mendelian ratios, indicating a survival adversity, similar to the homozygous pdx1sa280 mutant (24). Surprisingly, we found no hyperglycemia after overnight fasting in our pdx1−/− mutants (Fig. 4A). Adult homozygous pdx1sa280 mutants showed a 2.7-fold blood glucose increase; however, fasting periods before measurement were not defined, while the wild-type values are indicating ∼2 h of fasting (24,38). We devised a nutrition challenge (10) and measured postprandial blood glucose levels 2 h after feeding overnight-fasted zebrafish, which uncovered a significant 1.6-fold glucose elevation in pdx1−/− zebrafish with 71 vs. 45 mg/dL at the 2-h cutoff compared with wild-type siblings (Fig. 4B). Therefore, the pdx1−/− mutants shift to impaired glucose tolerance in adulthood with repeated daily postprandial hyperglycemia under routine feeding conditions.

Hypersprouting and hyperbranching was further aggravated in the adult pdx1−/− retina, and significantly more interconnections in between the vascular arcades were measured as well as a twofold increase in free-ending sprouts (Fig. 4C and D). Middle- and high-vascularity areas of the pdx1−/− retina respond with activation of retinal angiogenesis, while low vascularity areas with higher intervascular distances show no reactivity (Supplementary Fig. 4). Intervascular distance itself is not altered (Fig. 4E). Increased vascular diameter as an initial sign of retinopathy was not persistent in the mature pdx1−/− IOC (Fig. 4F). Together, this suggests a progressive state of the proangiogenic retinal phenotype on the basis of the repeated postprandial hyperglycemia in the adult pdx1−/− mutants (Fig. 4G and H). Additionally, heterozygous pdx1+/− zebrafish of old age are also susceptible to developing impaired glucose tolerance and retinal alterations compared with wild-type fish of similar age (Fig. 5A and B). We found a 1.9-fold increase in postprandial blood glucose levels (Fig. 5C) and a similar phenotype as in younger pdx1−/− mutants, with significant hyperbranching and hypersprouting in pdx1+/− zebrafish at 20 months postfertilization (mpf) (Fig. 5D and E). Similar retinal areas are affected, but effect strength is lower compared with pdx1−/− (Supplementary Fig. 5). Again, IOC diameter showed no dilatation in mature states (Fig. 5F), while an increase in intervascular distance in old pdx1+/− fish was found (Fig. 5G).

Hyaloidal Vascular Phenotype in pdx1−/− Zebrafish Is Mediated by Hyperglycemia and Responds to Pharmacological Interventions

We selected two antihyperglycemic drugs, namely metformin and PK11195, to prove a causal relationship between hyperglycemia and vascular alterations and to rule out specific side effects influencing the retinal phenotype (3941). PK11195 showed stronger effects on glucose levels compared with metformin in hyperglycemic zebrafish larvae at 5 dpf (Supplementary Fig. 6). Antihyperglycemic treatments at 10 μmol/L concentration significantly rescued the proangiogenic aspects of hyperbranching and hypersprouting (Fig. 6A–C) as well as the increased vascular diameters in the pdx1−/− hyaloid network (Fig. 7A and B). Vascular alterations in the pdx1−/− hyaloid system are therefore unlikely caused by potential genetic off-target effects or altered transcriptional responses on the basis of the deletion of Pdx1.

Pharmacological Modulation of VEGF and Nitic Oxide Signaling Specifically Rescues Hyperglycemia-Mediated Vascular Alterations

VEGF promotes neovascularization in PDR, and hyperglycemia is classically considered a main activator (42,43). Hyaloid vessel dilation under high-glucose incubation in zebrafish is accompanied by increased VEGF mRNA expression, and our own findings uncovered that PTK787 (vatalanib) can rescue hypersprouting induced by either external high glucose or methylglyoxal incubation through inhibition of VEGF receptor 2 in the trunk vasculature in zebrafish (14,44). Similar effects of PTK787 in the hyaloid vasculature have also been proven recently in zebrafish (45). Antiangiogenic treatment with PTK787 at a 0.1 μmol/L concentration was correspondingly able to significantly rescue the hypersprouting, hyperbranching (Fig. 6A and B), and vascular diameter increase (Fig. 7A and B) at 5 dpf in the pdx1−/− hyaloid vascular network, while prior work has further shown that in this concentration range, PTK787 does not impair physiological angiogenesis (46). This suggests VEGF as mechanism of the proangiogenic phenotype and pathophysiological similarity between molecular mechanisms in the zebrafish retina under diabetic conditions and DR.

Endogenous nitric oxide (NO) production and VEGF signaling are strongly linked, and elevated serum NO levels (NOx) are commonly found in patients with PDR (47,48). To identify whether vasodilation was responsible for the increased vascular diameters in pdx1−/− larvae, we blocked NO-mediated vasodilation through NO synthase (NOS) inhibitor l-NAME. The pharmacological intervention revealed a significant reduction in vascular diameter back to wild-type levels in the pdx1−/− group (Fig. 7A and B), highlighting a hyperglycemia-induced vasodilatory effect mediated through NO signaling in the hyaloid vasculature (Fig. 7C). However, no significant reduction in hyperbranching or hypersprouting could be found (Fig. 6A–C). This shows that the proangiogenic aspect is mediated rather by VEGF signaling and not affected by NOS inhibition (Fig. 7C).

pdx1−/− Zebrafish Larvae Exhibit Distinct Changes in Their Amino Acid Metabolism Closely Linked to NO Production

Multiple changes in the amino acid metabolism were found through ultra-performance liquid chromatography fluorescence at 5 dpf, when both diabetic condition and hyaloid phenotype are present (Fig. 8A). Tyrosine, ornithine, and spermidine were significantly decreased, while proline, glutamate, and taurine showed increased levels (Fig. 8B–G). This indicates a metabolic activation in diabetic pdx1−/− larvae at 5 dpf as an adaption response to sustain NO production (Fig. 8A) through reduced degradation of arginine through ornithine to the polyamides spermine and spermidine and, on the other hand, increased bioavailability of proline and glutamate, which can be used to sustain arginine levels for NO production. The remaining amino acids and adenosine compounds showed no significant changes (Supplementary Figs. 7 and 8).

In this study, we established for the first time that a hyperglycemic condition induced by pdx1 knockout leads to a proangiogenic vascular phenotype mimicking aspects of PDR in zebrafish in both the short-term setting in zebrafish larvae and in fully matured adult zebrafish retinas. The phenotype responds to antiangiogenic and antihyperglycemic interventions as expected of DR and suggests the use of pdx1 mutant zebrafish to study hyperglycemia-induced retinal blood vessel alterations (Fig. 7C).

The pdx1 mutant shows induction of vasodilation in the larval hyaloid vasculature. Pharmacological interventions in the pdx1−/− larvae show that this phenotype is mediated by increased NO signaling reversible by NOS inhibition. The metabolic state of pdx1−/− larvae at 5 dpf supports this hypothesis and shows a shift in the amino acid metabolism related to arginine as substrate for NOS. Degradation of ornithine into the endogenous polyamine pathway is reduced, while amino acids convertible to arginine are increased in their total concentration. Similarly to NOS inhibition, treating the diabetic condition through antihyperglycemic drugs rescues vasodilation and shows that the increased NO production in zebrafish is mediated by hyperglycemia. The metabolic state in conjunction with the vascular phenotype implies that ornithine is depleted through the production of arginine to keep bioavailability constant as an early metabolic adaptive response in zebrafish larvae in a state of hyperglycemia-mediated increased NO production.

Corresponding to the human pathophysiology, inhibition of VEGF signaling by PTK787 (vatalanib) could rescue the proangiogenic vessel alterations in the hyaloid vasculature of the pdx1−/− larvae at 5 dpf. Inhibition of NO production on the other hand does not affect the hyperbranching and hypersprouting in the hyaloid vasculature but selectively leads to attenuation of the hyperglycemia-mediated vasodilation. Conflicting evidence in the literature is found for the relationship between VEGF and NO in the diabetic retina and their role regarding neoangiogenesis. Increased NO production is seen in patients with PDR (49), and several studies have postulated increased NO production as a downstream mechanism of VEGF signaling to play a role in VEGF-mediated angiogenesis. Our results, however, indicate that vasodilation as a sign of DR is mediated through increased NO production by VEGF in zebrafish, but NO signaling does not mediate angiogenesis in the hyaloid vasculature.

Recently, Singh et al. (50) found that the adult zebrafish retina could be primed to exhibit increased vascular sprouting upon larval high-glucose incubation, but no hyperbranching as indication of novel vessel formation was reported. Hypoxia-mediated vascular hyperbranching is only seen under strong hypoxia or prolonged incubation, while weaker hypoxic stimuli only lead to increased hypersprouting (12). This indicates the need for a prolonged diabetic condition to progress from initial hypersprouting to hyperbranching in the zebrafish retina and highlights one advantage of a genetic diabetes model. A progressive vascular phenotype is especially relevant since progressive alterations in the retinal blood vessels with present neovascularization are also rarely seen in rodents (4). In addition, increased vascular permeability is a characteristic element of DR indicated by increased extravasation in the diabetic hyaloid network.

The pdx1 mutant as a novel genetic model for DR offers the advantage of a proangiogenic condition while retaining similar handling and effort as needed for rodents. The application, however, is limited by the heavily impaired survival of pdx1−/− mutants into adulthood. Furthermore, not all vascular changes seen in DR are recapitulated by the phenotype. Heterozygous pdx1+/− zebrafish at an aged state show similar vascular phenotypes with reduced effect strength, but since they can be raised without impaired survival, their use might be an additional alternative in adult long-term studies.

Several other models of diabetes have now been established in zebrafish using dietary, chemical, or transgenic induction (7). Diet-induced zebrafish models have been studied the most (9) but have not shown a phenotype mimicking the later stages of DR as expected from a hypoxic influence, and elsewhere, unphysiologically high concentrations of glucose are needed to induce vascular phenotypes. In the pdx1 mutant, we see hypersprouting and hyperbranching mediated by an initial phase of hyperglycemia in the larvae and repeated postprandial hyperglycemic spikes in adulthood.

Use of zebrafish will allow researchers to screen for effects in the larval stage while having the option to investigate the effect on disease progression in the adult animal. In conclusion, this study identified the pdx1−/− zebrafish mutant as a novel model for the study of hyperglycemia-induced blood vessel alterations.

Funding. The study was supported by grants from Deutsche Forschungsgemeinschaft (CRC 1118 and IRTG 1874/2 DIAMICOM). The authors thank the Metabolomics Core Technology Platform of the Excellence Cluster “CellNetworks” (University of Heidelberg) and the Deutsche Forschungsgemeinschaft (grant ZUK 40/2010-3009262) for support with ultra-performance liquid chromatography–based metabolite quantification. The authors acknowledge the support of the Core Facility Live Cell Imaging Mannheim at the Centre for Biomedicine and Medical Technology Mannheim (DFG INST 91027/10-1FUGG) and of the Zebrafish Core Unit of Medical Faculty Mannheim.

Duality of Interest. No potential conflicts of interest relevant to this article were reported.

Author Contributions. L.M.W. performed experiments, analyzed data, and wrote the manuscript. H.Q., S.J.S., L.M., and K.B. performed experiments and analyzed data. G.P. performed metabolome experiments and analyzed data. G.K., J.-L.H., and H.-P.H. gave conceptual and technological advice. J.K. conceived and designed the study and wrote the manuscript. J.K. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Prior Presentation. Parts of this study were presented at the 53rd Annual Diabetes Congress of the German Diabetes Association (DDG), Berlin, Germany, 9–12 May 2018, and the 54th Annual Meeting of the European Association for the Study of Diabetes, Berlin, Germany, 1–5 October 2018.

1.
Ting
DS
,
Cheung
GC
,
Wong
TY
.
Diabetic retinopathy: global prevalence, major risk factors, screening practices and public health challenges: a review
.
Clin Exp Ophthalmol
2016
;
44
:
260
277
2.
Cheung
N
,
Mitchell
P
,
Wong
TY
.
Diabetic retinopathy
.
Lancet
2010
;
376
:
124
136
3.
Stitt
AW
,
Curtis
TM
,
Chen
M
, et al
.
The progress in understanding and treatment of diabetic retinopathy
.
Prog Retin Eye Res
2016
;
51
:
156
186
4.
Lai
AK
,
Lo
AC
.
Animal models of diabetic retinopathy: summary and comparison
.
J Diabetes Res
2013
;
2013
:
106594
5.
Robinson
R
,
Barathi
VA
,
Chaurasia
SS
,
Wong
TY
,
Kern
TS
.
Update on animal models of diabetic retinopathy: from molecular approaches to mice and higher mammals
.
Dis Model Mech
2012
;
5
:
444
456
6.
Lawson
ND
,
Weinstein
BM
.
In vivo imaging of embryonic vascular development using transgenic zebrafish
.
Dev Biol
2002
;
248
:
307
318
7.
Gut
P
,
Reischauer
S
,
Stainier
DYR
,
Arnaout
R
.
Little fish, big data: zebrafish as a model for cardiovascular and metabolic disease
.
Physiol Rev
2017
;
97
:
889
938
8.
Heckler
K
,
Kroll
J
.
Zebrafish as a model for the study of microvascular complications of diabetes and their mechanisms
.
Int J Mol Sci
2017
;
18
:
2002
9.
Wiggenhauser
LM
,
Kroll
J
.
Vascular damage in obesity and diabetes: highlighting links between endothelial dysfunction and metabolic disease in zebrafish and man
.
Curr Vasc Pharmacol
2019
;
17
:
476
490
10.
Lodd
E
,
Wiggenhauser
LM
,
Morgenstern
J
, et al
.
The combination of loss of glyoxalase1 and obesity results in hyperglycemia
.
JCI Insight
2019
;
4
:
e126154
11.
Schmöhl
F
,
Peters
V
,
Schmitt
CP
, et al
.
CNDP1 knockout in zebrafish alters the amino acid metabolism, restrains weight gain, but does not protect from diabetic complications
.
Cell Mol Life Sci
2019
;
76
:
4551
4568
12.
Cao
R
,
Jensen
LD
,
Söll
I
,
Hauptmann
G
,
Cao
Y
.
Hypoxia-induced retinal angiogenesis in zebrafish as a model to study retinopathy
.
PLoS One
2008
;
3
:
e2748
13.
Carnovali
M
,
Luzi
L
,
Banfi
G
,
Mariotti
M
.
Chronic hyperglycemia affects bone metabolism in adult zebrafish scale model
.
Endocrine
2016
;
54
:
808
817
14.
Jung
SH
,
Kim
YS
,
Lee
YR
,
Kim
JS
.
High glucose-induced changes in hyaloid-retinal vessels during early ocular development of zebrafish: a short-term animal model of diabetic retinopathy
.
Br J Pharmacol
2016
;
173
:
15
26
15.
Kinkel
MD
,
Prince
VE
.
On the diabetic menu: zebrafish as a model for pancreas development and function
.
BioEssays
2009
;
31
:
139
152
16.
Maddison
LA
,
Joest
KE
,
Kammeyer
RM
,
Chen
W
.
Skeletal muscle insulin resistance in zebrafish induces alterations in β-cell number and glucose tolerance in an age- and diet-dependent manner
.
Am J Physiol Endocrinol Metab
2015
;
308
:
E662
E669
17.
Marín-Juez
R
,
Jong-Raadsen
S
,
Yang
S
,
Spaink
HP
.
Hyperinsulinemia induces insulin resistance and immune suppression via Ptpn6/Shp1 in zebrafish
.
J Endocrinol
2014
;
222
:
229
241
18.
Moss
JB
,
Koustubhan
P
,
Greenman
M
,
Parsons
MJ
,
Walter
I
,
Moss
LG
.
Regeneration of the pancreas in adult zebrafish
.
Diabetes
2009
;
58
:
1844
1851
19.
Helker
CSM
,
Mullapudi
ST
,
Mueller
LM
, et al
.
A whole organism small molecule screen identifies novel regulators of pancreatic endocrine development
.
Development
2019
;
146
:
dev172569
20.
Weng
J
,
Macfarlane
WM
,
Lehto
M
, et al
.
Functional consequences of mutations in the MODY4 gene (IPF1) and coexistence with MODY3 mutations
.
Diabetologia
2001
;
44
:
249
258
21.
Stoffers
DA
,
Zinkin
NT
,
Stanojevic
V
,
Clarke
WL
,
Habener
JF
.
Pancreatic agenesis attributable to a single nucleotide deletion in the human IPF1 gene coding sequence
.
Nat Genet
1997
;
15
:
106
110
22.
Schwitzgebel
VM
,
Mamin
A
,
Brun
T
, et al
.
Agenesis of human pancreas due to decreased half-life of insulin promoter factor 1
.
J Clin Endocrinol Metab
2003
;
88
:
4398
4406
23.
Thomas
IH
,
Saini
NK
,
Adhikari
A
, et al
.
Neonatal diabetes mellitus with pancreatic agenesis in an infant with homozygous IPF-1 Pro63fsX60 mutation
.
Pediatr Diabetes
2009
;
10
:
492
496
24.
Kimmel
RA
,
Dobler
S
,
Schmitner
N
,
Walsen
T
,
Freudenblum
J
,
Meyer
D
.
Diabetic pdx1-mutant zebrafish show conserved responses to nutrient overload and anti-glycemic treatment
.
Sci Rep
2015
;
5
:
14241
25.
Flanagan-Steet
H
,
Fox
MA
,
Meyer
D
,
Sanes
JR
.
Neuromuscular synapses can form in vivo by incorporation of initially aneural postsynaptic specializations
.
Development
2005
;
132
:
4471
4481
26.
Blum
Y
,
Belting
HG
,
Ellertsdottir
E
,
Herwig
L
,
Lüders
F
,
Affolter
M
.
Complex cell rearrangements during intersegmental vessel sprouting and vessel fusion in the zebrafish embryo
.
Dev Biol
2008
;
316
:
312
322
27.
Traver
D
,
Paw
BH
,
Poss
KD
,
Penberthy
WT
,
Lin
S
,
Zon
LI
.
Transplantation and in vivo imaging of multilineage engraftment in zebrafish bloodless mutants
.
Nat Immunol
2003
;
4
:
1238
1246
28.
Kimmel
CB
,
Ballard
WW
,
Kimmel
SR
,
Ullmann
B
,
Schilling
TF
.
Stages of embryonic development of the zebrafish
.
Dev Dyn
1995
;
203
:
253
310
29.
Jurczyk
A
,
Roy
N
,
Bajwa
R
, et al
.
Dynamic glucoregulation and mammalian-like responses to metabolic and developmental disruption in zebrafish
.
Gen Comp Endocrinol
2011
;
170
:
334
345
30.
Jao
LE
,
Wente
SR
,
Chen
W
.
Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system
.
Proc Natl Acad Sci U S A
2013
;
110
:
13904
13909
31.
Hill
JT
,
Demarest
BL
,
Bisgrove
BW
,
Su
YC
,
Smith
M
,
Yost
HJ
.
Poly peak parser: method and software for identification of unknown indels using Sanger sequencing of polymerase chain reaction products
.
Dev Dyn
2014
;
243
:
1632
1636
32.
Stoll
SJ
,
Bartsch
S
,
Augustin
HG
,
Kroll
J
.
The transcription factor HOXC9 regulates endothelial cell quiescence and vascular morphogenesis in zebrafish via inhibition of interleukin 8
.
Circ Res
2011
;
108
:
1367
1377
33.
Zang
L
,
Shimada
Y
,
Nishimura
Y
,
Tanaka
T
,
Nishimura
N
.
Repeated blood collection for blood tests in adult zebrafish
.
J Vis Exp
2015
;(102):
e53272
34.
Wiggenhauser
LM
,
Kohl
K
,
Dietrich
N
,
Hammes
HP
,
Kroll
J
.
Studying diabetes through the eyes of a fish: microdissection, visualization, and analysis of the adult tg(fli:EGFP) zebrafish retinal vasculature
.
J Vis Exp
2017
(
130
):
e56674
35.
Wendik
B
,
Maier
E
,
Meyer
D
.
Zebrafish mnx genes in endocrine and exocrine pancreas formation
.
Dev Biol
2004
;
268
:
372
383
36.
Alvarez
Y
,
Cederlund
ML
,
Cottell
DC
, et al
.
Genetic determinants of hyaloid and retinal vasculature in zebrafish
.
BMC Dev Biol
2007
;
7
:
114
37.
Bek
T
.
Diameter changes of retinal vessels in diabetic retinopathy
.
Curr Diab Rep
2017
;
17
:
82
38.
Eames
SC
,
Philipson
LH
,
Prince
VE
,
Kinkel
MD
.
Blood sugar measurement in zebrafish reveals dynamics of glucose homeostasis
.
Zebrafish
2010
;
7
:
205
213
39.
Gut
P
,
Baeza-Raja
B
,
Andersson
O
, et al
.
Whole-organism screening for gluconeogenesis identifies activators of fasting metabolism
.
Nat Chem Biol
2013
;
9
:
97
104
40.
Zang
L
,
Shimada
Y
,
Nishimura
N
.
Development of a novel zebrafish model for type 2 diabetes mellitus
.
Sci Rep
2017
;
7
:
1461
41.
Capiotti
KM
,
Antonioli
R
 Jr
.,
Kist
LW
,
Bogo
MR
,
Bonan
CD
,
Da Silva
RS
.
Persistent impaired glucose metabolism in a zebrafish hyperglycemia model
.
Comp Biochem Physiol B Biochem Mol Biol
2014
;
171
:
58
65
42.
Duh
EJ
,
Sun
JK
,
Stitt
AW
.
Diabetic retinopathy: current understanding, mechanisms, and treatment strategies
.
JCI Insight
2017
;
2
:
e93751
43.
Wang
W
,
Lo
ACY
.
Diabetic retinopathy: pathophysiology and treatments
.
Int J Mol Sci
2018
;
19
:
1816
44.
Jörgens
K
,
Stoll
SJ
,
Pohl
J
, et al
.
High tissue glucose alters intersomitic blood vessels in zebrafish via methylglyoxal targeting the VEGF receptor signaling cascade
.
Diabetes
2015
;
64
:
213
225
45.
Li
Y
,
Zhao
Y
,
Sang
S
,
Leung
T
.
Methylglyoxal-induced retinal angiogenesis in zebrafish embryo: a potential animal model of neovascular retinopathy
.
J Ophthalmol
2019
;
2019
:
2746735
46.
Hollenbach
M
,
Stoll
SJ
,
Jörgens
K
,
Seufferlein
T
,
Kroll
J
.
Different regulation of physiological and tumor angiogenesis in zebrafish by protein kinase D1 (PKD1)
.
PLoS One
2013
;
8
:
e68033
47.
Kimura
H
,
Esumi
H
.
Reciprocal regulation between nitric oxide and vascular endothelial growth factor in angiogenesis
.
Acta Biochim Pol
2003
;
50
:
49
59
48.
Izumi
N
,
Nagaoka
T
,
Mori
F
,
Sato
E
,
Takahashi
A
,
Yoshida
A
.
Relation between plasma nitric oxide levels and diabetic retinopathy
.
Jpn J Ophthalmol
2006
;
50
:
465
468
49.
Tsai
DC
,
Charng
MJ
,
Lee
FL
,
Hsu
WM
,
Chen
SJ
.
Different plasma levels of vascular endothelial growth factor and nitric oxide between patients with choroidal and retinal neovascularization
.
Ophthalmologica
2006
;
220
:
246
251
50.
Singh
A
,
Castillo
HA
,
Brown
J
,
Kaslin
J
,
Dwyer
KM
,
Gibert
Y
.
High glucose levels affect retinal patterning during zebrafish embryogenesis
.
Sci Rep
2019
;
9
:
4121
Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered. More information is available at https://www.diabetesjournals.org/content/license.

Supplementary data