IDH1-mutated transgenic zebrafish lines: An in-vivo model for drug screening and functional analysis

Introduction The gene encoding isocitrate dehydrogenase 1 (IDH1) is frequently mutated in several tumor types including gliomas. The most prevalent mutation in gliomas is a missense mutation leading to a substitution of arginine with histidine at the residue 132 (R132H). Wild type IDH1 catalyzes oxidative decarboxylation of isocitrate to α-ketoglutarate (α-KG) whereas mutant IDH1 converts α-KG into D2-hydroxyglutarate (D2HG). Unfortunately, there are few in vivo model systems for IDH-mutated tumors to study the effects of IDH1 mutations in tumor development. We have therefore created transgenic zebrafish lines that express various IDH1 mutants. Materials and methods IDH1 mutations (IDH1R132H, IDH1R132C and loss-of-function mutation IDH1G70D), IDH1wildtype or eGFP were cloned into constructs with several brain-specific promoters (Nestin, Gfap or Gata2). These constructs were injected into fertilized zebrafish eggs at the one-cell stage. Results In total more than ten transgenic zebrafish lines expressing various brain-specific IDH1 mutations were created. A significant increase in the level of D2HG was observed in all transgenic lines expressing IDH1R132C or IDH1R132H, but not in any of the lines expressing IDH1wildtype, IDH1G70D or eGFP. No differences in 5-hydroxymethyl cytosine and mature collagen IV levels were observed between wildtype and mutant IDH1 transgenic fish. To our surprise, we failed to identify any strong phenotype, despite increased levels of the oncometabolite D2HG. No tumors were observed, even when backcrossing with tp53-mutant fish which suggests that additional transforming events are required for tumor formation. Elevated D2HG levels could be lowered by treatment of the transgenic zebrafish with an inhibitor of mutant IDH1 activity. Conclusions We have generated a transgenic zebrafish model system for mutations in IDH1 that can be used for functional analysis and drug screening. Our model systems help understand the biology of IDH1 mutations and its role in tumor formation.


Introduction
The gene encoding isocitrate dehydrogenase 1 (IDH1) is frequently mutated in several tumor types including gliomas. The most prevalent mutation in gliomas is a missense mutation leading to a substitution of arginine with histidine at the residue 132 (R132H). Wild type IDH1 catalyzes oxidative decarboxylation of isocitrate to α-ketoglutarate (α-KG) whereas mutant IDH1 converts α-KG into D2-hydroxyglutarate (D2HG). Unfortunately, there are few in vivo model systems for IDH-mutated tumors to study the effects of IDH1 mutations in tumor development. We have therefore created transgenic zebrafish lines that express various IDH1 mutants.

Materials and methods
IDH1 mutations (IDH1 R132H , IDH1 R132C and loss-of-function mutation IDH1 G70D ), IDH1 wildtype or eGFP were cloned into constructs with several brain-specific promoters (Nestin, Gfap or Gata2). These constructs were injected into fertilized zebrafish eggs at the one-cell stage.

Results
In total more than ten transgenic zebrafish lines expressing various brain-specific IDH1 mutations were created. A significant increase in the level of D2HG was observed in all transgenic lines expressing IDH1 R132C or IDH1 R132H , but not in any of the lines expressing IDH1 wildtype , IDH1 G70D or eGFP. No differences in 5-hydroxymethyl cytosine and mature collagen IV levels were observed between wildtype and mutant IDH1 transgenic fish. To our surprise, we failed to identify any strong phenotype, despite increased levels of the oncometabolite D2HG. No tumors were observed, even when backcrossing with tp53-mutant fish which suggests that additional transforming events are required for tumor formation. Elevated D2HG levels could be lowered by treatment of the transgenic zebrafish with an inhibitor of mutant IDH1 activity. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111

Introduction
Somatic missense mutations in the gene encoding isocitrate dehydrogenase 1 (IDH1) or IDH2 are frequently identified in various malignancies including gliomas, acute myeloid leukemia, cholangiocarcinoma, chondrosarcoma and sporadically in various other cancer types [1][2][3][4][5][6][7][8]. In gliomas, IDH1 mutations are one of the earliest genetic changes identified, preceding other common genetic aberrations such as 1p19q co-deletion, and are therefore present in virtually all tumor cells [9][10][11]. IDH1 and IDH2 mutations are almost always mutually exclusive. For glioma patients, presence of IDH mutations is of clinical relevance as patients harboring IDH mutated gliomas have a better survival compared to those with wildtype IDH. The prognostic significance of IDH mutations has led to its incorporation in the WHO 2016 update to classify gliomas [12]. Mutations in IDH1 are almost always heterozygous point mutations affecting the arginine at position 132 (R132). Approximately 90% of these mutations in gliomas are IDH1 R132H .
Wildtype IDH1 is a cytoplasmic enzyme that catalyzes the oxidative decarboxylation of isocitrate to α-ketoglutarate (αKG) and uses NADP + as a co-factor [13,14]. The mutant enzyme however, uses αKG as a substrate to produce D-2-hydroxyglutarate (D2HG) with concomitant consumption of NADPH [15]. The resulting accumulation of D2HG then competitively inhibits a spectrum of αKG-dependent enzymes including TET2, JMJD2 and various prolyl hydroxylases [16][17][18]. This inhibition ultimately facilitates carcinogenesis by retaining cells in an undifferentiated and stem-like state. Because of the oncogenic role of mutant IDH1, several groups have developed compounds that specifically inhibit the activity of the mutant enzyme [19,20]. These inhibitors are currently being tested in clinical trials.
Several IDH1 R132H conditional knock-in (KI) mouse models were recently generated to further study the role of the mutant enzyme in an in vivo model system. Unfortunately most mice in which IDH1 mutations were conditionally expressed either died before birth or rapidly after induction of expression of the mutant enzyme [21]. Nevertheless, expression of mutant IDH1 results in a retention of cells in an undifferentiated state or induces pre-cancerous lesions in cartilage or the SVZ [22][23][24]. Despite these signs of early tumor formation, no gliomas in any of the three mouse models were thus-far identified, also not when backcrossing into a Tp53 -mutant background [23,25].
To further study the effects of IDH1 mutations in tumor development, we have generated transgenic zebrafish that express IDH1 mutants under the control of various CNS-specific promoters.

Generation of transgenic zebrafish
All experiments with zebrafish (Tupfel long fin or TL) were conducted according to the protocols approved by the Animal Experimentation Committee of the Erasmus Medical center and EU guidelines. To generate transgenic zebrafish lines expressing GFP-IDH1 wildtype and GFP-IDH1 R132H driven by different promoters (Nestin, Gata2 and Gfap), we injected various constructs into the cells of fertilized zebrafish eggs at the one-cell stage. Embryos that showed GFP expression at 1 day post fertilization (dpf) were collected and raised to adulthood (3 months, F0) and then individually crossed with non-transgenic wildtype TL (F1). GFP expression in the F1 fish indicated that the constructs were integrated into the fish genome. The GFP-positive F1s were separately raised to adults and then interbred to generate homozygous F2. Although we did not actually test for homozygosity of our transgenic lines, we inferred this by the observation that all F2 inbred offspring expressed GFP. All F2 progenies were further inbred. The experiments were performed mainly on likely homozygous F4 zebrafish. tp53 mutant fish (tp53 M214K ) were described by Berghmans et al and obtained from ZFIN (ZFIN. org) [27].

Histology and Immunohistochemistry
Zebrafish embryos were fixed overnight in 4% paraformaldehyde (PFA) at 4˚C and then embedded in paraffin for further histological analysis. Paraffin sections (6 μm) were stained with hematoxylin and eosin (HE). For immunohistochemistry (IHC), paraffin sections were dewaxed and hydrated followed by boiling in 10 mM sodium citrate for eight minutes and 2 times 3 minutes of boiling in a microwave oven. Prior to immunostaining, the endogenous peroxidase activity was blocked by 30% hydrogen peroxide and 12.5% sodiumazide in PBS for 30 minutes. The slides were washed in PBS and PBS+ which contained 0.5% g/ml protifar and 0.15% g/ml glycine and then incubated with primary antibody overnight at 4˚C. The primary antibodies used were anti-GFP (1:2000) monoclonal antibody (Roche, Woerden, the Netherlands), 5hmC (1: 200, Active Motif, La Hulpe, Belgium) and anti-human IDH1 R132H (1:200) monoclonal antibody (Dianova, clone DIA H09, Huissen, the Netherlands), diluted in PBS+. The sections were then washed in PBS+ and incubated with BrightVision Poly-HRP-Anti IgG (immunologic) for 60 minutes at room temperature (RT). The slides were washed in PBS + and PBS and then treated with 1:50 diluted DAB-substrate (DAKO Liquid DAB substratechromogen system) for 4-8 minutes, followed by counterstaining with haematoxylin and mounted in entellan. Histological images were captured using an Olympus BX40 camera.

Real-time PCR
To examine the expression of IDH1 and GFP during envelopment, total RNA was extracted by dissolving embryos in 500 μl TRIzol 1 (Life technologies, Carlsbad, USA) and 100 μl chloroform followed by centrifugation at 12.000 g for 15 min at 4˚C. RNA in the aqueous phase was precipitated with 250 μl isopropanol and collected at 12 000 g for 10 minutes at 4˚C. The pellet was washed twice in 250 μl 75% ethanol, centrifuged at 12 000 g for 5 minutes at 4˚C, dried and dissolved in 10 μl nuclease free water (Ambion, Thermo Scientific, Rochester, USA). For cDNA synthesis, each reaction contained 1000 ng RNA, 1 μl hexamers, 1 μl 10 mM dNTP's and milliQ water to 13 μl and was heated to 65˚C for 5 minutes and left on ice for at least 1 minute. The RNA was then treated with 4 μl 5x Firststrand buffer, 1 μl 0.1M DTT, 0.5 μl RNaseOUT and 0.5 μl DNase. The samples were heated to 37˚C for 40 minutes and further heated to 65˚C for 10 minutes. The RNAs were then reverse-transcribed by adding 1 μl Superscript III (Invitrogen, Breda, the Netherlands) and 0.5 μl RNase OUT followed by incubation at 25˚C for 5 minutes and 42˚C for one hour. 1 μl cDNA was used in a 15 μl reaction containing 7.5 μl Syber Select Mastermix (Life technologies), 1mM primers and MilliQ. The primer sequences are described in S1 Table. The reactions were performed in triplicate using a CFX96 Real-Time PCR System (Bio-Rad). The threshold cycle (Cq) for each reaction was obtained and the values were averaged. The standard deviation (SD) had to be below 0.2. The relative expression levels, of different time points in zebrafish life, were calculated. First the ΔCq of a sample was calculated; ΔCq = IDH1 Cq meanβ-actin Cq mean. Then one time point was set as a reference (= 1.00) and the ΔΔCq was calculated as ΔΔCq = ΔCq reference-ΔCq unknown sample. To calculate the relative expression levels the formula 2^Δ ΔCq was used.

Protein extraction and immunoblotting
Zebrafish embryos were lysed in 500 μl HEPES-buffer containing 1x protease inhibitor (cOmplete, Thermo Scientific) and 3 μM DTT followed by homogenization by a PRO 200 homogenizer and incubated on ice for 30 minutes. 50 μg of protein was separated by loading onto a precast SDS gel (Thermo Scientific) and electrophoresis at 150 V till loading buffer reached the bottom of the gel. Protein was then transferred to a nitrocellulose membrane in transfer buffer at 100 V, 380 mA, for 1 hour. The membrane was blocked with blocking buffer containing 1% BSA in PBS for 1 hour at room temperature and incubated with primary antibody overnight at 4˚C with agitation. Primary antibodies used were anti-Collagen Type IV (1:1000, Abcam, Hilversum, The Netherlands) and 5hmC (1: 1000).

Microinjection of wildtype zebrafish embryos with Gfap constructs
20 μl of injection solution containing 350 ng of Gfap constructs, 30 ng/μl Tol2 transposase RNA and 0.1% pheno-red was freshly prepared before injection. 4.2 nl of injection solution was injected into the cell of 1-cell stage embryos using a Pneumatic PicoPump (PV820, WPI). For each construct, injection was performed on 100 eggs in three independent experiments. The fertilization rate was calculated based on 30 uninjected embryos collected on the same day. The number of GFP + , GFP -, healthy and abnormal embryos were counted on 1dpf.

5hmC assay
Total DNA was extracted using whole fish embryos. A nitrocellulose membrane was presoaked in 20X SSC for 1 hour. 250 ng DNA was diluted in 150μl H2O and 150μl 20X SSC. The membrane and two layers of thick filter papers were placed on a manifold (manifold II slotblot manifold, Cole-Parmer, Wertheim, Germany) and equilibrated with 10X SSC. DNA samples were then loaded and fixed on the membrane using a vacuum pump for 5 minutes. The membrane was air-dried and processed as described above in the immunoblotting section. Blots were stained using the 5hmC antibody (1:1000) and analyzed using ECL.

IDH1 mutant inhibitor test
Five transgenic zebrafish embryos from each line were screened for GFP expression at 1 or 2 dpf and removed from the chorion and raised in 2 ml tap water for 48 hours with either 10μM AGI-5198 (Xcess Biosciences, Inc.) in 0.1% DMSO or 0.1% DMSO. Zebrafish embryos were collected at 3 dpf in 25 μl of HBSS buffer for 2HG measurement.

Quantification of D/L2HG in zebrafish
To quantify the level of D-and L2HG in the zebrafish, five embryos were collected at 1, 2, 3, 5 and 6 dpf in HBSS buffer (5μl per embryo). The embryos were homogenized with a PRO 200 homogenizer and lysed with sonication before LC-MS/MS. The D and L forms of 2HG were separately measured and quantified as described previously [28].

Statistics
Differences in D-and L-2HG levels between experimental conditions were evaluated using the students t-test. A Fisher's exact test was used to compare differences between frequencies.

Generation and characterization of transgenic zebrafish lines
We firstly generated transgenic lines for two constructs, eGFP-IDH1 wildtype and eGF-P-IDH1 R132H , in which the transgene was expressed under control of a Nestin promoter. These constructs are referred to as Nes IDH1wt and Nes R132H . At least two independent lines per construct were generated to control for integration-site dependent effects.
Transgene expression was detected in the brain and spinal cord on 1, 3 and 6 days post fertilization (dpf) by fluorescent imaging (Fig 1A and S1A Fig) and by immunohistochemistry ( Fig 1B) using anti-GFP antibodies. Expression of Nes R132H was confirmed using an IDH1 R132H -mutant specific antibody (S2 Fig). As expected, this antibody did not show staining in the Nes IDH1wt -fish. Expression of transgenes on 1, 2, 3 and 6 dpf was also detected on the RNA level by RT-QPCR ( Fig 1C). We then measured D2HG levels to monitor the activity of the neomorphic enzyme. Consistent with RNA and protein expression, the D2HG level in Nes R132H mutant transgenic fish was elevated compared to controls (non-transgenic and Nes IDH1wt ) on 1-5 dpf (Fig 1D). The increase in D2HG was virtually identical when using macro-dissected embryos (head region) compared to whole fish (S3 Fig). L2HG levels in all the transgenic lines were similar to the nontransgenic controls ( Fig 1E); indicating expression of IDH1 R132H only affects D2HG levels. D2HG levels returned to normal by 21 dpf. These experiments demonstrate CNS-specific expression of IDH1 wt or IDH1 R132H in our transgenic zebrafish lines during development. This temporal expression pattern in the CNS is consistent with the Nestin promoter activity [29][30][31].
It has been reported that accumulation of D2HG results in DNA hypermethylation by inhibition of TET enzymes [32]. In our transgenic fish, DNA methylation as determined by 5hmC antibody staining was however not affected (S4 Fig). We next screened for collagen maturation defects, as these were observed in an IDH1 R132H -KI mouse model [21]. However, western blot analysis failed to detect the presence of immature isoforms of collagen in our transgenic fish lines (S5 Fig). In summary, despite expression of the transgene (and the elevated levels of D2HG in lines expressing Nes R132H ), all of the zebrafish lines remained healthy without presenting any identifiable developmental abnormalities (S6 Fig). Given the short temporal expression of IDH1 constructs driven by the Nestin promoter, we cloned constructs under the control of a brain specific Gata2 promoter. This promoter was previously used for constructing a transgenic zebrafish model for neurodegeneration [33]. Three transgenic lines were generated, pGata2-GFP-IDH1 wt , pGata2-GFP-IDH1 R132H and pGa-ta2-GFP. These constructs are referred to as Gata IDH1wt , Gata R132H and Gata GFP . Unfortunately, we failed to observe any transgene expression in the developing (or adult) CNS in any of the lines generated. We did however observe expression in the notochord from 1 for up to 5 dpf (S7 Fig), but, despite expression of a D2HG-producing IDH1 mutant, all fish were viable, developed normally and produced offspring. Similar to the Nes IDH1wt and Nes R132H fish, no gross abnormalities or (pre-) neoplastic lesions were observed. As we failed to observe expression in the CNS we did not further investigate these lines.
Because of the temporal limitations of the Nestin promoter and the lack of expression in the CNS using the Gata2 promoter, we generated six additional lines, one for eGFP-IDH1 wildtype , eGFP-IDH1 R132H , eGFP-IDH1 G70D and eGFP, and two for eGFP-IDH1 R132C , in which the transgene was expressed under control of a Gfap promoter. Constructs used for these lines are referred to as Gfap IDH1wt , Gfap R132H , Gfap R132C , Gfap G70D and Gfap GFP . The expression vector (gfap:GFP) has been demonstrated to have glial-specific expression in the zebrafish CNS, detectable during the embryonic stage [34]. The IDH1 G70D mutation is an enzymatic null mutation that was included to serve as a non-D2HG-producing control [15,35]. The IDH1 R132C mutation was generated to study potential differences between IDH1 R132H and IDH1 R132C , and is a mutant with reportedly higher neomorphic enzymatic activity [36]. Expression of the transgenes was confirmed using fluorescent imaging on 1, 3 and 5 dpf (Fig  2A and S1B Fig). GFP was observed in the brain and spinal cord in all the Gfap transgenic zebrafish lines. CNS-specific expression of the transgene was further confirmed by immunohistochemistry using anti-GFP antibodies (Fig 2B). Expression of the transgene on the RNA level was confirmed by RT-QPCR till at least 20 dpf (Fig 2C). There were no obvious differences in results from these assays between IDH1 R132H and IDH1 R132C transgenic zebrafish. The D2HG levels were markedly elevated in both Gfap R132C zebrafish lines (line 84 and 85) on 3 dpf, which are about 8 and 18 times higher compared to the Gfap GFP and Gfap IDH1wt lines (line 73 and 92, Fig 2D). D2HG levels in Gfap G70D zebrafish remained similar as in Gfap GFP and Gfap IDH1wt lines. L2HG levels of all the IDH1 transgenic lines were similar to the Gfap GFP control, confirming that expression of transgene only affects the D2HG level ( Fig 2E).When crossing Gfap R132C line 85, an average of 21.1% of generated embryos showed abnormal tail development on 1dpf, which was higher than in the Gfap GFP (3%) and Gfap IDH1wt (0%) control lines (Fig 2F and 2G). The tail defects may be explained by the fact that the first detectable expression of Gfap is at 10 h post fertilization in the developing tail bud [34]. However, no clear (pre-) cancerous lesion was observed in any of the zebrafish lines studies.
It is possible that IDH1 R132H/R132C induced a pathologic phenotype due to the site of integration of our transgene. To correct for potential integration site artifacts, we directly injected fertilized zebrafish eggs at the one-cell stage with various constructs and monitored zebrafish development. We specifically monitored tail development in our transgenic fish. In three independent experiments, we injected fertilized eggs with Gfap GFP , Gfap IDH1wt or Gfap R132C constructs. While most embryos injected with Gfap GFP remained healthy (n = 105/129, S8A and S8D Fig), most embryos injected with the Gfap R132C construct were not (n = 75/85, P<0.001, Fisher's exact test). In line with our transgenic lines, many showed an abnormal development of the tail on 1dpf. However, zebrafish embryos injected with Gfap IDH1wt constructs also sometimes had abnormal tail development, though the frequency was significantly lower than that of Gfap R132C (n = 13/24, P<0.001).

tp53 deficient Transgenic zebrafish crossed with IDH1 mutant fish
TP53 mutations often co-occur in IDH1-mutated astrocytomas. To determine whether Gfap R132C affects tumor formation, we crossed the homozygous tp53 M214K mutant transgenic zebrafish with our transgenic zebrafish lines. It was previously reported that homozygous tp53 mutant zebrafish developed tumors (Schwannomas) at~8 months post fertilization with an incidence of 28% [27]. Although we find that heterozygous tp53 mutant fish developed tumors (Table 1 and S9 Fig, incidence = 15%, n = 2/13) with an average age of onset~1 year post fertilization, this incidence was not increased when the fish were crossed into a pGfap: GFP-IDH1 R132H (or wt ) background, with incidence between 6 to 14.3% regardless of the IDH1 variants or GFP controls (P>0.3 for all comparisons, Fisher's exact test). The non-CNS tumors we observe in our transgenic lines are most likely Schwannomas, as previously described [27]. They are mainly in the abdominal cavity, an area where we do not see expression of our transgene. Our results therefore demonstrate that expression of mutant IDH1 does not promote tumor formation in tp53 mutant zebrafish.

IDH1 mutated transgenic zebrafish as an in vivo model for drug screening
AGI-5198 is a specific inhibitor for the IDH1 mutant enzymatic activity [19]. To determine whether this inhibitor also affects D2HG production in vivo, we applied it to our transgenic zebrafish lines harboring different IDH1 variants under control of the Nestin and Gfap promoters at 1dpf for 48 hours. Dose-response analysis indicates maximal inhibition at 10μM AGI-5198 on the Nes IDH1 transgenic zebrafish (S10 Fig). The inhibitor did not show any overt toxicity, even after prolonged (2 days) treatment. The accumulated D2HG in our Nes R132H transgenic zebrafish was decreased by 10 μM AGI-5198 to 41% of the levels prior to treatment whereas D2HG levels in the non-transgenic and Nes IDH1wt transgenic zebrafish were not affected (Fig 3A). The D2HG levels were also markedly reduced in the Gfap R132C zebrafish line from 34.86 reduced to 8.77 pmol (25% of the D2HG level in the untreated fish (Fig 3B)). Levels of D2HG in control transgenic lines remained low and were not affected by the inhibitor. The L2HG level in all of the treated transgenic lines was not altered (Fig 3C and 3D). These data demonstrate that our transgenic zebrafish lines can be used to screen the efficacy and toxicity of drugs that inhibit IDH1 mutant enzyme activity.

Discussion
Studying IDH1 mutations in gliomas has been hampered by the difficulty in generating appropriate model systems. For example, IDH-mutated gliomas are notoriously difficult to propagate in vitro [37] and mouse models for IDH1 mutations often have a lethal phenotype when IDH1 R132H is expressed at early stages of development [21,23,25]. Mice can survive when the mutant protein is expressed at later stages, but often with severe phenotypes (e.g. hydrocephalus). A Drosophila model with UAS-Idh-R195H resulted in defects in wing expansion [38]. Here we report on transgenic zebrafish model systems for IDH1 mutations, and show that expression of IDH1 mutations and subsequent accumulation of D2HG does not overtly affect zebrafish development in the majority of offspring. However, tail development defects were observed in a subset of offspring in one line and also following direct injection of mutant constructs in wildtype zebrafish embryos (S8 Fig). Expression of mutant, but not wildtype IDH1 may therefore affect the cells required for normal tail development.
In contrast to the Nestin or GFAP-R132H KI mouse models, we did not observe any overt phenotype in any of the Nes R132H transgenic zebrafish and the majority of Gfap transgenic fish. In mice, the brain hemorrhage phenotype is caused by collagen maturation defects (caused by inhibition of prolyl hydroxylases by D2HG) [21]. Alternatively, brain hemorrhages may be secondary to D2HG mediated coagulation defects [39]. In our transgenic lines, we failed to detect fish embryos were not affected (E). About 21% Gfap R132C embryos showed defects in tail development on 1dpf (F and G). Arrow heads: the site of abnormal tail development in the Gfap R132C embryos. For Q-PCR experiments, we used a pool of five (3dpf) or three (20 dpf) fish per time-point; D2HG and L2HG measurements were averages of two replicates using five (3 and 5 dpf) or three (20 dpf) fish per replicate. Scale bar: 500 μm.
https://doi.org/10.1371/journal.pone.0199737.g002 signs of collagen maturation defects or brain hemorrhage, which may provide an explanation why our fish are able to survive into adulthood. The absence of collagen maturation defects in our fish may be related to the level of D2HG accumulation in our model system, the expression level of IDH1 R132H in our transgenic fish, and/or to the more limited temporal expression of our constructs. D2HG accumulation may also be limited as it is likely able to diffuse out of the fish into the water of the tank. This may explain that only a modest increase in the D2HG level was detected in the IDH1-mutated fish. We failed to detect changes in 5hmc levels in the IDH1-mutated embryos which may also be caused by insufficient accumulation of local D2HG within the fish. In addition, any potential effects on fish 'fitness' is selected against in the process of generating transgenic lines: only healthy fish (despite elevated D2HG levels) are used to generate stable lines. We were unable to detect CNS-specific tumors in our transgenic zebrafish lines. This is in line with data from mouse models in which brain tumors were thus far not detected. This supports the notion that IDH1 mutation alone may be insufficient to promote tumor formation and other genetic alterations are required [40]. In most astrocytomas, TP53 and IDH mutations often co-occur [41]. However, the combination of IDH1 and tp53 mutations did not induce gliomas in our zebrafish model. Similarly, IDH1 mutations also did not increase tumor incidence in Tp53 mutant mouse model, despite the observation that IDH1 mutations induce pre-cancerous lesions such as proliferative subventricular nodules in one mouse model, but interestingly not in an almost identical other model [23,25]. Similarly, mutations in IDH1/2 in combination with Tp53 deficiency were insufficient to induce gliomagenesis in RCAS/tva mouse models [40]. Since mutations in IDH and TP53 are the most common genetic alterations in astrocytomas, it remains unclear which additional mutations are required to induce glioma formation in our zebrafish model. Candidate genes should include oncogenic drivers ATRX and/or PDGFRA as they are present at significant frequency in lower grade gliomas. Of note, zebrafish has been appreciated as a valid model to study tumorigenesis, for example, tp53mutant fish develop schwannomas and gliomas can also be generated in zebrafish by activating akt1 alone [42]. Our data also show that D2HG can be present at high levels throughout the development of zebrafish without any overt signs of pathology (although a minority of our transgenic fish did show defects in tail development). These data are in line with the observation that some D2HG aciduria patients, which have high levels of D2HG due to mutations in IDH2 or D2HGDH, do not have any overt phenotype. Moreover, D2HG aciduria patients do not have an increased incidence of brain tumors [43].
In conclusion, we developed various transgenic zebrafish models with CNS-specific expression of IDH1 mutation. We identified tail defects in a subset of IDH1-mutant fish, but we thus-far failed to identify tumors. Nevertheless, our transgenic zebrafish are a suitable model to functionally study the IDH1 mutation in vivo or to use as a drug screening model.