Kdm2aa but not Kdm2ab loss of function zebrafish show growth deficiency and reduced viability

We assessed the in vivo function of KDM2A using zebrafish mutants generated by the Zebrafish Mutation Project [32]. KDM2A has two paralogues in zebrafish, kdm2aa (ENSDARG00000059653) on chromosome 1 (chr1) and kdm2ab (ENSDARG00000078133) on chr14 (Fig 1A). Embryonic expression of kdm2ab peaks during blastula stages, whereas kdm2aa expression is highest later in embryogenesis, during gastrula and early segmentation stages (Fig 1I). We raised two premature stop codon alleles affecting kdm2aa and one premature stop codon allele affecting kdm2ab (Fig 1A). kdm2aa^sa898 and kdm2ab^sa1479 are assumed to produce non-functional protein. kdm2aa^sa9360 may produce a partially functional protein lacking the F-box and LRRs.

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Fig 1. Loss of Kdm2aa function leads to growth and oogenesis defects.

(A) The zebrafish paralogues Kdm2aa and Kdm2ab share the protein domain structure with their human orthologue KDM2A. The mutations kdm2aa^sa898 and kdm2aa^sa9360 produce premature stop codons in the JmjC and F-box domain, respectively. The allele kdm2ab^sa1479 produces a premature stop codon in the JmjC domain. (B) Juvenile fish compound heterozygous for kdm2aa^sa898 and kdm2aa^sa9360 are shorter and thinner at 30 d.p.f. than their heterozygous siblings. Scale bar: 5 mm. (C) Box plots showing length in mm of each of the three sibling groups for a representative family at 30, 90 and 180 d.p.f. Fish homozygous for kdm2aa^sa898 are smaller than their siblings. (D) Fish homozygous for premature stop codons in kdm2aa represent less than the expected 25% of the group. (E) A small proportion of maternal zygotic kdm2aa^sa9360 embryos survive to 5 d.p.f. with mild (arrow in second panel) or no malformations (bottom panel). (F) Embryos derived from kdm2aa^{sa9360/sa9360} intercrosses show severe defects and lethality during the first 24 h.p.f. whereas kdm2aa^{sa9360/sa9360} male outcrosses are normal. (G) Two examples of embryos derived from kdm2aa^{sa9360/sa9360} female outcrosses displaying severe defects at 24 h.p.f. (H) TRITC-Phalloidin and DAPI staining on MZkdm2aa^-/- embryos derived from intercrosses of kdm2aa^sa898/sa9360 fish at the 8–16 cell stage showed that despite cell asymmetry and asynchronous division, nuclei seemed overall normal. (I) Expression data from Expression Atlas http://www.ebi.ac.uk/gxa/experiments/E-ERAD-475 showing normalised counts (FPKMs) from RNA-seq data for kdm2aa and kdm2ab. There is maternal contribution of both genes, but kdm2ab polyA transcript abundance peaks at early blastula stages, whereas kdm2aa polyA transcript abundance peaks later at the gastrula shield stage.

https://doi.org/10.1371/journal.pgen.1006959.g001

Fish homozygous for kdm2ab^sa1479 showed no phenotype by 5 days post fertilisation (d.p.f.), grew to adulthood in the expected Mendelian ratio and had healthy offspring. We therefore concluded that kdm2ab loss of function (LOF) does not produce an overt embryonic or adult phenotype. Equally, both kdm2aa^sa898 and kdm2aa^sa9360 homozygous embryos did not display morphological defects at 5 d.p.f. (S1A and S1B Fig). We also generated double mutants between kdm2aa^sa898 and kdm2ab^sa1479 to test whether there was compensation between the paralogues, but embryos homozygous mutant for both genes also showed no phenotypic difference to their siblings at 5 d.p.f. (S1 Table).

However, by 30 d.p.f. juvenile fish homozygous for either kdm2aa allele were thinner and shorter compared to their siblings (Fig 1B and 1C, S2 Table and S1 File). The size difference persisted into adulthood at 180 d.p.f. (Fig 1C). We confirmed that this phenotype was due to the loss of kdm2aa function by raising two clutches containing compound heterozygous kdm2aa^sa898/sa9360 fish (Fig 1B and S1 File). In addition, survival of homozygotes was reduced at 30 d.p.f. from the expected 25% to below 20% and fell further by 90 d.p.f. (Fig 1D, S2 Table and S1 File). Furthermore, incrosses for either kdm2aa allele produced at most two or three females out of a maximum of 20 homozygotes.

kdm2aa^-/- females have a progressive oogenesis defect

We next assessed whether homozygous mutant kdm2aa adults were fertile. Initial intercrosses of kdm2aa^{sa9360/sa9360} or compound heterozygous kdm2aa^sa898/sa9360 adults produced phenotypically diverse clutches in which some embryos successfully inflated their swimbladders and either developed phenotypically normally (Fig 1E bottom panel), or with only mild defects (Fig 1E middle panel). Later crosses of the same females produced clutches in which over half of the eggs either failed to fertilise or did not divide beyond four cells (S1C Fig). The remaining eggs showed severe cleavage defects with asymmetric division, detaching cells, and slower division rate (Fig 1F). By 24 hours post fertilisation (h.p.f.) about a third of the maternal-zygotic mutant (MZ) kdm2aa^-/- embryos had died and those that survived displayed degrees of generalised developmental defects (Fig 1G). This indicated that subsequent intercrosses from the same females displayed a progressive worsening of egg quality, with fewer eggs being fertilised and fewer embryos surviving beyond 24 h.p.f. Double labelling with DAPI and TRITC-conjugated phalloidin of 8–32 cell wild-type and MZkdm2aa^-/- embryos confirmed asymmetric cells and unsynchronised division (Fig 1H).

To confirm that this phenotype was caused by kdm2aa LOF in the female, we outcrossed male and female kdm2aa^{sa9360/sa9360} fish to wild-type fish of the same genetic background. Offspring from three kdm2aa^{sa9360/sa9360} males were normal (Fig 1F and S1C Fig). By contrast, the majority of embryos from initial outcrosses of two kdm2aa^{sa9360/sa9360} females died before 5 d.p.f., however some (12/64) embryos survived to 5 d.p.f. with 6 out of 12 showing no obvious phenotype (S1D Fig top panel) and the remaining 6 displaying only localised malformations (S1D Fig). Subsequent homozygous female outcrosses produced clutches with low fertilisation rates and embryos with severe defects very similar to MZkdm2aa^-/- embryos (S1C and S1E Fig).

This demonstrates that embryos from oocytes devoid of functional kdm2aa mRNA or protein can develop normally and that the maternally deposited mRNA (Fig 1I) [33] does not explain the lack of phenotype in zygotic homozygous mutants. Instead the increase in unfertilized eggs and severity of the phenotype in the remaining embryos point to a role for Kdm2aa in maintaining the production of healthy oocytes.

Kdm2aa-deficient fish develop melanoma

From the age of 7 months, we observed that kdm2aa-deficient fish (homozygotes for either allele and also compound heterozygous fish) began to develop suspected cancers. We observed aberrant melanocytic pigmentation at the base of the tail extending into the tail fin (Fig 2Ai), masses behind one eye causing it to protrude (Fig 2Aii) and masses on the body (S2B Fig). 23/92 (25%) of kdm2aa^sa898/sa898 (Fig 2B) and 20/204 (10%) of kdm2aa^{sa9360/sa9360} (S2A Fig) fish developed these suspected cancers within the first 28 months, whereas none of the heterozygous or wild-type siblings did. Of the 43 fish with potentially cancerous phenotypes, 10 fish had excessive melanocytic pigmentation on their tail, 12 fish had a tumour behind one eye causing it to protrude, 18 fish had a mass on their body, and 3 fish were found to have both excessive melanocytic pigmentation on their tail and a mass on their body.

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Fig 2. Kdm2aa-deficient fish develop melanoma.

Aberrant melanocytic pigmentation on the tail (Ai, arrow) and eye tumours (Aii, arrow) are observed in Kdm2aa-deficient fish. (B) Survival graph for kdm2aa^sa898 showing incidence of suspected cancer. Each (*) indicates a single culled fish due to suspected cancer. No wild-type or heterozygous siblings developed any suspected cancers. (C) H and E stained section of a tail tumour showing a biphasic appearance with pseudoglandular features (arrow shows an example) alternating with areas of spindle cell growth. (D) Section of tail tumour staining positive for the melanoma marker Melan-A (brown). (E) Section of tail tumour negative for the melanoma marker S100. (F) Section of tail tumour staining negative for the melanoma marker HMB-45. (G) Section of tail tumour staining positive for mitotic marker phospho-histone H3 (brown). (H) Section of tail tumour showing negative staining for the neuroendocrine marker Synaptophysin. Arrow points to example of pseudoglandular structures. (I) Section of tail tumour showing positive staining for the epithelial marker Cytokeratin (brown) in both the pseudoglandular and spindle cell elements. (J) Section of tail tumour with positive phospho-ERK staining (brown). (K) Section of tail tumour with positive phospho-AKT staining (brown) (L) Section of a second tail tumour which did not stain positive for phospho-AKT. (M) H and E stained section from an eye tumour revealing spindle cell morphology. (N) Section of eye tumour staining positive for Melan-A (brown). (O) Eye tumour section showing negative staining for the S100. (P) Section of eye tumour negative for HMB-45. (Q) Section of eye tumour staining positive for phospho-histone H3 (brown). (R) Phospho-ERK staining of eye tumour sections was negative. (S) Section of eye tumour showing positive staining for phospho-AKT (brown). Scale bar in C-S is 100 μm.

https://doi.org/10.1371/journal.pgen.1006959.g002

To confirm that these growths were cancerous, tissue sections from 10 affected fish and 2 control siblings were haematoxylin and eosin (H&E) stained and analysed by two independent clinical histopathologists. Seven of the fish had excessive melanocytic pigmentation on their tails, and all of these fish were diagnosed with spindle cell malignant melanoma on the tail, invading the surrounding skeletal muscle and bone to varying degrees (Fig 2C). Furthermore pigmented melanophages were present in half of the tumours and these cells have previously been reported in zebrafish melanomas [34]. Two of the fish analysed had eye tumours, which confirmed as either spindle cell, or epithelioid and spindle cell melanoma and pigmented melanophages were present in one of the two tumours. A single fish was analysed with a suspected abdominal tumour and this was found to have a nodular lesion around the ultimobranchial body, vena cava and pancreas, composed of epithelioid and spindle cells (S2C Fig). No pigmented melanophages were present. Additionally in internal sections from one of the fish with excessive melanocytic pigmentation on the tail an abnormal spindle-cell proliferation within the proximal intestinal epithelium and the pancreas was found. Given the pigmentation, spindle cell morphology and malignant proliferation these two abdominal tumours are consistent with melanoma, but further analysis would be required for a firm diagnosis.

Three additional affected fish, two with excessive melanocytic pigmentation on the tail and one with an eye tumour, were analysed further using immunohistochemistry. H&E staining of both tail tumours revealed a biphasic appearance, with pseudoglandular or rosette-like structures alternating with areas of spindle cell growth (Fig 2C) and both the pseudoglandular and spindle cell elements stained positive for the melanoma marker Melan-A (Fig 2D) but negative for two alternative melanoma markers S100 and HMB-45 (Fig 2E and 2F). These tumours were also diagnosed as melanoma showing divergent differentiation. Both tumours stained positive for phospho-histone H3 (Fig 2G) indicating that they were mitotically active. To further characterise the pseudoglandular differentiation the tail tumours were stained for the neuroendocrine marker Synaptophysin (Fig 2H) which was negative and for the epithelial marker Cytokeratin (Fig 2I) which was positive. The eye tumour shared many characteristics with the tail tumours and was diagnosed as invasive melanoma; H&E staining revealed spindle cell morphology (Fig 2M), Melan-A and phospho-histone H3 were positive (Fig 2N and 2Q) and S100 and HMB-45 were negative (Fig 2O and 2P). Interestingly, both tail tumours stained positively for phospho-ERK (Fig 2J) indicating activation of the MAPK signalling pathway, whereas the eye tumour was phospho-ERK negative (Fig 2R). One tail tumour and the eye tumour stained positive for phospho-AKT (Fig 3K and 3S) indicating that PI3K signalling was activated, whereas the second tail tumour was phospho-AKT negative (Fig 2L).

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Fig 3. RNA-seq profiling of individual kdm2aa-deficient embryos and their siblings.

(A) Summary of experimental design. TruSeq stranded mRNA sequencing libraries were prepared from four individual embryos from each genotype at both 5 d.p.f. and 12 d.p.f., totalling 48 libraries. Paired end sequencing with a read length of 75 bp was performed on four lanes of Illumina HiSeq 2500 machines. Sequence was aligned to the GRCz10 reference genome with TopHat and read counts were obtained with HTseq-count. Quality control was performed using QoRTs and outliers removed. 32,261 genes were detected. DESeq2 was used to determine DE genes in each individual clutch, DE genes at each stage by combining the two clutches for each stage whilst controlling for clutch in the DESeq2 model, and also in a combined analysis combining all 4 clutches whilst controlling for stage and clutch. (B) Venn diagram showing the overlap in DE genes between the two 5 d.p.f. experiments and the combined 5 d.p.f. analysis. The increase in power due to the increased sample size enabled an additional 1106 DE genes to be detected. (C) Venn diagram displaying the overlap in DE genes between the two 12 d.p.f. experiments and the combined 12 d.p.f. analysis. An additional 1102 DE genes were detected in the combined analysis. (D) Volcano plot of all detected genes in the combined RNA-seq analysis with the 3752 DE genes shown in red and kdm2aa circled. (E) Box plot of normalised counts for the chromatin modifier nsd1b, with adjusted p-values for individual experiments, stage-specific and combined analysis as indicated by horizontal bars. Data for heterozygous and wild-type siblings are combined. In the figure legend ‘s’ denotes siblings and ‘m’ homozygous mutants. (F) Box plot of normalised counts for dnmt3bb.2, a de novo DNA methyl transferase, with adjusted p-values for individual experiments, stage-specific and combined analysis as indicated by horizontal bars. Data for heterozygous and wild-type siblings are combined. In the figure legend ‘s’ denotes siblings and ‘m’ homozygous mutants. (G) Table of enriched Gene Ontology (GO) terms of the biological process (BP) GO domain which overlap in both 5 d.p.f. experiments and also the 5 d.p.f. combined analysis. P-values shown are for the combined analysis. (H) Table of enriched GO terms that overlap in both individual 12 d.p.f. experiments and the 12 d.p.f. combined analysis, with p-values for the combined analysis shown.

https://doi.org/10.1371/journal.pgen.1006959.g003

Melanoma development in kdm2aa-deficient zebrafish is independent of coding mutations in common oncogenes and tumour suppressors

To assess the mutational landscape in kdm2aa-deficient fish melanomas, we performed whole exome sequencing on four dissected tumours, adjacent non-tumour control tissue and sibling tissue, and called the single nucleotide variants (SNVs) and small insertions/deletions (indels) present. Across the 11 samples we obtained exome coverage of 50x (S3 Table). Laboratory zebrafish are not inbred and consequently there is a high level of natural variation [35]. We therefore used exome data from 3,811 individual fish generated in the Zebrafish Mutation Project [32] to define a common variant catalogue of 61,276,211 SNVs and filtered the SNVs found in sibling, control and tumour tissues using this variant set. This revealed on average 951 SNVs between sibling fish and control tissues (Table 1). Tumour tissues harboured on average 517 SNVs compared to non-tumour tissue from the same fish demonstrating that the tumours had accumulated mutations and increased their SNV burden by the equivalent of 50% of the normal sibling variation. By contrast the tumours had not increased their indel frequency with each tumour only showing one additional indel when compared to control tissue (Table 1).

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Table 1. Mutations present in tumour samples.

https://doi.org/10.1371/journal.pgen.1006959.t001

Next, we sought to identify potential protein-disrupting mutations. Filtering the mutations for those which are predicted to disrupt the protein (for details see Methods) revealed between 9 and 21 disruptive mutations per tumour compared to control tissue, and there was no overlap between these acquired disruptive mutations in the different tumours (S3 Table). Whole genome sequencing would be needed in order to assess whether mutations were present in non-coding regions.

Although we did not detect a common mutational signature in the tumours analysed, the exome data showed that none of the oncogenes or tumour suppressors most commonly mutated in human melanomas had accumulated exonic mutations (S3 Table). We looked specifically at the 13 significantly mutated genes in human cutaneous melanomas identified by The Cancer Genome Atlas, as well as HRAS and KRAS which were used to categorise the samples and also GNAQ, GNA11, KIT, CTNNB1 and EZH2 which were found to carry mutations at low frequencies in the triple wild-type set of melanomas [36]. We used the Ensembl Compara database [37] to detect orthologues for these genes in our whole exome sequencing data, but none were found to carry potentially disruptive exonic mutations (S3 Table). Analysis of four tumours is not enough to prove conclusively that kdm2aa-deficient melanomas never harbour mutations in common oncogenes. However if mutations were present at the same frequency as in human melanomas there is, for example, a 94.7% probability that we would have detected a braf mutation (present in 52% of human melanomas [36]) and a 73.1% probability that we would have found nras mutations which are present in 28% of human melanomas [36]. Since BRAF and NRAS hotspot mutations are almost mutually exclusive [36] there is close to a 97.6% probability that we would have found either a BRAF or an NRAS hotspot mutation in at least one of the tumours. Therefore the absence of potentially disruptive mutations in any of the 20 genes assessed across the four melanomas supports a role for Kdm2aa in melanoma development independent of common oncogenes and tumour suppressors.

mRNA expression profiling in individual embryos reveals widespread effects on transcript abundances in kdm2aa LOF fish

Given the role of Kdm2aa in chromatin regulation [20, 21, 26] we investigated whether loss of Kdm2aa resulted in an altered transcriptional profile which might explain the observed phenotypes. We performed a comparative transcriptome analysis (polyA RNA-seq) on four individual homozygous mutant, heterozygous and homozygous wild-type siblings each from both alleles at 5 d.p.f. and 12 d.p.f. giving us four mRNA expression profiles (Fig 3A). We chose these time points and individual embryos from two different alleles to capture changes at the mRNA level in morphologically normal individuals rather than secondary transcriptional deviations due to size differences, developmental delay and genetic background. Using DESeq2 [38] we determined differential transcript abundance as significant at an adjusted p-value <0.05. This revealed that kdm2aa transcripts were present at lower levels in fish homozygous for either allele and at both stages (log₂ fold change between -0.34 and -0.73), indicating nonsense-mediated decay [39] had occurred. We tested for haploinsufficiency in heterozygous animals by running differential analysis between heterozygous and wild-type embryos in both alleles at 5 d.p.f. and 12 d.p.f. This yielded 0 and 1 differentially expressed (DE) genes for kdm2aa^sa9360/+ at 5 d.p.f. and 12 d.p.f., respectively. By contrast, kdm2aa^sa898/+ heterozygosity led to 29 (5 d.p.f.) and 80 (12 d.p.f.) DE genes, suggesting a mild haploinsufficiency effect of that allele on mRNA levels (S4 Table). When comparing homozygous mutants with siblings (Fig 3B–3D and S4 Table) between 539 and 1433 genes out of 32,261 detected genes were significantly differentially abundant in the four clutches. The four DE gene lists only had 19 genes in common (S3A and S3B Fig) with an additional 76 DE genes being significant in at least three of the four clutches (S3A Fig). The discrepancy between the clutches could either be due to clutch-specific and/or stochastic effects on transcript abundance or the fact that four biological replicates per condition do not provide sufficient power to detect differential gene expression above individual embryo variability. To test this we combined the samples for each stage and ran the differential analysis of homozygous mutants against siblings while controlling for clutch in the DESeq2 model (Fig 3A and Methods). The combined stage-specific analyses showed large overlap with their respective individual experiments (77% and 72% at 5 d.p.f., 57% and 75% at 12 d.p.f.), confirming that the majority of discrepancy was due to detection power rather than clutch difference (Fig 3B and 3C). The increase in power due to the larger sample size also enabled us to detect over 1100 additional DE genes for each stage (Fig 3B and 3C). Combining all four experiments in the analysis while controlling for stage and clutch identified 3,752 DE genes (Fig 3D). These results are consistent with previous findings that the number of biological replicates is the main factor in the ability to identify differentially expressed genes [40].

Genes involved in translation, DNA replication and chromatin regulation respond to loss of kdm2aa

Gene ontology (GO) analysis of DE genes using topGO [41] revealed enrichment of a large number of terms relating to translation, DNA replication, energy metabolism, and chromosome organisation in the biological process (BP) domain in the four separate and the combined 5 d.p.f. RNA-seq analyses (S5 Table). The translation enrichment was driven mostly by upregulation of genes encoding ribosomal proteins (19/72 contributing genes in the 5 d.p.f. analysis), translation elongation or initiation factors (14/72) and mitochondrial ribosomal proteins (9/72), which is consistent with KDM2A’s described function in repressing ribosomal RNA genes [23]. This upregulation of ribosomal genes and energy generation processes together with differential expression of DNA replication genes suggests cellular stress.

We also found stage-specific differences. While different terms relating to translation, chromosome organisation and metabolism appeared in all individual analyses, the GO enrichment at 12 d.p.f. also included a large number of terms related to development of different tissues. This is very likely to reflect the emerging growth retardation observed morphologically from 30 d.p.f. onwards. To visualise this stage difference we filtered the lists for terms that are present in the stage analysis as well as their individual experiments (Fig 3G and 3H and S3C and S3D Fig). This showed a dominance of translation, DNA replication and chromosome segregation at 5 d.p.f., whereas the list at 12 d.p.f. contains mostly translation- and development-related terms.

In accordance with the role of Kdm2aa in chromatin regulation the theme of DNA replication and chromatin remodelling represents the core gene expression profile even in the comparatively small set of 95 DE genes that overlapped between at least three individual clutches across both stages and alleles (S3E Fig). Included in this core set are chromatin modifiers such as nsd1b, a methyltransferase for the KDM2A target H3 lysine 36, and the de novo DNA methyltransferase dnmt3bb.2 which is recruited to DNA by H3K36me3 [42], both of which were downregulated (Fig 3E and 3F). By contrast, the gene encoding the Snf2-related CREBBP activator protein Srcap, the catalytic subunit of a protein complex that incorporates the histone variant H2A.Z at promoters and eu- and heterochromatin boundaries was upregulated (S3F Fig) (S2 File for all count plots). This gene expression signature suggests a compensation for loss of H3K36-demethylase activity and a wider concerted response to chromatin disruption.

Kdm2aa represses specific genes in heterochromatic regions

When plotting up- and downregulated genes separately onto their chromosomes, we noticed enrichment of upregulated genes on the long arm of chr4 (Fig 4A). This region is repeat rich (Fig 4A), and contains extensive constitutive heterochromatin [43, 44]. We therefore speculated that kdm2aa LOF causes generalised de-repression of genes located within this heterochromatin stretch. However, of the 208 genes that were upregulated on the long arm of chr4 in the combined analysis, 183 genes were annotated as containing a zinc finger (ZnF) domain. ZnF domain-containing genes represented 49.2% of detected genes on the long arm of chr4, but rose to 85.5% in the DE gene set and thus demonstrated specific enrichment (Fig 4B). Furthermore, none of the 183 DE ZnF genes on the long arm were downregulated whereas this was the case for 6 of the other 31 DE genes (Fig 4C). Kdm2aa therefore seems to have a function in repressing heterochromatic ZnF genes on the long arm of chr4 in a gene-specific manner. We have shown previously that these genes are normally expressed in a sharp peak at zygotic genome activation [33], pointing to a role for these genes in regulating zygotic transcription.

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Fig 4. Specific de-repression of a group of ZnF genes on the long arm of chromosome 4.

(A) Ideogram of all DE genes in the combined analysis. Upregulated genes are plotted on the left, down-regulated genes on the right of each chromosome. Genes containing ZnF domains are indicated in red. Red shading of chromosomes shows type I transposons. (B) Table of genes detected and differentially expressed on the long arm of chr4 showing enrichment for ZnF domain-containing genes in the differentially expressed sets. (C) Volcano plots of genes on the long arm of chr4 demonstrate that genes without a ZnF domain are both up- and downregulated, whereas ZnF genes are exclusively upregulated.

https://doi.org/10.1371/journal.pgen.1006959.g004

Husbandry

Zebrafish were maintained in accordance with UK Home Office regulations, UK Animals (Scientific Procedures) Act 1986, under project licence 70/7606, which was reviewed by the Wellcome Trust Sanger Institute Ethical Review Committee. Embryos were obtained either through natural matings or in vitro fertilisation and maintained in an incubator at 28.5°C up to 5 days post fertilisation (d.p.f.). The mutant alleles kdm2aa^sa898, kdm2aa^sa9360 and kdm2ab^sa1479 were obtained from the Zebrafish Mutation Project [32].

Length measurements

Standard length (SL) and height at the anterior margin of the anal fin (HAA) of anaesthetised offspring from heterozygous intercrosses were measured at 30, 90 and 180 d.p.f. Measurements were taken as previously described [78]. Tissue samples were taken from each measured fish for genotyping either by sacrificing whole individuals at 30 d.p.f. or by caudal fin biopsies at 90 and 180 d.p.f. To test whether there is a difference in SL or HAA as a function of genotype, we performed ANOVA on each clutch to check for significant differences between the three genotype groups of homozygous mutant, heterozygous and homozygous wild-type fish. Post-hoc testing (Tukey HSD) was used to assess which groups differed significantly.

Genotyping

DNA from embryos or fin biopsies was extracted and DNA samples were genotyped for kdm2aa^sa898, kdm2aa^sa9360 or kdm2ab^sa1479 using KASP genotyping as previously described [79].

Histology

Fish samples were either collected in formalin and sent to Advance Histopathology Laboratory Ltd, 75 Harley Street, London, UK, for H&E staining and analysis, or fixed, processed and stained as described in [80]. Briefly, fish tissue was fixed in 4% PFA at 4°C for 3 days, decalcified in 0.5M EDTA (pH 8) at 4°C for 5 days and transferred to 70% ethanol. It was then processed in 95% ethanol, absolute alcohol, xylene and paraffin wax, embedded in wax blocks, cut into 5 μm thick sections and placed onto glass slides.

Hematoxylin and eosin staining and immunohistochemistry were performed as described in [80]. The slides were de-waxed by xylene and ethanol washes, stained, dehydrated and mounted with DPX. Antigen retrieval for IHC was performed in 0.01 M citrate buffer (1.8 mM citric acid, 8.2 mM sodium citrate, distilled water—pH 6) in a microwave pressure cooker. The samples were stained with the primary antibody (monoclonal mouse anti-human Melan-A clone A103, DAKO, Cat. No. M7196 concentration 1:75, anti-phospho-Histone 3, Cell Signalling Technology, rabbit, 1:200, anti-phospho-p44/42 MAPK (Erk1/2), Cell Signalling Technology, rabbit, 1:400 and anti-phospho-Akt, Cell Signalling Technology, rabbit, 1:50) overnight at 4°C and secondary antibody (HRP rabbit/mouse, DAKO) for 30 min at room temperature. DAKO Real EnVision Detection System (Peroxidase/DAB+, Rabbit/Mouse, Cat. No. K5007) was used to visualise the IHC staining. S100, HMB-45, Cytokeratin AE1/3 and Synaptophysin antibody stainings were performed under standard laboratory conditions at the Immunohistochemistry Laboratory in the Department of Pathology, Royal Infirmary of Edinburgh.

The stained slides were imaged using Pathology Nanozoomer SlideScanner and the images were processed using NDP.2 software.

Whole mount staining of zebrafish embryos

For DAPI and TRITC-Phalloidin staining, embryos at the 8–32 cell stage were fixed in 4%PFA/PBS overnight at 4°C, washed in PBST (0.1% Tween-20 in PBS) and dechorionated. After 4 x 30 minute washes in 2% Triton/PBS they were incubated with 4',6-diamidino-2-phenylindole (DAPI) (1:300) in PBST and TRITC-Phalloidin (1:200) in PBST in the dark at 4°C overnight. Embryos were washed 3-4x in PBST, mounted in Vectashield Antifade Mounting Medium and imaged using a Leica SP5 confocal microscope.

Illumina library preparation for RNA-seq

Using Sera Mag beads, total nucleic acid was isolated from 96 larvae from heterozygous sibling intercrosses for both kdm2aa alleles at 5 d.p.f. and 12 d.p.f. resulting in four experiments. KASP genotyping was performed on all samples to identify 4 individual homozygous mutant, heterozygous and wild-type sibling samples for each of the four experiments. From these 48 samples 300 ng total RNA were used to prepare sequencing libraries with Ambion ERCC spike-in mix 1 (Cat. No. 4456740) according to the manufacturer’s instructions using the Illumina TruSeq Stranded mRNA Sample Prep Kit Set A and B (RS-122-2101 and RS-122-2102). Paired end sequencing with a read length of 75 bp was performed on four lanes of Illumina HiSeq 2500 machines.

Read mapping and differential expression analysis

Quality control of sequenced samples was performed using QoRTs [81] and 7 libraries showing characteristics of RNA degradation were excluded from further analysis. Sequence was aligned to the GRCz10 reference genome with TopHat 2.0.13, using a known transcripts file from Ensembl v87 (ftp://ftp.ensembl.org/pub/release-87/gtf/danio_rerio/Danio_rerio.Zv9.87.gtf.gz) and the "fr-firststrand" library type option. Read counts were obtained with htseq-count and used as input for differential expression analysis with DESeq2. For the analyses of individual clutch experiments, the DESeq2 model was “~ condition” where the condition is either “hom” or “het_wt”. For the stage-specific analyses, the model was “~ group + condition” with the same conditions as previously and where the group is either “sa898” or “sa9360”, corresponding to the different alleles. For the combined analysis, the model was also “~ group + condition” with the same conditions as previously and where the groups are “sa898_day5”, “sa898_day12”, “sa9360_day5” or “sa9360_day12”, corresponding to the different alleles and stages. Enrichment analysis for Gene Ontology terms from Ensembl v87 annotation was performed with topGO [41] using the Kolmogorov-Smirnov test and the "elim" algorithm with a nodeSize of 10. RNA-seq data were submitted to ENA under Study Accession Number: ERP007082 and to ArrayExpress under Accession Number: E-ERAD-326.

Whole exome sequencing

Biopsies were taken from tumours and adjacent non-tumour control tissue of homozygous mutants and from corresponding tissues of wild-type or heterozygous siblings. Dissected tissues were placed in 400 μl of 100 μg/ml proteinase K overnight at 55°C, followed by 30 min at 80°C to heat inactivate the proteinase K. DNA was precipitated by adding 400 μl of isopropanol and centrifuging for 40 min at 4100 rpm at room temperature. DNA pellets were washed twice with 400 μl of 70% ethanol followed by centrifugation at 4100 rpm for 25 min and 10 min, and resuspended in ddH₂0. The isolated DNA was whole exome enriched using Agilent SureSelect and used to generate standard Illumina sequencing libraries, which were paired end sequenced with a read length of 75 bp using two lanes of Illumina HiSeq 2500 machines. SNVs were called using MuTect [82] and indels were called using Strelka [83]. Known SNPs, obtained from the Zebrafish Mutation Project [32], were removed from the MuTect output. Potential protein-disrupting SNVs were identified using the Ensembl Variant Effect Predictor (VEP) [84] and filtering the output for stop_gained, missense_variant, transcript_ablation, splice_acceptor_variant, splice_donor_variant and frameshift_variant consequences. Whole exome sequencing data were submitted to ENA under Study Accession Number: ERP016095.