Zebrafish kmt2d null mutants exhibit phenotypes observed in human Kabuki Syndrome patients

Zebrafish kmt2d (ENSDARG00000037060) on Chromosome 23 contains 53 exons, with a 17.6 kb mRNA encoding a 362.7 KDa protein. Although the zebrafish Kmt2d protein (UniProt E7F2F7) only has 44.3% amino acid identity with the human KMT2D (UniProt O14686), BLAST analysis of the individual protein domains showed that the Plant Homeo Domain (PHD) located at the N terminus of the zebrafish Kmt2d has 86.1% identity with the human PHD (UniProt O14686: aa 5030 to 5137 and 126 to 217) and the Su-Enhancer-of-zeste and Trithorax (SET) and Post-SET domains located at the C terminus of the zebrafish protein have 99.1% and 100% identity with the human SET (O14686: aa 5398 to 5513) and Post-Set domains (O14686: aa 5521 to 5537), respectively. Important for functional analysis using reverse genetic approaches, this is the only ortholog for the human Kmt2d gene found in zebrafish, and there are no known paralogues.

To evaluate Kmt2d functional roles in zebrafish, we used CRISPR/Cas9 genome editing to generate zebrafish kmt2d null mutants. Exon 8 (ENSDARE00001117370), which contains the coding sequence for a PHD domain of the Kmt2d protein (Fig 1A), was targeted with a single guide RNA (sgRNA). On-target mutagenesis of injected embryos (F0) was confirmed by High Resolution Melt Analysis (HRMA) of the predicted target region for the sgRNA. Germline transmission of mutant alleles was confirmed by F1 genotyping. Genotyping of individual F1 adults revealed multiple kmt2d alleles, 3 of which were selected for propagation and additional analysis: kmt2d^zy58 (Fig 1B [panels b, e, h; 1bp deletion]), kmt2d^zy59 (Fig 1B [panels a, d, g; 19 bp deletion]), and kmt2d^zy60 (Fig 1B [panels c, f, I; 2 bp deletion]). These mutant alleles are predicted to result in a premature stop codon leading to truncation in one of the PHD domain located at the N terminus of the protein. Loss of full-length Kmt2d protein was confirmed by immunohistochemistry (S1A Fig and S1B Fig).

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Fig 1. Generation and transcriptome profiling of kmt2d zebrafish mutants.

(A) Schematic of zebrafish kmt2d gene showing the 19 bp deletion allele (zy59) and its predicted amino acid change in the protein. The gRNA was designed to target exon 8 (red shaded exon) at the 5ʹ end of the gene. The resulting 19 bp deletion is predicted to cause an early stop codon at the level of the PHD tandem domains (blue) located at the N terminus of the protein. The anti-Kmt2d antibody epitope is located after the PHD domains and before the HMG domain (green), allowing the validation of the early stop in kmt2d^zy59 zebrafish null mutant. (B) Lateral views of zebrafish wild-type sibling embryos (a–c) and kmt2d mutants for 3 different alleles: kmt2d^zy59 (a, d, g), kmt2d^zy58 (b, e, h), and kmt2d^zy60 (c, f, i) at 3 dpf. All 3 alleles shared the same phenotypic characteristics: microcephaly (arrowhead), heart edema (arrow), and mild to moderate body axis defects. Variable expressivity was observed in all the analyzed mutant alleles (Classes I to III). Scale bar = 500 μm. (C) Variable expressivity was analyzed in 3 different embryo clutches resulting from a heterozygous by heterozygous cross for each mutant allele. Embryos were ranked in 3 different classes based on the severity of the phenotype. Class I, wild-type and heterozygous siblings with no phenotype; class II, mutants with microcephaly and heart edema; class III, mutants with microcephaly, heart edema, and shorten body axes. Percentages of different clutches were calculated per the total number of living embryos for each genotype. Chi-square test (p = 0.14) and binomial test (p = 0.09) were performed to assess mendelian ratios considering heterozygous embryos within the Class I category. There was no significant discrepancy between obtained and expected percentages of embryo phenotype. Values for each data point can be found in S1 Data. (D) MA plot of differentially expressed genes from RNA-seq of individual kmt2d^zy59 mutants (n = 6) versus wild-type (n = 6) sibling embryos at 1 dpf. The log₁₀ of mean normalized counts are plotted against the log₂ fold changes for each gene tested. Green horizontal lines represent 2-fold change differences. Negative log₂ fold changes represent genes with reduced expression in the mutants relative to wild type. Both light pink points (no outline) and color-coded points (with outline) represent significantly differentially expressed genes (FDR adjusted p < 0.05). The top 50 genes (ranked by FDR adjusted p-value) were classified into 6 categories based on expression data, bibliography, phenotype information, and gene ontology (S1 Table). Raw data used for this analysis can be found in the following repository: https://b2b.hci.utah.edu/gnomex/gnomexFlex.jsp?requestNumber=475R. (E) GSEA. Analysis was performed by converting zebrafish gene names to human gene names using genes with a one-to-one ortholog relationship retrieved from Ensembl Compara database. The number of resulting genes identifiers analyzed was 9,128 out of 33,737 (Genome build Zv9, Ensembl annotation released version 79). Adjusted p-values were calculated per category. NES of gene sets with a FDR of 5% are plotted to summarized GSEA results. dpf, days post fertilization; ECM, extracellular matrix; FDR,; GO, gene ontology; GSEA, gene set enrichment analysis; HMG, High Mobility Group domain; Kmt2d, Histone-Lysine N-Methyltransferase 2D; NES, Normalized Enrichment Score; PHD, Plant Homeo Domain; RNA-seq, RNA sequencing; SET: Su-Enhancer-of-zeste and Trithorax domain.

https://doi.org/10.1371/journal.pbio.3000087.g001

In order to assess the phenotypes of kmt2d mutations and to determine whether any identified phenotypic traits followed expected mendelian ratios, we performed 3 different heterozygous by heterozygous crosses for each of 3 mutant alleles and screened for phenotypes, with an emphasis on organs and systems that are affected in KS patients. Interestingly, embryonic development was grossly normal until 2 days post fertilization (dpf). Starting at 3 dpf, embryos showed signs of cardiac edema (Fig 1B, arrow), microcephaly (Fig 1B, arrowhead), and body axis defects (Fig 1B [panels g, h, i])—attributes similar to KS clinical manifestations. Whole body edema was evident at 3 to 4 dpf (S2A Fig, S2B Fig, S2C Fig), and these phenotypes continued to develop until approximately 7 dpf.

To assess whether these phenotypes segregated with the mutant alleles, we selected 16 embryos with wild-type phenotypes and 32 embryos with KS phenotypes at 3 dpf and processed them individually for DNA extraction and HRMA analysis. All embryos with KS phenotypes were homozygous for the kmt2d mutant alleles, whereas embryos that appeared phenotypically normal were homozygous wild type or heterozygous at the kmt2d locus. Moreover, the percentage of embryos from a het by het cross with an abnormal phenotype was approximately 25% in 3 different clutches for each of the 3 alleles assayed (Fig 1C). These results were verified by performing Chi-square and binomial distribution tests against an expected mendelian ratio of 75% wild-type and heterozygous embryos (no phenotype) and 25% mutant embryos (with phenotype; Fig 1C).

Humans with KS have wide variation in phenotypes [7,31]. Similarly, zebrafish kmt2d mutants had a range of variable expressivity of phenotypic traits. To assess whether the observed variable expressivity occurred for different parental crosses and mutant alleles, we defined 3 phenotypic categories as follow: class I, WT phenotype corresponding to homozygous wild-type and heterozygous siblings (Fig 1B [panels a, b, c]); class II, homozygous mutant embryos exhibiting heart edema and microcephaly (Fig 1B [panels d, e, f]); class III, homozygous mutant embryos exhibiting heart edema, microcephaly, and shorter body axis (Fig 1B [panels g, h, i]). Embryos from 3 different parental crosses for each of 3 mutant alleles were screened for each category. Our results showed that variable expressivity of the phenotypic traits is a consistent characteristic in zebrafish kmt2d homozygous mutants (Fig 1C).

Our protein expression data showed that Kmt2d was strongly and ubiquitously expressed during development (S1C Fig, S1D Fig, S1E Fig, and S1F Fig), consistent with phenotypes in multiple organ systems. In order to discover pathway perturbations that lead to KS phenotypes, we pursued the kmt2d-dependent alterations in transcriptional profiles that immediately precede the onset of the KS phenotypes, reasoning that transcriptome changes that are proximal to the earliest onset of phenotypes would be most informative and not confounded by downstream secondary effects of transcriptomes. RNA-sequencing (RNA-seq) analysis was performed on single 1 dpf embryos, prior to the appearance of a KS phenotype. The transcriptome was analyzed in 6 individual homozygous kmt2d^zy59 mutants and 6 individual homozygous wild-type siblings (total n = 12). For gene differential expression analysis, we performed DESeq2 using Likelihood Ratio Test (LRT) [32] at a false discovery rate (FDR) of 5%. Our results showed that, at 1 dpf, 276 genes were differentially expressed in kmt2d^zy59 mutants when compared with their wild-type siblings (Fig 1D, MA plot pink and colored dots). Text-mining analysis (further described in Methods) revealed that 72.1% of the top 50 genes are associated with neural and/or cardiovascular system, whereas the remaining genes have been associated with reproductive system, muscle, and pharyngeal arches development (Fig 1D, MA plot color-coded dots; S1 Table). Furthermore, gene set enrichment analysis (GSEA) revealed a small group of gene sets that were enriched in kmt2d^zy59 mutants (S2 Table). Interestingly, all the gene sets with a 5% FDR were exclusively associated with structural extracellular matrix (ECM) glycoproteins (Fig 1E; S2 Table), suggesting essential changes in ECM composition or topography even before phenotype manifestation. In addition, a specific Notch downstream target gene, her4.4, appeared to be up-regulated in kmt2d mutants (Log2 fold change = 044, p = 0.039), consistent with our subsequent analysis of Notch signaling.

Our gross examination of kmt2d mutants at 3 dpf and transcriptome analysis supports the hypothesis of a relatively small group of Kmt2d-dependent genes affected early in development that could explain the molecular etiology of kmt2d^zy59 phenotype observed at later stages. Together, these data introduce a zebrafish kmt2d null mutant model and demonstrate its phenotypic and molecular utility as a model for studying Kabuki Syndrome.

kmt2d^zy59 mutants exhibit anomalous palate development and middle ear structural defects

Our morphological and transcriptome analyses suggested that abnormal pharyngeal arch development might contribute to the spectrum of cranial defects observed in kmt2d^zy59 embryos (Fig 1B and 1D). Additionally, palate and middle ear structures are derived from pharyngeal arches and are affected in KS patients, serving as diagnostic features for the syndrome [2,4–6]. Zebrafish anterior neurocranium and lower jaw structures are well established models for mammal palate and middle ear development, respectively [33]. To assess whether Kmt2d loss affects pharyngeal arch development, we analyzed craniofacial skeleton phenotypes by Alcian Blue/Red Alizarin staining in kmt2d^zy59 mutants and wild-type siblings. Craniofacial cartilage architecture was strongly affected in kmt2d^zy59 mutants (Fig 2) and in kmt2d^zy58 and kmt2d^zy60 mutants (S2D Fig, S2E Fig, S2F Fig).

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Fig 2. kmt2d mutants exhibit anomalous palate development and middle ear structures defects.

(A-D) Alcian Blue/Alizarin Red staining of cartilage and bone showing zebrafish homologous structures for mammalian palate (neurocranium) and middle ear (jaw structures). Ventral view of zebrafish sibling control (A) and kmt2d^zy59 mutant (C) embryos at 4 dpf with corresponding simplified cartoons (B, D). kmt2d^zy59 mutants had severe hypoplasia of visceral cartilages (C; dark blue, D) and neurocranium (C; light blue, D) when compared with wild-type siblings (A, B). The ethmoid plate was present but displayed abnormal development (eth). The cartilages that pattern the jaw in the mandibular (m, pq) and hyoid (ch, hm) arches were absent (m*) or drastically reduced. Pharyngeal arches were absent (cb1-5). The specific structures that are considered mammalian homologs of palate and middle ear are highlighted in orange (eth, me). (E) Quantification of width/length ratio of the ethmoid plate in wild-type siblings and kmt2d^zy59 mutants. In all mutant embryos analyzed, the ethmoid plate was present but had a significantly reduced length/width ratio when compared with wild-type siblings. Statistical analysis was carried out using two-tailed t test, p < 0.0001, n = 10 per genotype. Values for each data point can be found in S1 Data. (F) Embryos were classified according to the degree of development of homolog structures for mammalian middle ear. The categories were present, mispatterned, or absent. Qualitative assessment was plotted for percentage of embryos per genotype (n = 10 per genotype). Values for each data point can be found in S1 Data. bh, basihyal; cb, ceratobranchial; ch, ceratohial; dpf, days post fertilization; eth: ethmoid plate; hm, hyomandibula; ie, inner ear; m, Meckel’s; me, middle ear mammalian homologs; ot, otolith; pq, palatoquadrate.

https://doi.org/10.1371/journal.pbio.3000087.g002

The neurocranium (Fig 2B and 2D; light blue) was underdeveloped, and the ethmoid plate (henceforth palate) failed to grow distally (Fig 2A and 2C, arrow). For wild-type siblings, the average length/width ratio of the palate was 1.03 (Fig 2E; n = 10), whereas kmt2d^zy59 mutants exhibited a significantly shorter and wider configuration with an average length/width ratio of 0.85 (Fig 2E; n = 10). These results demonstrate that Kmt2d is essential for normal palate development in zebrafish embryos. Comparably, visceral cartilages were significantly affected in kmt2d^zy59 embryos (Fig 2B and 2D; dark blue). The pharyngeal arch–derived structures that contribute to the support of the delicate gill tissues (Fig 2B–2D; cb1-5) were completely absent in kmt2d mutants. The cartilage that patterns the lower jaw was absent (Fig 2C and 2D; Meckel’s cartilage, asterisk) or drastically reduced (Fig 2C and 2D; palatoquadrate, hyomandibula, ceratohyal). This represents mispatterning of the structures that are homologous to the mammalian middle ear (Fig 2D; marked as me* in orange). To evaluate reproducibility of this phenotype, we categorized embryos based on the degree of development of Meckel’s, palatoquadrate, and hyomandibula cartilages and calculated percentage of individuals in each category in wild-type siblings and kmt2d^zy59 embryos (Fig 2F). Our results showed that 90% of kmt2d^zy59 embryos had absent or mispatterned cartilages in the lower jaw. Together, these results demonstrate that Kmt2d is required for normal development of palate and lower jaw in zebrafish, consistent with clinical findings in KS patients.

kmt2d^zy59 mutants have hypoplastic hearts and occluded lumens because of aberrant endocardial cell morphology

Congenital heart defects (CHD) is diagnosed in 28% to 80% of Kabuki Syndrome patients [34]. The spectrum of CHD is wide, with prevalence of aortic coarctation, hypoplastic left heart, and other left-sided obstructive defects [14,15,34]. Previous studies of a mouse cardiomyocyte conditional kmt2d deletion demonstrated that Kmt2d is required in cardiac precursors and cardiomyocytes [22]. Additionally, zebrafish kmt2d morphants were reported to have heart looping defects and abnormal development of atrium and/or ventricle [35]. Despite the growing body of evidence that Kmt2d functions during myocardium development, it has not yet been possible in mice to assess the effects of Kmt2d loss in all cardiac tissues in a null mutant context. To investigate the effects of Kmt2d loss during heart development, we first asked whether Kmt2d protein was expressed in both myocardial and endocardial tissues in zebrafish hearts. Whole mount immunofluorescence in wild-type zebrafish embryos revealed that Kmt2d was ubiquitously expressed in the nucleus of both myocardium and endocardium at 2 dpf (Fig 3A and 3B). In our kmt2d mutants, the cardiac edema that appears at 3 dpf could be the result of accumulated defects in heart morphogenesis or secondary effects from other altered embryological processes. To address whether heart edema could be the result of impaired cardiac function, the heart beat was recorded at 1, 2, 3, and 4 dpf in kmt2d^zy59 embryos and wild-type siblings. We found statistically significant bradycardia in kmt2d^zy59 embryos compared with their wild-type siblings (S3C Fig; ANOVA significant p = 0.000264, F (1,76) = 14.647). Interestingly, the mean difference between wild type and mutants was constant throughout this developmental period (ANOVA, interaction effect p = 0.746), suggesting bradycardia was not due to later-onset secondary effects but is a constitutive phenotype of kmt2d^zy59 embryos.

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Fig 3. kmt2d mutants exhibit hypoplastic heart and aberrant endocardial cells morphology.

(A) Confocal images of whole mount immunofluorescence for wild-type zebrafish Kmt2d protein expression at 2 dpf (ventral view). Kdrl (endothelium and endocardium) and MF20 (myosin) were use as context marker for cardiovascular tissues and myocardium. Kmt2d expression was found in the nuclei of myocardial (Aʹ, asterisk in example cells) and endocardial (A", arrows) cells in zebrafish heart (Aʹ and A" are zoomed images in ventricle area). Scale bar = 100 μm. (B) Cartoon showing Kmt2d nuclear expression in both myocardium and endocardium tissue of zebrafish heart. Arrows in Aʹ, A", and F are showing Kmt2d endocardial expression in the same set of cells. (C–D) Confocal images of wild-type tg(kdrl:GFP) sibling (C, Cʹ) and kmt2d^zy59;tg(kdrl:GFP) (D, Dʹ) embryos at 5 dpf. Ventral view. kmt2d^zy59 mutant had aberrant cardiac morphology with significantly reduced ventricle size (D). Myocardial cell labeled with Alcama antibody (C–D, red) showed normal cell morphology in both genotypes. Maximum intensity projections of GFP channel evidenced abnormal morphology of endocardial cells (Dʹ). Some endocardial cells exhibited cell protrusions (Dʹ, arrowheads). (E, F) High magnification images of the heart ventricle chamber in a middle-plane view from the three-dimentional data set in wild-type sibling embryo (E, higher magnification of C) and kmt2d^zy59 mutant (F, higher magnification of D) at 5 dpf. Z-stack analysis of the data set revealed that endocardial cells from the ventricle are organized in concentric layers (F, arrows) filling up the cardiac lumen in kmt2d^zy59;tg(kdrl:GFP) mutants (E, F, asterisk). (G) Ventricle cavity volume measurements in 5 embryos per genotype. Statistical analysis was carried out using two-tailed t test, p < 0.0001. Scale bar = 25 μm. Values for each data point can be found in S1 Data. (H, I) Ventricle myocardial (H) and endocardial (I) cells quantification at 3 and 4 dpf in zebrafish wild-type siblings and kmt2d^zy59 mutants. Embryos were processed for immunofluorescence against myosine heavy chain (MF20) and GFP (Kdrl). Nuclei were stained with DAPI for cell quantification with Imaris software. (H) Myocardial cells, t tests per time point, p-values as follow: 3 dpf p = 0.129, effect size = 24.6. 4 dpf p = 0.906, effect size = −2. Bonferroni corrected p-values for 2 t tests p adjusted values, 3 dpf = 0.258 and 4 dpf = 1.000. (I) Endocardial cells, t tests per time point, p-values as follow: 3 dpf p- = 0.0007, effect size = 29. 4 dpf p = 0.4139, effect size = 14.6. Bonferroni corrected p-values for 2 t tests p adjusted values, 3 dpf = 0.0015 (value reported in figure) and 4 dpf = 0.8278. Values for each data point can be found in S1 Data. avc, atrio-ventricular canal; dpf, days post fertilization; kdrl, kinase insert domain receptor like; kmt2d, Histone-lysine N-methyltransderase 2D; oft, outflow tract; ventr, ventricle.

https://doi.org/10.1371/journal.pbio.3000087.g003

Next, we analyzed myocardium and endocardium patterning using tg(kdrl:GFP) crossed into the kmt2d^zy59 line to mark endocardium and endothelium. Cardiac morphology was significantly altered in kmt2d^zy59 at 5 dpf, with overall heart size reduced and a hypoplastic ventricle (Fig 3C and 3D). To assess whether the hypoplastic ventricle was due to abnormal myocardium development, we analyzed ventricle cardiomyocyte cell numbers at 3 and 4 dpf in wild-type siblings and kmt2d^zy59 mutants. Additionally, myocardial cell architecture was evaluated with the marker Alcama at 3 dpf. The numbers of ventricle myocardial cells in wild-type siblings and kmt2d^zy59 mutants were equivalent (Fig 3H; t tests per time point, p-values as follow: 3 dpf p = 0.129, effect size = 24.6. 4 dpf p = 0.906, effect size = −2. Bonferroni corrected p-values for 2 t tests p adjusted values, 3 dpf = 0.258 and 4 dpf = 1.000.), suggesting that the hypoplastic ventricle phenotype in zebrafish kmt2d^zy59 is not due to a reduction in the myocardial cell population.

At 3 dpf, zebrafish heart ventricle undergoes the tightly regulated process of trabeculation [36,37], whereby myocardial cells extrude and expand into the lumen of the ventricle [38] forming a network of luminal projections consisting of myocardial cells lined by endocardial cells [38,39]. After this process, myocardial cells of the outer ventricle curvature have a characteristic elongated shape [40]. In order to assess whether cardiomyocyte cell shape was affected in kmt2d^zy59 mutants, we measured outer curvature ventricle myocardial cell area and sphericity in wild-type siblings and kmt2d^zy59 at 3 dpf as previously described by Auman and colleagues [40]. Our results showed that ventricle myocardial cells morphology was equivalent in wild-type siblings and kmt2d^zy59 mutants at 3 dpf (S3A Fig, t test p = 0.583 not significant [n.s.] t = 0.59 dF = 5 for area; S3D Fig t test, p = 0.946 n.s.t = 0.71, dF = 6 for myocardial cells sphericity). Overall these results suggest that the hypoplastic heart ventricle in kmt2d^zy59 is not driven by a reduction in size, shape, or absolute numbers of myocardial cells.

In contrast to what we observed in myocardium, the endocardium of kmt2d mutant hearts displayed strikingly abnormal morphology (Fig 3Cʹ and 3Dʹ), with endocardial cells in the ventricle forming discrete aggregates in which some individual cells show protruding borders resembling mesenchyme-like morphology (Fig 3Dʹ; arrowheads). In kmt2d^zy59 embryos, endocardial cells lose their tight interaction with the adjacent myocardium and form concentric layers (Fig 3E and 3F; arrows) that ultimately occlude the lumen of the ventricle (Fig 3E and 3F; asterisk). Thus, endocardium mispatterning led to lumen volume reduction in all analyzed mutant samples (Fig 3G; t test p = 0.0001, t = 7.54, dF = 8). Interestingly, quantification of ventricle endocardial cells in wild-type versus kmt2d^zy59 mutants at 3 dpf showed a significant reduction in the number of EC cells in kmt2d mutants (Fig 3I, t tests per time point, p-values as follow: 3 dpf p- = 0.0007, effect size = 29. 4 dpf p = 0.4139, effect size = 14.6. Bonferroni corrected p-values for 2 t tests p adjusted values, 3 dpf = 0.0015 and 4 dpf = 0.8278). However, this reduction in endocardial cell number did not appear to be due to cell death, because anti active caspase3 IF did not find significant differences in the number of apoptotic cells in kmt2d mutant hearts in the ventricle (S3B Fig; arrowhead). Additionally, the observed difference in endocardial cell number at 3 dpf was not sustained at 4 dpf (Fig 3I, 4 dpf, not significant; 4 dpf p = 0.4139, effect size = 14.6. Bonferroni corrected p-values for 2 t tests p adjusted values, 3 dpf = 0.0015 and 4 dpf = 0.8278). This result indicates that the overall endocardium phenotype is due to (a) slower cell division rate and (b) an aberrant endocardial cell behavior that results in abnormal endocardial tissue patterning and occlusion of the ventricle lumen. This is consistent with the absence of higher rates of apoptosis (S3B Fig; arrowhead) and the observation that at 4 dpf the endocardial cell number was recovered. Altogether, these results indicate that Kmt2d is required for endocardium pattering during zebrafish cardiogenesis. Loss of kmt2d reduced overall heart size, resulting in hypoplastic ventricle with occluded cavity. These results suggest a previously unknown role of Kmt2d in endocardial patterning might contribute the cardiac phenotype observed in KS patients.

kmt2d^zy59 mutants are defective in vasculogenesis and angiogenesis

Although KS patients frequently manifest abnormalities of the aortic arches, including hypoplastic aortic arch or coarctation, a mechanistic understanding between KMT2D mutations and vascular anomalies is lacking. During early development, common progenitor cells (angioblasts) give rise to the cardiac endocardium and to the primary vascular endothelium through vasculogenesis [41–43]. After the primary vascular plexus is formed, a more complex vascular network is established through angiogenesis (production of vessels from preexisting ones) [44,45]. Considering their common developmental origin and our results demonstrating abnormal endocardium patterning, we assessed whether kmt2d^zy59 mutants have normal vascular patterning. To do so, we analyzed general vasculature integrity through o-dianisidine staining [46] in wild-type siblings and kmt2d^zy59 embryos at 6 dpf (S4A Fig, S4B Fig, S4C Fig, S4D Fig) and found vasculature mispatterning reflected by aggregates of red blood cells in the head and aortic arches (S4B Fig and S4D Fig; white arrowheads).

To assess whether the vascular phenotype in kmt2d^zy59 mutants is driven by defects in vasculogenesis or angiogenesis, we analyzed vascular architecture in kmt2d^zy59;tg(kdrl:GFP) mutants and tg(Kdrl:GFP) wild-type siblings by focusing on vasculogenesis of aortic arches (AAs) and angiogenesis of the cranial vessels network at 3, 4, 5, and 7 dpf. AA patterning, thickness, and development of general vasculature was dramatically altered in kmt2d^zy59 mutants (Fig 4A and 4D; S4F Fig and S4H Fig) when compared with their wild-type siblings (Fig 4A and 4C; S4E Fig, S4F Fig and S4G Fig). Additionally, kmt2d mutants exhibited a primitive mouth with underdeveloped vasculature that failed to elongate anteriorly (Fig 4B, arrowhead). AAs were reduced or completely absent in kmt2d mutants. Consequently, all the vessels that branch into the gills were missing (Fig 4B; vessels on both sides of the heart). Three-dimensional volume rendering of confocal imaging confirmed that AA1 was fully formed but in a primitive state, whereas AAs 3, 4, 5, and 6 were atrophic on one side of the left-right symmetry axis and missing on its specular side (Fig 4Aʹ and 4Bʹ and S1 and S2 Videos). This result indicates that while the formation of the vascular plexus of the AAs was initiated, the overall vasculogenesis process is markedly abnormal in kmt2d^zy59 mutants.

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Fig 4. kmt2d^zy59 mutants fail to develop AAs and exhibit misspatterned cephalic vasculature.

(A-B) Ventral view of vasculature in wild-type tg(kdrl:GFP) sibling (A) and kmt2d^zy59;tg(kdrl:GFP) mutants at 5 dpf. Cephalic vascular architecture in kmt2d^zy59 embryos was abnormal with reduced elongation in the anteroposterior body axis, loss of bilateral symmetry, and reduced HA (A, B). In kmt2d^zy59 mutants, AA1 (A, B) was shorter with minimum elongation towards the anterior area of the embryo, the AA3 (A, B) was rudimentary,whereas AA4−6 were reduced or absent (A, B). Mouth and eyes in kmt2d^zy59 showed primitive characteristics with differences in the thickness of the OV in particular (A–B). Note the abnormal endocardial component of the heart (A, B; he). Aʹ and B are simplified cartoons of the main differences in the AAs development in wild-type (Aʹ) and mutant (Bʹ) backgrounds. Branchial vasculature loops were removed to allow better visualization of aortic arch points of origin. (C-D) Lateral view of cephalic vasculature in wild-type tg(kdrl:GFP) sibling and kmt2d^zy59;tg(kdrl:GFP) mutants at 7 dpf. kmt2d^zy59 exhibited a complete absence of the vascular loops associated with AAs 3–6 with only a rudiment of the AA3 present (D, AA*). The general cranial vascular network was mispatterned with a particular strong impact in the CtA, ACeV, and VA (D; in orange). In kmt2d^zy59 mutant, most vessels had reduced lumens with the exception of the NCA (C, D), DVC (C, F) and IOC (C, F) that show thicker vessel diameter. Note the reduced HA from a lateral view (D, HA in orange; scale bars = 100 μm). (E) Cell cycle profile analysis by FACS for 7 individual embryos per genotype at 7 dpf. The gates set up for nuclear staining (DAPI) in kdrl positive cells (endothelial and endocardial cells) are shown. (F) Cell cycle profiles for Kdrl positive cells in kmt2d^zy59 mutants showed no significant difference in the percentage of G2/M cells at 3 dpf. In contrast, at 7 dpf, there was a significant decrease in number of dividing cells in kmt2d^zy59 mutants when compared with wild-type siblings. Unpaired two-tailed t test, p = 0.407 n.s. for 3 dpf; p = 0.0002 for 7 dpf, n = 7 per genotype. Values for each data point can be found in S1 Data. AA, aortic arch; AA1, mandibular arch; AA2, hyoid arch; AA3, first branchial arch; AA4, second branchial arch; AA5 third branchial arch; AA6, fourth branchial arch; ACeV, anterior cerebral vein; CtA, central artery; DCV, dorsal ciliary vein; DLV, dorsal longitudinal vein; dpf, days post fertilization; ey, eye; FACS, fluorescent activated cell sorting; GFP, green fluorescent protein; HA, hypobranchial artery; kdrl, kinase insert domain receptor like; IOC, inner optic circle; MsV, mesencephalic vein; NCA, nasal ciliary artery; ORA, opercular artery; OV, optic vein; VA, ventral aorta.

https://doi.org/10.1371/journal.pbio.3000087.g004

The vascular plexus of the brain in kmt2d^zy59 mutants had all the major vessels present but with aberrant patterning. Defects were particularly evident in the central artery (CtA), anterior cephalic vein (ACeV), hypobranchial artery (HA) and ventral artery (VA; Fig 4C and 4D; orange segments indicate altered patterning). These observations suggest that even when blood vessels sprout and form, they fail to establish a normal vascular patterning (S4E Fig, S4F Fig, S4G Fig and S4H Fig).

To address whether the kmt2d^zy59 vascular defects were driven by abnormal endothelial cell proliferation, we profile endothelial cell cycle by fluorescent activated cell sorting (FACS). In order to assess this, single embryos with distinguishable wild-type or mutant phenotypes were dissociated for FACS, using kmt2d^zy59;tg(kdrl:GFP) embryos and tg(Kdrl:GFP) wild-type siblings at 3 dpf and 7 dpf (Fig 4E and 4F; S4 Fig). Strikingly, our results showed no significant difference in endothelial cell cycle profile at 3 dpf (unpaired t test, p = 0.4, t = 0.86 dF = 12 n.s.). In contrast, at 7 dpf, kmt2d^zy59;tg(kdrl:GFP) embryos showed a significant reduction in proliferative endothelial cells (Fig 4F; t test, p = 0.0001, t = 5.56 dF = 12; n = 7 per genotype). Endothelial/endocardial cell proliferation was also analyzed by confocal analysis of phospho S10 Histone 3 (pH3) marker at 3dpf and 5dpf (S8 Fig and S7A Fig and S7C Fig). At 3 dpf, kmt2d^zy59 mutants did not show statistically significance alterations in the number of endothelial/endocardial mitotic cells (S8 Fig, t test p = 0.076, dF = 7.92, N = 5 embryos per genotype). However, at 5 dpf, the difference in mitotic endothelial and endocardial cells between wild-type siblings and kmt2d^zy59 mutants was statistically significance (Fig 7G and 7H, p = 0.001 in AA and p = 0.0307 in endocardium and S7A Fig and S7C Fig). These data indicate that the vascular misspatterning in kmt2d^zy59 mutants is not driven by reduced cell proliferation at early developmental stages when the cardiovascular system is being established but by a reduction in proliferation at later stages. In contrast to normal proliferation at 3 dpf, there was a strong reduction in EC proliferation at 5 dpf and 7 dpf, particularly evident in the area of the vasculature that irrigates the gills (S7A Fig; white dashed line), which are normally established from 5 dpf to 7 dpf through active cell proliferation from vessels derived from AA3 to AA6 [47]. Overall, these results demonstrate key roles of Kmt2d in zebrafish for vascular patterning, with mutants displaying defects in both vasculogenesis and angiogenesis.

kmt2d^zy59 mutants fail to initiate AA3 to AA6 formation

In zebrafish, AA1 forms at 24 hpf, coincident with the time when circulation begins. A vestigial AA2 also forms at this time point, which is immediately replaced by the opercular artery (ORA). AA3 to AA6 emerge later, between 2 dpf and 2.5 dpf [47,48]. As described above, kmt2d^zy59 mutants are able to form AA1 and the ORA, but they fail to form the subsequent AAs. Additionally, our data indicate that endothelial cell proliferation is normal in kmt2d^zy59 mutants at 3 dpf, the time point at which the gross embryological phenotypes begin to appear. We explored 2 possible mechanisms for the absence of AA3 through AA6 in kmt2d^zy59 mutants: (1) arch patterning fails to initiate at 2 dpf, or (2) alternatively, AA3 to AA6 vascular patterniing is initiated normally but at a later stage the arches become atrophic and regress. To test these possibilities, we performed live time-lapse analysis of AA development in wild-type tg(kdrl:GFP) siblings and kmt2d^zy59;tg(kdrl:GFP) mutants from 2 dpf to 3 dpf.

At 2 dpf, kmt2d^zy59 mutants have normal gross morphology, but time-lapse imaging revealed thinner blood vessels and abnormal AA sprouting when compared with their wild-type siblings (Fig 5A–5D). Interestingly, kmt2d^zy59 mutants were not able to form the primary AA sprouts during the duration of data acquisition (Fig 5B–5B‴, 5D and 5Dʹ, S4 Video), whereas wild-type embryos succeeded in developing and extending AA3 through AA6 (Fig 5A–5A‴, 5C and 5Cʹ, S3 Video). This result suggests that kmt2d mutants do not initiate the formation AA3 to AA6 within the same time interval as their wild-type counterpart. On the other hand, the time-lapse videos showed unusually high levels of endothelial cell activity in the area were the AAs should emerge (ventral boundary of the lateral dorsal aorta [LDA]), suggesting that AA could develop at a later time point. To investigate this possibility, embryos subjected to time-lapse analysis were recovered for 5 hours and processed for IF and confocal analysis. At approximately 3 dpf, kmt2d mutants showed primary sprouting of abnormal AA3 to AA6 that were not present at earlier stages (Fig 5E and 5F). This indicates that kmt2d mutants eventually form atretic AAs around 3 dpf that will ultimately become vestigial at later time points (Fig 4A and 4B).

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Fig 5. kmt2d^zy59 mutants fail to develop AA3 to AA6.

(A–B) Still images (MIP) from time-lapse live imaging performed from 2 dpf to 3 dpf. Cranial-lateral view at the level of AA development from wild-type sibling (A–A‴, S3 Video) and kmt2d^zy59 mutant (B–B‴, S4 Video). Images were selected at the 0, 4, and 11 hour time points. Images were converted to grayscale and inverted for better visualization (A–A", B–B"). A‴ and B‴ show A" and B" without grayscale processing. Red asterisk (B") denotes abnormal vascular development of AA sprouts at the level of the ventral border of LDA. (scale bars = 50 μm). (C-D) Schematic cartoons highlighting the development of AA in wild-type embryos (C, Cʹ) and kmt2d^zy59 mutants (D, Dʹ). C corresponds to A, Cʹ correspond to A", D corresponds to B and Dʹ to B". Red asterisk indicates ectopic AA sprouting. (E–F) Cranial-lateral view of vasculature in wild-type tg(kdrl:GFP) sibling (A) and kmt2d^zy59;tg(kdrl:GFP) mutants at 3 dpf. After time-lapse experiment (A, B), embryos were released from agarose and processed for IF and confocal imaging. Wild-type embryos showed correct patterning and secondary sprouting of AA3 to AA6 (E). At 3 dpf, kmt2d^zy59 mutants had abnormal development of primary sprouts of AA3 to AA6, which are thinner and atrophic. In contrast, AA1 and ORA were thicker and exhibit abnormal morphogenesis and endothelial cell protrusions (F, white arrowhead; scale bars = 25 μm). (G-I) Still images (MIP) from time-lapse live imaging performed from 3 dpf to 4 dpf. Cranial-lateral view at the level of AA development from wild-type sibling and kmt2d^zy59 mutant. Images were selected at the 0, 10, 11.25, and 15 hour time points. Asterisks (G–G‴) denote normal vascular development in the area between AA1 and ORA. No blood vessel forms in this area in wild-type background. Arrowheads (H–H‴, I–I‴) indicate endothelial cells extending multiple filopodia and forming a new and ectopic blood vessel. White dashed rectangle (H) specifies zoomed area in (I). Images were set on grayscale and the look-up table was inverted for better contrast of tip cell-like endothelial cells in kmt2d^zy59 mutant (I–I‴; scale bars = 100 μm). AA, aortic arch; AA1, mandibular arch; AA2, hyoid arch; AA3, first branchial arch; AA4, second branchial arch; AA5, third branchial arch; AA6, fourth branchial arch; dpf, days post fertilization; he, heart; hpf, hours post fertilization; kdrl, kinase insert domain receptor like; LDA, lateral ventral aorta; MIP, maximum intensity projection; ORA, opercular artery.

https://doi.org/10.1371/journal.pbio.3000087.g005

Ectopic endothelial sprouting in kmt2d^zy59 mutants

Our superresolution confocal analysis of the 3 dpf embryos found distinctive aberrant sprouting of endothelial cells from AA1 and ORA in kmt2d^zy59 embryos (Fig 5F, white arrowheads). This observation led us to test the hypothesis that angiogenesis defects in in kmt2d mutants involve ectopic blood vessels formation. To test this hypothesis, we performed time-lapse analysis of AA development in wild-type tg(kdrl:GFP) siblings and kmt2d^zy59;tg(kdrl:GFP) mutants at a later time point, from 3 dpf to 4 dpf. At 3 dpf (72 hpf), aberrant blood vessel phenotypes were apparent in kmt2d mutants, with both decreased vessel lumens and abnormal patterning (Fig 5G and 5H). Strikingly, by 10 hours later (82 hpf), kmt2d^zy59;tg(kdrl:GFP) mutants showed ectopic endothelial cell sprouting, emerging from the AA1 and ORA (Fig 5G, 5G", 5H' and 5H"; arrowheads; Fig 5I and 5I", red arrowheads). These cells possessed many long and dynamic filopodia extending towards apposing cells with similar comportment. This aberrant hyperactive sprouting resembles tip cell morphology seen during normal angiogenesis [49]. However, in the context of kmt2d^zy59 mutants, the observed tip cell-like behavior occurred in aberrant (ectopic) positions, ultimately producing ectopic blood vessels (Fig 5H‴ and 5I‴, S5 Video, wild-type control; S6 Video kmt2d mutant). This ectopic and abnormal endothelial cell behavior was also observed at the level of AA3 as well as in other regions of the recorded area (S6 Video kmt2d mutant), suggesting that the tip-like phenotype can potentially occur in any endothelial cell of kmt2d^zy59 mutants.

Ectopic blood vessel formation is a well described response to hypoxia conditions [50]. Considering the reduced lumen size in kmt2d mutant blood vessels, we asked whether ectopic blood vessel formation was a consequence of hypoxia response mechanisms. To test this, we induced hypoxia by treatment with dimethyloxalylglycineinduced (DMOG), an inhibitor of HIF-prolyl hydroxylases, which thereby stabilizes Hif-1 and triggers hypoxia response even in normoxia conditions [51]. Tg(kdrl:GFP) wild-type siblings and kmt2d^zy59;tg(kdrl:GFP) mutant embryos were treated from 3 dpf to 4 dpf with DMOG and DMSO as control. After treatment, embryos were washed and processed for IF and confocal imaging. At 4 dpf, wild-type embryos treated with DMOG showed ectopic vessel sprouting at the level of the optic vein (OV; S5A Fig and S5B Fig, white arrow). In contrast, AA1 and ORA were not altered by DMOG treatment (S5A Fig and S5B Fig), suggesting that these vessels have a higher resistance threshold to hypoxia. Interestingly, kmt2d mutant embryos treated with DMOG did not show any additional vessel sprouting (S5B Fig and S5D Fig) beyond the ectopic blood vessel sprouting from the AA1 and ORA in normoxia (S5B Fig and S5D Fig; white arrow), indicating that hypoxia does not increase or alter the ectopic blood vessels formed in kmt2d mutants. These results suggest that the aberrant endothelial cell behavior and ectopic blood vessel sprouting seen in kmt2d mutants are not due to hypoxia response and that Kmt2d functions to suppress ectopic or hyperactive angiogenesis. Altogether, these results demonstrate that Kmt2d is required for the timely and normal development of AA3 to AA6 in zebrafish.

Notch signaling is hyperactivated in endocardial and endothelial cells of kmt2d mutants

The ectopic tip cell-like phenotype led us to investigate an iconic molecular mechanism involved in tip-stalk cell identity as well as in endocardium patterning during cardiogenesis: Notch signaling pathway. Multiple studies in mice, zebrafish, cell culture, and tumor models have shown that Notch pathway is a key regulator of vertebrate vasculogenesis and angiogenesis [52–58]. Considering our discovery of ectopic angiogenesis in kmt2d mutants and previous observations that Notch signaling regulates endocardial and endothelial cell growth, differentiation, and patterning, we investigated whether kmt2d mutants have altered Notch signaling activity. We injected kmt2d CRISPR/Cas9 into a Notch signaling reporter line tg(tp1:EGFP)^um14 [59] and performed F0 kmt2d mosaic mutants analysis [60] (S6 Fig) to assess Notch signaling activity in kmt2d mutants. At 3 dpf, Notch signaling in the zebrafish heart was strongly active in endocardial cells of the outflow tract and AV canal, with weaker activity in some endocardial cells of the ventricle (Fig 6A; GFP positive cells). Interestingly, in kmt2d mutants, the number of endocardial cells with positive Notch activity was significantly increased in both ventricle and atrium (Fig 6B and 6C t test, p = 0.0001 in ventricle t = 4.95 dF = 36, atrium p = 0.0001, t = 10.99 dF = 36 n = 7 per treatment). These transgene reporter results indicate that Notch signaling is cell autonomously hyperactivated in kmt2d mutant endocardial cells. To validate that endogenous Notch signaling is altered in kmt2d mutants, we assayed endogenous components of the Notch pathway in kmt2d^zy59 mutants by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and IF at 3 dpf. Of these components, only transcripts of Notch-specific transcription factor rbpja were increased in kmt2d^zy59 mutants, whereas the notch1b receptor and the downstream target hes1 mRNA levels were comparable to the transcripts level found in wild-type siblings (Fig 6D). To investigate whether elevated expression of rbpja mRNA resulted in elevated Rbpj protein expression, we performed immunofluorescence against Rbpj Notch transcription factor and compared protein levels in the heart in wild-type tg(Kdrl:GFP) siblings and kmt2d^zy59;tg(Kdrl:GFP) mutants. At 5 dpf, Rbpj protein was present in all heart tissues, with enhanced Rbpj protein accumulation in kmt2d^zy59 mutants, with well-defined nuclear localization. To quantify this elevated expression in endothelial/endocardial cells and to determine whether this increased Rbpj protein expression started at earlier stages, we performed single embryo FACS of tg(Kdrl:GFP) wild-type siblings and kmt2d^zy59;tg(Kdrl:GFP) mutants at 3dpf. Embryos were processed for immunofluorescence against GFP (driven by Kdrl in endothelial/endocardial cells) and Rbpj. After gating for single cells, samples were gated for GFP to exclusively study the endothelial/endocardial single cell population. Subsequently we measured in EC the percentage of Rbpj positive cells and the Rbpj Median Fluorescence Intensity (MFI) as an indicative of protein expression levels (Fig 6D). Consistent with our previous cell counting results, we observed that the overall number of Rbpj positive cells in EC from wild-type sibling and kmt2d^zy59 mutants is equivalent at 3 dpf (Fig 5A, % of EC Rbpj positive, unpaired t test, p = 0.69, t = 0.42 dF = 6 n.s.). Crucially, the MFI values in kmt2d^zy59 mutant ECs were significantly higher, indicating elevated Rbpj protein levels in Kmt2d-deficient endothelial/endocardial cells (Fig 6D, t test p = 0.0015, t = 5.51, dF = 6).

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Fig 6. Notch signaling is hyperactive in kmt2d mutant endothelium/endocardium.

(A-B) Confocal images of the heart of wild-type noninjected control tg(tp1:EGFP)^um14 embryo (A) and F0 kmt2d mosaic mutants tg(tp1:EGFP)^um14 embryo injected with CRISPR/Cas9 targeting kmt2d (B). F0 kmt2d mosaic mutants injected embryos showed hypoplastic heart as seen in kmt2d^zy59 null mutants (B). Notch positive cells (green) were mostly distributed in the atrio-ventricular valve of a 3 dpf embryonic heart. Some endocardial cells in the ventricle and outflow tract were also observed (A). In F0 kmt2d mosaic mutants, hearts showed a significant increase in the number of Notch positive cells in both ventricle and atrium. Ventral view of the heart, only middle sections of the whole data set are shown. Scale bar = 25 μm. (C) Quantification of the amount of Notch positive cells in ventricle and atrium of control and injected embryos. N = 7 per group, unpaired two-tailed t test, p = 0.0001 in ventricle t = 4.95, dF = 36, atrium p = 0.0001, t = 10.99, dF = 36. Values for each data point can be found in S1 Data. (D) RT-qPCR analysis of wild-type sibling control embryos and kmt2d^zy59 mutants for some components of the Notch signaling pathway. The Notch transcription factor rbpja was significantly up-regulated kmt2d^zy59 embryos, corroborating the results obtained in the F0 kmt2d mosaic mutant analysis. There were no significant differences found for notch1b and hes1. N = 4 per genotype with 2 technical replicates per gene and per genotype assessed; elfα was use as control gene. Ct values were normalized using α-tubulin as gene of reference; fold change of relative expression was calculated using the ΔΔCt method. Multiple t test p < 0.0001 for rbpja, t = 6.04, dF = 24. Values for each data point can be found in S1 Data. (E–F) Confocal images of wild-type sibling control embryos (E) and kmt2d^zy59 mutants (F) showing Rbpj protein expression levels and patterning at 5 dpf. Ventral view of the heart, only MIP of half data set is shown. Scale bar = 25 μm. (G) Summarized data and statistics from FACS experiment performed in 14 individual samples (7 wild-type siblings and 7 kmt2d mutants). Wild-type tg(kdrl:GFP) siblings (A) and kmt2d^zy59;tg(kdrl:GFP) mutants were collected at 3 dpf, were processed for IF, were digested, and were prepared for FACS. Unpaired two-tailed t test, % of EC cells Rbpj positives p = 0.63, n.s. t = 0.49, dF = 12; Rbpj MFI p = 0,0009, t = 4.35, df = 12. Values for each data point can be found in S1 Data. Altogether our results show that Notch pathway is hyperactivated in endocardial cells of kmt2d^zy59 mutants and demonstrate that this increased Notch activity is consequence of up-regulated Notch pathway transcription factor Rbpj specifically in EC cells. To our knowledge, these results provide the first evidence of a regulatory link between Kmt2d and Notch signaling during developmental processes in vertebrates. Ct, cycle threshold; ΔΔCt, delta-delta cycle thrshold; dpf, days post fertilization; EC, endocardial cells; F0, filial 0; FACS, fluorescent activated cell sorting; G0, generation 0; IF, immunofluorescense; MIP, maximum intensity projection; oft, out flow tract; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; ven, ventricle.

https://doi.org/10.1371/journal.pbio.3000087.g006

Pharmacological inhibition of Notch signaling rescues cardiovascular development in Kabuki Syndrome zebrafish

Our results showed hyperactivation of Notch activity in endocardial and endothelial cells of kmt2d mutants, suggesting a molecular mechanism for the cardiovascular phenotype in KS. To test whether interference with this mechanism could rescue the cardiovascular phenotype in kmt2d^zy59, we inhibited the Notch pathway at the level of the Notch receptor cleavage by blocking y-secretase activity with DAPT (Fig 7E) [58,61]. kmt2d^zy59;tg(kdrl:GFP) embryos and wild-type tg(kdrl:GFP) siblings were treated with DAPT or DMSO (control group) from 1 dpf to 2 dpf and were washed and assessed at 5 dpf for cardiovascular development. Wild-type embryos treated with DAPT had other phenotypes predicted from inhibiting Notch as previously described by Arslanova and colleagues [61]: abnormal somite development, heart edema, disrupted vasculature. Confocal images of wild-type DAPT-treated embryos revealed aberrant heart morphology with hypoplastic ventricle and stretched cardiac tube, likely a consequence of pericardic edema (Fig 7A and 7B) evidenced by myosin staining of the sternohyoideus muscle (Fig 7A–7D asterisks, magenta labeling). Strikingly, DAPT treatment partially rescued AA development and heart morphology in kmt2d^zy59 mutant embryos (Fig 7D). Heart ventricle volume was significantly increased in kmt2d^zy59 DAPT-treated embryos compared with the control groups: DMSO kmt2d^zy59 embryos and wild-type DAPT-treated embryos (Fig 7F; two-way ANOVA multiple comparison test, n = 10 per condition, adjusted p-values per group; see figure legend for p-value details).

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Fig 7. Inhibition of Notch pathway with DAPT rescues cardiovascular phenotypes in kmt2d mutants by enhancing cell proliferation in endothelial and endocardial cells.

(A-D) Confocal images of wild-type sibling (A, B) and kmt2d^zy59 mutant (C, D) embryos at 5 dpf. DMSO as solvent control (A, C) and DAPT for Notch signaling inhibition (B, D) were applied to embryos of indicated genotypes. DAPT treatment affected cardiovascular development in wild-type embryos (B) as a consequence of Notch signaling inhibition. kmt2d^zy59 mutants that were treated with DAPT had rescued AA development and partially rescued heart development (D) when compared with the kmt2d^zy59 DMSO control group (C). Arrowheads indicate normal (A), disrupted (B, C), and rescued (D) AA. Note that cardiovascular phenotype was rescued in DAPT-treated mutants despite cardiac edema, as evidenced by the sternohyoideus deformation (asterisk; B, C, D, MF20 in magenta). (E) Schematic of Notch signaling pathway showing DAPT inhibition of γ-secretase activity, the second cleavage step of Notch receptor processing. DAPT thus prevents NICD release to the cytoplasm for nuclear import. (F) Cardiac ventricle volume measurements for DMSO control groups (wild type and kmt2d^zy59) and DAPT treatment groups (wild type and kmt2d^zy59). The volume of the ventricle chamber was significantly rescued in kmt2d^zy59 mutant embryos after Notch pathway inhibition with DAPT. Embryos were randomly selected from 3 different clutches; measurements were blind to embryo genotypes, which were assayed by HRMA after measurement. Two-way ANOVA multiple comparison test adjusted p-values per each condition as follow: wild-type DMSO versus wild-type DAPT, p < 0.0001; wild-type DMSO versus kmt2d^zy59 DMSO, p < 0.0001; wild-type DMSO versus kmt2d^zy59 DAPT, p = 0.0159; wild-type DAPT versus kmt2d^zy59 DMSO, p = 0.5482; wild-type DAPT versus kmt2d^zy59 DAPT, p < 0.0001; kmt2d^zy59 DMSO versus kmt2d^zy59 DAPT, p = 0.0001. Values for each data point can be found in S1 Data. (G) Number of endothelial cells proliferating in the AA region in each group treatment. There were significantly more proliferating endothelial cells in the AA region of kmt2d^zy59 mutant after DAPT treatment, indicating that the phenotype was rescued by increasing cell proliferation. Two-way ANOVA multiple comparison test adjusted p-values per each condition as follow: wild-type DMSO versus wild-type DAPT, p = 0.0001; wild-type DMSO versus kmt2d^zy59 DMSO, p = 0.0001; wild-type DMSO versus kmt2d^zy59 DAPT, p = 0.0006; wild-type DAPT versus kmt2d^zy59 DMSO, p = 0.6043; wild-type DAPT versus kmt2d^zy59 DAPT, p = 0.0091; kmt2d^zy59 DMSO versus kmt2d^zy59 DAPT, p = 0.0047. Values for each data point can be found in S1 Data. (H) Number of endocardial cells proliferating in the heart per experimental group. There were significantly more proliferating endocardial cells in kmt2d^zy59 mutant hearts after DAPT treatment, indicating that the phenotype was rescued by increasing endocardial cell proliferation. Two-way ANOVA multiple comparison test adjusted p-values per each condition as follow: wild-type DMSO versus wild-type DAPT, p = 0.0051; wild-type DMSO versus kmt2d^zy59 DMSO, p = 0.0307; wild-type DMSO versus kmt2d^zy59 DAPT, p = 0.0013; wild-type DAPT versus kmt2d^zy59 DMSO, p = 0.8106; wild-type DAPT versus kmt2d^zy59 DAPT, p < 0.0001; kmt2d^zy59 DMSO versus kmt2d^zy59 DAPT, p < 0.0001. Cell count was blind and embryos were randomly selected from 2 different clutches (E, F). Genotype was confirmed after measurement by HRMA. Values for each data point can be found in S1 Data. AA, aortal arch; ADAM, containing a disintegrin and metalloprotease; dpf, days post fertilization; ER, endoplasmic reticulum; HRMA, High Resolution Melt Analysis; kdrl, kinase insert domain receptor like; MF20, Myosin Heavy Chain Antibody; NICD, Notch intracellular domain.

https://doi.org/10.1371/journal.pbio.3000087.g007

We then asked whether the rescue of AA and heart development by Notch inhibition was due to a change in the proliferative capability of endothelial/endocardial cells. To test this, we analyzed cell proliferation by pH3 labeling and counted GFP positive cells (endothelial and endocardial cells) co-localizing with pH3 in a delineated area (S7A Fig; white dashed line). Our analysis showed that DAPT treatment of kmt2d mutants induces a significant increase in the number of proliferating endothelial and endocardial cells in the AA area and heart, respectively.

Pharmacological inhibition of Notch signaling reestablishes Rbpj protein levels in kmt2d mutant endothelial and endocardial cells

Since kmt2d^zy59 mutants showed increased levels of the Notch transcription factor Rbpj in endocardial and endothelial cells (Fig 6E–6G), we asked whether inhibition of Notch signaling with DAPT could rescue this phenotype. To test this, kmt2d^zy59;tg(kdrl:GFP) embryos and wild-type tg(kdrl:GFP) siblings were treated with DAPT (or DMSO as a control) from 1 dpf to 2 dpf and were washed and grown until 3 dpf. Embryos were then fixed and genotyped by tail-clipping followed by DNA extraction and HRMA. Samples were processed for immunofluorescence against Rbpj and GFP (Kdrl:GFP transgenic enhancement) and analyzed by confocal imaging and FACS.

In agreement with our previous confocal imaging analysis in 5 dpf embryos (Fig 6E and 6F), Rbpj protein levels were increased at 3 dpf in kmt2d^zy59 mutants when compared with their wild-type siblings (Fig 8A, 8Aʹ, 8C and 8Cʹ). Remarkably, Rbpj protein expression was locally enhanced in the area corresponding to endocardial cells (Fig 8Cʹ, arrow). DAPT-treated kmt2d^zy59 embryos showed a substantial general reduction in Rbpj signal (Fig 8D and 8Dʹ) that is particularly distinctive in endocardial cells (Fig 8D and 8Dʹ). In order to quantify this, we proceeded with FACS analysis to measure Rbpj MFI exclusively in endothelial and endocardial cells (GFP positive cells) in individual embryos as described above. Also, to assess the temporal resolution of Rbpj protein levels rescue, we performed this FACS experiment with 2 dpf and 3 dpf embryos.

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Fig 8. Pharmacological inhibition of Notch signaling reestablishes normal Rbpj protein levels in kmt2d mutant endothelial and endocardial cells.

(A–D) Confocal images of wild-type sibling (A, B) and kmt2d^zy59 mutant (C, D) embryos at 3 dpf. Cephalic ventral views. DMSO as solvent control (A, C) and DAPT for Notch signaling inhibition (B, D) were applied to embryos of indicated genotypes. Rbpj protein levels (Red channel) are higher in DMSO-kmt2d^zy59 mutants (C, Cʹ). Arrow in Aʹ–Dʹ is indicating endocardial Rbpj expression—note higher endocardial Rbpj levels in Cʹ. DAPT treatment restored Rbpj signal levels in kmt2d^zy59 embryos (D, Dʹ) as a consequence of Notch signaling inhibition. Dashed line in Aʹ–Dʹ indicates heart outline. Red, Rbpj; green, GFP (to enhance endocardial/endothelial Kdrl:GFP transgenic label). (E-F) Summarized data and statistics from FACS experiments performed in 16 individual samples per time point (4 DMSO wild-type siblings and 4 DMSO kmt2d mutants; 4 DAPT wild-type siblings and 4 DAPT kmt2d mutants per time point). Wild-type tg(kdrl:GFP) siblings (A) and kmt2d^zy59;tg(kdrl:GFP) mutants from each treatment (DMSO or DAPT) were collected at 2 dpf (E) and 3 dpf (F), processed for IF, digested, and prepared for FACS. Two-way ANOVA multiple comparison test adjusted p-values per each condition at 2 dpf as follow: DMSO:wild-type versus DMSO:kmt2d^zy59: p = 0.7762, DMSO:wild type versus DAPT:wild type: p = 0.9179, DMSO:wild type versus DAPT:kmt2d^zy59: p = 0.9849, DMSO:kmt2d^zy59 versus DAPT:wild type: p = 0.9823, DMSO:kmt2d^zy59 versus DAPT:kmt2d^zy59: p = 0.9186, DAPT:wild type versus DAPT:kmt2d^zy59: p = 0.9910. Two-way ANOVA multiple comparison test adjusted p-values per each condition at 3 dpf as follow: DMSO:wild type versus DMSO:kmt2d^zy59: p = 0.0221 (significant *), DMSO:wild type versus DAPT:wild type: p = 0.5394, DMSO:wild type versus DAPT:kmt2d^zy59: p = 0.9263 (no significant difference, DAPT rescue was to similar control wild type values), DMSO:kmt2d^zy59 versus DAPT:wild type: p = 0.0012, DMSO:kmt2d^zy59 versus DAPT:kmt2d^zy59: p = 0.0039 (significant *, DAPT rescues kmt2d mutant Rbpj levels), DAPT:wild type versus DAPT:kmt2d^zy59: p = 0.9839. ANOVA summary results: Interaction F (1, 12) = 4.173, p = 0.0637; Treatment F (1, 12) = 19.37, p = 0.0009, Genotype F (1, 12) = 9.202, p = 0.0104. Values for each data point can be found in S1 Data. dpf, days post fertilization; FACS, fluorescent activated cell sorting; GFP, green fluorescent protein; kdrl, kinase insert domain receptor like.

https://doi.org/10.1371/journal.pbio.3000087.g008

Our results showed that at 2 dpf, Notch pathway inhibition with DAPT does not affect Rbpj protein levels in any of the analyzed groups, as reflected in similar MFI values (Fig 8E). In contrast, at 3 dpf, Rbpj MFI values were significantly increased in DMSO (control-treated) kmt2d^zy59 compared with DMSO-treated wild-type siblings. DAPT treatment resulted in reducing these values to basal Rbpj MFI levels, similar to those observed in wild-type control group (Fig 8F, two-way ANOVA multiple comparison test, n = 4 per condition, adjusted p-values per group; see figure legend for p-value details). These results substantiate our findings that Notch signaling is hyperactivated in kmt2d mutants, resulting in increased Rbpj expression in endocardial/endothelial cells and further support the hypothesis of a regulatory link between the Notch pathway and Kmt2d.

Pharmacological inhibition of Notch signaling rescues endothelial patterning and ectopic blood vessel formation in zebrafish kmt2d mutants

Our endothelial cell analysis in kmt2d mutants revealed an aberrant endothelial cell behavior as early as 2 dpf that precedes disruption of normal AA development (Fig 5A–5C). Moreover, we showed that some endothelial cells located in the region of AA development display tip cell-like phenotypes and generate ectopic blood vessel initiation (Fig 5G–5I). Considering that DAPT treatment was able to partially rescue AA development (Fig 7) and dampen down Notch signaling levels in kmt2d^zy59 mutants to wild-type levels (Fig 8), we decided to test whether DAPT would be able to rescue the early endothelial phenotype and reduce ectopic blood vessel sprouting in the AA development area.

We treated kmt2d^zy59;tg(kdrl:GFP) embryos and wild-type tg(kdrl:GFP) siblings with DAPT or DMSO from 1 dpf to 2 dpf. At 2 dpf, embryos were washed and immediately processed for in vivo time-lapse confocal microscopy. A total number of 24 embryos (12 DMSO controls and 12 DAPT treated) were assigned unique identifiers and imaged laterally, focusing on the AA development region. Images were taken every 20 minutes for a period of 10 hours. Cohorts were imaged from 2 dpf to 2.5 dpf and from 3 dpf to 3.5 dpf. After imaging period, embryos were recovered and individually genotyped.

Our results indicated that Notch pathway inhibition with DAPT from 1 dpf to 2 dpf was capable of substantially rescuing AA formation in kmt2d^zy59 mutants, including early endothelial cell behavior in the ventral border of the lateral dorsal aorta (LDA) where AA structures emerge (S5–S8 Videos, Fig 9A–9D), followed by substantially rescued formation of AA3 through AA6 by 3.5 dpf (S9–S12 Videos, Fig 9H). Interestingly, observed occasional ectopic blood vessel formation in kmt2d^zy59 DAPT-treated mutants at 2 dpf but at lower frequencies than untreated mutants. This indicated that Notch signaling inhibition was not able to completely rescue this phenotype at early stages (Fig 9D, arrowhead). However, this lower frequency of ectopic sprouts did not result in ectopic vessels, because 3 dpf kmt2d^zy59 DAPT-treated mutants did not show ectopic blood vessels (S9–S12 Videos, Fig 9H). Moreover, the hyperactive endothelial cell behavior that is observable in the junction between LDA and AA1 in kmt2d^zy59 mutant from the DMSO control group was not observed in DAPT-treated kmt2d^zy59 mutants (S14 Video, Fig 9G, arrowhead and Fig 9H). These results emphasize the utility of this Kabuki Syndrome model in discovering fine details of cardiovascular development and strengthen the hypothesis of a regulatory link between Notch signaling and Kmt2d in vasculogenesis.

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Fig 9. Pharmacological inhibition of Notch signaling rescues early endothelial phenotypes and suppresses ectopic blood vessel formation in zebrafish kmt2d mutants.

(A–H) Still images (MIP) from time-lapse live imaging performed from 2 dpf to 2.5 dpf (A–D) and 3 dpf to 3.5 dpf (E–H). wild-type tg(Kdrl:GFP) and kmt2d^zy59;tg(Kdrl:GFP) samples were treated with DMSO (n = 12; A, C, E, and G) or DAPT (n = 12; B, D, F, and H) from 1 dpf to 2 dpf. After treatment, samples were washed and prepared for in vivo time-lapse imaging. Cranial-lateral view at the level of AA development corresponding to video as follow: A, S7 Video; B, S9 Video; C, S8 Video; D, S10 Video; E, S11 Video; F, S13 Video; G, S12 Video; H, S14 Video. Images were selected at last time points recorded. Videos and images were converted to grayscale and inverted for better visualization. Asterisk (B, C, and G = AA*) denotes abnormal or missing vascular development of AA sprouts at the level of the ventral border of LDA. (scale bars = 50 μm). AA, aortic arch; AA1, mandibular arch; AA2, hyoid arch; AA3, first branchial arch; AA4, second branchial arch; AA5 third branchial arch; AA6, fourth branchial arch; dpf, days post fertilization; hpf, hours post fertilization; kdrl, kinase insert domain receptor like; LDA, lateral ventral aorta; MIP, maximum intensity projection; ORA, opercular artery.

https://doi.org/10.1371/journal.pbio.3000087.g009

Ethics statement

Zebrafish embryos, larvae, and adults were produced, grown, and maintained according to standard protocols approved by the Institutional Animal Care and Use Committee of University of Utah, protocol number 18–05006. For experiments, zebrafish embryos ranging from 1 dpf to 7 dpf were used. Adults were maintained at approximately 5 fish per liter for all experiments in aquarium with controlled light cycle and water temperature at 28°C. Animals were fed 3 times a day. Published strains used in this study include: wild-type AB, tg(kdrl:EGFP)^la116 [72] and Tg(EPV.Tp1-Mmu.Hbb:EGFP)^um14 referred in this manuscript as tg(tp1:EGFP)^um14 [73].

Method details

Quantification and statistical analysis