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The dyslexia susceptibility KIAA0319 gene shows a specific expression pattern during zebrafish development supporting a role beyond neuronal migration

Abstract Dyslexia is a common neurodevelopmental disorder caused by a significant genetic component. The KIAA0319 gene is one of the most robust dyslexia susceptibility factors but its function remains poorly understood. Initial RNA‐interference studies in rats suggested a role in neuronal migration whereas subsequent work with double knock‐out mouse models for both Kiaa0319 and its paralogue Kiaa0319‐like reported effects in the auditory system but not in neuronal migration. To further understand the role of KIAA0319 during neurodevelopment, we carried out an expression study of its zebrafish orthologue at different embryonic stages. We used different approaches including RNAscope in situ hybridization combined with light‐sheet microscopy. The results show particularly high expression during the first few hours of development. Later, expression becomes localized in well‐defined structures. In addition to high expression in the brain, we report for the first time expression in the eyes and the notochord. Surprisingly, kiaa0319‐like, which generally shows a similar expression pattern to kiaa0319, was not expressed in the notochord suggesting a distinct role for kiaa0319 in this structure. This observation was supported by the identification of notochord enhancers enriched upstream of the KIAA0319 transcription start site, in both zebrafish and humans. This study supports a developmental role for KIAA0319 in the brain as well as in other developing structures, particularly in the notochord which, is key for establishing body patterning in vertebrates.

a phonological deficit is the most commonly accepted cause for dyslexia, sensory dysfunction in the visual and auditory systems have also been observed in a number of studies (Goswami, 2014;Ramus & Ahissar, 2012;Shaywitz & Shaywitz, 2005).
The DYX1C1, DCDC2, ROBO1, and KIAA0319 genes are known as the classical dyslexia susceptibility genes and they are supported by a number of independent replication studies (Carrion-Castillo, Franke, & Fisher, 2013;Newbury, Monaco, & Paracchini, 2014). A role in cortical development, and specifically in neuronal migration, has been proposed for these genes (Paracchini, Scerri, & Monaco, 2007), in line with earlier postmortem observations that reported subtle cortical defects in individuals with dyslexia (Galaburda, LoTurco, Ramus, Fitch, & Rosen, 2006;Humphreys, Kaufmann, & Galaburda, 1990). In particular, KIAA0319 variants have been found to be associated with dyslexia and reading abilities in multiple clinical and epidemiological cohorts (Newbury et al., 2014;Paracchini, 2011). A specific dyslexiaassociated variant was shown to affect KIAA0319 transcription regulation and gene expression levels, providing a mechanism to link genetic variation with the disorder (Dennis et al., 2009;S Paracchini et al., 2006). Its paralogous gene, KIAA0319-LIKE or KIAA0319L, has also been reported to be associated with dyslexia but with weaker evidence (Couto et al., 2008). Both KIAA0319 and KIAA0319L are transmembrane proteins (Velayos-Baeza, Toma, da Roza, , but their exact cellular functions remain unclear. A new role in cilia biology is emerging for dyslexia candidate genes (Brandler & Paracchini, 2014;Paracchini, Diaz, & Stein, 2016). A transcriptome study showed differential regulation for KIAA0319, DCDC2, and DYX1C1 in ciliated tissues (Ivliev, t Hoen, van Roon-Mom, Peters, & Sergeeva, 2012). Functional studies for Dyx1c1 and Dcdc2 showed a role in ciliogenesis in different biological models, including zebrafish, (Chandrasekar, Vesterlund, Hultenby, Tapia-Paez, & Kere, 2013;Massinen et al., 2011;Tarkar et al., 2013) and some patients with ciliopathies have been found to harbor mutations in both genes (Schueler et al., 2015;Tarkar et al., 2013). While there is no direct evidence supporting a role for KIAA0319 in cilia, the presence of five PKD domains in KIAA0319 lends support to this notion (Velayos-Baeza, Toma, Paracchini, & Monaco, 2008). Mutations in PKD genes, which play key roles in cilia, lead to ciliopathies and laterality defects (Hildebrandt, Benzing, & Katsanis, 2011). KIAA0319 has been shown to be a target of T-Brain-1 (TBR1), a transcription factor implicated in autism which regulates different brain developmental processes, such as neuronal migration, axon guidance (Chuang, Huang, & Hsueh, 2015) and the determination of left-right asymmetries in bilaterians (Kitaguchi, Mizugishi, Hatayama, Aruga, & Mikoshiba, 2002). KIAA0319 has been shown to be involved in axon growth and regeneration supporting a role in the adult peripheral nervous system (Franquinho et al., 2017).
Both KIAA0319 and KIAA0319L have been implicated in neuronal migration following knockdown experiments that specifically targeted neurons at the early stages of brain development using in utero shRNA in rats Paracchini et al., 2006;Peschansky et al., 2010;Platt et al., 2013;Szalkowski et al., 2012). However, knockout (KO) mouse models do not display any cortical abnormalities that could be explained by defective neuronal migration Martinez-Garay et al., 2017). Instead, the KO mice presented auditory system defects  in line with observations reported in adult rats that underwent KIAA0319 knock-down in utero Szalkowski et al., 2012). Therefore, while a role for KIAA0319 in neurodevelopment is supported by different lines of evidence, its exact function remains largely unclear.
Here, we report a gene expression study for the kiaa0319 gene in zebrafish to further understand the role of this gene during vertebrate development. We observed a spatiotemporal expression pattern beyond the brain including in the eyes and the notochord. Surprisingly, kiaa0319-like, which presents a widespread expression in other species, was not expressed in the notochord, suggesting role specific to kiaa0319.
These data support a role for KIAA0319 both in the brain and in other structures and suggest for the first time a function in the notochord.

| Fish care
All the experimental procedures were approved by the Animal Welfare Ethics Committee at the University of St Andrews in compliance with the Home Office regulations. All researchers who conducted work with animals held a Personal License issued by the Home Office.
Wild type zebrafish (Danio rerio) (WIK and AB/TU) and the double transgenic Tg(gfap:GFP);Tg(Oligo2:dsRed) were raised at The Queen's Medical Research Institute at the University of Edinburgh according to standard procedures in a home office approved facility. Developmental stages, maintained at 28.5 C, were identified as previously described (Kimmel, Ballard, Kimmel, Ullmann, & Schilling, 1995). Animals were handled in accordance with the guidelines from European Directive 2010/63/EU and euthanised in accordance with Schedule 1 procedures of the Home Office Animals (Scientific Procedures) Act 1986. Zebrafish embryos were obtained using the Mass Embryo production system (MEPs) of the wild type line Wik.

| PCR and qPCR
Total RNA from developmental stages between 16 and 32 cells, up to 120 hour post-fertilization (hpf) was extracted using the RNeasy Mini kit according to the manufacturer's instructions (QIAGEN) using at least 50 embryos at each stage. The heart, liver and brain were dissected from five adult fish, flash-frozen on dry ice and stored at −80 C until the RNA was extracted. Eyes were dissected (N = 40 eyes total) at 48 hpf stage and flash frozen on dry ice.
The PrimeScript RT reagent kit (Takara) was used to transcribe the RNA into the cDNA following the manufacturer's protocol. The presence of kiaa0319 transcripts was verified by electrophoresis following PCR amplification. Gene expression was assessed by quantitative PCR  Table S1. Differences in gene expression were evaluated with the Wilcoxon-Mann-Whitney test implemented in R (RStudioTeam, 2015).

| Whole-mount in situ hybridization
Whole-mount in situ hybridization (WISH) was carried out following a previously described protocol (Thisse & Thisse, 2008). Briefly, a DIGlabeled riboprobe targeting kiaa0319 was transcribed using a T3 Polymerase with a DIG RNA Labeling Mix (Roche) according to the manufacturer's instructions and, as a template, a 1,066 bp PCR fragment amplified from cDNA (Table S1). Zebrafish embryos were collected and processed at 3 somite, 14 somite, 30 hpf, and 48 hpf. Embryos were imaged with a Leica MZ16F or MZFLIII bright field microscopes following treatment with an anti-DIG antibody (Roche, diluted 1:5000 in blocking buffer) and a staining solution (NBT and BCIP, Roche).

| Sequence analysis
The zebrafish orthologues of the human KIAA0319 and KIAA0319L genes were identified in the zebrafish genome using the UCSC genome browser (Kent et al., 2002).

| Exploratory analysis
As a first step of our analysis, we verified the expression of kiaa0319 during early zebrafish development using RT-PCR ( Figure 1a). The expression of kiaa0319 was observed across all the developmental stages that were analyzed. In the adult, expression was much higher in the brain compared to heart and liver, where it is barely detectable.
The high expression in the brain is consistent with the expression profile observed for KIAA0319 in humans in contrast to KIAA0319L, which is widespread across human tissues ( Figure S1). Quantification of expression by qPCR confirmed kiaa0319 expression at different developmental stages and showed that the highest level was detected in the earliest stages of development (Figure 1b), whereas the lower expression was observed at 12 hpf. Expression at the first development stages (up to 5 hpf) was significantly higher compared to later time points (p < .0001).
To localize kiaa0319 expression patterns we conducted WISH ( Figure S2). Consistent with the qPCR data, we observed high where a role for KIAA0319 has not been described before.

| RNAscope analysis
To verify this expression pattern and to achieve higher resolution, and specificity we used the highly sensitive RNAscope fluorescent multiplex assay (Figure 2). In particular, we focused on tissues other than brain. These included the body midline, the otic vesicles and the eyes following the observations made by WISH. For comparison, we included in the analysis the kiaa0319-like gene. Consistent with the qPCR and the WISH results, at 24 hpf kiaa0319 expression is widespread but stronger in the brain and body midline ( with DsRed (Shin, Park, Topczewska, Mawdsley, & Appel, 2003) and therefore is useful to distinguish the developing neural tube.
Among the elements controlling gene expression in the notochord, FOXA2 is a key transcription factor (Tamplin et al., 2011). We scanned the genomic sequences upstream of the KIAA0319 and KIAA0319L TSS in both humans and zebrafish for FOXA2 consensus motifs ( Figure 3; Table S3). We analyzed the 10 kb region upstream of the TSS, where regulatory elements for downstream genes would most likely reside (Metzakopian et al., 2012). In zebrafish, six and three FOXA2 motifs were found upstream of kiaa0319 and kiaa0319-like, respectively. Three of the six motifs upstream of kiaa0319 were within the 6 kb upstream of the TSS, while all three of the FOXA2 motifs were more distant to the kiaa319-like TSS. In humans, FOXA2 motifs were found only upstream of KIAA0319 (N = 2) and not of KIAA0319L. Figure 3b shows the position of these two motifs relatively to KIAA0319 and to the dyslexia-associated SNPs (Newbury et al., 2014;Paracchini et al., 2006).
Both the WISH and RNAscope analyses suggested expression of kiaa0319 in the otic vesicles ( Figure S4), which is of interest in the context of previous reports of a possible role of Kiaa0319 in the auditory system of rodents Guidi et al., 2017;Szalkowski et al., 2012). However, this structure tends to accumulate nonunspecific signal when conducting in situ hybridization because of technical artifacts, such as probe trapping. Detailed RNAscope analysis showed a signal for both kiaa0319 (Figure 4b) and kiaa0319-like (Figure 4c) in the otic vesicles, however a signal was detected also in the negative controls (Figure 4f, g). In comparison to the controls, both genes showed stronger expression and a signal characterized by a speckled pattern including within the main structures, suggestive of a genuine expression. In contrast, the controls showed a weaker signal, mainly localized along the contour of the otic vesicles suggesting probe trapping. However, given this background noise, we cannot conclude with confidence that kiaa0319 and kiaa0319l are expressed at these structures.
The WISH analysis also suggested expression in the eyes, another structure that might lead to unspecific signals ( Figure S2). The F I G U R E 1 kiaa0319 is expressed during different stages of development. (a) The top panel shows RT-PCR amplicons generated with primers targeting different kiaa0319 exons using cDNA prepared at different zebrafish developmental stages (hours postfertilization, hpf) and in selected adult tissues. The amplicons have the expected size of 1,024 bp. The lower panel shows fragments (322 bp) for bactin2 used as a control for cDNA quality at the corresponding stages and tissues. Genomic DNA (gDNA) in the last lane demonstrates the specificity of the assay with no band for the kiaa0319 reaction and a fragment of 407 bp for the bactin2 as expected. kiaa0319 is expressed throughout the different development stages and in the adult brain but with only weak signal in the hearth and liver. The adult data are consistent with the expression profiles observed in human adult tissue ( Figure S1). The top and lower panel are images from two separate gels where samples were loaded in the same order. (b) Quantification of the expression of kiaa0319 by qPCR measured during the first 5 days of development. Expression is measured against the eef1a1l2 gene, used as reference. Mean values are derived from biological triplicates and error bars indicate standard deviations. The first two time points showed significantly higher expression compared to later stages (p < .0001) RNAscope analysis at the eyes showed expression for both kiaa0319 (Figure 4b,j) and kiaa0319-like (Figure 4c,l). Both genes are expressed on the surface of the eyes and, most strongly, around the eye lens. The negative controls had no signal confirming the specificity of the probes.
Expression in the eyes was further confirmed by qPCR (Figure 4m).

| DISCUSSION
We conducted the first zebrafish characterization of the dyslexia susceptibility KIAA0319 gene. We found that kiaa0319 is highly expressed at early developmental stages and, in addition to the expected expression in the brain; we show that it is expressed in the notochord, the eyes, and possibly the otic vesicles. For comparison, we analyzed the kiaa0319-like gene, which showed a similar expression pattern but presented a very weak signal in the notochord. This observation is surprising given the generally higher and ubiquitous expression of KIAA0319-LIKE reported in human tissues ( Figure S1) and suggests a specific role for kiaa0319. To the best of our knowledge, this is the first study reporting expression of kiaa0319 during the very first hours of development and clearly showing its expression in specific structures other than the brain.
The function of the KIAA0319 protein has been studied in human cell lines and in rodent models, however it is not yet fully understood.
The first functional characterization was conducted in rats and suggested a role in neuronal migration (Paracchini et al., 2006) while more recent studies in mice indicate an involvement in biological processes beyond brain development (Franquinho et al., 2017;Guidi et al., 2017Guidi et al., , 2018. Consistent with the latest studies, we observed expression in the brain, but also observed expression in other organs. Guidi and colleagues ) generated a double KO mouse model for the Kiaa0319 and Kiaa0319l genes and the most notable phenotype reported was an impairment of the auditory system. Analysis of individual KO for both genes showed mild effects for Kiaa0319l but no effects for Kiaa0319 alone. Rodent models for other dyslexia candidate genes (i.e., Dcdc2 and Dyx1c1) have also suggested an impairment in auditory processing (Szalkowski et al., 2013;Truong et al., 2014). The potential F I G U R E 2 kiaa0319 is specifically expressed in the notochord. The expression of kiaa0319 (a) and kiaa0319l (b) was examined at three embryonic stages (12, 24, and 48 hpf; WT zebrafish) using the RNAScope fluorescent multiplex assay with results shown for the head (a1, a2, a3, b1, b2, and b3) and the body (a4, a5, a6, b4, b5, and b6). kiaa0319 (red) is expressed throughout the three stages. High expression is detected in the developing brain and body midline. At 48 hpf, kiaa0319 is still highly expressed in the brain and strong signal is detected in the otic vesicles (a3; white arrows corresponding to the red arrow in the reference image) and in the notochord (a6). kiaa0319l shows a similar pattern of expression, including a strong signal in the otic vesicles (b3), but the expression in the notochord at 48hpf is very weak (b6). Black area in the brain at 48 hpf correspond to the pigmentation of the embryo (a3 and b3). (c) Longitudinal view of the body with 3D reconstructions from light-sheet microscopy images at three developmental stages 72 (c1), 96 (c2), and 120(c3) hpf. The samples from a WT zebrafish are labeled with the kiaa0319 probe. kiaa0319 expression is localized to the notochord (white arrow). The signal diminishes as development progresses and the notochord regresses. See a 3D animation at 72hpf that allows assessing the signal from different orientations (Video SV1). See Figure S3 for the positive and negative controls acquired with light sheet microscopy (d) kiaa0319 expression in the notochord was confirmed with the Tg(gfap:GFP);Tg(Oligo2:dsRed) transgenic line, which express GFP (green) in the spinal cord (d1). No signal was detected for the negative control (d2) myoD1 (myogenic differentiation 1, a universal target for myogenic cells [Weinberg et al., 1996]) was used as positive control and demonstrates the specificity of the assay images were collected at 42 hpf by confocal microscopy. The scale bar indicates 50 μm in all panels expression of both kiaa0319 and kiaa0319l in the otic vesicles ( Figure 2, Figures S2 and S4) would be interesting in the context of the rodent data. However, further work will be required to establish whether these genes are expressed at the otic vesicles given possible probe trapping in these structures ( Figure S4). Instead, given the eyespecific expression observed in our study (Figure 4), it would be valuable to assess visual phenotypes in rodent models.
Whether dyslexia is the result of a deficit in sensory systems, as predicted by the magnocellular theory (Stein, 2001), remains highly debated (Paracchini et al., 2016). Defects in both the visual and auditory systems have been reported in individuals with dyslexia across different studies, but heterogeneity and inconsistency across studies remain significant challenges (Goswami, 2014). The kiaa0319 expression in the eyes during zebrafish development could be considered in line with a role in sensory organs. While it would be tempting to reach conclusions, it is worth noting that it is not possible to generalize and make strong assumptions based on observations for genes analyzed in isolation. Moreover, the KIAA0319 genetic associations (as with most genetic associations and with complex traits) explains only a small fraction of dyslexia heritability (Paracchini et al., 2016).
The most compelling observation of our study is the strong notochord expression of kiaa0319, which, in comparison, was only very weak for kiaa0319-like (Figure 2). The notochord is a transient embryonic structure in zebrafish essential for guiding the development and patterning of the early embryo (Stemple, 2005). Because of the important functions of the notochord, the identification of the notochordexpressed genes is important to understand these developmental processes. The notochord is a source of signaling to the surrounding tissues to guide structural development, particularly for the spinal cord. For example, the notochord is the source of sonic hedgehog (SHH) signaling which controls many processes including the development of motor neurons, the establishment of the dorsal-ventral axis and left/right asymmetries (Echelard et al., 1993;Schilling, Concordet, & Ingham, 1999). The notochord also controls, in a highly specific spatiotemporal manner, the trajectories of dorsal root ganglion (DRG) axons through repressive signals mediated by aggrecan, one of the chondroitin sulfate proteoglycans (CSPGs) specifically found in the cartilage (Masuda et al., 2004). A similar repressive role has been described for Kiaa0319 in mice, including the repression of axon growth in hippocampal and DRG neurons (Franquinho et al., 2017). The same study also showed that Kiaa0319 was expressed in sensory and spinal cord neurons in postnatal and adult mice. Our data are consistent with these findings suggesting an evolutionary conserved function for kiaa0319 beyond brain development.
While it is not possible to directly infer information about gene function from expression patterns, our data represent a useful resource to guide and interpret follow up functional studies. First of all, the specific spatial/temporal pattern suggests that zebrafish would be a valuable model for knock-down and knock-out studies. Furthermore, these results will help to formulate well-defined hypotheses on which assays and which lines or mutants might be particular useful at dissecting specific roles at well-defined structures.
A specific expression pattern is likely to result from a fine-tuned regulation. Most of the markers associated with dyslexia map to the KIAA0319 regulatory regions (Figure 3; Newbury et al., 2014;Paracchini et al., 2006). Previously, we showed that a dyslexiaassociated allele (rs9406145) at this region affects the affinity for a transcription factor and reduce the expression of KIAA0319 (Dennis et al., 2009). FOXA2 is a key transcription factor involved in the regulation of genes expressed in the notochord (Tamplin et al., 2011).
However, FOXA2 motifs are not sufficient to predict expression in the notochord as other transcription factors (e.g., Brachyury, GLIS3, and F I G U R E 4 kiaa0319 and kiaa0319-like are expressed in the eyes. Results for RNAscope analysis in the eyes for kiaa0319 (labeled in red; panels [b,j]) and kiaa0319-like (labeled in green; [c,l]). The triple negative control shows no signal ([f,g] DAPI [a,e,i]) shows nuclear staining. Panels (d,h,k) show the merged signal for all channels. Panel (i) to (l) provide a different view of the eye. All images show the left side of animals oriented with brain on the left and tail on the right at 48 hpf (WT zebrafish). The scale bar is 50 μm in all panels. (m) Quantification of expression by qPCR of kiaa0319 and kiaa0319-like. Expression is measured as 1/ΔCt referenced against the eef1a1l2 gene. The measurement is derived after pooling a total of 40 eyes collected at 48 hpf. The mean values were derived from three technical replicates. The error bars indicate standard deviations RFX3) might be required and would function through enhancers located at specific distances (Farley, Olson, Zhang, Rokhsar, & Levine, 2016). Nevertheless, the FOXA2 consensus motifs upstream of KIAA0319 TSS support the patterns observed in Figure 2 showing a much stronger expression for kiaa0319 in the notochord compared to kiaa0319l. The region upstream of kiaa0319 had more FOXA2 consensus sequences and in more proximal position compared to kiaa0319l. In humans, FOXA2 motifs were found for KIAA0319 only, suggesting a conserved role for KIAA0319 across vertebrates. These observations also provide a framework to interpret the genetic associations with dyslexia reported in KIAA0319 noncoding regions ( Figure 3) (Dennis et al., 2009;Paracchini et al., 2006). Genetic variation at this locus might affect not only gene expression levels, as previously shown (Dennis et al., 2009), but also perturb the regulation of a specific spatiotemporal pattern.
In summary, our characterization of the KIAA0319 dyslexia susceptibility gene in zebrafish reveals a specific pattern of expression during development. In addition to the expected expression in the brain, we show for the first time high embryonic expression during the first hours of development and, later on, at specific structures such as the eyes and the notochord. Our study therefore supports a developmental role for KIAA0319 which is not restricted to the brain and may contribute to the ongoing discussion around the role of neuronal migration in dyslexia. While our data do not exclude a role in the developing brain and in neuronal migration, they suggest an involvement in other developmental processes as well.

CONFLICT OF INTEREST
The authors declare no competing interests.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are presented in the main text and supplementary material. Additional data and images are available from the corresponding author.