Jmjd6a regulates GSK3β RNA splicing in Xenopus laevis eye development

It has been suggested that Jmjd6 plays an important role in gene regulation through its demethylation or hydroxylation activity on histone and transcription factors. In addition, Jmjd6 has been shown to regulate RNA splicing by interaction with splicing factors. In this study, we demonstrated that Jmjd6a is expressed in developing Xenopus laevis eye during optic vesicle formation and retinal layer differentiation stages. Knockdown of Jmjd6a by an antisense morpholino resulted in eye malformation including a deformed retinal layer and no lens formation. We further found down-regulation of gene expression related to eye development such as Rx1, Otx2, and Pax6 in Jmjd6a morpholino injected embryos. Jmjd6 interacts with splicing factor U2AF25 and GSK3β RNA in the anterior region of Xenopus embryos. Knockdown of Jmjd6a led to deletion of GSK3β RNA exon 1 and 2, which resulted in generation of N’-terminal truncated GSK3β protein. This event further caused decreased phosphorylation of β-catenin and subsequently increased β-catenin stability. Therefore, our result may suggest that Jmjd6a plays an important role in Xenopus eye development through regulation of GSK3β RNA splicing and canonical Wnt/β-catenin signaling.


Introduction
During vertebrate eye development, spatiotemporal orchestration of eye-specific gene expression occurs between different cell types from different embryonic origins including the neural ectoderm, surface ectoderm, and periocular mesenchyme [1]. Eye development is initiated at the onset of gastrulation by the determination of the eye field in the anterior neuroectoderm [2]. As gastrulation proceeds, the eye field is expanded by proliferation and the retinal primordium is formed. Evagination of the neuroectoderm forms optic vesicles and subsequently the optic vesicles are transformed into optic cups with retinal differentiation [3]. Ocular development begins with the formation of the optic vesicles, which come into close contact with the surface ectoderm where the lens placode is formed. The optic cups are further subdivided into the outer retinal pigment epithelium (RPE), the neural retina, and the ciliary marginal zone (CMZ), which is located at the edge of the retina and contains a proliferative population of undifferentiated cells such as retinal stem and progenitor cells. After maturation, the neural retina forms three well-structured layers consisting of the outer nuclear layer (ONL), the inner nuclear layer (INL), and the ganglion cell layer (GCL). The ONL, which is the outermost retinal layer, contains the cell bodies of cone and rod photoreceptors. In the INL, the cell bodies of bipolar, horizontal, and amacrine interneurons exist. The GCL, which is the innermost retinal layer, contains the cell bodies of ganglion cells. Müller glial cells span all retinal layers. These developmental events are tightly regulated by signaling cascades, which control cellular proliferation and differentiation [1].
Among cellular signaling pathways, the canonical Wnt/β-catenin signaling pathway plays an essential role in eye development [4][5][6]. Wnt/β-catenin signaling is initiated by binding of Wnt to the Frizzled/LRP5/6 receptor, which leads to the accumulation and nuclear translocation of β-catenin. In the nucleus, β-catenin interacts with transcription factors and regulates the expression of their target genes. In the absence of Wnt, β-catenin is phosphorylated at Thr 41, Ser 33, Ser 37, and Thr 41 by GSK3β, which is a member of the β-catenin destruction complex composed of multiple proteins including APC, axin, and CK1α. This subsequently triggers β-catenin destabilization by ubiquitination [7,8]. In vertebrate eye development, Wnt/βcatenin signaling is activated in distinctive regions of the optic vesicle and the optic cup for dorsoventral patterning [9]. It is subsequently restricted to the peripheral RPE [5]. Wnt/β-catenin signaling is also activated in the CMZ to maintain the population of retinal progenitors [10].
In this study, we demonstrated that Jmjd6a is expressed highly in the eye and brain originated from anterior neural tissue during Xenopus development. Knockdown of Jmjd6a by antisense morpholino led to augmented canonical Wnt/β-catenin signaling through generation of aberrant GSK3β RNA, which resulted in the production of N-terminal truncated GSK3β protein. In turn, these events may cause abnormal eye development in Xenopus embryos.

Animal manipulation
Xenopus laevis fertilized eggs were dejellied in 2% cysteine (pH 8.0) and grown in 0.1×MMR (Marc's Modified Ringer). Developmental stages of embryo were determined in accordance with Nieuwkoop and Faber's staging system [30]. Animals were handled in accordance with a standard protocol approved by the animal care committee of Sogang University.

RT-PCR
Total RNA was extracted from Xenopus embryos using RNAiso Plus (Takara). First-strand cDNA was synthesized from 5μg of total RNA with PrimeScript RT Master Mix (Takara). For real time-PCR, the resulting cDNAs were amplified using 2×PreMIX SYBR kit (Enzynomics) with Stratagen Mx3000p (Agilent Technologies). PCR conditions entailed an initial denaturation at 95˚C for 10 min followed by 40 cycles of denaturation (95˚C for 15 sec), annealing (60˚C for 40 sec) and elongation (72˚C for 60 sec), with a final elongation at 72˚C for 10 min. Expression was calculated from the cycle threshold (Ct) value using the ΔCt method for quantification. Expression of EF1α was used for normalization. For conventional PCR, cDNAs were amplified with GeneAmp PCR system 2700 (Applied Biosystems). PCR conditions entailed an initial denaturation at 95˚C for 2 min followed by 33 cycles of denaturation (95˚C for 30 sec), annealing (60˚C for 30 sec) and elongation (72˚C for 30 sec), with a final elongation at 72˚C for 5 min. Expression of EF1α was used for normalization. Oligonucleotides used for RT-PCR are listed in Supporting Information (S1 Table).

5' RACE and RNA synthesis
To identify the 5' end of Xenopus GSK3β RNA, 5' RACE was performed with mRNA extracted from Jmjd6a MO injected embryos using 5'-full RACE core kit (Takara). The cDNAs were subcloned into pGEM T-easy plasmid vector. After sequencing, GSK3β cDNA without exon 1 and 2 was subcloned into pCS4+ plasmid vector. Xenopus Jmjd6a cDNA without 5' UTR (Δ5'UTR Jmjd6a) was subcloned into pSC2+ basic plasmid vector. For in vitro transcription, capped mRNA was synthesized using SP6 mMESSAGE mMACHINE Kit (AM2075, Ambion). Oligonucleotides used for 5' RACE and subcloning are listed in Supporting Information (S1 Table).

TOP-Flash assay
TOP-Flash reporter plasmid (50 pg) containing multiple copies of Tcf-binding site was injected with CMV-Renilla luciferase plasmid (5 pg) into two blastomeres of the animal-dorsal region at the 8-cell stage. The anterior regions of the injected embryos were collected at stage 26 and a luciferase assay was performed using the Dual-Luciferase Assay System (Promega). Renilla luciferase activity served as an internal control for normalizing the firefly luciferase activity.

Statistical analyses
All quantitative data are presented as mean ± standard deviation (SD). for three independent experiments. For examination of gene expression, more than 30 embryos were analyzed in each experiment. The differences between two groups were evaluated by a paired t-test. Significance values were � P � 0.05 and �� P � 0.01.

Expression pattern of Jmjd6 in Xenopus laevis development
We first examined temporal and spatial expression patterns of Jmjd6a and Jmjd6b, which are expressed maternally and zygotically [28]. Quantitative RT-PCR demonstrated that expressions of Jmjd6a and Jmjd6b were gradually decreased during Xenopus early development (S1 Fig). The expression level of Jmjd6a was higher than that of Jmjd6b and changed more drastically (S1 Fig). We further analyzed Jmjd6 expression by whole-mount in situ hybridization using antisense Jmjd6 riboprobes. Jmjd6a was expressed broadly in the anterior neural tissues including the eye and brain primordia at stage 20 (Fig 1A). At stage 26, increased Jmjd6a expression was detected in the eye primordia, brain primordia, and neural tube (Fig 1B and  1C), and the elevated expression of Jmjd6a was maintained in the developing eye and brain at stage 30 ( Fig 1D). However, Jmjd6a expression was restricted in the forebrain, midbrain and hindbrain at stage 40 ( Fig 1E). To confirm Jmjd6 expression, immunohistochemistry was performed using anti-Jmjd6 antibody, which recognizes Xenopus Jmjd6, in transverse sections. Jmjd6 was detected consistently in the developing eye and brain region (Fig 1C'-1E'). Also, Jmjd6 expression was detected in the retinal layers at stage 30 (Fig 1D'). Although the expression pattern of Jmjd6b was similar to that of Jmjd6a in the developing eye and brain region, its expression was greatly decreased after stage 30 (S2 Fig).

Jmjd6a is required for Xenopus eye development
To examine whether knockdown of Jmjd6a affects Xenopus eye development, we designed antisense morpholino oligonucleotide (MO) against Xenopus Jmjd6a (Fig 2A). Knockdown of Jmjd6a by MO efficiently reduced the endogenous level of Jmjd6 protein (Fig 2B). However, coinjection of Jmjd6a mRNA without 5'-UTR (Δ5'UTR Jmjd6a), which cannot be paired with Jmjd6a MO, restored Jmjd6 expression (Fig 2B). To examine the effect of Jmjd6a knockdown on eye development, MO was injected into a single animal-dorsal blastomere of 8-cell stage embryos [34]. Proper MO injection was confirmed by co-injection of plasmid containing RFP (red fluorescence protein) cDNA as a lineage tracer (S3 Fig). Knockdown of Jmjd6a by MO injection resulted in smaller or partially developed eyes (Fig 2C-2F). We further analyzed 468 embryos and categorized them in accordance with the extent of abnormal eye phenotypes such as the size and shape of retinal pigment epithelium (Fig 3A and 3B). Twenty one % (100/468) of embryos showed mild abnormality with smaller eye size compared to normal eyes. Twenty six % (123/468) of embryos showed moderate abnormalities with partially developed eyes. Twenty six % (120/468) of embryos had severe phenotypes including poor eye development or no eye formation. Histological observation further supported abnormal eye development. In normal developing eye, the RPE, ONL, INL, and GCL were well formed. However, some retinal layers were missing or were indistinguishable in Jmjd6a MO-injected embryos (Fig 3A, 3 rd row). In severe cases, no lens was formed (Fig 3A, 2 nd and 3 rd row). In addition, an ectopic eye, which developed incompletely, was found in embryos with no eye formation (Fig 3D). We also examined expression of Islet1, a  marker of Xenopus retinal development, using anti-Islet1 antibody [35]. In normal developing eye, Islet1 was expressed in most of the cells in the GCL and a few cells of the INL. However, the expression level of Islet1 was decreased in defected eyes induced by Jmjd6a MO injection (Fig 3A,  4 th row). As expected, co-injection of Jmjd6a mRNA without 5'-UTR (Δ5'UTR Jmjd6a) with Jmjd6a MO resulted in a reduced number of embryos with abnormal eye (Fig 3B). In addition, we found malformation of anterior brain structures in Jmjd6a MO-injected embryos (Fig 3C).

Knockdown of Jmjd6a affects expression of genes related to Xenopus eye development
To investigate the effect of Jmjd6a MO on expression of genes related to Xenopus eye development, we analyzed the expression of Otx2 (developmental marker for forebrain, eye, and anterior midbrain), Rx1 (developmental marker for eye), and Pax6 (developmental marker for forebrain and eye) at tailbud stages (stage 22, 26 and 33). We found that expression of Otx2, Rx1, and Pax6 decreases in the eye primordia at stage 22 in the Jmjd6a MO injected side compared with the un-injected or control MO-injected side of embryos ( Fig 4A). However, expression of Otx2 and Pax6 was not affected in the brain region (Fig 4A). At stages 26 and 33, decreased expressions of Otx2, Rx1, and Pax6 gene were maintained (Fig 4B).

Jmjd6a acts as RNA splicing regulator of Xenopus GSK3β
Mammalian Jmjd6 regulates RNA splicing by interacting with specific SR-related proteins or catalyzing lysyl-hydroxylation of splicing factor U2AF65 (U2 small nuclear ribonucleoprotein auxiliary factor 65-kilodalton subunit) [19,21,22]. Thus, first we examined the interaction between Jmjd6 and U2AF65 by an immunoprecipitation assay using lysate from Jmjd6a expressing anterior region of embryos at stage 26. Consistently, we found that Jmjd6 interacts with Xenopus U2AF65 (Fig 5A). To identify the possible RNA target of Jmjd6a in Xenopus eye development, we searched a mouse database that presented Jmjd6-associated RNAs using an RNA-immunoprecipitation assay [22]. Among them, we chose GSK3β as a candidate because of its critical role in vertebrate eye development [36][37][38]. To confirm Jmjd6 interaction with Xenopus GSK3β RNA, RNA-immunoprecipitation was performed using lysate from the anterior region of Xenopus embryos. RT-PCR analysis indicated that Jmjd6 associates with GSK3β RNA (Fig 5B). However, no or weak interaction was detected in the posterior region of embryos, where the expression level of Jmjd6a was low. In addition, Jmjd6 showed no interaction with EF1α RNA, which was used as a negative control. Based on these results, we next examined whether knockdown of Jmjd6a affects the splicing pattern of GSK3β RNA, which consists of 11 exons, in developing Xenopus embryos. We performed RT-PCR analysis with exon specific oligonucleotides in control or Jmjd6a MO-injected embryos at stage 26. The Jmjd6a MO injection resulted in decreased levels of GSK3β exon 1 and 2 compared with the control MO-injected embryos (Fig 5C and 5D). However, other exons were not changed in either the control or Jmjd6a MO-injected embryos (S4 Fig). We confirmed the generation of GSK3β RNA without exon 1 and 2 by 5' RACE (rapid amplification of cDNA ends) analysis in Jmjd6a MO-injected embryos at stage 26 ( Fig 5E). The aberrant GSK3β RNA started with the exon 3 containing ATG start codon (Fig 5E). Because the absence of GSK3β exon 1 and exon 2 is expected to result in the loss of 94 N'-terminal amino acids of the protein, we next performed western blot analysis using a lysate from Xenopus embryos injected with GSK3β mRNA started with exon 3. We found an extra 35 kDa band of GSK3β protein as well as endogenous full length proteins (S5 Fig). Consistently, knockdown of Jmjd6a by MO injection resulted in the production of a 35 kDa band of GSK3β protein in Xenopus embryos, with no such band being detected in control MO injected embryos (Fig 5F). Moreover, an extra 35 kDa band was not detected in western blot analysis with anti-GSK3β antibody recognizing N'-terminal protein (Fig 5F). These results may suggest that Jmjd6a interacts with splicing factor U2AF65 and regulates GSK3β RNA splicing in Xenopus eye development.

Knockdown of Jmjd6a affects canonical Wnt/β-catenin signaling in Xenopus laevis development
Given that Jmjd6a regulates GSK3β RNA splicing, we next investigated the effect of Jmjd6a knockdown on canonical Wnt/β-catenin signaling using a TOP-flash luciferase reporter containing multiple TCF binding sites. We injected the TOP-flash reporter with Jmjd6a MO into Xenopus embryos at stage 26. Although basal activity of luciferase was observed in the TOP-flash reporter-injected embryos, Jmjd6a MO co-injection resulted in an increase of luciferase activity (Fig 6A). However, co-injection of Jmjd6a mRNA without 5'-UTR (Δ5'UTR Jmjd6a) suppressed increased luciferase activity by Jmjd6a knockdown. Consistently, phosphorylation of β-catenin was decreased and stability of β-catenin was increased in Jmjd6a MO-injected embryos (Fig 6B). Taken together, our results demonstrate that knockdown of Jmjd6a results in aberrant GSK3β RNA splicing and generation of an N-terminal truncated form of GSK3β protein, which induces increased β-catenin stability.

Discussion
The developmental process of eukaryotes requires tightly regulated gene expression at multiple levels including RNA splicing. It has been reported that mammalian Jmjd6 regulates gene expression by modification of histones or transcription factors [13,18,39]. Jmjd6 also regulates RNA splicing by interaction of splicing factors [19,21,40]. We and others found that two pseudoalleles of Jmjd6, Jmjd6a and Jmjd6b, are expressed in developing Xenopus eye, brain, and neural tube [28]. Immunohistochemical examination demonstarted that the expression pattern of Jmjd6 ptrotein was similar to that of Jmjd6 RNA, and Jmjd6 protein was also expressed in retinal layers at stage 30. Similar spatiotemporal expression of Jmjd6 has been reported in developing mouse embryos [29]. These results suggest that Jmjd6 may play an important role in Xenopus eye developmental processes such as optic vesicle formation and retinal layer differentiation. In this study, we found that knockdown of Jmjd6a induces abnormal eye development with mild to severe abnormalities. Histological examination further revealed deformed retinal layer formation as well as no lens formation. Immunohistochemical examination of Islet1, a marker of Xenopus retina development, showed aberrant retinal cell differentiation in Jmjd6a MO injected embryos. Islet1 is expressed in ganglion, amacrine, bipolar, and horizontal cells in the GCL and INL [35]. However, decreased expression of Islet1 was detected in Jmjd6a MO injected embryos. Consistent with our result, Jmjd6 knockout embryos showed eye malformations that ranged from abnormal differentiation in retinal cell layers including the INL to anophthalmia (no eye formation). Moreover, ectopic eyes were found in case of anophthalmia [29]. We further found that knockdown of Jmjd6a altered gene expression related to eye development including Otx2, Rx1, and Pax6. Otx2 is expressed in the entire optic vesicle at the initial stage of eye development, and its expression is restricted to the presumptive RPE at later stages [41]. Rax, which is a mouse homolog of Rx1, is intensively expressed in the developing retina in mouse [42] and the ONL of Xenopus eyes [43]. Pax6 is an important transcription factor for eye development because ectopic expression of Pax6 alone is sufficient to induce ectopic eyes in fly and frog embryos [44,45]. Pax6 is expressed highly in the early optic vesicles and the surface ectoderm, and its expression remains in all eye components at the optic-cup stage. Its expression becomes restricted to the lens, corneal and conjunctive epithelia, iris, and inner portion of the neuroretina [46].
Previous studies have shown that Jmjd6 regulates RNA splicing by interaction with splicing factors [19,22,40]. We confirmed the interaction of Jmjd6 with U2AF65 splicing factor and GSK3β RNA in Xenopus embryos. Knockdown of Jmjd6a resulted in GSK3β RNA lacking exon 1 and 2, thereby generating an N'-terminal truncated form of GSK3β protein with a molecular weight of approximately 35 kDa. A few studies have been described the importance of the N'-terminal region of GSK3β, for instance the N'-terminal of GSK3β may regulate its kinase activity on β-catenin because of the presence of critical lysine resides (K85 and K86) located at the ATP binding site of GSK3β [47][48][49]. Moreover, kinase dead mutant of GSK3β (GSK3β K85M) has been shown to abolish the interaction with axin, indicating that kinase actitivy is required for β-catenin destruction [50,51]. Thus, generation of an N'-terminal truncated form of GSK3β protein induced by the knockdown of Jmjd6a may induce decreased βcatenin phosphorylation and consequently increased β-catenin stability.
It is well known that canonical Wnt/β-catenin signaling pathway plays a critical role in vertebrate eye development. For instance, canonical Wnt/β-catenin signaling is active in the dorsal optic vesicle and presumptive RPE at the optic vesicle stage. It is subsequently restricted to the peripheral RPE [5,52,53]. At the optic cup stage, RPE transdifferentiates into the neural retina in the absence of β-catenin [53,54]. Mis-regulation of canonical Wnt/β-catenin signaling results in multiple eye malformations because of defects in the process of cell fate determination and differentiation [6]. Consistent with our results demonstrating increased level of βcatenin protein by Jmjd6a knockdown, overexpression of constitutively active β-catenin results in disorganization of the retina layers in mouse [53,55].
In our model, Jmjd6a and U2AF65 bind to GSK3β RNA, resulting in inclusion of exon 1 and 2. Subsequently, GSK3β phosphorylates β-catenin and β-catenin is degraded during Xenopus optic vesicle formation and retinal cell differentiation (Fig 6C). However, knockdown of Jmjd6a results in loss of GSK3β RNA exon 1 and 2, thereby generating an N'-terminal truncated form of GSK3β protein. In turn, the truncated form of GSK3β may not phosphorylate βcatenin, leading to increased β-catenin stability (Fig 6C). These events further result in abnormal development of eye as well as of brain. In this study, we demonstrated Jmjd6a-mediated RNA splicing of GSK3β, which is highly a conserved kinase for cellular signaling pathways such as PI3 kinase, Wnt, Hedgehog, and Notch signaling in embryonic development, cellular differentiation, and several human diseases [56,57]. Therefore, our findings may expand our knowledge of the functions of Jmjd6 in animal development as well as multiple human diseases including cancer [58].