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Jmjd6a regulates GSK3β RNA splicing in Xenopus laevis eye development

  • Jee Yoon Shin,

    Roles Conceptualization, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing – original draft, Writing – review & editing

    Affiliation Department of Life Science, Sogang University, Seoul, Korea

  • Jeongin Son,

    Roles Investigation, Methodology

    current address: Department of Veterinary and Biomedical Sciences, Center for Molecular Toxicology and Carcinogenesis, Pennsylvania State University, University Park, Pennsylvania, United States of America

    Affiliation Department of Life Science, Sogang University, Seoul, Korea

  • Won Sun Kim,

    Roles Conceptualization, Formal analysis, Supervision, Validation, Writing – review & editing

    Affiliation Department of Life Science, Sogang University, Seoul, Korea

  • Jungsug Gwak ,

    Roles Conceptualization, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing

    bgju@sogang.ac.kr (BGJ); gwakjs@sogang.ac.kr (JG)

    Affiliation Department of Life Science, Sogang University, Seoul, Korea

  • Bong-Gun Ju

    Roles Conceptualization, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing

    bgju@sogang.ac.kr (BGJ); gwakjs@sogang.ac.kr (JG)

    Affiliation Department of Life Science, Sogang University, Seoul, Korea

Jmjd6a regulates GSK3β RNA splicing in Xenopus laevis eye development

  • Jee Yoon Shin, 
  • Jeongin Son, 
  • Won Sun Kim, 
  • Jungsug Gwak, 
  • Bong-Gun Ju
PLOS
x

Abstract

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 [46]. 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/β-catenin 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].

Jumonji domain-containing protein 6 (Jmjd6) (previously known as phosphatidylserine receptor) is a member of the large family of JmjC domain-containing metalloenzymes. Jmjd6 is an iron- and 2–oxoglutarate-dependent dioxygenase that catalyzes the demethylation or hydroxylation of various proteins [11, 12]. For example, Jmjd6 demethylates histone H3 at arginine 2 and histone H4 at arginine 3 [13], estrogen receptor (ERα) [14], RNA helicase A [15], Pax3 [16], and TRAF6 [17]. Recently, the tyrosine kinase activity of Jmjd6 on histone H2A.X has been reported [18]. Also, the role of Jmjd6 in RNA splicing has been reported [1923]. Jmjd6 catalyzes hydroxylation of splicing factor U2AF65 and regulates alternative RNA splicing [19]. In addition, Jmjd6 plays important roles in multiple biological processes including cell differentiation, proliferation, migration, and apoptosis [2426]. Therefore, knockdown or knockout of Jmjd6 demonstrates multiple defects in the developing brain, heart, notochord, and somite in zebrafish, frog, and mouse [2729].

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.

Materials and methods

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).

Whole-mount in situ hybridization, histology, and immunohistochemistry

For whole-mount in situ hybridization, Xenopus embryos were fixed in MEMFA (3.7% formaldehyde, 100 mM MOPS, 2 mM EGTA, 1 mM MgSO4) for overnight at 4°C. After serial dehydration in ethanol, the embryos were processed to whole-mount in situ hybridization in accordance with the standard protocol with minor modifications [31]. Antisense probes were transcribed by T7 or SP6 RNA polymerase using linearized pGEM T-easy plasmid vector containing Jmjd6a, Jmjd6b, Otx2, Rx1, or Pax6 cDNA with DIG-labeling kit (Boehringer Mannheim). Oligonucleotides used for subcloning are listed in Supporting Information (S1 Table). Hybridization was carried out with 1 μg/ml of riboprobes in reaction solution [50% formamide, 5× SSC (pH 4.5), 1% SDS, 50 μg/ml yeast tRNA, and 50 μg/ml heparin] at 70°C for overnight. The hybridized embryos were washed consecutively with 5X SSC, 2X SSC, and 0.2X SSC containing 50% formamide for 15 min at 62°C. Anti-DIG antibody (Roche) was incubated with the hybridized embryos in PBST solution (0.1% Tween 20 in PBS) for overnight at 4°C. Color reaction was carried out using BM purple alkaline phosphatase substrate (Boehringer Mannheim). The color reaction was terminated with stop solution (1mM EDTA, 0.1% Tween 20 in PBS). For histology, embryos were fixed in MEMFA (3.7% formaldehyde, 100 mM MOPS, 2 mM EGTA, 1 mM MgSO4) for overnight at 4°C, dehydrated in ethanol, embedded in paraffin, and sectioned at 7 μm. The tissue sections were deparaffinized and stained with hematoxylin and eosin. For immunohistochemistry, paraffin embedded embryos were sectioned at 5 μm and dried for overnight. After deparaffinization of tissue sections, antigen retrieval was performed with sodium citrate buffer (10mM sodium citrate, 0.05% Tween 20, pH 6.0). Tissue sections were immunostained with anti-Jmjd6 (LS-C30343, LSbio, 1:200) and anti-Islet1 (AB_1157901, Developmental Studies Hybridoma Bank, 1:50) antibodies. TRITC-conjugated goat anti-mouse IgG (T5393, Sigma‐Aldrich) or TRITC-conjugated goat anti-rabbit IgG (T2402, Sigma-Aldrich) was used as the secondary antibody.

Injection of morpholino antisense oligonucleotide and RNA

Jmjd6a morpholino antisense oligonucleotide (Jmjd6a MO, 5’-ATCCCCGGTGTTTCC TGACCACCTC-3’) against the Xenopus laevis Jmjd6a (NM_001092479) was designed and synthesized (Gene Tools). For negative control, 5’-mismatched oligonucleotide of Jmjd6a MO (Control MO, 5’-ATGCCCCGTGTTTGCTGAGCAGCTC-3’) was synthesized (Gene Tools). Morpholino (16 ng) was injected into one or two animal-dorsal blastomeres of 8-cell stage embryos. To test the specificity of Jmjd6a MO, 10 ng of synthesized Jmjd6a mRNA without 5’-UTR (Δ5’UTR Jmjd6a) was co-injected into 2-cell stage embryos. For translation of GSK3β RNA, 5 ng of synthesized GSK3β mRNA without exon 1 and 2 was injected into 2-cell stage embryos.

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).

Immunoprecipitation and western blot analysis

Embryos were homogenized in RIPA buffer [10 mM Tris-Cl (pH 8.0), 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 140 mM NaCl] in the presence of complete protease inhibitors (Roche) and 1mM phenylmethylsulphonylfluoride. Yolk was removed by centrifugation for 10 min at 4°C. The extract was incubated with anti-Jmjd6 antibody (LS-C30343, LSbio) for overnight at 4°C and subsequently incubated with protein A/G agarose beads (Sigma‐Aldrich) for 4 hr at 4°C. The beads were washed extensively and mixed with SDS sample buffer. For negative control, rabbit IgG (12–370, Millipore) was used. Western blotting was performed with anti-Jmjd6 (LS-C30343, LSbio, 1:1000), anti-α/β tubulin (2148, Cell Signaling, 1:1000), anti-U2AF65 (ab151582, Abcam, 1:1000), anti-β-catenin (610154, BD Transduction Laboratories, 1:1000), anti-phospho-β-catenin (Ser33/37/Thr41) (9561S, Cell Signaling Technology, 1:1000), and anti-GSK3β (SC-81462, SC-71186, Santa Cruz Biotechnology, 1:1000) antibodies.

RNA-immunoprecipitation

RNA-immunoprecipitation was performed in accordance with the standard protocols with minor modifications [32, 33]. Embryos were cross-linked with 1% formaldehyde in PBS and incubated in ice-cold nuclear isolation buffer [0.32 M sucrose, 10 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 1% Triton X-100] for 20 min with frequent mixing. After centrifugation for 15 min at 2500g, the resulting nuclei pallet was incubated with RIP buffer [150 mM KCl, 25 mM Tris (pH 7.4), 5 mM EDTA, 0.5 mM DTT, 0.5% NP40, 120 U/ml RNase inhibitor, protease inhibitor cocktail]. Anti-Jmjd6 antibody (LS-C30343, LSbio) or rabbit IgG (12–370, Millipore) coupled to protein G agarose bead (SC-2002, Santa Cruz Biotechnology) was incubated with the nuclear extracts for overnight at 4°C. After washing the beads with ice-cold RIP buffer, DNase I (2270A, Takara) was treated. The RNAs were extracted with phenol/chloroform and precipitated by ammonium acetate/ethanol. Oligonucleotides 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.

Result

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).

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Fig 1. Spatiotemporal expression of Jmjd6a in Xenopus laevis development.

Whole-mount in situ hybridization with antisense riboprobe against Xenopus Jmjd6a and immunohistochemistry with anti-Jmjd6 antibody were performed at indicated stages (n = 3). (A) At late neurula stage (stage 20), Jmjd6a is expressed broadly in the anterior neural tissue including the eye primordia (white arrow) and brain primordia (green arrow). Posterior view is shown. (B and C) At early tailbud stage (stage 26), Jmjd6a expression is increased in the eye primordia (white arrow), brain primordia (green arrow), and neural tube (white bracket). Anterior and lateral views are shown. (C’) In transverse section of C (white dotted line), Jmjd6 protein is detected in the brain (green arrow), optic cup (white arrow), and boundary of the pharyngeal cavity (pc) at stage 26. (D) At stage 30, Jmjd6a is expressed in the forebrain (green arrow), hindbrain (red arrow) and eye (white arrow). Lateral view is shown. (D’) In transverse section of D (white dotted line), Jmjd6 protein is localized in the brain (green arrow), retinal layer (white arrow), notochord (nt), and boundary of the pharyngeal cavity (pc) at stage 30. (E) At stage 40, Jmjd6a expression is detected mainly in the forebrain (green arrow), midbrain (blue arrow), and hindbrain (red arrow). Lateral view is shown. (E’) In transverse section of E (white dotted line), Jmjd6 protein is detected in the dorsal region of brain (green arrow), optic stalk (os), and boundary of the pharynx (p) at stage 40. Nuclei were stained with DAPI. Scale bars: 50 μm.

https://doi.org/10.1371/journal.pone.0219800.g001

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, co-injection 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, 3rd row). In severe cases, no lens was formed (Fig 3A, 2nd and 3rd 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, 4th 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).

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Fig 2. Knockdown of Jmjd6a affects Xenopus laevis eye development.

(A) Antisense morpholino oligonucleotide against Jmjd6 (Jmjd6 MO) was designed to cover 5’ UTR and the translation start site of Xenopus Jmjd6 gene (red). Underline denotes translation start codon. Asterisk denotes conserved nucleotide between Xenopus Jmjd6a and Jmjd6b. (B) Knockdown efficiency of Jmjd6 MO was evaluated by western blot analysis (n = 3). To test the specificity of Jmjd6a MO, Jmjd6a mRNA without 5’UTR (Δ5’UTR Jmjd6a) was co-injected. Control or Jmjd6 MO was injected into one blastomere at the 8-cell stage. Lysates from Xenopus embryos (stage 26) were immunoblotted with anti-Jmjd6 or anti-tubulin antibodies. Western blots were analyzed quantitatively. Data represent mean ± SD. Significance value is **P ≤ 0.01. (C~F) Compared with the un-injected side of embryos, smaller eye at stage 30 (white arrow) and partially developed eye (white arrow) were observed in Jmjd6 MO-injected sides (n = 3). Lateral views are shown.

https://doi.org/10.1371/journal.pone.0219800.g002

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Fig 3. Knockdown of Jmjd6a leads to various eye defects in Xenopus laevis development.

(A) Injection of Jmjd6 MO results in various eye defects (normal to severe) in the injected side of embryos (red arrows) at stage 40 (n = 3). Dorsal (1st row) and lateral (2nd row) views are shown. In transverse sections (3rd row), retinal layers including the RPE, ONL, INL and GCL are deformed in the Jmjd6 MO-injected side of embryos. In severe cases, the lens is not formed. Immunohistochemical examination (4th row) reveals that expression of Islet1, which is expressed in the GCL and INL in normal Xenopus eye, is decreased in Jmjd6 MO injected embryos. In severe cases, expression of Islet1 is barely detected. Scale bars: 100 μm. (B) Quantification of abnormal eye phenotypes. Abnormal eye phenotypes are rescued by co-injection of Jmjd6a mRNA without 5’UTR (Δ5’UTR Jmjd6a). (C) In longitudinal section of embryo at stage 40, Jmjd6 MO-injected sides (red arrow) show abnormal brain (*) structure. Scale bars: 20 μm. (D) In longitudinal section of embryo at stage 40, Jmjd6 MO-injected sides (red arrow) shows formation of an ectopic eye, which developed incompletely (**) in embryos without eye formation. Scale bars: 20 μm. RPE: retinal pigment epithelium, ONL (O): outer nuclear layer, INL (I): inner nuclear layer, GCL (G): ganglion cell layer, L: lens, C: ciliary marginal zone.

https://doi.org/10.1371/journal.pone.0219800.g003

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).

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Fig 4. Knockdown of Jmjd6a reduces the expression of genes related to Xenopus eye development.

Expression of genes related to Xenopus eye development was analyzed by whole-mount in situ hybridization (n = 3). (A) At stage 22, expressions of Otx2, RX1, and Pax6 (white bracket) are decreased in the Jmjd6a MO-injected side (red arrow). However, the gene expression (white bracket) is not altered in the control MO-injected sides (red arrow) of embryos. Anterior view is shown. (B) At stages 26 and 33, the gene expression (white bracket) is also decreased in the Jmjd6a MO-injected side (red arrow). Lateral (left and right) and dorsal (middle) views are shown.

https://doi.org/10.1371/journal.pone.0219800.g004

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 [3638]. 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.

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Fig 5. Jmjd6a regulates GSK3β RNA splicing in Xenopus laevis development.

(A) Jmjd6 interacts with U2AF65. Lysate from the anterior region of embryos (stage 26) was subjected to immunoprecipitation with anti-Jmjd6 antibody, and immunoblot with anti-U2AF65 antibody (n = 3). As a negative control, IgG was used. (B) Jmjd6 interacts with Xenopus GSK3β RNA. RNA-immunoprecipitation was performed with Jmjd6 antibody using lysates from the anterior and posterior region of embryos (stage 26) (n = 3). Xenopus GSK3β and EF1α RNA were analyzed by RT-PCR. As a negative control, IgG was used. (C) Knockdown of Jmjd6a induces generation of Xenopus GSK3β RNA without exon 1 and 2. RT-PCR was performed in the control or Jmjd6a MO-injected anterior region of embryos using exon-specific oligonucleotides (n = 3). (D) Real time-PCR analysis for Xenopus GSK3β RNA splicing in the control or Jmjd6a MO-injected anterior region of embryos using exon-specific oligonucleotides (n = 3). Relative expressions were normalized with GSK3β RNA exon 10. Data represent mean ± SD. Significance values are **P ≤ 0.01. (E) 5’RACE (rapid amplification of cDNA ends) analysis indicates that GSK3β RNA starts with exon 3 in Jmjd6a MO-injected embryos. (F) Knockdown of Jmjd6a results in generation of an N’-terminal truncated form of Xenopus GSK3β protein with a molecular weight of approximately 35kDa (*). Endogenous full length of GSK3β proteins are denoted as **. Western blot analysis using lysate from the anterior region of embryos at stage 26 was performed with antibodies recognizing full-length or N-terminal of GSK3β (n = 3). Anti-tubulin antibody was used as a loading control.

https://doi.org/10.1371/journal.pone.0219800.g005

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.

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Fig 6. Altered splicing of GSK3β by Jmjd6a knockdown augments canonical Wnt/β-catenin signaling in Xenopus laevis development.

(A) Knockdown of Jmjd6a activates canonical Wnt/β-catenin signaling. TOP-Flash plasmid containing TCF binding sites was co-injected with control or Jmjd6a MO into Xenopus embryos at stage 26 (n = 3). Firefly luciferase assay was performed using lysate from the anterior region of injected embryos. Renilla luciferase activity was used to normalize firefly luciferase. Over-expression of Jmjd6a (Δ5’UTR Jmjd6a) suppresses up-regulation of TOP-flash activity in Jmjd6a MO-injected embryos. Data represent mean ± SD. Significance values are **P ≤ 0.01. (B) Knockdown of Jmjd6a induces decreased phosphorylation of β-catenin and increased stability of β-catenin. Lysates from the anterior region of the control or Jmjd6a MO-injected embryos (stage 26) were immunoblotted with anti-Jmjd6, anti-β-catenin, and anti-phospho β-catenin (Ser33, Ser37, Thr41) antibodies (n = 3). Anti-Tubulin antibody was used as a loading control. (C) Proposed model for Jmjd6a-mediated regulation of canonical Wnt/β-catenin signaling in Xenopus eye development.

https://doi.org/10.1371/journal.pone.0219800.g006

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 [4749]. 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 β-catenin 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 β-catenin 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 β-catenin, 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].

Supporting information

S1 Fig. Temporal Jmjd6a and b gene expression in Xenopus laevis development.

Quantitative RT-PCR was performed using whole Xenopus embryos from 8cell stage to stage 30. Data represent mean ±SD. Significance values were *P ≤ 0.05 and **P ≤ 0.01.

https://doi.org/10.1371/journal.pone.0219800.s001

(TIF)

S2 Fig. Spatiotemporal expression of Jmjd6b in Xenopus laevis development.

Whole-mount in situ hybridization of Jmjd6b was performed at indicated stages (n = 3). (A) At late neurula stage (stage 20), Jmjd6b is expressed in the eye primordia (white arrow), brain primordia (green arrow), and neural tube (red arrow). Posterior view is shown. (B) At early tailbud stage (stage 25), Jmjd6b expression is detected in the eye (white arrow) and brain region (red arrow). Lateral view is shown. (C) At stage 30, Jmjd6b is expressed in the eye (white arrow). Lateral view is shown. (D) At stage 40, Jmjd6b expression is not detected. Lateral view is shown.

https://doi.org/10.1371/journal.pone.0219800.s002

(TIF)

S3 Fig. Confirmation of MO injection.

To confirm proper MO injection, plasmid containing RFP (red fluorescence protein) cDNA was co-injected into one blastomere at the 8-cell stage. (A) Lateral view of un-injected side of embryo. (B) Dorsal view of embryo. RFP-injected side of embryo is shown to the right. (C) Lateral view of RFP-injected side of embryo. Note the red fluorescence in RFP-injected side of embryo.

https://doi.org/10.1371/journal.pone.0219800.s003

(TIF)

S4 Fig. Splicing pattern of Xenopus GSK3β in Jmjd6a MO-injected embryos.

Each GSK3β exon (4~11) was amplified by real time-PCR in the anterior region of Jmjd6a MO-injected embryos (stage 26) using exon specific oligonucleotides. Relative expressions were normalized with GSK3β RNA exon 10 because its expression was not changed based on EF1a expression. Data represent mean ± SD (Exon 3, p = 0.22; Exon 4, p = 0.25; Exon 5, p = 0.11; Exon 6, p = 0.12; Exon 7, p = 0.15; Exon 8, p = 0.55; Exon 9, p = 0.13; Exon 11, p = 0.39).

https://doi.org/10.1371/journal.pone.0219800.s004

(TIF)

S5 Fig. Translation of N-terminal truncated form of Xenopus GSK3β.

Synthesized GSK3β RNA without exon1 and 2 was injected into Xenopus embryos and the lysate was analyzed by western blotting using an antibody recognizing the full length of GSK3β. An extra 35 kDa band of GSK3β is detected (*). Endogenous full length of GSK3β is denoted as **.

https://doi.org/10.1371/journal.pone.0219800.s005

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S1 Table. Oligonucleotides used in this study.

https://doi.org/10.1371/journal.pone.0219800.s006

(PPTX)

S1 File. Raw values for statistical analysis.

https://doi.org/10.1371/journal.pone.0219800.s007

(XLSX)

Acknowledgments

The authors thank to Drs. Chang-Yeol Yeo and Hosung Jung for providing us reagents.

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