Shin JY, Son J, Kim WS, Gwak J, Ju BG.

             retina layer formation

	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.
	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.
	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.
	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.
	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.
	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.
	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
	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.
	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.
	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).
	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 **.

Álvarez-Hernán, Islet-1 immunoreactivity in the developing retina of Xenopus laevis. 2013, Pubmed, Xenbase

Álvarez-Hernán, Islet-1 immunoreactivity in the developing retina of Xenopus laevis. 2013, Pubmed , Xenbase
Aprelikova, The epigenetic modifier JMJD6 is amplified in mammary tumors and cooperates with c-Myc to enhance cellular transformation, tumor progression, and metastasis. 2016, Pubmed
Barman-Aksözen, Iron availability modulates aberrant splicing of ferrochelatase through the iron- and 2-oxoglutarate dependent dioxygenase Jmjd6 and U2AF(65.). 2013, Pubmed
Bertrand, Structural characterization of the GSK-3beta active site using selective and non-selective ATP-mimetic inhibitors. 2003, Pubmed
Beurel, Glycogen synthase kinase-3 (GSK3): regulation, actions, and diseases. 2015, Pubmed
Boeckel, Jumonji domain-containing protein 6 (Jmjd6) is required for angiogenic sprouting and regulates splicing of VEGF-receptor 1. 2011, Pubmed
Böse, The phosphatidylserine receptor has essential functions during embryogenesis but not in apoptotic cell removal. 2004, Pubmed
Chang, JMJD6 is a histone arginine demethylase. 2007, Pubmed
Chow, Early eye development in vertebrates. 2001, Pubmed
Chow, Pax6 induces ectopic eyes in a vertebrate. 1999, Pubmed , Xenbase
Clifton, Structural studies on 2-oxoglutarate oxygenases and related double-stranded beta-helix fold proteins. 2006, Pubmed
Eldar-Finkelman, Expression and characterization of glycogen synthase kinase-3 mutants and their effect on glycogen synthase activity in intact cells. 1996, Pubmed
Eom, GSK3 beta N-terminus binding to p53 promotes its acetylation. 2009, Pubmed
Fadok, A receptor for phosphatidylserine-specific clearance of apoptotic cells. 2000, Pubmed
Farago, Kinase-inactive glycogen synthase kinase 3beta promotes Wnt signaling and mammary tumorigenesis. 2005, Pubmed
Ferkey, Glycogen synthase kinase-3 beta mutagenesis identifies a common binding domain for GBP and Axin. 2002, Pubmed , Xenbase
Fuhrmann, Wnt signaling in eye organogenesis. 2008, Pubmed
Fujimura, WNT/β-Catenin Signaling in Vertebrate Eye Development. 2016, Pubmed
Fujimura, Spatial and temporal regulation of Wnt/beta-catenin signaling is essential for development of the retinal pigment epithelium. 2009, Pubmed
Gao, JMJD6 Licenses ERα-Dependent Enhancer and Coding Gene Activation by Modulating the Recruitment of the CARM1/MED12 Co-activator Complex. 2018, Pubmed
Hägglund, Canonical Wnt/β-catenin signalling is essential for optic cup formation. 2013, Pubmed
Halder, Induction of ectopic eyes by targeted expression of the eyeless gene in Drosophila. 1995, Pubmed
Heavner, Eye development and retinogenesis. 2012, Pubmed
Heim, Jumonji domain containing protein 6 (Jmjd6) modulates splicing and specifically interacts with arginine-serine-rich (RS) domains of SR- and SR-like proteins. 2014, Pubmed
Heisenberg, A mutation in the Gsk3-binding domain of zebrafish Masterblind/Axin1 leads to a fate transformation of telencephalon and eyes to diencephalon. 2001, Pubmed
Hendrickson, Concordant regulation of translation and mRNA abundance for hundreds of targets of a human microRNA. 2009, Pubmed
Hong, Phosphatidylserine receptor is required for the engulfment of dead apoptotic cells and for normal embryonic development in zebrafish. 2004, Pubmed
Ikeda, Axin, a negative regulator of the Wnt signaling pathway, forms a complex with GSK-3beta and beta-catenin and promotes GSK-3beta-dependent phosphorylation of beta-catenin. 1998, Pubmed
Khalil, Many human large intergenic noncoding RNAs associate with chromatin-modifying complexes and affect gene expression. 2009, Pubmed
Kwok, Jmjd6, a JmjC Dioxygenase with Many Interaction Partners and Pleiotropic Functions. 2017, Pubmed
Lawrence, Redistribution of demethylated RNA helicase A during foot-and-mouth disease virus infection: role of Jumonji C-domain containing protein 6 in RHA demethylation. 2014, Pubmed
Lee, JMJD6 is a driver of cellular proliferation and motility and a marker of poor prognosis in breast cancer. 2012, Pubmed
Liu, JMJD6 regulates histone H2A.X phosphorylation and promotes autophagy in triple-negative breast cancer cells via a novel tyrosine kinase activity. 2019, Pubmed
Liu, Ciliary margin transdifferentiation from neural retina is controlled by canonical Wnt signaling. 2007, Pubmed
Liu, JMJD6 promotes melanoma carcinogenesis through regulation of the alternative splicing of PAK1, a key MAPK signaling component. 2017, Pubmed
Liu, Mapping canonical Wnt signaling in the developing and adult retina. 2006, Pubmed
Mantri, Crystal structure of the 2-oxoglutarate- and Fe(II)-dependent lysyl hydroxylase JMJD6. 2010, Pubmed
Maretto, Mapping Wnt/beta-catenin signaling during mouse development and in colorectal tumors. 2003, Pubmed
Martinez-Morales, Otx genes are required for tissue specification in the developing eye. 2001, Pubmed
Meyers, β-catenin/Wnt signaling controls progenitor fate in the developing and regenerating zebrafish retina. 2012, Pubmed
Moody, Segregation of fate during cleavage of frog (Xenopus laevis) blastomeres. 1990, Pubmed , Xenbase
Muranishi, An essential role for Rax in retina and neuroendocrine system development. 2012, Pubmed
Peifer, Phosphorylation of the Drosophila adherens junction protein Armadillo: roles for wingless signal and zeste-white 3 kinase. 1994, Pubmed
Phukan, GSK3beta: role in therapeutic landscape and development of modulators. 2010, Pubmed
Sinn, An eye on eye development. 2013, Pubmed
Stachel, Lithium perturbation and goosecoid expression identify a dorsal specification pathway in the pregastrula zebrafish. 1993, Pubmed
Tikhanovich, Dynamic Arginine Methylation of Tumor Necrosis Factor (TNF) Receptor-associated Factor 6 Regulates Toll-like Receptor Signaling. 2015, Pubmed
Vangimalla, Bifunctional Enzyme JMJD6 Contributes to Multiple Disease Pathogenesis: New Twist on the Old Story. 2017, Pubmed
Viczian, Expression of Xenopus laevis Lhx2 during eye development and evidence for divergent expression among vertebrates. 2006, Pubmed , Xenbase
Walther, Pax-6, a murine paired box gene, is expressed in the developing CNS. 1991, Pubmed
Webby, Jmjd6 catalyses lysyl-hydroxylation of U2AF65, a protein associated with RNA splicing. 2009, Pubmed
Westenskow, Beta-catenin controls differentiation of the retinal pigment epithelium in the mouse optic cup by regulating Mitf and Otx2 expression. 2009, Pubmed
Wu, Loading of PAX3 to Mitotic Chromosomes Is Mediated by Arginine Methylation and Associated with Waardenburg Syndrome. 2015, Pubmed
Yi, JMJD6 and U2AF65 co-regulate alternative splicing in both JMJD6 enzymatic activity dependent and independent manner. 2017, Pubmed
Yost, The axis-inducing activity, stability, and subcellular distribution of beta-catenin is regulated in Xenopus embryos by glycogen synthase kinase 3. 1996, Pubmed , Xenbase
Zhang, JmjC Domain-containing Protein 6 (Jmjd6) Derepresses the Transcriptional Repressor Transcription Factor 7-like 1 (Tcf7l1) and Is Required for Body Axis Patterning during Xenopus Embryogenesis. 2015, Pubmed , Xenbase