Vacca B , Sanchez-Heras E , Steed E , Balda MS , Ohnuma SI , Sasai N , Mayor R , Matter K .

	Fig. 1. MD3 structure and expression in Xenopus embryo. (A) Xenopus laevis MD3 (MD3; Genbank: BC 068841) protein includes three intracellular (IC, red), four transmembrane (TM, green) and two extracellular (EC, blue) domains. Numbers correspond to the amino acids. Spatial analysis of expression of MD3 transcript (B,D,D′) and an eye marker Rx1 (C,E) was performed by whole-mount in situ hybridization (WISH) in neurulas (stage 15, B) and tadpoles (stage 38, D,D′). Scale bar: 500 μm; black arrowhead, eye; white arrowhead, lens. (F) MD3 expression analysis by semi-quantitative RT-PCR using RNA isolated from dissected optic vesicles (stage 25) and eyes (stage 42). GAPDH was amplified as a control. bp, base pairs. (G) MD3 signal was detected by WISH in the inner and outer plexiform layers (red arrowheads) and in the lens (white arrowhead) on eye sections (stage 42).
	Fig. 4. The MD3 MO-induced eye phenotype originates from neural plate and crest but not mesoderm. (A) Alteration of eye morphology was quantified in embryos injected with MD3 MOs in one dorsal blastomere at 8-cell stage. NS, non significant; **P<0.01. Student's t-test (P values, CT MO=0.34; MD3AB MO=1*10−2). (B) Scheme representing the dorsal blastomeres A1, A3 and A4 of an embryo at 32-cell stage. A, animal pole; V, vegetal pole; V, ventral; D, dorsal (scheme adapted from Xenbase). Alteration of eye morphology was analysed and quantified in embryos injected with MD3 MOs in one dorsal blastomere A1, A2 or A4 at 32-cell stage (C-E′). Note the disrupted eye morphology in A1- and A3-injected embryos. Scale bar: 500 µm; numbers indicate the embryos analysed at stage 42; red asterisk, injected side of the embryo.
	Fig. 5. MD3 KD affects early eye-field formation. Eye-field specification was analysed by WISH against the eye markers Rx1, Pax6, Otx2 in neurulas (stage 17; anterior view; A,A′,C,C′,E,E′) and tailbuds (stage 30; B,B′,D,D′,F,F′) injected with CT or MD3 MOs. Black dotted line, midline of embryo; arrowhead, enlarged (A′,C′,E′) and reduced (B′,D′,F′) expression domain of the eye markers; red asterisks, injected side of the embryo; numbers indicate the embryos analysed. Scale bar: 500 µm.
	Fig. 6. MD3 KD stimulates cell proliferation and apoptosis in the eye field. Eye-field proliferation was analysed by WISH against cyclinD1 in neurula (stage 17; anterior view; A-A′) and tailbud (stage 30; B-B′) injected with CT or MD3 MOs. Black dotted line, midline of embryo; arrowhead, enlarged (A′) and reduced (B,B′) expression domain of cyclinD1; red asterisks, injected side of the embryo; numbers indicate the embryos analysed. Scale bar: 500 µm. Cell proliferation in the anterior region of the neurula was quantified with a BrdU staining (C) and cell death with a TUNEL assay in tailbud (D) as described in the Materials and methods; Student's t-test (P values, BrdU in C, CT MO=0.16, MD3AB MO=9.4*10−6; TUNEL in D, CT MO=7*10−2, MD3AB MO=5.66*10−9). The numbers indicate the embryos analysed (A-B′,D) or the sections counted (C). NS, non significant; ***P<0.001.
	Fig. 7. MD3 overexpression affects the balance between cell proliferation and apoptosis in the eye field. Eye-field specification was analysed by WISH against the eye markers Rx1, Pax6, Otx2 in neurulas (stage 17; anterior view; A,A′,C,C′,E,E′) and tailbuds (stage 30; B,B′,D,D′,F,F′) injected with GFP or MD3 RNAs. Black dotted line, midline of embryo; green and red lines, enlarged expression domain of the eye markers; red asterisks, injected side of the embryo; numbers indicate the embryos analysed. Scale bar: 500 µm. Cell proliferation in the anterior region of the neurula was quantified with a BrdU staining (G) and cell death with a TUNEL assay in tailbud (H) as described in the Materials and methods; Student's t-test (P values, BrdU in G, GFP RNA=0.94, FL MD3 RNA=2.1*10−5; TUNEL in H, GFP RNA=0.8, FL MD3 RNA=2*10−3). The numbers indicate the embryos analysed (H) or the sections counted (G). NS, non significant; **P<0.01 and ***P<0.001.
	Fig. 8. JNK inhibition disrupts eye morphogenesis. (A-E) The morphology of the eye was analysed in wild-type embryos treated from stage 11 to 22 with DMSO or increasing concentrations of SP600125, a JNK inhibitor. Scale bar: 500 µm. (F) The eye size was quantified from the embryos (A-E) as described in the Materials and methods; Mann–Whitney test (P values, 0.25 μg ml-1=0.93; 0.5 μg ml-1=7.8*10−5; 1 μg ml-1=1.02*10−8; 2.5 μg ml-1=1.07*10−5). The numbers indicate the embryos analysed. Note, the embryos show reduced eye formation in a dose-dependent manner. NS, non significant; ***P<0.001.
	Fig. 9. MD3-induced JNK activation is required for eye morphogenesis. (A) Activity of the reporter plasmid AP1-Luc (JNK pathway) was determined by Luciferase assay from whole-embryos (stage 12) co-injected with Renilla (as a control), c-Jun (as positive control) or FL MD3 mRNA. The morphology of the eye was analysed at stage 42 (B-C′), quantified (D) and the eye size determined (E) in embryos injected with MD3 MOs, co-injected with wt JNK or CA-JNK mRNA. Scale bars: 500 µm. The numbers indicate the embryos analysed. Statistical significance was assessed by Student's t-test (P values in A, positive CT=2.6*10−3; MD3 RNA=3.5*10−3) and Mann–Whitney test (P values in D, MD3AB MO=0.02; MD3AB MO+wt JNK RNA=0.06; MD3AB MO+CA-JNK RNA=0.19; P values in E, MD3AB MO=1.65*10−9; MD3AB MO+wt JNK=1.4*10−3; MD3AB MO+CA-JNK=4.37*10−6). *P<0.05; **P<0.01; ***P<0.001; NS, non significant.
	marveld3 (MARVEL domain containing 3) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 15, anterior view, dorsal up. Keye: eye primordium and anterior placodal area (black arrowheads)

Auger, A bromodeoxyuridine (BrdU) based protocol for characterizing proliferating progenitors in Xenopus embryos. 2012, Pubmed, Xenbase

Auger, A bromodeoxyuridine (BrdU) based protocol for characterizing proliferating progenitors in Xenopus embryos. 2012, Pubmed , Xenbase
Bailey, Regulation of vertebrate eye development by Rx genes. 2004, Pubmed , Xenbase
Balda, Tight junctions at a glance. 2008, Pubmed
Bilitou, The role of cell cycle in retinal development: cyclin-dependent kinase inhibitors co-ordinate cell-cycle inhibition, cell-fate determination and differentiation in the developing retina. 2010, Pubmed , Xenbase
Cavodeassi, Early stages of zebrafish eye formation require the coordinated activity of Wnt11, Fz5, and the Wnt/beta-catenin pathway. 2005, Pubmed
Chiang, Cyclopia and defective axial patterning in mice lacking Sonic hedgehog gene function. 1996, Pubmed
Collier, The effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the mortality and growth of two amphibian species (Xenopus laevis and Pseudacris triseriata). 2008, Pubmed , Xenbase
Cording, In tight junctions, claudins regulate the interactions between occludin, tricellulin and marvelD3, which, inversely, modulate claudin oligomerization. 2013, Pubmed
Eckert, Tight junction biogenesis during early development. 2008, Pubmed , Xenbase
Erickson, Vascular permeability in ocular disease and the role of tight junctions. 2007, Pubmed
Fisher, Synaptic arrays of the inner plexiform layer in the developing retina of Xenopus. 1976, Pubmed , Xenbase
Fleming, Junctional complexes in the early mammalian embryo. 2000, Pubmed
Fleming, Assembly of tight junctions during early vertebrate development. 2000, Pubmed , Xenbase
Fuhrmann, Wnt signaling in eye organogenesis. 2008, Pubmed
Garriock, Wnt11-R, a protein closely related to mammalian Wnt11, is required for heart morphogenesis in Xenopus. 2005, Pubmed , Xenbase
Hao, Tubeimoside-1 (TBMS1) inhibits lung cancer cell growth and induces cells apoptosis through activation of MAPK-JNK pathway. 2015, Pubmed
Harada, Molecular regulation of visual system development: more than meets the eye. 2007, Pubmed
Hirai, Expression of MUK/DLK/ZPK, an activator of the JNK pathway, in the nervous systems of the developing mouse embryo. 2005, Pubmed
Huet, EMMPRIN modulates epithelial barrier function through a MMP-mediated occludin cleavage: implications in dry eye disease. 2011, Pubmed
Itoh, Axis determination by inhibition of Wnt signaling in Xenopus. 1999, Pubmed , Xenbase
Iwamoto, Localization of angulin-1/LSR and tricellulin at tricellular contacts of brain and retinal endothelial cells in vivo. 2014, Pubmed
Kibardin, Metastasis-associated kinase modulates Wnt signaling to regulate brain patterning and morphogenesis. 2006, Pubmed , Xenbase
Kim, The assembly of POSH-JNK regulates Xenopus anterior neural development. 2005, Pubmed , Xenbase
Lee, Dishevelled mediates ephrinB1 signalling in the eye field through the planar cell polarity pathway. 2006, Pubmed , Xenbase
Luehders, The small leucine-rich repeat secreted protein Asporin induces eyes in Xenopus embryos through the IGF signalling pathway. 2015, Pubmed , Xenbase
Maurus, Noncanonical Wnt-4 signaling and EAF2 are required for eye development in Xenopus laevis. 2005, Pubmed , Xenbase
McDonough, Dissection, culture, and analysis of Xenopus laevis embryonic retinal tissue. 2012, Pubmed , Xenbase
Moody, Fates of the blastomeres of the 32-cell-stage Xenopus embryo. 1987, Pubmed , Xenbase
Muthusamy, Ischemia-reperfusion injury induces occludin phosphorylation/ubiquitination and retinal vascular permeability in a VEGFR-2-dependent manner. 2014, Pubmed
Pushpavalli, Drosophila MOF regulates DIAP1 and induces apoptosis in a JNK dependent pathway. 2016, Pubmed
Raleigh, Tight junction-associated MARVEL proteins marveld3, tricellulin, and occludin have distinct but overlapping functions. 2010, Pubmed
Ripley, Bves is expressed in the epithelial components of the retina, lens, and cornea. 2004, Pubmed
Roessler, Mutations in the human Sonic Hedgehog gene cause holoprosencephaly. 1996, Pubmed
Shen, Tight junction pore and leak pathways: a dynamic duo. 2011, Pubmed
Steed, MarvelD3 couples tight junctions to the MEKK1-JNK pathway to regulate cell behavior and survival. 2014, Pubmed
Steed, Identification of MarvelD3 as a tight junction-associated transmembrane protein of the occludin family. 2009, Pubmed
Takabatake, Conserved expression control and shared activity between cognate T-box genes Tbx2 and Tbx3 in connection with Sonic hedgehog signaling during Xenopus eye development. 2002, Pubmed , Xenbase
Tserentsoodol, Colocalization of tight junction proteins, occludin and ZO-1, and glucose transporter GLUT1 in cells of the blood-ocular barrier in the mouse eye. 1998, Pubmed
Valesio, Exposure to the JNK inhibitor SP600125 (anthrapyrazolone) during early zebrafish development results in morphological defects. 2013, Pubmed
Wei, Insights into the role of cell-cell junctions in physiology and disease. 2013, Pubmed
Weston, The JNK signal transduction pathway. 2002, Pubmed
Yamanaka, JNK functions in the non-canonical Wnt pathway to regulate convergent extension movements in vertebrates. 2002, Pubmed , Xenbase
Zuber, Giant eyes in Xenopus laevis by overexpression of XOptx2. 1999, Pubmed , Xenbase
Zuber, Eye field specification in Xenopus laevis. 2010, Pubmed , Xenbase