Vacca B et al. (2016), MarvelD3 regulates the c-Jun N-terminal kinase ...

XB-ART-52791

Biol Open 2016 Nov 15;511:1631-1641. doi: 10.1242/bio.018945.

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MarvelD3 regulates the c-Jun N-terminal kinase pathway during eye development in Xenopus.

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

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Ocular morphogenesis requires several signalling pathways controlling the expression of transcription factors and cell-cycle regulators. However, despite a well-known mechanism, the dialogue between those signals and factors remains to be unveiled. Here, we identify a requirement for MarvelD3, a tight junction transmembrane protein, in eye morphogenesis in Xenopus MarvelD3 depletion led to an abnormally pigmented eye or even an eye-less phenotype, which was rescued by ectopic MarvelD3 expression. Altering MarvelD3 expression led to deregulated expression of cell-cycle regulators and transcription factors required for eye development. The eye phenotype was rescued by increased c-Jun terminal Kinase activation. Thus, MarvelD3 links tight junctions and modulation of the JNK pathway to eye morphogenesis.

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Species referenced: Xenopus
Genes referenced: ccnd1 jun mapk8 marveld3 mmut otx2 pax6 rax rho
GO keywords: eye development
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	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=110−2). (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=9.410−6; TUNEL in D, CT MO=710−2, MD3AB MO=5.6610−9). 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=2.110−5; TUNEL in H, GFP RNA=0.8, FL MD3 RNA=210−3). 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=7.810−5; 1 μg ml-1=1.0210−8; 2.5 μg ml-1=1.0710−5). 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=2.610−3; MD3 RNA=3.510−3) 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=1.6510−9; MD3AB MO+wt JNK=1.410−3; MD3AB MO+CA-JNK=4.3710−6). 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)

References [+] :

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

	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=110−2). (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=9.410−6; TUNEL in D, CT MO=710−2, MD3AB MO=5.6610−9). 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=2.110−5; TUNEL in H, GFP RNA=0.8, FL MD3 RNA=210−3). 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=7.810−5; 1 μg ml-1=1.0210−8; 2.5 μg ml-1=1.0710−5). 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=2.610−3; MD3 RNA=3.510−3) 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=1.6510−9; MD3AB MO+wt JNK=1.410−3; MD3AB MO+CA-JNK=4.3710−6). 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)