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<t>EGFL7/miR-126</t> is a target of GATA2. (A) miR-126 was expressed in the dorsal aorta and in the LVV-ECs (arrow) of control embryos. (B) Expression of miR-126 was downregulated in the LVV-ECs (arrow) of E12.0 Lyve1-Cre;Gata2 f/f embryos. However, no obvious difference in miR-126 expression was observed in the dorsal aorta of mutants. Red dashed line indicates the endothelial layer of the lymph sac. (C) EGFL7 was expressed in the mesenteric arteries, veins and lymphatic vessels of E18.5 control embryos. The strongest expression of EGFL7 was observed in LVs (arrow). (D) Expression of EGFL7 was dramatically downregulated in the mesenteric lymphatic vessels of mice lacking GATA2 in LECs. Also, notice the absence of LVs in the mutant. (E) ChIP revealed that GATA2 strongly associates with the promoter element of the EGFL7/miR-126 locus. The top gel shows PCR performed using primers flanking the GATA2-binding site. The lower gel shows PCR performed using primers for a non-specific site. The graph compares qPCR signals generated by primers flanking the GATA2-binding site. A, artery; DA, dorsal aorta; L, lymphatic vessel; LS, lymph sac; V, vein. (A,B) n =3 embryos and 6 LVV complexes per genotype; (C,D) n =3 embryos per genotype; (E) n =4. ** P <0.01. Scale bars: 250 μm (A,B); 200 μm (C,D).
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<t>EGFL7/miR-126</t> is a target of GATA2. (A) miR-126 was expressed in the dorsal aorta and in the LVV-ECs (arrow) of control embryos. (B) Expression of miR-126 was downregulated in the LVV-ECs (arrow) of E12.0 Lyve1-Cre;Gata2 f/f embryos. However, no obvious difference in miR-126 expression was observed in the dorsal aorta of mutants. Red dashed line indicates the endothelial layer of the lymph sac. (C) EGFL7 was expressed in the mesenteric arteries, veins and lymphatic vessels of E18.5 control embryos. The strongest expression of EGFL7 was observed in LVs (arrow). (D) Expression of EGFL7 was dramatically downregulated in the mesenteric lymphatic vessels of mice lacking GATA2 in LECs. Also, notice the absence of LVs in the mutant. (E) ChIP revealed that GATA2 strongly associates with the promoter element of the EGFL7/miR-126 locus. The top gel shows PCR performed using primers flanking the GATA2-binding site. The lower gel shows PCR performed using primers for a non-specific site. The graph compares qPCR signals generated by primers flanking the GATA2-binding site. A, artery; DA, dorsal aorta; L, lymphatic vessel; LS, lymph sac; V, vein. (A,B) n =3 embryos and 6 LVV complexes per genotype; (C,D) n =3 embryos per genotype; (E) n =4. ** P <0.01. Scale bars: 250 μm (A,B); 200 μm (C,D).
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<t>EGFL7/miR-126</t> is a target of GATA2. (A) miR-126 was expressed in the dorsal aorta and in the LVV-ECs (arrow) of control embryos. (B) Expression of miR-126 was downregulated in the LVV-ECs (arrow) of E12.0 Lyve1-Cre;Gata2 f/f embryos. However, no obvious difference in miR-126 expression was observed in the dorsal aorta of mutants. Red dashed line indicates the endothelial layer of the lymph sac. (C) EGFL7 was expressed in the mesenteric arteries, veins and lymphatic vessels of E18.5 control embryos. The strongest expression of EGFL7 was observed in LVs (arrow). (D) Expression of EGFL7 was dramatically downregulated in the mesenteric lymphatic vessels of mice lacking GATA2 in LECs. Also, notice the absence of LVs in the mutant. (E) ChIP revealed that GATA2 strongly associates with the promoter element of the EGFL7/miR-126 locus. The top gel shows PCR performed using primers flanking the GATA2-binding site. The lower gel shows PCR performed using primers for a non-specific site. The graph compares qPCR signals generated by primers flanking the GATA2-binding site. A, artery; DA, dorsal aorta; L, lymphatic vessel; LS, lymph sac; V, vein. (A,B) n =3 embryos and 6 LVV complexes per genotype; (C,D) n =3 embryos per genotype; (E) n =4. ** P <0.01. Scale bars: 250 μm (A,B); 200 μm (C,D).
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<t>EGFL7/miR-126</t> is a target of GATA2. (A) miR-126 was expressed in the dorsal aorta and in the LVV-ECs (arrow) of control embryos. (B) Expression of miR-126 was downregulated in the LVV-ECs (arrow) of E12.0 Lyve1-Cre;Gata2 f/f embryos. However, no obvious difference in miR-126 expression was observed in the dorsal aorta of mutants. Red dashed line indicates the endothelial layer of the lymph sac. (C) EGFL7 was expressed in the mesenteric arteries, veins and lymphatic vessels of E18.5 control embryos. The strongest expression of EGFL7 was observed in LVs (arrow). (D) Expression of EGFL7 was dramatically downregulated in the mesenteric lymphatic vessels of mice lacking GATA2 in LECs. Also, notice the absence of LVs in the mutant. (E) ChIP revealed that GATA2 strongly associates with the promoter element of the EGFL7/miR-126 locus. The top gel shows PCR performed using primers flanking the GATA2-binding site. The lower gel shows PCR performed using primers for a non-specific site. The graph compares qPCR signals generated by primers flanking the GATA2-binding site. A, artery; DA, dorsal aorta; L, lymphatic vessel; LS, lymph sac; V, vein. (A,B) n =3 embryos and 6 LVV complexes per genotype; (C,D) n =3 embryos per genotype; (E) n =4. ** P <0.01. Scale bars: 250 μm (A,B); 200 μm (C,D).
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EGFL7/miR-126 is a target of GATA2. (A) miR-126 was expressed in the dorsal aorta and in the LVV-ECs (arrow) of control embryos. (B) Expression of miR-126 was downregulated in the LVV-ECs (arrow) of E12.0 Lyve1-Cre;Gata2 f/f embryos. However, no obvious difference in miR-126 expression was observed in the dorsal aorta of mutants. Red dashed line indicates the endothelial layer of the lymph sac. (C) EGFL7 was expressed in the mesenteric arteries, veins and lymphatic vessels of E18.5 control embryos. The strongest expression of EGFL7 was observed in LVs (arrow). (D) Expression of EGFL7 was dramatically downregulated in the mesenteric lymphatic vessels of mice lacking GATA2 in LECs. Also, notice the absence of LVs in the mutant. (E) ChIP revealed that GATA2 strongly associates with the promoter element of the EGFL7/miR-126 locus. The top gel shows PCR performed using primers flanking the GATA2-binding site. The lower gel shows PCR performed using primers for a non-specific site. The graph compares qPCR signals generated by primers flanking the GATA2-binding site. A, artery; DA, dorsal aorta; L, lymphatic vessel; LS, lymph sac; V, vein. (A,B) n =3 embryos and 6 LVV complexes per genotype; (C,D) n =3 embryos per genotype; (E) n =4. ** P <0.01. Scale bars: 250 μm (A,B); 200 μm (C,D).

Journal: Development (Cambridge, England)

Article Title: GATA2 controls lymphatic endothelial cell junctional integrity and lymphovenous valve morphogenesis through miR-126

doi: 10.1242/dev.184218

Figure Lengend Snippet: EGFL7/miR-126 is a target of GATA2. (A) miR-126 was expressed in the dorsal aorta and in the LVV-ECs (arrow) of control embryos. (B) Expression of miR-126 was downregulated in the LVV-ECs (arrow) of E12.0 Lyve1-Cre;Gata2 f/f embryos. However, no obvious difference in miR-126 expression was observed in the dorsal aorta of mutants. Red dashed line indicates the endothelial layer of the lymph sac. (C) EGFL7 was expressed in the mesenteric arteries, veins and lymphatic vessels of E18.5 control embryos. The strongest expression of EGFL7 was observed in LVs (arrow). (D) Expression of EGFL7 was dramatically downregulated in the mesenteric lymphatic vessels of mice lacking GATA2 in LECs. Also, notice the absence of LVs in the mutant. (E) ChIP revealed that GATA2 strongly associates with the promoter element of the EGFL7/miR-126 locus. The top gel shows PCR performed using primers flanking the GATA2-binding site. The lower gel shows PCR performed using primers for a non-specific site. The graph compares qPCR signals generated by primers flanking the GATA2-binding site. A, artery; DA, dorsal aorta; L, lymphatic vessel; LS, lymph sac; V, vein. (A,B) n =3 embryos and 6 LVV complexes per genotype; (C,D) n =3 embryos per genotype; (E) n =4. ** P <0.01. Scale bars: 250 μm (A,B); 200 μm (C,D).

Article Snippet: Primary antibodies for immunohistochemistry were: rabbit anti-PROX1 (11-002, Angiobio), goat anti-human PROX1 (AF2727, R&D Systems), sheep anti-mouse FOXC2 (AF6989, R&D Systems), goat anti-mouse VEGRF3 (AF743, R&D Systems), rat anti-mouse CD31 (553370, BD Pharmingen), goat anti-mouse ITGA9 (AF3827, R&D Systems), rat anti-mouse VE-cadherin (550548, BD Pharmingen), hamster anti-mouse PDPN (127401, Biolegend), rat anti-mouse ITGA5 (553319, BD Pharmingen), goat anti-mouse GATA2 (AF2046, R&D Systems), rabbit anti-mouse CX37 (40-4200, Life Technologies), rabbit anti-mouse LAMA5 (Ab11575, Abcam), rabbit anti-human fibronectin (ab2413, Abcam), goat anti-human ANGPT2 (AF623, R&D Systems), goat anti-mouse EGFL7 (AF3089, R&D Systems), rabbit anti-mouse CLDN5 (34-1600, Thermo Fisher Scientific), rabbit anti-mouse LYVE-1 (11-034, Angiobio).

Techniques: Control, Expressing, Mutagenesis, Binding Assay, Generated