Figure 5
Figure 5. The double knockdown of sox18 and sox7 affects vascular development. We carefully analyzed the development of the vascular tree in the double morphants, using 2 different vascular-specific transgenic lines (ie, tg(fli1:EGFP)y1 and tg(flk1:EGFP)). (A,A′-D,D′) lateral views, anterior to the left, of 2.5-dpf embryos; for each embryo, both brightfield (A-D) and fluorescence (A′-D′) stereomicroscopic images are shown. (A,A′,B,B′) control std-MO– and DMO-injected embryos, respectively, of the tg(fli1:EGFP)y1 line. (C,C′-D,D′) control std-MO– and DMO-injected embryos, respectively, of the tg(flk1:EGFP) line. The expression of the transgene is not substantially reduced in the fli1:EGFP line, while it is dramatically reduced in the flk1:EGFP line upon double knockdown of sox18 and sox7. (E-H) Staining of the endogenous AP activity was performed to analyze angiogenesis in the region of the subintestinal vessels (SIV) basket at 3 dpf. (E,F) control std-MO–injected embryos in dorsal and lateral views, respectively, anterior to the left. (G,H) DMO-injected embryos in dorsal and lateral views, respectively, anterior to the left. The SIV basket is severely disorganized and enlarged in the double morphants, showing thinner vessels and anomalous spikes pointing toward the yolk. (I,P) Confocal images of fli1:EGFP double morphants and control embryos showing the SIV basket (I-L) and the caudal intersomitic vessels (ISVs) (M-P) at 3 dpf. All lateral views, anterior to the left. (I,M) Control std-MO–injected embryos. (J,N) Embryos coinjected with sox7-MO1 and sox18-MO1, 125 fmol each. (K,O) Embryos coinjected with sox7-MO1 and sox18-MO1, 250 fmol each. (L,P) Control embryos treated with 2,3-BDM to block heartbeat and blood circulation. The alterations in the SIV basket and in the caudal ISVs visible in the double morphants are clearly distinct from the mere effect of absent blood flow. (J,K) Consistent with AP staining, confocal analysis reveals in the double morphants a global disorganization of the SIV basket, which is highly enlarged, particularly in the posterior aspect (white arrow). The caliber of most vessels in the basket, and especially of the subintestinal vein, is reduced in the double morphants (white arrowhead), which also present abnormal spikes toward the yolk (white asterisks). (L) The SIV basket appears instead reduced (white arrow) in the embryos treated with 2,3-BDM. (N,O) The caudal vein plexus (white arrowhead) is severely reduced in the double morphants, but not in 2,3-BDM–treated controls (P). In the double morphants (N,O), anomalous branching (white asterisks) is visible in the dorsal aspect of the caudal ISVs, pointing to defective proliferation/guidance mechanisms in angiogenic development. No abnormal branching of the ISVs is caused by a mere block in blood flow (P).

The double knockdown of sox18 and sox7 affects vascular development. We carefully analyzed the development of the vascular tree in the double morphants, using 2 different vascular-specific transgenic lines (ie, tg(fli1:EGFP)y1 and tg(flk1:EGFP)). (A,A′-D,D′) lateral views, anterior to the left, of 2.5-dpf embryos; for each embryo, both brightfield (A-D) and fluorescence (A′-D′) stereomicroscopic images are shown. (A,A′,B,B′) control std-MO– and DMO-injected embryos, respectively, of the tg(fli1:EGFP)y1 line. (C,C′-D,D′) control std-MO– and DMO-injected embryos, respectively, of the tg(flk1:EGFP) line. The expression of the transgene is not substantially reduced in the fli1:EGFP line, while it is dramatically reduced in the flk1:EGFP line upon double knockdown of sox18 and sox7. (E-H) Staining of the endogenous AP activity was performed to analyze angiogenesis in the region of the subintestinal vessels (SIV) basket at 3 dpf. (E,F) control std-MO–injected embryos in dorsal and lateral views, respectively, anterior to the left. (G,H) DMO-injected embryos in dorsal and lateral views, respectively, anterior to the left. The SIV basket is severely disorganized and enlarged in the double morphants, showing thinner vessels and anomalous spikes pointing toward the yolk. (I,P) Confocal images of fli1:EGFP double morphants and control embryos showing the SIV basket (I-L) and the caudal intersomitic vessels (ISVs) (M-P) at 3 dpf. All lateral views, anterior to the left. (I,M) Control std-MO–injected embryos. (J,N) Embryos coinjected with sox7-MO1 and sox18-MO1, 125 fmol each. (K,O) Embryos coinjected with sox7-MO1 and sox18-MO1, 250 fmol each. (L,P) Control embryos treated with 2,3-BDM to block heartbeat and blood circulation. The alterations in the SIV basket and in the caudal ISVs visible in the double morphants are clearly distinct from the mere effect of absent blood flow. (J,K) Consistent with AP staining, confocal analysis reveals in the double morphants a global disorganization of the SIV basket, which is highly enlarged, particularly in the posterior aspect (white arrow). The caliber of most vessels in the basket, and especially of the subintestinal vein, is reduced in the double morphants (white arrowhead), which also present abnormal spikes toward the yolk (white asterisks). (L) The SIV basket appears instead reduced (white arrow) in the embryos treated with 2,3-BDM. (N,O) The caudal vein plexus (white arrowhead) is severely reduced in the double morphants, but not in 2,3-BDM–treated controls (P). In the double morphants (N,O), anomalous branching (white asterisks) is visible in the dorsal aspect of the caudal ISVs, pointing to defective proliferation/guidance mechanisms in angiogenic development. No abnormal branching of the ISVs is caused by a mere block in blood flow (P).

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