Figure 2
Figure 2. Hh requires VEGF for arterial identity and endothelial tube formation. (A) Whole-mount in situ hybridization for Vegf mRNA in E8.5 Smo mutant embryos. Representative embryos and all yolk sacs from 8 somite stage Smo+/+ (n = 2) and Smo−/− (n = 3) littermates are shown. Brackets indicate the occipital somite (OS) and presomitic/primitive streak (PS) regions of the embryo microdissected for Q-RT-PCR in panel B. (B) Q-RT-PCR for Vegf mRNA in Smo control (Smo+/+, n = 2 and Smo+/−, n = 2) and Smo−/− (n = 4) embryos. Q-RT-PCR was performed on distinct regions of 8-somite stage littermates corresponding to the occipital somites region (labeled OS in panel A) and presomitic region (labeled PS in panel A) in control or mutant embryos. Data are presented as Vegf expression relative to the loading control, Polr2a. Asterisk indicates statistical significance (t test) between control and mutant sample groups, P < .05. (C) Whole-mount PECAM1 staining of E8.5 (approximately 8-somite stage) Smo;Flt1 compound mutant embryos. Representative examples of littermate Smo+/+;Flt1+/+ (n = 2), Smo+/+;Flt1+/− (n = 2), Smo−/−;Flt1+/+ (n = 1), and Smo−/−;Flt1+/− (n = 4) embryos are shown. Brackets indicate the region of the dorsal aorta affected in Smo−/−;Flt1+/+ embryos and rescued in Smo−/−;Flt1+/− embryos. The affected region corresponds exactly to the location of the somites in each embryo. Scale bar represent 500 μm. (D) Whole-mount PECAM1 staining of E8.5 (approximately 8-somite stage) Smo;Flt1 compound mutant yolk sacs. Representative examples of littermate Smo+/+;Flt1+/+ (n = 2), Smo+/+;Flt1+/− (n = 2), Smo−/−;Flt1+/+ (n = 1), Smo−/−;Flt1+/− (n = 4) embryos are shown. Brackets indicate the position of the blood islands. Scale bar represents 500 μm. (E) Whole-mount in situ hybridization for Dll4 mRNA in E8.5 (approximately 8-somite stage) Smo+/−;Flt1+/− (n = 1), Smo−/−;Flt1+/+ (n = 3), and Smo−/−;Flt1+/− (n = 2) littermate embryos. Bracket indicates occipital somite region. Scale bar represents 500 μm.

Hh requires VEGF for arterial identity and endothelial tube formation. (A) Whole-mount in situ hybridization for Vegf mRNA in E8.5 Smo mutant embryos. Representative embryos and all yolk sacs from 8 somite stage Smo+/+ (n = 2) and Smo−/− (n = 3) littermates are shown. Brackets indicate the occipital somite (OS) and presomitic/primitive streak (PS) regions of the embryo microdissected for Q-RT-PCR in panel B. (B) Q-RT-PCR for Vegf mRNA in Smo control (Smo+/+, n = 2 and Smo+/−, n = 2) and Smo−/− (n = 4) embryos. Q-RT-PCR was performed on distinct regions of 8-somite stage littermates corresponding to the occipital somites region (labeled OS in panel A) and presomitic region (labeled PS in panel A) in control or mutant embryos. Data are presented as Vegf expression relative to the loading control, Polr2a. Asterisk indicates statistical significance (t test) between control and mutant sample groups, P < .05. (C) Whole-mount PECAM1 staining of E8.5 (approximately 8-somite stage) Smo;Flt1 compound mutant embryos. Representative examples of littermate Smo+/+;Flt1+/+ (n = 2), Smo+/+;Flt1+/− (n = 2), Smo−/−;Flt1+/+ (n = 1), and Smo−/−;Flt1+/− (n = 4) embryos are shown. Brackets indicate the region of the dorsal aorta affected in Smo−/−;Flt1+/+ embryos and rescued in Smo−/−;Flt1+/− embryos. The affected region corresponds exactly to the location of the somites in each embryo. Scale bar represent 500 μm. (D) Whole-mount PECAM1 staining of E8.5 (approximately 8-somite stage) Smo;Flt1 compound mutant yolk sacs. Representative examples of littermate Smo+/+;Flt1+/+ (n = 2), Smo+/+;Flt1+/− (n = 2), Smo−/−;Flt1+/+ (n = 1), Smo−/−;Flt1+/− (n = 4) embryos are shown. Brackets indicate the position of the blood islands. Scale bar represents 500 μm. (E) Whole-mount in situ hybridization for Dll4 mRNA in E8.5 (approximately 8-somite stage) Smo+/−;Flt1+/− (n = 1), Smo−/−;Flt1+/+ (n = 3), and Smo−/−;Flt1+/− (n = 2) littermate embryos. Bracket indicates occipital somite region. Scale bar represents 500 μm.

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