Fig. 9.

Endothelial-specific knockout of Alk3 results in loss of venous identity in mice. a, b. Immunofluorescent analysis of CD31, EPHB4 and ALK3 (a) and in situ hybridization for Bmp4 (b) in transverse sections from E9.5 mouse embryos. Scale bars are 50 μm. c Representative E10.5 whole-mount images from wild-type Alk3^+/+ (n = 16), heterozygous Alk3^EC/+; (n = 7) and homozygous Alk3^EC/EC (n = 7) embryos expressing the arterial Dll4in3:LacZ transgene (Nine litters). Robust transgene expression, specific to arterial endothelial cells, was seen in all embryos although Alk3^EC/EC embryos were often significantly growth retarded. Grey numbers on bottom right denote the number of embryos similar to picture shown; for Alk3^EC/EC, two different images show Dll4in3:LacZ expression in the range of morphological defects. Grey scale bars are 500 μm. d Representative E10.5 whole-mount images from wild-type Alk3^+/+ (n = 34), heterozygous Alk3^EC/+; (n = 21) and homozygous Alk3^EC/EC (n = 16) embryos expressing venous Ephb4^LacZ (21 litters, for littermates, see Supplementary Fig. 16). Robust X-gal activity is detected in the veins of Alk3^+/+ embryos but is reduced in Alk3^EC/+ embryos and absent in Alk3^EC/EC regardless of extend of growth retardation and morphological defects. Grey numbers on bottom right denote the number of embryos similar to picture shown; for Alk3^EC/EC, two different images are shown to indicate Ephb4^LacZ expression in the range of morphological defects. Grey scale bars are 500 μm. e Representative transverse sections from E10.5 Alk3^EC/EC embryos transgenic for either arterial Dll4in3:LacZ or venous Ephb4^LacZ at two different levels. Some vessels were clearly seen in both; these expressed Dll4in3:lacZ but not Ephb4^LacZ and were located in arterial positions, suggesting the presence of dorsal aorta but no cardinal vein. Black scale bars are 100 μm. f, g Representative arterial endothelial-specific Alk3^+/+ and Alk3^ART/ART whole-mount E10.5 embryos (f) and transverse sections stained with CD31 (g). Loss of Alk3 in arterial endothelial cells had no effect on vascular development at E10.5. White scale bars are 100 μm. h Observed frequency of Alk3^fl/fl;Dll4in3:Cre embryos at E10.5 and P5. Mendelian ratios were present at both time points. For all panels, ^EC is Tie2:Cre-mediated deletion, ^ART is Dll4in3:Cre-mediated deletion. ^+/+ indicates Cre−, ^EC/+ indicates Cre+,Alk3^fl/+ and ^EC/EC indicates Cre+;Alk3^fl/fl. da = dorsal aorta, ica = internal carotid artery, isa = intersomitic arteries, isv = intersomitic vessels, baa = branchial arch arteries, nt = neural tube, cv = cardinal vein, cev = branches of cerebral venous plexus, mda = midline dorsal aorta