Figure 2.
Figure 2. Characterization of mice with EC-specific S1P1 deletion. (A) Section of X-gal stained Tie2-Cre/ROSA26 E10.5 brain vessel showing lacZ staining specifically in ECs. (B) Tissue specificity of Tie2-Cre-mediated recombination of S1P1loxP using E12.5 embryo tissue DNA samples and PCR primers as shown in Figure 1B. In embryos carrying the Tie2-Cre gene, substantially more recombination of the S1P1loxP allele to produce the S1P1ΔEx2 allele was found in yolk sac and aorta as compared with embryonic neural tissue. No evidence of recombination was found in embryos without the Tie2-Cre gene. S1P 1 loxP, S1P1 allele containing loxP sites; S1P1ΔEx2, S1P1 allele after Cre-mediated recombination; S1P1WT, wild-type S1P1 allele; S1P 1Ko, null S1P1 allele.15 (C) S1P1 expression in ECs in control and S1P1loxP/Ko Tie2-Cre embryonic brains at E12.5. Sections were stained with both isolectin B4-fluorescein isothiocyanate (FITC; Vector Laboratories, Burlingame, CA), a specific marker for mouse ECs, and the anti-S1P1 antibody. Note that in control section essentially all ECs that are stained with isolectin B4 also stain with the antibody to S1P1. In the section from the S1P1loxP/Ko Tie2-Cre conditional mutant, only a few ECs stain strongly with the S1P1 antibody. Midbrain neuroepithelia from control and S1P1-conditional E12.5 embryos shown as positive control for S1P1 expression; both show about equal staining. (D) Phenotype of S1P1-conditional mutant embryos. At E13.5, the yolk sacs of S1P1loxP/Ko Tie2-Cre conditional mutant embryos show normal vasculature but less blood compared with control embryos (top, arrowhead). The mutant embryos display an enlarged pericardial cavity (PCC; right), undeveloped limbs, and intraembryonic hemorrhages (bottom; FL, front limb; HL, hind limb). (E) Vascular maturation in S1P1-conditional mutant embryos. Transverse sections of aortae (top) from an S1P1loxP/KO Tie2-Cre embryo and a control embryo stained with anti-SM α-actin (DAKO, Carpinteria, CA). Note VSMCs are clustered to the ventral side of the aorta and fail to surround the vessel completely in the mutant. Also, ECs on the dorsal side of the aorta appear discontinuous (arrows). Cranial arteries (bottom) from S1P1loxP/Ko Tie2-Cre and control embryos stained with anti-SM α-actin. Note that VSMCs cluster on one side of the vessel of the S1P1-conditional mutant embryo. Scale bars = 50 μm.

Characterization of mice with EC-specific S1P1deletion. (A) Section of X-gal stained Tie2-Cre/ROSA26 E10.5 brain vessel showing lacZ staining specifically in ECs. (B) Tissue specificity of Tie2-Cre-mediated recombination of S1P1loxP using E12.5 embryo tissue DNA samples and PCR primers as shown in Figure 1B. In embryos carrying the Tie2-Cre gene, substantially more recombination of the S1P1loxP allele to produce the S1P1ΔEx2 allele was found in yolk sac and aorta as compared with embryonic neural tissue. No evidence of recombination was found in embryos without the Tie2-Cre gene. S1P1 loxP, S1P1 allele containing loxP sites; S1P1ΔEx2, S1P1 allele after Cre-mediated recombination; S1P1WT, wild-type S1P1 allele; S1P1Ko, null S1P1 allele.15  (C) S1P1 expression in ECs in control and S1P1loxP/KoTie2-Cre embryonic brains at E12.5. Sections were stained with both isolectin B4-fluorescein isothiocyanate (FITC; Vector Laboratories, Burlingame, CA), a specific marker for mouse ECs, and the anti-S1P1 antibody. Note that in control section essentially all ECs that are stained with isolectin B4 also stain with the antibody to S1P1. In the section from the S1P1loxP/KoTie2-Cre conditional mutant, only a few ECs stain strongly with the S1P1 antibody. Midbrain neuroepithelia from control and S1P1-conditional E12.5 embryos shown as positive control for S1P1 expression; both show about equal staining. (D) Phenotype of S1P1-conditional mutant embryos. At E13.5, the yolk sacs of S1P1loxP/KoTie2-Cre conditional mutant embryos show normal vasculature but less blood compared with control embryos (top, arrowhead). The mutant embryos display an enlarged pericardial cavity (PCC; right), undeveloped limbs, and intraembryonic hemorrhages (bottom; FL, front limb; HL, hind limb). (E) Vascular maturation in S1P1-conditional mutant embryos. Transverse sections of aortae (top) from an S1P1loxP/KOTie2-Cre embryo and a control embryo stained with anti-SM α-actin (DAKO, Carpinteria, CA). Note VSMCs are clustered to the ventral side of the aorta and fail to surround the vessel completely in the mutant. Also, ECs on the dorsal side of the aorta appear discontinuous (arrows). Cranial arteries (bottom) from S1P1loxP/KoTie2-Cre and control embryos stained with anti-SM α-actin. Note that VSMCs cluster on one side of the vessel of the S1P1-conditional mutant embryo. Scale bars = 50 μm.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal