Extended Data Figure 2 : Cpt1a gene excision in Prox1∆CPT1A embryos and additional phenotypic information and kinetics of loss of CPT1A in vivo and in vitro.

From: The role of fatty acid β-oxidation in lymphangiogenesis

Extended Data Figure 2

a, Genotyping for the presence of the Cpt1a floxed (2,200 bp) and excision band (300 bp) in E14.5 Prox1∆CPT1A embryos. b, Cartoon of anatomical orientation of the dorsal aorta (DA) and jugular vein (JV) with respect to the jugular lymphatic sacs (JLS). The red dotted lines in the embryo drawing indicate the jugular region of focus in wild-type E14.5 mouse embryos. SC, spinal cord; VB, vertebral body. c, d, Staining for CPT1A (magenta) and VEGFR3 (green) in wild-type (Prox1) and Prox1∆CPT1A embryos at E14.5. CPT1A immunoreactivity (magenta) is seen within VEGFR3+ LECs (green) of the JLS in wild-type embryos (c). In the JLS of the E14.5 Prox1∆CPT1A embryo (d), CPT1A immunoreactivity is undetectable. For the CPT1A signal channel only (left panels), a higher magnification is shown than for the VEGFR3 signal channel (top right panels) or the merged signal (bottom right panels). The white dashed lines denote endothelium in the JLS. Arrows denote LECs without apparent CPT1A immunoreactivity. Scale bars, 5 μm. e, Genotyping for the presence of the Cpt1a floxed (2,200 bp) and excision band (300 bp) in E9.5 Prox1FK∆CPT1A embryos. f, g, Representative micrographs of staining for CPT1A (magenta) and VEGFR3 (green) in LECs within the JLS at E11.5 in a wild-type (f) and Prox1∆CPT1A (g) embryo. Top panels show the single channel CPT1A signal. The insets represent an overlay of CPT1A and VEGFR3 signals (bottom left f, g) and the single channel signal for VEGFR3 (bottom right f, g). Dashed white lines outline the endothelium of the JLS, as identified by VEGFR3 staining. Arrows highlight areas within the lymphatic endothelium with strong CPT1A immunoreactivity in VEGFR3+ cells within the JLS of a wild-type embryo (f) and weaker but still detectable CPT1A-immunoreactive signal in VEGFR3+ cells within the JLS in a Prox1∆CPT1A embryo (g). h, Primary antibody omission for CPT1A and VEGFR3. Isolectin B4 (blue) was used to highlight vascular structures in the negative control (wild-type E14.5 embryo), and the JLS characteristically was more weakly stained compared to the jugular vein (upper panel). As expected, primary antibody omission (VEGFR3 (488, middle panel); CPT1A (Cy5, lower panel)) revealed no aspecific immunoreactivity. i, j, Representative micrographs of staining for CPT1A (magenta) and VEGFR3 (green) in LECs within the JLS at E11.5 in Prox1FK∆CPT1A (i) and Flt4∆CPT1A (j) embryo. The left panels show the single channel CPT1A signal. The insets represent an overlay of CPT1A and VEGFR3 signals (upper i, j), the single channel signal for VEGFR3 (bottom left i, j) and the primary antibody omission for CPT1A (bottom right i, j). As expected, primary antibody omission revealed no aspecific immunoreactivity. Dashed white lines outline the endothelium of the JLS, as identified by VEGFR3 staining. Arrows highlight areas within the lymphatic endothelium with still detectable CPT1A-immunoreactive signal in VEGFR3+ cells within the JLS in a Prox1FK∆CPT1A (i) and Flt4∆CPT1A (j) embryo. Scale bars, 10 μm. k, Genotyping for the presence of the Cpt1a floxed (2,200 bp) and excision band (300 bp) in E9.5 Prox1∆CPT1A embryos. l, Quantitative RT–PCR analysis of CPT1A mRNA expression at 1–3 days after CPT1AKD in VECs (n = 3). m, Densitometric quantification of immunoblotted CPT1A protein relative to β-ACTIN at 1–4 days after CPT1AKD in VECs, illustrating that, probably owing to the CPT1A protein stability, as much as 35–40% residual CPT1A protein levels were still detectable at 3 days after silencing CPT1A (n = 3). n, Representative stereomicroscope images of E10.5 wild-type (left) and VEcad∆CPT1A (right) embryos after tamoxifen administration from E7.5–E9.5, showing severe developmental and vascular abnormalities. o, p, Representative micrograph of staining for CPT1A (magenta) and VEGFR3 (green) in an E16.5 Prox1FK∆CPT1A (o) and Flt4∆CPT1A (p) embryo. Compared to CPT1A immunoreactivity seen within the VEGFR3+ LECs of the JLS in wild-type embryos (Extended Data Fig. 2c), CPT1A immunoreactivity is undetectable in LECs of E16.5 Prox1FK∆CPT1a (o) and Flt4∆CPT1A (p) embryos (left panels). The right panels represent the single channel signal for VEGFR3 (top right) and the merged signal for CPT1A and VEGFR3 (bottom right), highlighting the lack of detectable CPT1A immunoreactivity in mutant embryos. The white dashed lines denote the lymphatic endothelium in the JLS. Arrows denote LECs without apparent CPT1A immunoreactivity. Of note, owing to our TSA-based amplification method for all of our immunostaining protocols of embryo sections (see Methods), the VEGFR3 immunoreactivity seen in o and p was maximally enhanced, which is why VEGFR3 immunoreactivity does not appear lower for the gene-deficient embryos compared to wild type shown in Extended Data Fig. 2c. Scale bars, 5 μm. Mean ± s.e.m. Statistical test: t-test was used for comparison of two groups. *P < 0.05; NS, not statistically significant.