Foxc1 and Foxc2 deletion causes abnormal lymphangiogenesis and correlates with ERK hyperactivation
Anees Fatima, … , Yoh-suke Mukouyama, Tsutomu Kume
Anees Fatima, … , Yoh-suke Mukouyama, Tsutomu Kume
Published May 23, 2016
Citation Information: J Clin Invest. 2016;126(7):2437-2451. https://doi.org/10.1172/JCI80465.
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Research Article Angiogenesis Vascular biology

Foxc1 and Foxc2 deletion causes abnormal lymphangiogenesis and correlates with ERK hyperactivation

Abstract

The lymphatic vasculature is essential for maintaining interstitial fluid homeostasis, and dysfunctional lymphangiogenesis contributes to various pathological processes, including inflammatory disease and tumor metastasis. Mutations in FOXC2 are dominantly associated with late-onset lymphedema; however, the precise role of FOXC2 and a closely related factor, FOXC1, in the lymphatic system remains largely unknown. Here we identified a molecular cascade by which FOXC1 and FOXC2 regulate ERK signaling in lymphatic vessel growth. In mice, lymphatic endothelial cell–specific (LEC-specific) deletion of Foxc1, Foxc2, or both resulted in increased LEC proliferation, enlarged lymphatic vessels, and abnormal lymphatic vessel morphogenesis. Compared with LECs from control animals, LECs from mice lacking both Foxc1 and Foxc2 exhibited aberrant expression of Ras regulators, and embryos with LEC-specific deletion of Foxc1 and Foxc2, alone or in combination, exhibited ERK hyperactivation. Pharmacological ERK inhibition in utero abolished the abnormally enlarged lymphatic vessels in FOXC-deficient embryos. Together, these results identify FOXC1 and FOXC2 as essential regulators of lymphangiogenesis and indicate a new potential mechanistic basis for lymphatic-associated diseases.

Authors

Anees Fatima, Ying Wang, Yutaka Uchida, Pieter Norden, Ting Liu, Austin Culver, William H. Dietz, Ford Culver, Meredith Millay, Yoh-suke Mukouyama, Tsutomu Kume

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Figure 1

FOXC1 and FOXC2 are coexpressed in LEC progenitors and lymphatic valve-forming cells during mouse development.

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FOXC1 and FOXC2 are coexpressed in LEC progenitors and lymphatic valve-f...
(A and B) Foxc1 expression in PROX1+ LEC progenitors of Foxc1lacZ/+ embryos as revealed by β-gal immunostaining. The middle and right panels show a higher magnification of the boxed regions indicated in the left panels (×40). (A) Triple immunostaining of PROX1, CD31, and β-gal (FOXC1) at E10.5. PROX1+β-gal+ LEC progenitors were detected in the cardinal vein (CV) and cells migrating out of the CV (white arrowheads). Scale bars: 100 μm. (B) Triple immunostaining of PROX1, LYVE-1, and β-gal (FOXC1) at E12.5. PROX1+β-gal+ LECs were detected in lymph sacs (LS) (white arrowheads). Scale bars: 100 μm. (C) Triple immunostaining of FOXC1, FOXC2, and PROX1 on an E10.5 sagittal section. FOXC1 and FOXC2 were coexpressed in PROX1-expressing LEC progenitors in the CV and cells migrating out of the CV (yellow arrowheads). (D) Triple immunostaining of FOXC1, FOXC2, and PROX1 at the level of the neck on an E12.5 longitudinal section. FOXC1 and FOXC2 were detected in PROX1-positive LS. (E and F) Whole mount triple immunostaining of mesenteric vessels at E17 (E) and P3 (F) for FOXC1, FOXC2, and PROX1. Note overlapping expression of FOXC1, FOXC2, and PROX1 in lymphatic valve-forming cells (VFCs). BV, blood vessel; LV, lymphatic vessel. Scale bars: 50 μm. (G) qPCR analysis of relative mRNA levels of Foxc1, Foxc2, and Prox1 in PROX1+LYVE-1+ LECs isolated from E15.5 dorsal skin. The value of Prox1 mRNA levels is set at 1-fold. (H) qPCR analysis of relative mRNA levels of FOXC1 and FOXC2 in human LECs isolated from foreskin samples of donors between 2 and 5 years of age (HLEC-1, HLEC-2, and HLEC-3) and human neonatal LECs from Lonza (HMVEC-dLy Neo).