Human FLT4 mRNA

The formation of the lymphatic system requires the coordinated expression of several key regulators: vascular endothelial growth factor C (VEGFC), its receptor FLT4, and the key transcriptional effector PROX1. However, the mechanisms regulating the expression of these signaling pathway components are unclear. Here, researchers combined genetic and molecular biological approaches to identify the transcription factor hematopoietic expression homeobox (HHEX) as an upstream regulator of VEGFC, FLT4, and PROX1 during angiogenic sprouting and lymphangiogenesis in vertebrates. By analyzing zebrafish mutants, researchers found that hhex is an essential factor for vascular sprouting of the posterior cardinal vein, which is required for lymphangiogenesis. In addition, mammalian HHEX was studied by tissue-specific gene deletion in mice and gene knockdown in cultured human endothelial cells, revealing its highly conserved functions during vascular and lymphatic development. These findings indicate that HHEX is essential for the regulation of the VEGFC/FLT4/PROX1 axis, providing insights into the molecular regulation of lymphangiogenesis.

Here, researchers assessed vegfc and flt4 expression at 24 and 32 hpf after fertilization by whole-mount in situ hybridization (Figure 1a-h). At 24 hpf after fertilization, hhex mutants showed reduced expression of flt4, while vegfc expression was slightly increased and ectopically expressed in the PCV (Figure 1a-d). These changes were more pronounced at 32 hpf, as flt4 expression was greatly reduced in hhex mutants, while vegfc expression was significantly increased in the PCV (Figure 1e-h). To test whether the phenotypes observed in hhex mutants were at least partially due to the regulation of the Vegfc/Flt4 signaling pathway, researchers performed a rescue experiment by injecting messenger RNA (mRNA) encoding full-length human FLT4. Confocal imaging of the main trunk vessels of hhex mutant larvae and quantitative analysis of vISV and thoracic duct (TD) extension showed that vISV and lymphatic vessel formation were partially restored after injection of FLT4 mRNA (Figure 1i-l), indicating that the Vegfc/Flt4 signaling pathway functions downstream of Hhex. Taken together, these results indicate that Hhex is a key regulator of flt4 and vegfc expression in PCVs during angiogenic sprouting.

Figure 1. The Vegfc/Flt4 pathway is affected in zebrafish hhex mutants. (Gauvrit S, et al., 2018)