Transcriptional regulation during lymphangiogenesis is strictly controlled, and recent evidence suggests the specific functions of several key transcription factors in lymphangiogenesis. The transcription factor V-maf musculoaponeurotic fibrosarcoma oncogene homolog B (MAFB), which is involved in the differentiation of various cell types, regulates the transcriptional changes invoked by VEGF-C in LECs (Dieterich et al., 2015; Dieterich et al., 2020; Rondon-Galeano et al., 2020; Arnold et al., 2022). MAFB induces the expression of PROX1, other transcription factors and markers of differentiated LECs, indicating the role of MAFB in the maintenance of the mature LEC phenotype (Dieterich et al., 2015). LEC-specific MAFB deficiency in mice causes increased lymphatic branching in the diaphragm at P7, enhanced tumor-induced lymphangiogenesis, increased perinatal lethality associated with cyanosis, and excessive smooth muscle cell coverage indicating a defect in the maturation of lymphatic networks. This suggests MAFB could be a potential target for therapeutic modulation of lymphangiogenesis (Dieterich et al., 2020; Rondon-Galeano et al., 2020). The transcription factor hematopoietically expressed homeobox (HHEX), an upstream regulator of VEGF-C/VEGFR3/PROX1 signaling during angiogenic sprouting and lymphatic formation, is required cell-autonomously in endothelial cells to promote venous and lymphatic sprouting. Mice deficient for HHEX exhibit severe vascular defects in blood and lymphatic vessel development (Gauvrit et al., 2018).

Forkhead box (Fox) O1, a member of the Fox transcription factor family, acts an essential role in developmental lymphangiogenesis by promoting LEC migration toward the chemokine (C-X-C motif) ligand (CXCL) 12 and regulating their proliferative activity (Niimi et al., 2020). The LEC-specific deletion of FOX O1 in mice decreases LEC migration toward CXCL12 by downregulating C-X-C chemokine receptor (CXCR) 4, induces excess LEC proliferation, and decreases LEC apoptosis, which leads to the disconnected and dilated structure of the lymphatic vessels (Niimi et al., 2020).

Brahma-related gene 1 (BRG1), a chromatin-remodeling enzyme, epigenetically regulates COUP-TFII expression, by remodeling chromatin within the COUP-TFII promoter and impacting the ability of transcriptional machinery to access the promoter (Davis et al., 2013). The EC-specific deletion of Brg1 in mice results in downregulation of COUP-TFII expression in developing veins (Davis et al., 2013).

Chromodomain helicase DNA-binding 4 (CHD4), an ATPase subunit of the nucleosome remodeling deacetylase (NuRD) chromatin-remodeling complex, regulates vascular integrity in the mid-gestation (Ingram et al., 2013). Specifically, CHD4 controls the development of lymphovenous valves, which regulates the return of lymph to the blood circulation by forming fibrin-rich thrombi that prevent blood from entering the lymphatic system (Crosswhite et al., 2016). The LEC-specific deletion of CHD4 in mice leads to increased transcription of the urokinase plasminogen activator receptor (uPAR), thereby facilitating activation of the fibrin-degrading protease plasmin and then degrading the fibrin near the lymphovenous valves (Crosswhite et al., 2016). In addition, CDH4 is functionally associated with the Hippo signaling pathway in lymphatic endothelial cells (Figure 2). The Hippo pathway final effectors Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) promote remodeling of lymphatic plexus patterning and postnatal lymphatic valve maintenance by negatively regulating Prox1 expression (Cho et al., 2019). LEC-specific deletion of YAP/TAZ in mice suppresses both lymphatic plexus patterning and valve initiation via upregulation of PROX1, whereas LEC-specific YAP/TAZ overexpression downregulates PROX1, disrupts lymphatic specification, and restricts lymphatic sprouting (Cho et al., 2019).

Disruptor of telomeric silencing 1-like (DOT1L), a histone H3 lysine (H3K) 79 methyltransferase, promotes transcription by histone methylation of chromatin and is a crucial factor in the homeostasis of various organs such as the heart and hematopoiesis (Jo et al., 2011; Nguyen et al., 2011). Vascular endothelial cell (VEC)-specific, but not LEC-specific, deletion of DOT1L causes fully penetrant lymphatic aplasia by altering the lymphatic transcription program and reducing H3K79me2 enrichment at lymphatic genes, including the transcription factors SOX18, SOX17, and FOXC2, which are critical for LEC differentiation and valve formation, as well as Vegfr3, which is critical for LEC proliferation and migration (Yoo et al., 2020) (Figure 2).

LECs use fatty acid β-oxidation for proliferation and epigenetic regulation of PROX1, which mediates epigenetic changes that promote lymphangiogenesis during LEC differentiation (Figure 2): 1) PROX1 upregulates carnitine palmitoyltransferase (CPT) 1A expression, which increases fatty acid β-oxidation-dependent acetyl coenzyme A production; 2) Acetyl coenzyme A is used by the histone acetyltransferase p300 to acetylate histones at lymphangiogenic genes; 3) histone acetyltransferase p300 interacts with Prox1 to facilitate preferential histone acetylation at the loci of PROX1-targeted genes (Wong et al., 2017). LEC-specific deletion of CPT1A in mice impairs lymphatic vessel development by exhibiting severe impairment of dermal lymphatic vessel outgrowth and branching at E16.5. (Wong et al., 2017). Other transcription factors expressed in LEC, E26 avian leukemia oncogene (ETS) 1 and 2, act as downstream effectors of the Ras/MAPK pathway and participate in VEGFR3 gene expression in LECs by recruiting the histone acetyltransferase p300 to the VEGFR3 locus and leading to histone acetylation and transcriptional activation of the VEGFR3 promoter (Ichise et al., 2012). In addition, ETS2 enhances inflammatory lymphangiogenesis and endothelial migration towards VEGF-C through the induction of VEGFR3 expression by binding to the VEGFR3 promoter in concert with PROX1 (Yoshimatsu et al., 2011). Additionally, mitochondrial complex III also regulates the critical PROX1-VEGFR3 feedback loop. The functional inactivation of mitochondrial complex III impairs lymphatic vessel development by disrupting the maintenance of the PROX1-VEGFR3 feedback loop through the reduction in H3K4me3 and H3K27ac histone modifications at the VEGFR3 and PROX1 promoters (Ma et al., 2021).

Lymph flow is essential for the development and maturation of lymphatic valves (Kume, 2015), which play a critical role in preventing the backflow of lymph fluid. The lymphatic valves are formed from lymphatic endothelial cells, a process that is occurred by flow-dependent lymphatic vessel remodeling caused by oscillatory share stress (OSS) at branching points in the lymphatic plexus during the early stage of lymphatic development (Sabine et al., 2012; Shin and Lawson, 2021). The OSS response leads to an increase in the expression of GATA-binding protein 2 (GATA2), Prox1, and Foxc2, which induce valve forming cells to the site of valve formation (Kazenwadel et al., 2015; Sweet et al., 2015; Shin and Lawson, 2021). In valve forming cells, Gata2 directly regulates PROX1 and FOXC2 expression, whereas Foxc2 regulates valve maturation in cooperation with Prox1 to control intraluminal invagination of LECs and reorganization into valve forming leaflets by postnatal day (P)1 (Kazenwadel et al., 2015). As an upstream epigenetic factor, the histone-modifying enzyme histone deacetylase 3 (HDAC3) regulates lymphatic valve formation. In response to OSS, Hdac3 is recruited to the Gata2 enhancer element and physically interacts with the transcription factors T-cell acute lymphocytic leukemia protein 1 (TAL1), Gata2, and Ets1/2 to promote Gata2 expression (Janardhan et al., 2017) (Figure 3).

Human FOXC2 is a causative gene whose mutations are dominantly associated with lymphedema-distichiasis syndrome characterized by failure of lymph drainage in limbs, venous valve failure, and the growth of an extra set of eyelashes (Fang et al., 2000; Traboulsi et al., 2002; Mellor et al., 2007; Tavian et al., 2016). Foxc2 regulates connexin 37 expression and activation of calcineurin/nuclear factor of activated T-cells (NFAT) signaling during lymphatic collecting vessel maturation and valve formation (Petrova et al., 2004; Norrmen et al., 2009; Sabine et al., 2012). Foxc2 is also identified as a crucial factor for lymphatic valve maintenance by regulating LEC junctional integrity and cellular quiescence under reversing flow conditions via restriction of TAZ-mediated proliferation (Sabine et al., 2015). Foxc1 is a closely related member of the Fox transcription factor family, and LEC-specific deletion of FOXC1, FOXC2, or both in mice leads to increased LEC proliferation, enlarged lymphatic vessels, and abnormal lymphatic vessel morphogenesis, accompanied by increased Ras/ERK signaling during embryonic lymphangiogenesis (Fatima et al., 2016). Unlike FOXC2, LEC-specific FOXC1 mutant mice normally develop initial mesenteric lymphatic valves; however, the formation of matured lymphatic vessels (v-shaped or semilunar bi-leaflet structures) is significantly impaired (Norden et al., 2020). Importantly, the number of mesenteric lymphatic valves is remarkably reduced in the LEC-specific deletion of FOXC1 and FOXC2 compared to LEC-specific FOXC2 deletion alone, suggesting that FOXC1 and FOXC2 function cooperatively in the maturation and maintenance of lymphatic valves (Norden et al., 2020).

Foxo1 is crucial for controlling the expression of valve forming genes including FOXC2, PROX1, and GATA2 as a key downstream effector of shear stress by regulating lymphatic valve maintenance, and LEC-specific deletion of FOXO1 in mice leads to the formation of additional lymphatic valves compared to control mice (Scallan et al., 2021). Another study also reveals the role of Foxo1 as a repressor for lymphatic valve formation and maintenance via the inhibition of OSS-induced upregulation of lymphatic valve-specific genes such as PROX1 and FOXC2 (Niimi et al., 2021).

A recent study has shown that Foxp2, another Fox transcription factor previously implicated in speech development, is expressed in lymphatic endothelial cells of collecting vessels and their valve-forming cells, and that Foxp2 is induced after initiation of lymph flow and upon OSS on LECs (Hernandez Vasquez et al., 2021). LEC-specific FOXP2 mutant mice exhibit enlarged collecting vessels and defective lymphatic valves characterized by loss of NFATc1 activity (Hernandez Vasquez et al., 2021).

Piezo type mechanosensitive ion channel component 1 (PIEZO1), a cation channel activated by mechanical forces such as fluid shear stress or membrane stretch, is a causative gene associated with congenital lymphedema with pleural effusion (Nonomura et al., 2018). The LEC-specific deletion of PIEZO1 in mice leads to a reduction in the number of lymphatic valves and impairments in lymphatic valve protrusion such as collective cell migration, actin polymerization, and remodeling of cell-cell junctions, whereas the expression patterns of Foxc2 and NFATc1, both of which are crucial factors for lymphatic valve development, are normally detected in these mutant mice (Nonomura et al., 2018). Another study demonstrated that PIEZO1 is the force sensor in the mechanotransduction pathway controlling lymphatic valve development and maintenance, although PIEZO1 knockdown in cultured LECs largely abrogated the OSS-induced upregulation of the lymphatic valve signature genes including FOXC2 and GATA2 (Choi et al., 2019). Moreover, overexpressing PIEZO1 in cultured LECs upregulates FOXC2 and GATA2 in the absence of OSS, demonstrating that ectopic expression of PIEZO1 can recapitulate the lymphatic valve gene profile (Choi et al., 2019). Treatment with Yoda1, a chemical agonist of PIEZO1, leads to changes in LEC morphology by inducing the remodeling of actomyosin and/or VE-cadherin⁺ cell–cell adhesion and activates the expression of some lymphatic valve genes such as FOXC2 and GATA2 in a PIEZO1-dependent manner (Nonomura et al., 2018; Choi et al., 2019). Together, these results suggest that the activation of mechanosensitive PIEZO1 can control, at least in part, transcriptional regulation of lymphatic valve forming cells under the absence of mechanical forces.