Choose your destiny: Make a cell fate decision with COUP-TFII.

Arterial, venous and lymphatic vessels are the three types of major conduits to circulate body fluid. Blood pumped out from cardiac ventricles travels through arteries to reach micro vessels in peripheral tissue and returns via veins to atria. Meanwhile, lymphatic vessels collect and return interstitial fluid to the general circulation via connecting to subclavian veins. Fates of arteries and veins are determined during embryonic development 68 . We and others show that COUP-TFII and Notch signaling serve as key regulators in specification of venous and arterial fates, respectively 25 , 69 - 72 . Artery-fated angioblasts receive signals from vascular endothelial growth factor (VEGF) via the receptor VEGFR2 and the co-receptor neuropilin 1 (NRP1) to activate Notch signaling for further differentiation toward the arterial phenotype. Additionally, forkhead transcription factors Foxc1 and Foxc2 also promote arterial gene expression through increasing expression of Notch signaling genes Dll4 and Hey2 73 , 74 . In contrast, vein-fated angioblasts express COUP-TFII, which suppresses notch signaling to confer vein identity. COUP-TFII directly suppresses expression of NRP1 and FOXC1, two upstream regulators of Notch signaling 7 . The resulting suppression of the Notch signaling is evidenced by reduced expression of Notch downstream effectors HES1, HES2 and HEY2, increased levels of venous marker gene EPHB4, and decreased expression of arterial marker EFNB2. Additionally, COUP-TFII also binds to the promoter of the HEY2 gene and directly represses its transcriptional activities 7 . Thus, COUP-TFII specifies the venous fate through suppression of the Notch signaling pathway at multiple points. Notably, Notch is shown to repress COUP-TFII expression in the dorsal aorta of zebrafish and cultured endothelial cells 37 , 38 , suggesting a reciprocal repression mechanism of COUP-TFII and Notch during vascular development. Recently, the chromatin remodeling enzyme Brg1 (also known as Smarca4) has been shown to positively regulate COUP-TFII expression, and is required for venous specification in mouse embryos 75 . Brg1 binds at the promoter and a −1.2-Kb upstream region of COUP-TFII gene. Brg1 depletion results in a reduced H3K9ac active enhancer mark and increased histone H3 binding at both Brg1 binding sites, leading to decreased RNA polymerase II recruitment at the COUP-TFII promoter region and reduced COUPTFII expression 75 . Veins of endothelial-specific Brg1 knockout mice exhibit decreased COUP-TFII expression and increased NRP1 and DLL4 levels, parallel to the COUP-TFII knockout phenotype 75 . Collectively, these findings indicate that Brg1 suppresses Notch signaling through epigenetically modifying COUP-TFII gene activities for venous fate specification. The lymphatic vessels arise from the anterior cardinal veins and intersomitic vessels 76 , 77 . In mid-gestation embryos, a subset of endothelial cells fated as lymphatic precursors migrate out of blood vessels to form lymphatic sac and subsequent lymphatic vasculature 76 - 78 . Specification of lymphatic precursors requires COUP-TFII and Sox18 to promote expression of the lymphatic master regulator Prox1, in which direct transcriptional regulation serves as the underlying mechanism of action 24 , 79 . In addition, ERK signaling also stimulates Sox18 and Prox1 expression for lymphatic specification 80 . The ERK signaling may work in parallel or downstream of COUP-TFII because constitutive activation of ERK does not affect COUP-TFII expression 80 . After fate specification, the Prox1-positive lymphatic precursors bud out and leave blood vessels to undergo further differentiation and sprouting in response to mesenchyme-derived vascular endothelial growth factor C (VEGFC) 77 . In lymphatic precursors, expression of the VEGFC receptor, VEGFR3, requires physical interaction between COUP-TFII and Prox1 to jointly promote its transcription 81 . Regulation of VEGFR3 by COUP-TFII continues to be essential during the process of lymphangiogenesis. Loss of COUP-TFII results in decreased VEGFR3 expression and impairs proliferation and migration of lymphatic endothelial cells 8 . In summary, COUP-TFII controls lymphatic system development by regulating cell fate specification, migration, proliferation and differentiation.

During embryogenesis, kidneys derive from renal progenitor cells in the metanephric mesenchyme. The metanephric mesenchyme arises from a subpopulation of intermediate mesodermal cells that are specified to adopt the renal progenitor fate 82 . The intermediate mesodermal cells express high levels of COUP-TFII and COUP-TFII deficient mice fail to develop metanephric mesenchyme 9 . These findings together indicate that specification of renal progenitors requires COUP-TFII. After formation of metanephric mesenchyme, COUP-TFII remains prominently expressed in these renal progenitors. At this stage, COUP-TFII works in parallel with Osr1, another master renal development gene, to regulate the Eya1-Pax2-Six2 axis that promotes expression of Gdnf for subsequent induction of nephron formation 83 . At the same time, COUP-TFII also promotes expression of the Wt1 gene that is essential for survival of renal progenitors cells in the metanephric mesenchyme. Mechanistically, COUP-TFII modulates these two pathways by Sp1-dependent binding at the Eya1 and Wt1 promoters to induce their transcriptional activities 9 . Taken together, COUP-TFII is central to a network of renal transcriptional regulators that control fate specification, cell survival and intercellular communication of renal progenitors. The nephron is the working unit of a kidney. Each nephron is segmented into four functional domains – glomerulus, proximal tubule, loop of Henle and distal tubule. During the process of nephron segmentation, Notch2 specifies the identity of proximal tubules. Deletion of Notch2 results in kidney hypoplasia where glomeruli and proximal tubules are absent but the distal tubules remain in place 84 . On the other hand, COUPTFII is expressed in distal tubules but not in proximal tubules of developing kidneys 85 , 86 . Interestingly, deletion of COUP-TFII in renal progenitor cells (CKO six2-cre ) also produces kidney hypoplasia with an opposite phenotype in which proximal tubules form with much less corresponding distal tubules (Yu et al., unpublished observations). Further analysis found that the COUP-TFII deficient tubule cells acquire the capacity to bind Lotus tetragonolobus lectin (LTL) that marks the proximal tubules 86 , suggesting that cells alter characters subsequent to loss of COUP-TFII, possibly through adaptation of the proximal tubule fate. These findings collectively implicate that, in contrast to Notch2 dependent specification of proximal tubule fate, COUP-TFII may play a critical role in specifying cells that are fated for distal tubules during nephron segmentation. In adult kidney, COUP-TFII is required to maintain expression of renin 87 , the enzyme that activates the renin-angiotensin system to regulate blood pressure. COUPTFII and renin are both expressed in the juxtaglomerular cells that are the primary renin-expression cells 87 . Deletion of COUP-TFII, after the kidney is fully matured, reduces the basal levels of renin expression in renin-expressing cells. At the molecular level, COUPTFII binds to a direct repeat at the proximal renin promoter and works with another transcription factor cAMP-binding protein to promote renin expression 87 . It is worthy to note that genome-wide association studies have linked COUP-TFII to hypertension in human 88 , 89 , supporting the clinical relevance of COUP-TFII in regulation of the reninangiotensin system. Intriguingly, the renin expressing cells also arise from progenitors in the metanephric mesenchyme where COUP-TFII is highly expressed 9 , while emerging evidence suggests that Notch signaling regulates the expansion of renin-expressing cell population under pathological conditions through cellular reprogramming 90 - 92 . Owing to the close relation between COUP-TFII and Notch signaling in cell fate determination, it is tempting to speculate that COUP-TFII might also participate in this reprogramming event at the adult stage.

Chicken Ovalbumin Upstream Promoter Transcription Factor II (COUP-TFII, also known as NR2F2) belongs to the steroid hormone receptor superfamily. COUP-TFII acts as a transcription factor to directly activate or repress transcriptional activities of target genes 1 , 2 . Alternatively, COUP-TFII can sequester other transcription regulators, such as Smad4, TR, RXR and RAR, to affect the expression of downstream targets 2 - 4 . At the cellular level, COUP-TFII promotes cell differentiation 5 , 6 , proliferation 4 , 7 , 8 , migration 7 , 8 , survival 9 and intercellular communication 10 - 12 . Analyses of genetically engineered mouse models reveal regulatory functions of COUP-TFII in the development of many organs and tissues, including cerebellum 13 , brain 14 , eye 15 , heart 16 - 18 , stomach 19 , diaphragm 20 , limb 21 , kidney 9 , adipose 22 , testis 23 and blood and lymphatic vessels 8 , 16 , 24 , 25 . In adult physiology, COUP-TFII modulates male and female fertility 12 , 23 , 26 as well as glucose and energy metabolism 22 . Pathologically, COUP-TFII facilitates tumor angiogenesis 10 , 27 , promotes tumorigenesis 4 and suppresses endometriosis progression 28 . Upstream regulators of COUP-TFII include retinoic acid 29 , 30 , Sonic hedgehog 31 - 33 , Indian hedgehog 34 , 35 , cyclic AMP 30 , IL-1β 28 , TNFα 28 , TGFβ1 28 , Wnt/β-catenin 36 , Sox7 37 , Sox18 37 , Notch 37 , 38 and microRNAs 28 , 39 - 41 . COUP-TFII’s relevance in human embryonic development finds support from numerous genetic studies. The human COUP-TFII genomic locus locates at 15q26 on chromosome 15. Mutations in the 15q26 region are shown to associate with congenital diaphragmatic hernia 42 - 44 , analogous to the phenotype observed in COUP-TFII conditional knockout mice 20 . In addition, patients with mutations in the COUP-TFII gene body and a subpopulation of patients with 15q26 mutations exhibit cardiac dysmorphogenesis 44 - 46 . Interestingly, kidney abnormalities are also seen in many patients of 15q26 mutations who also had congenital heart defects and/or congenital diaphragmatic hernia 47 . In developing embryos, cell fate specification occurs when progenitor cells proceed to derive various types of terminally differentiated cells. During this process, genomic profiles change drastically to produce designated cellular phenotypes, which require coordinated control by networks of transcription factors and other regulatory mechanisms. COUP-TFII has been shown to be essential for specifying cell fates of fat, bone, muscle and eye progenitors 6 . In the present article, we will summarize the role of COUP-TFII in cell fate decisions of cardiovascular and renal systems.

COUP-TFII serves as a master regulator for cell fate specification in multiple tissues. Numerous studies indicate that COUP-TFII establishes specific cell identities in conjunction with or working through various key transcriptional regulators ( Figure 2 ). Although compositions of COUP-TFII dependent regulatory networks may be context-dependent, control of common important cell fate determinants by COUP-TFII are conserved among tissues. For example, in hearts and blood vessels, COUP-TFII directly suppresses transcription of the Hey2 gene, which is a common regulator for ventricular and arterial fate specification 7 , 57 . Furthermore, COUP-TFs repress Pax2 gene expression in both renal and ocular progenitors, despite going through different effectors 9 , 15 . On the other hand, collaborations between COUP-TFII and other transcription regulators may increase COUP-TFII’s functional diversity. This mechanism has been shown to result in distinct genomic profiles that underlie individual cell fates 93 . Therefore, genome-wide motif analyses in COUP-TFII binding regions and unbiased proteomic analyses of COUP-TFII interacting proteins should facilitate the discovery of COUP-TFII dependent genetic codes that are utilized to determine cell fates. Interestingly, deletion of COUPTFII at the adult stage does not produce overt phenotypes, which suggests that COUPTFII may mainly operate at a permissive window of plasticity for cell fate specification 57 . This notion finds further support in previous studies that fate plasticity is time-sensitive in hearts and blood vessels 94 , 95 . With the emerging cell-based therapeutic strategies in sight, learning from COUP-TFII dependent regulatory mechanisms would help to better understand the time and molecular circuits that define cell fates.