Tsutomu Kume

The forkhead/winged helix gene Mf1 is disrupted in the pleiotropic mouse mutation congenital hydrocephalus.

Mf1 encodes a forkhead/winged helix transcription factor expressed in many embryonic tissues, including prechondrogenic mesenchyme, periocular mesenchyme, meninges, endothelial cells, and kidney. Homozygous null Mf1lacZ mice die at birth with hydrocephalus, eye defects, and multiple skeletal abnormalities identical to those of the classical mutant, congenital hydrocephalus. We show that congenital hydrocephalus involves a point mutation in Mf1, generating a truncated protein lacking the DNA-binding domain. Mesenchyme cells from Mf1lacZ embryos differentiate poorly into cartilage in micromass culture and do not respond to added BMP2 and TGFbeta1. The differentiation of arachnoid cells in the mutant meninges is also abnormal. The human Mf1 homolog FREAC3 is a candidate gene for ocular dysgenesis and glaucoma mapping to chromosome 6p25-pter, and deletions of this region are associated with multiple developmental disorders, including hydrocephaly and eye defects.

Retinoic acid from the meninges regulates cortical neuron generation

Extrinsic signals controlling generation of neocortical neurons during embryonic life have been difficult to identify. In this study we demonstrate that the dorsal forebrain meninges communicate with the adjacent radial glial endfeet and influence cortical development. We took advantage of Foxc1 mutant mice with defects in forebrain meningeal formation. Foxc1 dosage and loss of meninges correlated with a dramatic reduction in both neuron and intermediate progenitor production and elongation of the neuroepithelium. Several types of experiments demonstrate that retinoic acid (RA) is the key component of this secreted activity. In addition, Rdh10- and Raldh2-expressing cells in the dorsal meninges were either reduced or absent in the Foxc1 mutants, and Rdh10 mutants had a cortical phenotype similar to the Foxc1 null mutants. Lastly, in utero RA treatment rescued the cortical phenotype in Foxc1 mutants. These results establish RA as a potent, meningeal-derived cue required for successful corticogenesis.Extrinsic signals controlling generation of neocortical neurons during embryonic life have been difficult to identify. In this study we demonstrate that the dorsal forebrain meninges communicate with the adjacent radial glial endfeet and influence cortical development. We took advantage of Foxc1 mutant mice with defects in forebrain meningeal formation. Foxc1 dosage and loss of meninges correlated with a dramatic reduction in both neuron and intermediate progenitor production and elongation of the neuroepithelium. Several types of experiments demonstrate that retinoic acid (RA) is the key component of this secreted activity. In addition, Rdh10- and Raldh2-expressing cells in the dorsal meninges were either reduced or absent in the Foxc1 mutants, and Rdh10 mutants had a cortical phenotype similar to the Foxc1 null mutants. Lastly, in utero RA treatment rescued the cortical phenotype in Foxc1 mutants. These results establish RA as a potent, meningeal-derived cue required for successful corticogenesis.

Foxc Transcription Factors Directly Regulate Dll4 and Hey2 Expression by Interacting with the VEGF-Notch Signaling Pathways in Endothelial Cells

Background Recent studies have shown that in the developing embryo, arterial and venous identity is established by genetic mechanisms before circulation begins. Vascular endothelial growth factor (VEGF) signaling and its downstream Notch pathway play critical roles in arterial cell fate determination. We have recently shown that Foxc1 and Foxc2, two closely related Fox transcription factors, are essential for arterial cell specification during development by directly inducing the transcription of Delta-like 4 (Dll4), a ligand for Notch receptors. However, the basic mechanisms whereby the VEGF and Notch signaling pathways control transcriptional regulation of arterial-specific genes have yet to be elucidated. Methodologies/Principal Findings In the current study, we examined whether and how Foxc transcription factors are involved in VEGF and Notch signaling in induction of Dll4 as well as the Notch target gene Hey2 in endothelial cells. We found that Foxc1 and Foxc2 directly activate the Hey2 promoter via Foxc binding elements. Significantly, Foxc2 physically and functionally interacts with a Notch transcriptional activation complex containing Su(H) and Notch intracellular domain to induce Hey2 promoter activity. Moreover, activation of the Dll4 and Hey2 promoters is induced by VEGF in conjunction with either Foxc1 or Foxc2 more than by either component alone. VEGF-activated PI3K and ERK intracellular pathways modulate the transcriptional activity of Foxc proteins in Dll4 and Hey2 induction. Conclusions/Significance Our new findings demonstrate that Foxc transcriptional factors interact with VEGF and Notch signaling to regulate arterial gene expression in multiple steps of the VEGF-Dll4-Notch-Hey2 signaling pathway.

Foxc1 is a critical regulator of haematopoietic stem/progenitor cell niche formation

Haematopoietic stem and progenitor cells are maintained by special microenvironments known as niches in bone marrow. Many studies have identified diverse candidate cells that constitute niches for haematopoietic stem cells in the marrow, including osteoblasts, endothelial cells, Schwann cells, α-smooth muscle actin-expressing macrophages and mesenchymal progenitors such as CXC chemokine ligand (CXCL)12-abundant reticular (CAR) cells, stem cell factor-expressing cells, nestin-expressing cells and platelet-derived growth factor receptor-α (PDGFR-α)+Sca-1+CD45−Ter119− (PαS) cells. However, the molecular basis of the formation of the niches remains unclear. Here we find that the transcription factor Foxc1 is preferentially expressed in the adipo-osteogenic progenitor CAR cells essential for haematopoietic stem and progenitor cell maintenance in vivo in the developing and adult bone marrow. When Foxc1 was deleted in all marrow mesenchymal cells or CAR cells, from embryogenesis onwards, osteoblasts appeared normal, but haematopoietic stem and progenitor cells were markedly reduced and marrow cavities were occupied by adipocytes (yellow adipose marrow) with reduced CAR cells. Inducible deletion of Foxc1 in adult mice depleted haematopoietic stem and progenitor cells and reduced CXCL12 and stem cell factor expression in CAR cells but did not induce a change to yellow marrow. These data suggest a role for Foxc1 in inhibiting adipogenic processes in CAR progenitors. Foxc1 might also promote CAR cell development, upregulating CXCL12 and stem cell factor expression. This study identifies Foxc1 as a specific transcriptional regulator essential for development and maintenance of the mesenchymal niches for haematopoietic stem and progenitor cells.

Angiopoietin-1/Tie2 signal augments basal Notch signal controlling vascular quiescence by inducing delta-like 4 expression through AKT-mediated activation of β-catenin

Angiopoietin-1 (Ang1) regulates both vascular quiescence and angiogenesis through the receptor tyrosine kinase Tie2. We and another group previously showed that Ang1 and Tie2 form distinct signaling complexes at cell-cell and cell-matrix contacts. We further demonstrated that the former up-regulates Notch ligand delta-like 4 (Dll4) only in the presence of cell-cell contacts. Because Dll4/Notch signal restricts sprouting angiogenesis and promotes vascular stabilization, we investigated the mechanism of how the Ang1/Tie2 signal induces Dll4 expression to clarify the role of the Dll4/Notch signal in Ang1/Tie2 signal-mediated vascular quiescence. Under confluent endothelial cells, the basal Notch signal was observed. Ang1, moreover, induced Dll4 expression and production of the Notch intracellular domain (NICD). Ang1 stimulated transcriptional activity of β-catenin through phosphoinositide 3-kinase (PI3K)/AKT-mediated phosphorylation of glycogen synthase kinase 3β (GSK3β). Correspondingly, the GSK3β inhibitor up-regulated Dll4, whereas depletion of β-catenin by siRNA blocked Ang1-induced Dll4 expression, indicating the indispensability of β-catenin in Ang1-mediated up-regulation of Dll4. In addition, Dll4 expression by the GSK3β inhibitor was only observed in confluent cells, and was impeded by DAPT, a γ-secretase inhibitor, implying requirement of the Notch signal in β-catenin-dependent Dll4 expression. Consistently, we found that either Ang1 or NICD up-regulates Dll4 through the RBP-J binding site within intron 3 of the DLL4 gene and that β-catenin forms a complex with NICD/RBP-J to enhance Dll4 expression. Ang1 induced the deposition of extracellular matrix that is preferable for basement membrane formation through Dll4/Notch signaling. Collectively, the Ang1/Tie2 signal potentiates basal Notch signal controlling vascular quiescence by up-regulating Dll4 through AKT-mediated activation of β-catenin.

Novel insights into the differential functions of Notch ligands in vascular formation

The Notch signaling pathway is a critical component of vascular formation and morphogenesis in both development and disease. Compelling evidence indicates that Notch signaling is required for the induction of arterial-cell fate during development and for the selection of endothelial tip and stalk cells during sprouting angiogenesis. In mammals, two of the four Notch receptors (Notch1 and Notch4) and three of the five Notch ligands (Jagged1, Dll1, and Dll4) are predominantly expressed in vascular endothelial cells and are important for many aspects of vascular biology. During arterial cell-fate selection and angiogenesis, the roles of Notch1 and Notch4 are thought to be similar, and the function of Dll4 is well-characterized. However, the molecular mechanisms that determine the functional similarities and differences of Notch ligands in vascular endothelial cells remain largely unknown; consequently, additional research is needed to elucidate the ligand-specific functions and mechanisms associated with Notch activation in the vascular endothelium. Results from recent studies indicate that Dll1 and Dll4 have distinct roles in the specification and maintenance of arterial cell identity, while Dll4 and Jagged1 have opposing functions in tip- and stalk-cell selection during sprouting angiogenesis. This review will focus on the newly discovered, distinct functions of several Notch ligands in the regulation of blood vessel formation and will provide perspectives for future research in the field.

Pax3:Foxc2 Reciprocal Repression in the Somite Modulates Muscular versus Vascular Cell Fate Choice in Multipotent Progenitors

Maintenance of multipotency and how cells exit this state to adopt a specific fate are central questions in stem cell biology. During vertebrate development, multipotent cells of the dorsal somite, the dermomyotome, give rise to different lineages such as vascular smooth and skeletal muscle, regulated by the transcription factors Foxc2 and Pax3, respectively. Here we show reciprocal inhibition between Pax3 and Foxc2 in the mouse embryo. Using both genetic approaches and manipulation of external signals in somite explants, we demonstrate that the Pax3:Foxc2 ratio modulates myogenic versus vascular cell fates. This provides insight into how cell fate choices are orchestrated by these lineage genes in the dermomyotome.

Forkhead transcription factors regulate expression of the chemokine receptor CXCR4 in endothelial cells and CXCL12-induced cell migration.

Foxc1 and Foxc2 transcription factors are required for vascular development. However, the molecular mechanisms by which Foxc1 and Foxc2 control angiogenesis, the growth of new blood vessels from pre-existing vessels and capillaries, remain unknown. CXC chemokine ligand 12 (CXCL12) and its receptor, CXCR4, are critical for the process of angiogenesis, including the migration and tube formation of endothelial cells. Here we show that Foxc1 and Foxc2 directly induce CXCR4 expression by activating its promoter in endothelial cells. Furthermore, Foxc1-deficient endothelial cells show a significant reduction in CXCR4 expression as well as CXCL12-stimulated migration. Taken together, these results provide novel evidence that Foxc transcription factors are important regulators of the chemotactic motility of endothelial cells through the induction of CXCR4 expression.

The Foxc2 Transcription Factor Regulates Angiogenesis via Induction of Integrin β3 Expression

Forkhead transcription factor Foxc2 is an essential regulator of the cardiovascular system in development and disease. However, the cellular and molecular functions of Foxc2 in vascular endothelial cells are still not fully understood. Here, through gene expression profiling in endothelial cells, we identified molecules associated with cell-extracellular matrix interactions, integrin β3 (Itgb3), integrin β5 (Itgb5), and fibronectin, as downstream targets of Foxc2. We found that Itgb3 expression is directly regulated by Foxc2 through multiple Forkhead-binding elements within two high homology regions in the Itgb3 promoter. Because Itgb3 is known to regulate angiogenesis, we further tested whether Foxc2 is directly involved in angiogenesis by regulating Itgb3 expression by in vitro experiments. Overexpression of Foxc2 significantly enhanced endothelial cell migration and adhesion, whereas this effect was strongly inhibited by Itgb3 neutralization antibody. In accordance with these results, pulmonary microvascular endothelial cells isolated from Foxc2 heterozygous mutant mice showed a marked reduction in Itgb3 expression and cell migration. Finally, ex vivo aortic ring assay to test the sprouting and microvessel formation revealed enhanced microvessel outgrowth by Foxc2 overexpression. Conversely, microvessel outgrowth from aortas of Foxc2 heterozygous mutant mice was reduced. Taken together, these results suggest that Foxc2 directly induces Itgb3 expression and regulates angiogenesis by Itgb3-mediated endothelial cell adhesion and migration.

A lymphatic defect causes ocular hypertension and glaucoma in mice

Glaucoma is a leading cause of blindness, afflicting more than 60 million people worldwide. Increased intraocular pressure (IOP) due to impaired aqueous humor drainage is a major risk factor for the development of glaucoma. Here, we demonstrated that genetic disruption of the angiopoietin/TIE2 (ANGPT/TIE2) signaling pathway results in high IOP, buphthalmos, and classic features of glaucoma, including retinal ganglion degeneration and vision loss. Eyes from mice with induced deletion of Angpt1 and Angpt2 (A1A2Flox(WB) mice) lacked drainage pathways in the corneal limbus, including Schlemms canal and lymphatic capillaries, which share expression of the PROX1, VEGFR3, and FOXC family of transcription factors. VEGFR3 and FOXCs have been linked to lymphatic disorders in patients, and FOXC1 has been linked to glaucoma. In contrast to blood endothelium, in which ANGPT2 is an antagonist of ANGPT1, we have shown that both ligands cooperate to regulate TIE2 in the lymphatic network of the eye. While A1A2Flox(WB) mice developed high IOP and glaucoma, expression of ANGPT1 or ANGPT2 alone was sufficient for ocular drainage. Furthermore, we demonstrated that loss of FOXC2 from lymphatics results in TIE2 downregulation, suggesting a mechanism for ocular defects in patients with FOXC mutations. These data reveal a pathogenetic and molecular basis for glaucoma and demonstrate the importance of angiopoietin ligand cooperation in the lymphatic endothelium.