Post-endocytic sorting of Plexin-D1 controls signal transduction and development of axonal and vascular circuits.

Local endocytic events involving receptors for axon guidance cues play a central role in controlling growth cone behaviour. Yet, little is known about the fate of internalized receptors, and whether the sorting events directing them to distinct endosomal pathways control guidance decisions. Here, we show that the receptor Plexin-D1 contains a sorting motif that interacts with the adaptor protein GIPC1 to facilitate transport to recycling endosomes. This sorting process promotes colocalization of Plexin-D1 with vesicular pools of active R-ras, leading to its inactivation. In the absence of interaction with GIPC1, missorting of Plexin-D1 results in loss of signalling activity. Consequently, Gipc1 mutant mice show specific defects in axonal projections, as well as vascular structures, that rely on Plexin-D1 signalling for their development. Thus, intracellular sorting steps that occur after receptor internalization by endocytosis provide a critical level of control of cellular responses to guidance signals.

Chemokine-coupled ß2 integrin-induced macrophage Rac2-Myosin IIA interaction regulates VEGF-A mRNA stability and arteriogenesis.

Myeloid cells are important contributors to arteriogenesis, but their key molecular triggers and cellular effectors are largely unknown. We report, in inflammatory monocytes, that the combination of chemokine receptor (CCR2) and adhesion receptor (ß2 integrin) engagement leads to an interaction between activated Rac2 and Myosin 9 (Myh9), the heavy chain of Myosin IIA, resulting in augmented vascular endothelial growth factor A (VEGF-A) expression and induction of arteriogenesis. In human monocytes, CCL2 stimulation coupled to ICAM-1 adhesion led to rapid nuclear-to-cytosolic translocation of the RNA-binding protein HuR. This activation of HuR and its stabilization of VEGF-A mRNA were Rac2-dependent, and proteomic analysis for Rac2 interactors identified the 226 kD protein Myh9. The level of induced Rac2-Myh9 interaction strongly correlated with the degree of HuR translocation. CCL2-coupled ICAM-1 adhesion-driven HuR translocation and consequent VEGF-A mRNA stabilization were absent in Myh9(-/-) macrophages. Macrophage VEGF-A production, ischemic tissue VEGF-A levels, and flow recovery to hind limb ischemia were impaired in myeloid-specific Myh9(-/-) mice, despite preserved macrophage recruitment to the ischemic muscle. Micro-CT arteriography determined the impairment to be defective induced arteriogenesis, whereas developmental vasculogenesis was unaffected. These results place the macrophage at the center of ischemia-induced arteriogenesis, and they establish a novel role for Myosin IIA in signal transduction events modulating VEGF-A expression in tissue.

The H2.0-like homeobox transcription factor modulates yolk sac vascular remodeling in mouse embryos.

OBJECTIVE: The H2.0-like homeobox transcription factor (HLX) plays an essential role in visceral organogenesis in mice and has been shown to regulate angiogenic sprouting in vitro and in zebrafish embryos. We therefore examined the role of HLX in vascular development in mouse and avian embryos. APPROACH AND RESULTS: In situ hybridization showed that Hlx is expressed in a subset of sprouting blood vessels in postnatal mouse retinas and embryos. Hlx expression was conserved in quail embryos and upregulated in blood vessels at the onset of circulation. In vitro assays showed that Hlx is dynamically regulated by growth factors and shear stress alterations. Proangiogenic vascular endothelial growth factor induces Hlx expression in cultured endothelial cells, whereas signals that induce stalk cell identity lead to a reduction in Hlx expression. HLX was also downregulated in embryos in which flow was ablated, whereas injection of a starch solution, which increases blood viscosity and therefore shear stress, causes an upregulation in HLX. HLX knockdown in vitro resulted in a reduction in tip cell marker expression and in reduced angiogenic sprouting, but Hlx(-/-) embryos showed no defect in vascular sprouting at E8.5, E9.5, or E11.5 in vivo. Vascular remodeling of the capillary plexus was altered in Hlx(-/-) embryos, with a modestly enlarged venous plexus and reduction of the arterial plexus. CONCLUSIONS: Our findings indicate not only that Hlx regulates sprouting in vitro, but that its role in sprouting is nonessential in vivo. We find HLX is regulated by shear stress and a subtle defect in vascular remodeling is present in knockout embryos.

Endothelial cell-dependent regulation of arteriogenesis.

RATIONALE: Arteriogenesis is the process of formation of arterial conduits. Its promotion is an attractive therapeutic strategy in occlusive atherosclerotic diseases. Despite the functional and clinical importance of arteriogenesis, the biology of the process is poorly understood. Synectin, a gene previously implicated in the regulation of vascular endothelial cell growth factor signaling, offers a unique opportunity to determine relative contributions of various cell types to arteriogenesis. OBJECTIVE: We investigated the cell-autonomous effects of a synectin knockout in arterial morphogenesis. METHODS AND RESULTS: A floxed synectin knockin mouse line was crossbred with endothelial-specific (Tie2, Cdh5, Pdgfb) and smooth muscle myosin heavy chain-specific Cre driver mouse lines to produce cell type-specific deletions. Ablation of synectin expression in endothelial, but not smooth muscle cells resulted in the presence of developmental arterial morphogenetic defects (smaller size of the arterial tree, reduced number of arterial branches and collaterals) and impaired arteriogenesis in adult mice. CONCLUSIONS: Synectin modulates developmental and adult arteriogenesis in an endothelial cell-autonomous fashion. These findings show for the first time that endothelial cells are central to both developmental and adult arteriogenesis and provide a model for future studies of factors involved in this process.

Endothelial RAF1/ERK activation regulates arterial morphogenesis.

Arterial morphogenesis is one of the most critical events during embryonic vascular development. Although arterial fate specification is mainly controlled by the Notch signaling pathway, arterial-venous patterning is modulated by a number of guidance factors. How these pathways are regulated is still largely unknown. Here, we demonstrate that endothelial activation of RAF1/extracellular signal-regulated kinase (ERK) pathway regulates arterial morphogenesis and arterial-venous patterning via Δ/Notch and semaphorin signaling. Introduction of a single amino acid RAF1 mutant (RAF1 Ser259Ala), which renders it resistant to inhibition by phosphorylation, into endothelial cells in vitro induced expression of virtually the entire embryonic arteriogenic program and activated semaphorin 6A-dependent endothelial cell-cell repulsion. In vivo, endothelial-specific expression of RAF1(S259A) during development induced extensive arterial morphogenesis both in the yolk sac and the embryo proper and disrupted arterial-venous patterning. Our results suggest that endothelial ERK signaling is critical for both arteriogenesis and arterial-venous patterning and that RAF1 Ser(259) phosphorylation plays a critical role in preventing unopposed ERK activation.

Bmp2 is required for migration but not for induction of neural crest cells in the mouse.

Bone morphogenetic protein (BMP) signaling is essential for neural crest development in several vertebrates. Genetic experiments in the mouse have shown that Bmp2 is essential for the genesis of migratory neural crest cells. Using several markers and a transgenic reporter approach, we now show that neural crest cells are induced in Bmp2 null mutant embryos, but that these cells fail to migrate out of the neural tube. The absence of migratory neural crest cells in these mutants is not due to their elimination by cell death. The neuroectoderm of Bmp2-/- embryos fail to close and create abnormal folds both along the anterior-posterior and medio-lateral axes, which are associated with an apparent medio-lateral expansion of the neural tube. Finally, our data suggest that the molecular cascade downstream of BMP signaling in early neural crest development may be different in mouse and avian embryos.

Tbx1 is required for proper neural crest migration and to stabilize spatial patterns during middle and inner ear development.

Tbx1 belongs to the family of T-box containing transcription factors. In humans, TBX1 is implicated in the etiology of the DiGeorge syndrome. Inactivation of the Tbx1 gene in mice produces a variety of malformations including abnormal branching of the heart outflow tract, deficiencies in the branchial arch derivatives, agenesis of pharyngeal glands and abnormal development of the auditory system. We analyze here the middle and inner ear phenotypes of the Tbx1 null mice. The middle ear is strongly affected. Its skeletal components are malformed to varying degrees, some being slightly hypoplastic and others completely absent. However, a seemingly normal-looking tympanic membrane can still be recognized. Middle ear anomalies are associated with other skeletal deficiencies in the branchial arch-derived skeleton. These phenotypes derive from a combination of the failure of the posterior branchial arches to develop and the misrouting of neural crest cells. The inner ears of Tbx1(-/-) animals are hypoplastic. No vestibular or cochlear structures are detectable, but the endolymphatic duct, the cochleovestibular ganglia and residual sensory patches are still identifiable. Molecular analyses revealed a seemingly normal spatial distribution of a variety of patterning markers in the otic vesicles of Tbx1 null mutants at E9.0. However, 1 day later, several of these markers presented altered domains of expression in the otocysts of these mutant embryos, suggesting that Tbx1 is not required for the establishment of spatial patterns in the otocyst, but rather for their maintenance. The inability of the Tbx1(-/-) embryos to keep properly segregated functional domains in the otocyst is likely the cause of the strong inner ear phenotypes observed in these mutants.