Yoh-suke Mukouyama

Peripheral Nerve-Derived CXCL12 and VEGF-A Regulate the Patterning of Arterial Vessel Branching in Developing Limb Skin

In developing limb skin, peripheral nerves provide a spatial template that controls the branching pattern and differentiation of arteries. Our previous studies indicate that nerve-derived VEGF-A is required for arterial differentiation but not for nerve-vessel alignment. In this study, we demonstrate that nerve-vessel alignment depends on the activity of Cxcl12-Cxcr4 chemokine signaling. Genetic inactivation of Cxcl12-Cxcr4 signaling perturbs nerve-vessel alignment and abolishes arteriogenesis. Further in vitro assays allow us to uncouple nerve-vessel alignment and arteriogenesis, revealing that nerve-derived Cxcl12 stimulates endothelial cell migration, whereas nerve-derived VEGF-A is responsible for arterial differentiation. These findings suggest a coordinated sequential action in which nerve Cxcl12 functions over a distance to recruit vessels to align with nerves, and subsequent arterial differentiation presumably requires a local action of nerve VEGF-A in the nerve-associated vessels.

Rbfox3-regulated alternative splicing of Numb promotes neuronal differentiation during development

Rbfox3 is required to promote neuronal differentiation of postmitotic neurons through Numb alternative splicing.

TGFβ signaling is required for sprouting lymphangiogenesis during lymphatic network development in the skin

Dermal lymphatic endothelial cells (LECs) emerge from the dorsolateral region of the cardinal veins within the anterior trunk to form an intricate, branched network of lymphatic vessels during embryogenesis. Multiple growth factors and receptors are required for specification and maintenance of LECs, but the mechanisms coordinating LEC movements and morphogenesis to develop three-dimensional lymphatic network architecture are not well understood. Here, we demonstrate in mice that precise LEC sprouting is a key process leading to stereotypical lymphatic network coverage throughout the developing skin, and that transforming growth factor β (TGFβ) signaling is required for LEC sprouting and proper lymphatic network patterning in vivo. We utilized a series of conditional mutants to ablate the TGFβ receptors Tgfbr1 (Alk5) and Tgfbr2 in LECs. To analyze lymphatic defects, we developed a novel, whole-mount embryonic skin imaging technique to visualize sprouting lymphangiogenesis and patterning at the lymphatic network level. Loss of TGFβ signaling in LECs leads to a severe reduction in local lymphangiogenic sprouting, resulting in a significant decrease in global lymphatic network branching complexity within the skin. Our results also demonstrate that TGFβ signaling negatively regulates LEC proliferation during lymphatic network formation. These data suggest a dual role for TGFβ signaling during lymphatic network morphogenesis in the skin, such that it enhances LEC sprouting and branching complexity while attenuating LEC proliferation.

The Molecular Profiles of Neural Stem Cell Niche in the Adult Subventricular Zone

Neural stem cells (NSCs) reside in a unique microenvironment called the neurogenic niche and generate functional new neurons. The neurogenic niche contains several distinct types of cells and interacts with the NSCs in the subventricular zone (SVZ) of the lateral ventricle. While several molecules produced by the niche cells have been identified to regulate adult neurogenesis, a systematic profiling of autocrine/paracrine signaling molecules in the neurogenic regions involved in maintenance, self-renewal, proliferation, and differentiation of NSCs has not been done. We took advantage of the genetic inducible fate mapping system (GIFM) and transgenic mice to isolate the SVZ niche cells including NSCs, transit-amplifying progenitors (TAPs), astrocytes, ependymal cells, and vascular endothelial cells. From the isolated cells and microdissected choroid plexus, we obtained the secretory molecule expression profiling (SMEP) of each cell type using the Signal Sequence Trap method. We identified a total of 151 genes encoding secretory or membrane proteins. In addition, we obtained the potential SMEP of NSCs using cDNA microarray technology. Through the combination of multiple screening approaches, we identified a number of candidate genes with a potential relevance for regulating the NSC behaviors, which provide new insight into the nature of neurogenic niche signals.

Coronary veins determine the pattern of sympathetic innervation in the developing heart

Anatomical congruence of peripheral nerves and blood vessels is well recognized in a variety of tissues. Their physical proximity and similar branching patterns suggest that the development of these networks might be a coordinated process. Here we show that large diameter coronary veins serve as an intermediate template for distal sympathetic axon extension in the subepicardial layer of the dorsal ventricular wall of the developing mouse heart. Vascular smooth muscle cells (VSMCs) associate with large diameter veins during angiogenesis. In vivo and in vitro experiments demonstrate that these cells mediate extension of sympathetic axons via nerve growth factor (NGF). This association enables topological targeting of axons to final targets such as large diameter coronary arteries in the deeper myocardial layer. As axons extend along veins, arterial VSMCs begin to secrete NGF, which allows axons to reach target cells. We propose a sequential mechanism in which initial axon extension in the subepicardium is governed by transient NGF expression by VSMCs as they are recruited to coronary veins; subsequently, VSMCs in the myocardium begin to express NGF as they are recruited by remodeling arteries, attracting axons toward their final targets. The proposed mechanism underlies a distinct, stereotypical pattern of autonomic innervation that is adapted to the complex tissue structure and physiology of the heart.

Nf1 mutation expands an EGFR-dependent peripheral nerve progenitor that confers neurofibroma tumorigenic potential.

Defining growth factor requirements for progenitors facilitates their characterization and amplification. We characterize a peripheral nervous system embryonic dorsal root ganglion progenitor population using in vitro clonal sphere-formation assays. Cells differentiate into glial cells, smooth muscle/fibroblast (SM/Fb)-like cells, and neurons. Genetic and pharmacologic tools revealed that sphere formation requires signaling from the EGFR tyrosine kinase. Nf1 loss of function amplifies this progenitor pool, which becomes hypersensitive to growth factors and confers tumorigenesis. DhhCre;Nf1(fl/fl) mouse neurofibromas contain a progenitor population with similar growth requirements, potential, and marker expression. In humans, NF1 mutation predisposes to benign neurofibromas, incurable peripheral nerve tumors. Prospective identification of human EGFR(+);P75(+) neurofibroma cells enriched EGF-dependent sphere-forming cells. Neurofibroma spheres contain glial-like progenitors that differentiate into neurons and SM/Fb-like cells in vitro and form benign neurofibroma-like lesions in nude mice. We suggest that expansion of an EGFR-expressing early glial progenitor contributes to neurofibroma formation.

Excessive vascular sprouting underlies cerebral hemorrhage in mice lacking αVβ8-TGFβ signaling in the brain.

Vascular development of the central nervous system and blood-brain barrier (BBB) induction are closely linked processes. The role of factors that promote endothelial sprouting and vascular leak, such as vascular endothelial growth factor A, are well described, but the factors that suppress angiogenic sprouting and their impact on the BBB are poorly understood. Here, we show that integrin αVβ8 activates angiosuppressive TGFβ gradients in the brain, which inhibit endothelial cell sprouting. Loss of αVβ8 in the brain or downstream TGFβ1-TGFBR2-ALK5-Smad3 signaling in endothelial cells increases vascular sprouting, branching and proliferation, leading to vascular dysplasia and hemorrhage. Importantly, BBB function in Itgb8 mutants is intact during early stages of vascular dysgenesis before hemorrhage. By contrast, Pdgfbret/ret mice, which exhibit severe BBB disruption and vascular leak due to pericyte deficiency, have comparatively normal vascular morphogenesis and do not exhibit brain hemorrhage. Our data therefore suggest that abnormal vascular sprouting and patterning, not BBB dysfunction, underlie developmental cerebral hemorrhage.

Neuronal action on the developing blood vessel pattern.

The nervous system relies on a highly specialized network of blood vessels for development and neuronal survival. Recent evidence suggests that both the central and peripheral nervous systems (CNS and PNS) employ multiple mechanisms to shape the vascular tree to meet its specific metabolic demands, such as promoting nerve-artery alignment in the PNS or the development the blood brain barrier in the CNS. In this article we discuss how the nervous system directly influences blood vessel patterning resulting in neuro-vascular congruence that is maintained throughout development and in the adult.

Spatiotemporal mapping of vascularization and innervation in the fetal murine intestine

Background: In mice, the intestinal tube develops from the splanchopleure before embryonic day 9.5. Subsequent patterning of nerves and blood vessels is critical for normal digestive function. A hierarchical branching vascular network allows for efficient nutrient absorption, while the complex enteric nervous system regulates intestinal motility as well as secretion, absorption, and blood flow. Despite the well‐recognized significance of these systems, the precise mechanisms by which they develop have not been clearly established in mammals. Results: Using a novel whole‐mount immunohistochemical protocol, we visualize the pattern of intestinal neurovascular development in mice between embryonic day 10.5 and birth. In particular, we focus on the development and remodeling of the enteric vascular plexus, the migration and organization of enteric neural crest‐derived cells, and the integration of peripheral sympathetic nerves with the enteric nervous system. These correlative data lead us to hypothesize a functional interaction between migrating neural crest‐derived cells and endothelial cells of the primary capillary plexus, as well as a subsequent interaction between developing peripheral autonomic nerves and differentiated neural crest‐derived cells. Conclusions: These studies provide useful anatomical data for continuing investigations on the functional mechanisms underlying intestinal organogenesis. Developmental Dynamics 244:56–68, 2015. Published 2014. This article is a U.S. Government work and is in the public domain in the USA

Conditional deletion of Ccm2 causes hemorrhage in the adult brain: a mouse model of human cerebral cavernous malformations

Cerebral cavernous malformations (CCM) are irregularly shaped and enlarged capillaries in the brain that are prone to hemorrhage, resulting in headaches, seizures, strokes and even death in patients. The disease affects up to 0.5% of the population and the inherited form has been linked to mutations in one of three genetic loci, CCM1, CCM2 and CCM3. To understand the pathophysiology underlying the vascular lesions in CCM, it is critical to develop a reproducible mouse genetic model of this disease. Here, we report that limited conditional ablation of Ccm2 in young adult mice induces observable neurological dysfunction and reproducibly results in brain hemorrhages whose appearance is highly reminiscent of the lesions observed in human CCM patients. We first demonstrate that conventional or endothelial-specific deletion of Ccm2 leads to fatal cardiovascular defects during embryogenesis, including insufficient vascular lumen formation as well as defective arteriogenesis and heart malformation. These findings confirm and extend prior studies. We then demonstrate that the inducible deletion of Ccm2 in adult mice recapitulates the CCM-like brain lesions in humans; the lesions display disrupted vascular lumens, enlarged capillary cavities, loss of proper neuro-vascular associations and an inflammatory reaction. The CCM lesions also exhibit damaged neuronal architecture, the likely cause of neurologic defects, such as ataxia and seizure. These mice represent the first CCM2 animal model for CCM and should provide the means to elucidate disease mechanisms and evaluate therapeutic strategies for human CCM.