Senior Investigator Research Interests
Branching morphogenesis is a fundamental attribute of many organ systems. The overarching goal of Dr. Mukouyama’s laboratory is to uncover the molecular control of the morphologic processes underlying the patterning of tubular branching networks, particularly the vascular and nervous systems, which share several anatomic and functional characteristics, and are often patterned similarly in peripheral tissues. These characteristics suggest that there is interdependence between these two networks during tissue development and homeostasis. Thus, Dr. Mukouyama is studying neuronal influences on vascular branching patterns and vascular influences on both neuronal guidance and neural stem cell maintenance. His laboratory approaches these problems using a combination of high-resolution whole-mount imaging, molecular manipulations, advanced genetic perturbations, next-generation sequencing, and in vitro organ culture techniques.
By developing a model system of skin vasculature that allows for the direct observation of congruent vascular and neural networks in an anatomically recognizable pattern, Dr. Mukouyama and his colleagues have begun to unravel the neural control of vascular development. Their work has established two distinct developmental mechanisms: nerve-derived VEGF-A controls arterial differentiation, and nerve-derived CXCL12 controls vessel branching and nerve alignment. With a combination of high-resolution whole-mount imaging in vivo Ca2+ indicator mice, they are currently examining what happens to sensory activity in the mutants having neuro-vascular mis-patterning in developmental and pathological situations, including obesity-related nerve disorders. These studies will provide novel conceptual advances in the development and maintenance of functional neurovascular wiring.
Just as nerves influence vascular patterns in the skin, Dr. Mukouyama and his colleagues have also shown that blood vessels influence nerve patterns in the developing heart. Using whole-mount imaging, they demonstrated that sympathetic axons branch alongside large-diameter coronary veins in the sub-epicardial layer of the dorsal ventricular wall prior to innervating final targets such as coronary arteries and cardiomyocytes in the deeper myocardial layer. This novel neuro-vascular association is established by sequential expression of NGF in the subepicardial venous vascular smooth muscle cells (VSMCs) and myocardial arterial VSMCs of coronary vessels. With an ultra-high-frequency non-invasive imaging, they found abnormal cardiac functions such as irregular cardiac rhythms in the mutant embryos lacking autonomic nervous system. They are currently examining what happens to the myocardium development and conduction system architecture in the absence of autonomic nervous system and how autonomic nerves affect cardiomyocyte differentiation and maturation. These studies will provide a mechanistic framework for the functional consequence of sympathetic innervation in the developing heart.
No examination of neurovascular interactions could neglect the brain. The vasculature component of this specialized niche microenvironment helps to retain neural stem cell potential. Dr. Mukouyama and his colleagues have also found that brain vasculature influences unique distributions of embryonic macrophages including parenchymal microglia, as well as non-parenchymal meningeal and perivascular macrophages. They are currently examining how embryonic macrophages regulate radial glial cells, the primary source of cortical neurons in early brain development. These studies will provide insights into understanding the molecular and cellular basis of the neuro-vascular-immune interactions in the developing brain.
These three independent but thematically interrelated projects provide unique opportunities to advance understanding of the dynamic features of both vascular and neuronal branching networks.
A unique expression of hepatic leukemia factor (Hlf) in the cephalic paraxial mesenchyme
Triple RNA whole-mount in situ hybridization chain reaction of E8.5 wild-type embryos revealed the expression of Hlf (green), as well as the paraxial mesoderm markers Pax3 (blue) and Eomes (red) in the cephalic region. Koui et al. showed that Hlf-expressing cephalic paraxial mesenchymal cells contribute to the developing brain vascular cells such as endothelial cells and pericytes (Koui et al. Biol Open 11: bio059510, 2022).
Lymphatic vessels in the postnatal brain meninges.
Using the Prox1-GFP BAC transgenic reporter (green) and immunostaining with antibodies to lymphatic markers LYVE-1 (red) and pan-blood vessel marker PECAM-1 (blue), we have carried out whole-mount imaging of dural lymphatic vasculature at postnatal stages. We have found that between birth – postnatal day 13 (P)13, lymphatic vessels extend alongside dural blood vessels from the side of the skull towards the midline. The image was published in Developmental Dynamics (Izen et al. Dev Dyn 247:741-753, 2018).
Structural interdependence of blood vessels and nerves to form coordinated branching networks
EC, endothelial cells; VSMC, vascular smooth muscle cells. Modified from Fig. 3 in James & Mukouyama. Semin Cell Dev Biol 22: 1019-1027, 2011.
Nerve-artery alignment in developing skin
Sensory nerve (green) align with arteries (red) in the developing mouse skin. The image was published in Cell (Mukouyama et al. : 693-705, 2002).
Chemokine controls nerve-vessel co-patterning
Chemokine receptor expression (red) was restricted to nerve (green)-associated + arteries (blue). The image was published in Developmental Cell (Li et al. Dev Cell 24: 359-371, 2013).
Cardiac axons follow coronary vessels in embryonic heart
Cardiac sympathetic axons (green) follow VSMC-associated large-diameter coronary vessels in the subepicardium of the dorsal ventricular wall of the developing mouse heart. The image was selected as the cover of the issue in Development (Nam et al. Development 140: 1475-1485, 2013).
Intra-neural hemorrhage in the brain: a mouse model of human CCMs
The vascular lumen appears to have ruptured resulting in hemorrhage in conditional mutants as evidenced by erythrocytes (green) outside the vessel enclosure (red). The image was published in Human Molecular Genetics (Cunningham et al. Hum Mol Genet 20: 3198-3206, 2011)
Venous expression of EphB1 in embryonic limb skin vasculature
Whole-mount triple immunofluorescence analysis of forelimb skin of .5 embryos was performed with antibodies to (green) in combination with the pan-endothelial cell marker PECAM-1 (blue) and the vascular smooth muscle cell (VSMC) marker alpha SMA (red). By .5, arterial vessels are densely covered with VSMCs, whereas less or no VSMC coverage occurs in venous vessels. expression is restricted to the venous vasculature. No expression is detectable in the arterial and lymphatic vasculature. The image was published in Developmental Dynamics (Li et al. Dev Dyn 2013).
Whole-mount confocal microscopy reveals that + (blue)/LYVE-1+ (green) lymphatic vessels sprout toward the dorsal midline in .5 mouse dorsal skin. PECAM-1+ blood vessels (red) have reached the midline at this stage. The image was published in Development (James et al. Development 2013).
Gut angiogenesis and neurogenesis
An embryonic day (E)15.5 whole-mount small intestine stained with antibodies to PECAM1 (blood vessels, blue), TuJ1 (neuronal tubulin, neurons, green), and tyrosine hydroxylase (sympathetic neurons, red). Here, the enteric nervous system (ENS, green) can be seen surrounding the full length of the small intestine. Sympathetic nerves (red and green) project along arteries in the mesentery until they reach the intestinal wall. Upon reaching the ENS, these nerves defasciculate and extend throughout the existing enteric nerve plexus. The image was published in Developmental Dynamics (Hatch & Mukouyama, Dev Dyn 2014).
Sema3s-Nrp2 signaling is required for skin lymphangiogenesis
High-resolution whole-mount staining of back skin from E15.5 mutants and control littermate was performed with antibodies to lymphatic markers PROX1 (red) and LYVE1 (green). Sema3f;Sema3g double mutants exhibit increased lymphatic branching (middle image) compared with control littermate (top image). Nrp2 mutants exhibit decreased lymphatic branching and increased lymphatic endothelial cell growth. The image will be published in Biology Open (Uchida et al. Biol Open 2015).
Adult ear skin innervation and vascularization
High resolution whole-mount adult ear skin stained with antibodies to PECAM1 (blood vessels, blue), TuJ1 (neuronal tubulin, neurons, green), and alpha SMA (vascular smooth muscle cells, red). The alignment of Tuj1+ peripheral nerve bundles with large- or middle-diameter blood vessels covered with alpha SMA+ vascular smooth muscle cells. The image was the 8th place in the 2015 Nikon Photomicrography competition.
SV patient with Glenn physiology and APCs who had increased angiogenic activity in vitro
The image will be published in the Journal of Thoracic and Cardiovascular Surgery (Sandeep et al. J Thorac Cardiovasc Surg. 2015).
Some pericytes are of hematopoietic origin in embryonic skin vasculature
We discovered that the developmental sources of dermal pericytes are heterogeneous and a portion of dermal pericytes has a hematopoietic/myeloid origin. Confocal image obtained from fate-mapping experiments using embryo that have pan-hematopoietic Vav-Cre and R26R-EYFP reporter demonstrates that some NG2+ pericytes (red) associated with PECAM-1+ blood vessels (blue) are also EYFP+ (green), sugesting that EYFP+NG2+ pericytes originate from hematopoietic cells. The image was published in Cell Reports (Yamazaki et al. Cell Rep 2017).
Soluble APP negatively regulates NSC growth in culture
Whole-mount immunostaining of SVZ-derived neurospheres with antibodies to a NSC marker Sox2 (red) and a proliferation marker phospho-histone H3 (green). The treatment of soluble amyloid precursor protein (right) suppresses NSC growth in culture, compared to that of control alkaline phosphatase protein (left). The image was published in Development (Sato et al. Development 144: 2730-2736, 2017).
High resolution whole-mount tissue-cleared mouse embryo
High resolution whole-mount tissue-cleared mouse embryo (embryonic day 13.5) stained with antibodies to PECAM1 (blood vessels, blue) and TuJ1 (neuronal tubulin, neurons, green). The image was selected in the top 100 in the 2019 Nikon Small World competition.
High resolution whole-mount tissue-cleared mouse gut
High resolution whole-mount tissue-cleared mouse gut (embryonic day 13.5) stained with antibodies to PECAM1 (blood vessels, blue), TuJ1 (neuronal tubulin, neurons, red) and TH (tyrosine hydroxylase, sympathetic neurons, green). The image was selected in the top 100 in the 2019 Nikon Small World competition.
Whole-mount and ultrasound analysis of mutants lacking autonomic nerves
Whole-mount double immunofluorescence confocal microscopy was performed with the pan-neuronal marker Tuj1 (class III b-tubulin, green) and endothelial cell marker PECAM-1 (red). The Phox2b-/- ventricle has a near complete absence of autonomic neurons. M-mode ultrasound analysis of a Phox2b-/- embryo shows irregular ventricular contraction (arrows) both in rhythm and magnitude of contraction suggesting ectopic atrial or ventricular focus. The images were published in Ultrasound in Medicine & Biology (Mokshagundam et al. Ultrasound Med Biol 47:751-758, 2020).
Coronary Vasculature in Cxcr4 Gain-Of-Function Heart
Whole-mount immunofluorescence confocal microscopy of E15.5 heart was performed with antibodies to pan-endothelial cell marker PECAM-1 (green) and venous and capillary marker EMCN (red) to visualize coronary veins in the dorsal subepicardial layer of the ventricular wall. Endothelial cell-specific Cxcr4 gain-of-function embryo fails to form hierarchical coronary vascular network.
Meet the Team
Yosuke Mukoyama, Ph.D.
Yoh-suke Mukouyama, whose legal name is Yosuke Mukoyama, publishes as Yoh-suke Mukouyama. He received his A.B. in pharmacy studies from Tokyo University of Science and his M.S. and Ph.D. in developmental biology from the University of Tokyo. As a student in the laboratory of Atsushi Miyajima, he studied the properties of embryonic endothelial cells and hematopoietic stem cells (HSCs). He did postdoctoral research in the laboratory of David Anderson at the California Institute of Technology where he demonstrated that peripheral nerves direct arterial organization and formation by secreting VEGF; he also studied the properties of neural stem cells (NSCs). Dr. Mukouyama joined the NHLBI as a tenure-track Investigator in 2006. He also serves on the editorial board of Developmental Dynamics and is a member of the International Society for Stem Cell Research and the North American Vascular Biology Organization.
Krista Gill, B.S.
Wenling Li, Ph.D.
Linda Leatherbury, M.D.
William Kowalski, Ph.D.
Yuta Koui, Ph.D.
Ryo Sato, M.D., Ph.D.
Sara Gonzalez, PhD
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