Stem Cell and Neurovascular Research

The overarching goal of the Laboratory of Stem Cell and Neurovascular Research, led by Dr. Yoh-suke Mukouyama, is to uncover the molecular control of the morphologic processes underlying the branching morphogenesis and patterning of the vascular and nervous systems.

Yosuke Mukouyama

Research Interests

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.

Images

Lymphatic vessels in the postnatal brain meninges.
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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
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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
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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
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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
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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
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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
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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).

Skin lymphangiogenesis
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Skin lymphangiogenesis

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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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 Mukouyama

Yosuke Mukoyama, Ph.D.

Senior Investigator

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.

Shravani Vatti

Shravani Vatti

Postbaccalaureate IRTA
Katherine Lipsius

Katherine Lipsius

Postbaccalaureate IRTA
Yuto Koi

Yuta Koui, Ph.D.

Postdoctoral Fellow
Ryo Sato

Ryo Sato, M.D., Ph.D.

Postdoctoral Fellow
Sara Gonzalez

Sara Gonzalez, PhD

Postdoctoral Fellow
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Current Vacancy

Postdoctoral Fellow Position

A postdoctoral fellow position is available in Laboratory of Stem Cell and Neuro-Vascular Biology, Genetics and Developmental…

Alumni

Yutaka Uchida, Ph.D.

Visiting Fellow and Research Fellow
2008 - 2016
Yutaka Uchida, Ph.D. is currently a Research Assistant Professor, Department of Immunology and Cell Biology at Osaka University School of Medicine, Japan.

Nefthi Sandeep, M.D.

NIH-CNMC Clinical Fellow
2013 - 2015
Nefthi Sandeep, M.D. is currently a Pediatric and Fetal Cardiologist at Pediatric Cardiology, Northwest Congenital Heart Care.

Jennifer James, Ph.D.

Postdoctoral IRTA
2009 - 2014
Jennifer James, Ph.D. is currently a Biology teacher at Thomas Jefferson High School for Science and Technology.

Izumi Onitsuka, Ph.D.

Visiting Fellow
2009 - 2014
Izumi Onitsuka, Ph.D. is currently a Postdoctoral Associate, Department of Animal and Avian Sciences at University of Maryland.

Yoshiaki Kubota, M.D., Ph.D.

Guest Researcher
2012 - 2012
Yoshiaki Kubota, M.D., Ph.D. is currently a Professor, Department of Anatomy at Keio University School of Medicine, Japan.

Jingqiong Hu, Ph.D.

Visiting Fellow
2007 - 2009
Jingqiong Hu, Ph.D. is currently a Professor, in Stem Cell Center at Wuhan Union Hospital, Hubei, China.

Xuan Chi, Ph.D.

Postdoctoral IRTA
2008 - 2008
Xuan Chi, Ph.D. is currently a Toxicologist, Office of New Drugs, Center for Drug Evaluation and Research at FDA.

Bon-Nyeo Koo, M.D., Ph.D.

Visiting Fellow
2007 - 2008
Bon-Nyeo Koo, M.D., Ph.D. is currently a Professor in the Department of Anesthesiology at Yonsei University College of Medicine, Korea.

Heesuk Zang, Ph.D.

Visiting Fellow
2006 - 2008
Heesuk Zang, Ph.D. is currently a Partner at Innovation Consulting Services, Inc..

Steven Kim, Ph.D.

Postdoctoral IRTA Fellow
2006 - 2007
Steven Kim, Ph.D. is currently a Business at home.

Kevin Wu

Summer Student from JHU
2016 - 2016
Kevin Wu is currently a Undergraduate student in Chemical and Biomolecular Engineering major at Johns Hopkins University.

Jessica Choi

Postbaccalaureate IRTA
2014 - 2016
Jessica Choi is currently a Graduate Student at Duke University, Master of Science in Global Health.

Mano Soshi

Guest Researcher
2016 - 2016
Mano Soshi is currently a Student at Osaka Medical College, Japan.

David Kim

Guest Researcher from Colgate
2015 - 2015
David Kim is currently a Program Coordinator at The San Francisco Foundation.

Brian Shepherd

Postbaccalaureate IRTA
2013 - 2015
Brian Shepherd is currently a Graduate student at University of Oklahoma Health Sciences Center.

Aleksandra Kazimiera Staniszewska

Guest Research/Student from Aberdeen, U.k.
2015 - 2015
Aleksandra Kazimiera Staniszewska is currently a Academic Foundation Programme Trainnee at Academic Section of Vascular Surgery, Department of Surgery and Cancer, Imperial College London, U.K..

Aparna Sajja

Summer Student from GW
2012 - 2015
Aparna Sajja is currently a completing the accelerated seven-year BA/MD track at The George Washington University.

Tomoki Maetani

Guest Researcher/Student from KU, Japan
2013 - 2013
Tomoki Maetani is currently a Resident Doctor at Kyoto University Hospital, Japan.

Akihiro Kikuya

Guest Researcher/Student from KU, Japan
2013 - 2013
Akihiro Kikuya is currently a Resident Doctor at Tazuke Kofukai Medical Research Institute Kitano Hospital, Japan.

Suzanne Abdelazim

Postbaccalaureate IRTA
2012 - 2013
Suzanne Abdelazim is currently a Medical student at Philadelphia College of Osteopathic Medicine.

Ani Nalbandian

Postbaccalaureate IRTA
2010 - 2012
Ani Nalbandian is currently a Postdoctoral Residency Fellow at Columbia University Medical Center, New York Presbyterian Hospital.

John Hatch

Postbaccalaureate IRTA, Summer student and ARRA-funded Summer Student from UVA
2009 - 2014
John Hatch is currently a Graduate student at Harvard.

Joseph Nam

Postbaccalaureate IRTA
2007 - 2011
Joseph Nam is currently a Pediatric dental speciality residency at St. Barnabas Hospital in Bronx.

Lansdale Henderson

Guest Researcher/Student
2008 - 2010
Lansdale Henderson is currently a Graduate Student at Johns Hopkins School for Advanced International Studies (SAIS), Bologna, Italy.

Matan Setton

HHMI-NIH Research Scholar from Duke Medical School
2009 - 2010
Matan Setton is currently a Fellow at Pediatric Cardiology at Boston Children's Hospital.

Joseph Msefya

BRTP Postbaccalaureate IRTA and BRTP Summer student
2008 - 2010
Joseph Msefya is currently a Platoon Leader; Middle School Science Teacher at MDARNG, HHC 58 EXPED MI BDE; Newport Mill Middle School.

Erin O’Donnell

Postbaccalaureate IRTA
2008 - 2010
Erin O’Donnell is currently a Resident Pediatrics at Cincinnati Children's Hospital Medical Center.

Charity Lee

Summer student from UM
2009 - 2009
Charity Lee is currently a Medical student at University of Miami Miller School of Medicine.

Kosha Soneji

Postbaccalaureate IRTA
2006 - 2008
Kosha Soneji is currently a Obstetrics and Gynecology Physician at Kaiser Permanente, Panorama City.

Michelle Beauregard

Animal Technician
2008 - 2009
Michelle Beauregard is currently a Program Manager at Division of Veterinary Resources at the NIH Animal Center, SoBran Bioscience.

Bernadette Nebus

Animal Technician
2006 - 2008
Bernadette Nebus is currently a Supervisor of Primary Cell Manufacturing at Life Technologies.

Tomoko Yamazaki, Ph.D.

Visiting Fellow
2013 - 2017
Tomoko Yamazaki, Ph.D. is currently a Scientist at Tumor Microenvironment Laboratory, Earle A. Chiles Research Institute, Robert W. Franz Cancer Center, Providence Portland Medical Center.

Yoko Nishinaka-Arai, Ph.D.

Guest Researcher
2017 - 2017
Yoko Nishinaka-Arai, Ph.D. is currently a Postdoctoral Fellow at Department of Clinical Application, Center for iPS Cell Research and Application, Kyoto University, Japan.

Yoshihiko Morisue

Guest Researcher/Student from TU, Japan
2017 - 2018
Yoshihiko Morisue is currently a Medical Student at Tohoku University Medical School, Japan.

Honomi Kawase

Guest Researcher/Student from NCU, Japan
2017 - 2017
Honomi Kawase is currently a Medical Student at Nagoya City University Medical School, Japan.

Iris Garcia-Pak

Postbaccalaureate IRTA
2015 - 2018
Iris Garcia-Pak is currently a MD/PhD Candidate Student at UC San Diego School of Medicine.

Rebecca Izen

Postbaccalaureate IRTA
2015 - 2018
Rebecca Izen is currently a Graduate Student, Developmental and Regenerative Biology, Biological and Biomedical Sciences Program at Harvard Medical School.

Ryan Phelps

Summer Student from UCSF
2018 - 2018
Ryan Phelps is currently a Medical Student at UC San Francisco Medical School.

Aya Kudo

Guest Researcher/Student from KU
2018 - 2018
Aya Kudo is currently a Medical Student at Kumamoto University, School of Medicine, Japan.

Nathan Burns

Postbaccalaureate IRTA
2017 - 2019
Nathan Burns is currently a Graduate Student in the Molecular Biology Program at University of Utah.

Mishal Rao

Special Volunteer/Postbaccalaureate
2018 - 2019
Mishal Rao is currently a Graduate Student in the Integrative Systems Biology Graduate Program at University of Pittsburgh School of Medicine.

Yuya Sato, Ph.D.

Research Fellow
2016 - 2019
Yuya Sato, Ph.D. is currently a Tenure Track Lecturer at Department of Physiology and Cell Biology, Kobe University Graduate School of Medicine, Japan.

Deepa Mokshagundam, M.D.

NIH-CNMC Clinical Fellow
2016 - 2019
Deepa Mokshagundam, M.D. is currently a Advanced Fellow at Pediatric Heart Failure and Heart Transplant, St Louis Children's Hospital, Department of Pediatric Cardiology, One Children's Place, St Louis.

Mitsunori Nomura, Ph.D.

Visiting Fellow
2018 - 2019
Mitsunori Nomura, Ph.D. is currently a Senior Research Fellow at Buck Institute, University of California, San Francisco.

Atsufumi Yoshida

Guest Researcher/Student from KU
2019 - 2019
Atsufumi Yoshida is currently a Medical Student at Kumamoto University, School of Medicine, Japan.

Chang Liu

Visiting Fellow
2015 - 2019
Chang Liu is currently a scientist at Precision Biosciences, Inc. Durham.

Jeffrey Hayashi

Summer Student
Jeffrey Hayashi is currently a undergraduate at University of Portland.