Stem Cell and Neurovascular Research

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).

EC, endothelial cells; VSMC, vascular smooth muscle cells. Modified from Fig. 3 in James & Mukouyama. Semin Cell Dev Biol 22: 1019-1027, 2011.

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 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 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).

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)

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).

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).

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).

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.

The image will be published in the Journal of Thoracic and Cardiovascular Surgery (Sandeep et al. J Thorac Cardiovasc Surg. 2015).

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).

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 (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 (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 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).

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.