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Whole-mount Confocal Microscopy for Adult Ear Skin: A Model System to Study Neuro-vascular Branching Morphogenesis and Immune Cell Distribution

Here, we present a protocol of a whole-mount adult ear skin imaging technique to study comprehensive three-dimensional neuro-vascular branching ...

National Institutes of Health

Providence Portland Medical Center

Mural cells (pericytes and vascular smooth muscle cells) are essential for the regulation of vascular networks and maintenance of vascular integrity, but their origins are diverse in different tissues and not known in the organs that arise from the ectoderm, such as skin. Here, we show that tissue-localized myeloid progenitors contribute to pericyte development in embryonic skin vasculature. A series of in vivo fate-mapping experiments indicates that tissue myeloid progenitors differentiate into pericytes. Furthermore, depletion of tissue myeloid cells and their progenitors in PU.1 (also known as Spi1) mutants results in defective pericyte development. Fluorescence-activated cell sorting (FACS)-isolated myeloid cells and their progenitors from embryonic skin differentiate into pericytes in culture. At the molecular level, transforming growth factor-β (TGF-β) induces pericyte differentiation in culture. Furthermore, type 2 TGF-β receptor (Tgfbr2) mutants exhibit deficient pericyte development in skin vasculature. Combined, these data suggest that pericytes differentiate from tissue myeloid progenitors in the skin vasculature through TGF-β signaling.

Tissue Myeloid Progenitors Differentiate into Pericytes through TGF-β Signaling in Developing Skin Vasculature.

Obesity and type 2 diabetes are frequently associated with peripheral neuropathy. Though there are multiple methods for diagnosis and analysis of morphological changes of peripheral nerves and blood vessels, three-dimensional high-resolution imaging is necessary to appreciate the pathogenesis with an anatomically recognizable branching morphogenesis and patterning. Here we established a novel technique for whole-mount imaging of adult mouse ear skin to visualize branching morphogenesis and patterning of peripheral nerves and blood vessels. Whole-mount immunostaining of adult mouse ear skin showed that peripheral sensory and sympathetic nerves align with large-diameter blood vessels. Diet-induced obesity (DIO) mice exhibit defective vascular smooth muscle cells (VSMCs) coverage, while there is no significant change in the amount of peripheral nerves. The leptin receptor-deficient db/db mice, a severe obese and type 2 diabetic mouse model, exhibit defective VSMC coverage and a large increase in the amount of smaller-diameter nerve bundles with myelin sheath and unmyelinated nerve fibers. Interestingly, an increase in the amount of myeloid immune cells was observed in the DIO but not db/db mouse skin. These data suggest that our whole-mount imaging method enables us to investigate the neuro-vascular and neuro-immune phenotypes in the animal models of obesity and diabetes.

Whole-Mount Adult Ear Skin Imaging Reveals Defective Neuro-Vascular Branching Morphogenesis in Obese and Type 2 Diabetic Mouse Models.

Traditionally, the central nervous system (CNS) has been viewed as an immune-privileged environment with no lymphatic vessels. This view was partially overturned by the discovery of lymphatic vessels in the dural membrane that surrounds the brain, in contact with the interior surface of the skull. We here examine the distribution and developmental timing of these lymphatic vessels.

Article	Year
Tissue Myeloid Progenitors Differentiate into Pericytes through TGF-β Signaling in Developing Skin Vasculature. Cell reports\| PubMed ID: 28329690	2017
Whole-Mount Adult Ear Skin Imaging Reveals Defective Neuro-Vascular Branching Morphogenesis in Obese and Type 2 Diabetic Mouse Models. Scientific reports\| PubMed ID: 29323138	2018
Postnatal development of lymphatic vasculature in the brain meninges. Developmental dynamics : an official publication of the American Association of Anatomists\| PubMed ID: 29493038	2018

Tomoko Yamazaki

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