Whole-Mount Confocal Microscopy for Vascular Branching Morphogenesis

Mukouyama, Yoh‐suke; James, Jennifer M.; Nam, Joseph; Uchida, Yutaka

doi:10.1007/978-1-61779-523-7_7

Cited by 15 publications

(10 citation statements)

References 5 publications

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“…To improve imaging of the blood islands in mouse embryonic hearts, we used the method described by Mukouyama et al ( 2012 ) with our own modification. In summary, 12.5 dpc hearts were isolated under the stereomicroscope and then the atria were removed carefully using micro-dissecting scissors.…”

Section: Methodsmentioning

confidence: 99%

Vasculogenic and hematopoietic cellular progenitors are scattered within the prenatal mouse heart

Jankowska‐Steifer

Madej

Niderla-Bielińska

et al. 2014

Histochem Cell Biol

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Vasculogenesis and hematopoiesis are co-localized in the embryonic body, but precise phenotypes of the cells contributing to these processes are not defined. The aim of this study was to characterize phenotypic profiles and location of putative vasculogenic and hematopoietic cellular progenitors in the embryonic mouse heart. Confocal microscopy, as well as ultrastructural and stereomicroscopic analyses, was performed on immunohistochemical whole-mount-stained or sectioned hearts at stages 11.5–14 dpc. A FASC analysis was conducted to quantify putative vasculogenic and hematopoietic cells. We found subepicardial blood islands in the form of foci of accumulation of cells belonging to erythroblastic and megakaryocytic lineages at various stages of maturation, exhibiting phenotypes: GATA2+/CD41+, GATA2−/CD41+, GATA2+/CD71−, GATA2−/CD71+, Fli1+/CD71+, Fli1−/CD71+, with a majority of cells expressing the Ter119 antigen, but none of them expressing Flk1. The subepicardium and the outflow tract endothelium were recognized to be the areas where progenitor cells were scattered or adjoining the endothelial cells. These progenitor cells were characterized as possessing the following antigens: CD45+/Fli1+, CD41+/Flk1+, Flk1+/Fli1+. A FACS analysis demonstrated that the CD41/Flk1 double-positive population of cells constituted 2.68 % of total cell population isolated from 12.5 dpc hearts. Vessels and tubules were positive for CD31, Flk1, Fli1, Tie2, including blood islands endothelia. The endocardial wall endothelia were found to function as an anchoring apparatus for megakaryocytes releasing platelets into the cardiac cavities. Phenotypic characteristics of vasculogenic (Flk1+/Fli1+) and hematopoietic (GATA2+/CD71+, CD41+/GATA2+) progenitors, as well as the putative hemogenic endothelium (Flk1+/CD41+) in embryonic mouse hearts, have been presented. Cardiac blood islands, the subepicardium and endothelium of the outflow tract cushions have been defined as areas where these progenitor cells can be found.

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Section: Methodsmentioning

confidence: 99%

Vasculogenic and hematopoietic cellular progenitors are scattered within the prenatal mouse heart

Jankowska‐Steifer

Madej

Niderla-Bielińska

et al. 2014

Histochem Cell Biol

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“…IHC performed on embryonic limb skin was performed according to Mukouyama et al (2012) . Briefly, forelimbs from E14.5 embryos were removed in ice-cold 1× PBS and subsequently transferred to 4% PFA at 4°C overnight.…”

Section: Methodsmentioning

confidence: 99%

A novel reporter allele for monitoringDll4expression within the embryonic and adult mouse

et al. 2018

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Canonical Notch signaling requires the presence of a membrane bound ligand and a corresponding transmembrane Notch receptor. Receptor engagement induces multiple proteolytic cleavage events culminating in the nuclear accumulation of the Notch intracellular domain and its binding to a transcriptional co-factor to mediate gene expression. Notch signaling networks are essential regulators of vascular patterning and angiogenesis, as well as myriad other biological processes. Delta-like 4 (Dll4) encodes the earliest Notch ligand detected in arterial cells, and is enriched in sprouting endothelial tip cells. Dll4 expression has often been inferred by proxy using a lacZ knockin reporter allele. This is problematic, as a single copy of Dll4 is haploinsufficient. Additionally, Notch activity regulates Dll4 transcription, making it unclear whether these reporter lines accurately reflect Dll4 expression. Accordingly, precisely defining Dll4 expression is essential for determining its role in development and disease. To address these limitations, we generated a novel BAC transgenic allele with a nuclear-localized β-galactosidase reporter (Dll4-BAC-nlacZ). Through a comparative analysis, we show the BAC line overcomes previous issues of haploinsufficiency, it recapitulates Dll4 expression in vivo, and allows superior visualization and imaging. As such, this novel Dll4 reporter is an important addition to the growing Notch toolkit.

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“…IHC performed on embryonic limb skin was performed according to Mukouyama et al (Mukouyama et al, 2012). Briefly, forelimbs from E14.5 embryos were removed in ice-cold 1X PBS and subsequently transferred to 4% PFA at 4°C overnight.…”

Section: Immunohistochemistrymentioning

confidence: 99%

A novel reporter allele for monitoringDll4expression within the embryonic and adult mouse

Herman

Rhyner

Devine

et al. 2017

Preprint

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Canonical Notch signaling requires the presence of a membrane bound ligand and a corresponding transmembrane Notch receptor. Receptor engagement induces multiple proteolytic cleavage events culminating in the nuclear accumulation of the Notch intracellular domain and its binding to a transcriptional co-factor to mediate gene expression. Notch signaling networks are essential regulators of vascular patterning and angiogenesis, as well as myriad other biological processes. Delta-like 4 (Dll4) encodes the earliest Notch ligand detected in arterial cells, and is enriched in sprouting endothelial tip cells. Dll4 expression has often been inferred by proxy using a lacZ knockin reporter allele. This is problematic, as a single copy of Dll4 is haploinsufficient. Additionally, Notch activity regulates Dll4 transcription, making it unclear whether these reporter lines accurately reflect Dll4 expression. Accordingly, accurately defining Dll4 expression is essential for determining its role in development and disease. To address these limitations, we generated a novel BAC transgenic allele with a nuclear-localized β-galactosidase reporter (Dll4-BAC-nlacZ). Through a comparative analysis, we show the BAC line overcomes previous issues of haploinsufficiency, it recapitulates Dll4 expression in vivo, and allows superior visualization and imaging. As such, this novel Dll4 reporter is an important addition to the growing Notch toolkit.Summary Statement:We have developed a novel reporter line, free from complicating factors associated with previous alleles, for monitoring Dll4 expression, at a cellular resolution, in the developing and adult mouse.

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Whole-Mount Confocal Microscopy for Vascular Branching Morphogenesis

Cited by 15 publications

References 5 publications

Vasculogenic and hematopoietic cellular progenitors are scattered within the prenatal mouse heart

Vasculogenic and hematopoietic cellular progenitors are scattered within the prenatal mouse heart

A novel reporter allele for monitoringDll4expression within the embryonic and adult mouse

A novel reporter allele for monitoringDll4expression within the embryonic and adult mouse

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