Whole-Mount Confocal Microscopy for Vascular Branching Morphogenesis

We explored the relationship in chick embryos between somitogenesis and the onset of somite myogenesis by immunodetection of the muscle-specific intermediate filament protein desmin. Early somite desmin expression was detected by whole-mount in situ confocal microscopy. No detectable somite desmin was observed in embryos of 15 somites (Stage 12) or younger. In embryos having between 16 and 26 somites (Stages 12-15), desmin could be detected in somites positioned increasingly more caudal in the embryo. Finally, in embryos of 27 somites (Stage 16) and older, somite desmin expression was consistently present in all but the caudal-most six somites. Although the rate of somite formation is fairly constant, the rate of observed somite desmin expression progressing caudally in the embryo is greater initially than the rate of segmentation. After an embryo has formed about 27 somites, the rate of desmin appearance parallels the rate of segmentation at a distance of about six somites. This result suggests that very early somite myogenesis is not linked to somitogenesis.

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Identification and Characterization of Novel Perivascular Adventitial Cells in the Whole Mount Mesenteric Branch Artery Using Immunofluorescent Staining and Scanning Confocal Microscopy Imaging

International Journal of Cell Biology ◽

10.1155/2012/172746 ◽

2012 ◽

Vol 2012 ◽

pp. 1-6

Author(s):

Chandra Somasundaram ◽

Rahul K. Nath ◽

Richard D. Bukoski ◽

Debra I. Diz

Keyword(s):

Confocal Microscopy ◽

Neurite Growth ◽

Immunofluorescent Staining ◽

Neural Cells ◽

Microscopy Imaging ◽

Stress Injury ◽

Scanning Confocal Microscopy ◽

Adult Rat ◽

Calcium Sensing ◽

Whole Mount

A novel perivascular adventitial cell termed, adventitial neuronal somata (ANNIES) expressing the neural cell adhesion molecule (NCAM) and the vasodilator neuropeptide, calcitonin gene-related peptide (CGRP), exists in the adult rat mesenteric branch artery (MBA) in situ. In addition, we have previously shown that ANNIES coexpress CGRP and NCAM. We now show that ANNIES express the neurite growth marker, growth associated protein-43(Gap-43), palladin, and the calcium sensing receptor (CaSR), that senses changes in extracellular Ca(2+) and participates in vasodilator mechanisms. Thus, a previously characterized vasodilator, calcium sensing autocrine/paracrine system, exists in the perivascular adventitia associated with neural-vascular interface. Images of the whole mount MBA segments were analyzed under scanning confocal microscopy. Confocal analysis showed that the Gap-43, CaSR, and palladin were present in ANNIES about 37 ± 4%, 94 ± 6%, and 80 ± 10% respectively, comparable to CGRP (100%). Immunoblots from MBA confirmed the presence of Gap-43 (48 kD), NCAM (120 and 140 kD), and palladin (90–92 and 140 kD). In summary, CGRP, and NCAM-containing neural cells in the perivascular adventitia also express palladin and CaSR, and coexpress Gap-43 which may participate in response to stress/injury and vasodilator mechanisms as part of a perivascular sensory neural network.

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Retinal myeloid cells regulate tip cell selection and vascular branching morphogenesis via Notch ligand Delta-like 1

Scientific Reports ◽

10.1038/s41598-019-46308-3 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 2

Author(s):

Fabian Haupt ◽

Kashyap Krishnasamy ◽

L. Christian Napp ◽

Michael Augustynik ◽

Anne Limbourg ◽

...

Keyword(s):

Myeloid Cells ◽

Branching Morphogenesis ◽

Cell Selection ◽

Notch Ligand ◽

Tip Cell ◽

Vascular Branching

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Confocal microscopy of whole mount embryonic cartilage: Intracellular localization of F-actin, chick prolyl hydroxylase and type II collagen mRNA

Micron ◽

10.1016/0968-4328(93)90036-z ◽

1993 ◽

Vol 24 (6) ◽

pp. 587-594 ◽

Cited By ~ 12

Author(s):

Michelle S. Hirsch ◽

Kathy Kay Hartford Svoboda

Keyword(s):

Confocal Microscopy ◽

Intracellular Localization ◽

Type Ii Collagen ◽

Prolyl Hydroxylase ◽

Type Ii ◽

Collagen Mrna ◽

Whole Mount ◽

Embryonic Cartilage

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Cell influx and contractile actomyosin force drive mammary bud growth and invagination

The Journal of Cell Biology ◽

10.1083/jcb.202008062 ◽

2021 ◽

Vol 220 (8) ◽

Author(s):

Ewelina Trela ◽

Qiang Lan ◽

Satu-Marja Myllymäki ◽

Clémentine Villeneuve ◽

Riitta Lindström ◽

...

Keyword(s):

Mammary Gland ◽

Confocal Microscopy ◽

Branching Morphogenesis ◽

Early Growth ◽

Surface Epithelium ◽

Nonmuscle Myosin ◽

Cellular Mechanisms ◽

Surface Ectoderm ◽

Conditional Deletion ◽

Bud Growth

The mammary gland develops from the surface ectoderm during embryogenesis and proceeds through morphological phases defined as placode, hillock, bud, and bulb stages followed by branching morphogenesis. During this early morphogenesis, the mammary bud undergoes an invagination process where the thickened bud initially protrudes above the surface epithelium and then transforms to a bulb and sinks into the underlying mesenchyme. The signaling pathways regulating the early morphogenetic steps have been identified to some extent, but the underlying cellular mechanisms remain ill defined. Here, we use 3D and 4D confocal microscopy to show that the early growth of the mammary rudiment is accomplished by migration-driven cell influx, with minor contributions of cell hypertrophy and proliferation. We delineate a hitherto undescribed invagination mechanism driven by thin, elongated keratinocytes—ring cells—that form a contractile rim around the mammary bud and likely exert force via the actomyosin network. Furthermore, we show that conditional deletion of nonmuscle myosin IIA (NMIIA) impairs invagination, resulting in abnormal mammary bud shape.

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Ott1 (Rbm15) Is Essential for Placental Vascular Branching Morphogenesis and Embryonic Development of the Heart and Spleen

Molecular and Cellular Biology ◽

10.1128/mcb.00370-08 ◽

2008 ◽

Vol 29 (2) ◽

pp. 333-341 ◽

Cited By ~ 27

Author(s):

Glen D. Raffel ◽

Gerald C. Chu ◽

Jonathan L. Jesneck ◽

Dana E. Cullen ◽

Roderick T. Bronson ◽

...

Keyword(s):

Transcriptional Activation ◽

Cardiac Development ◽

Germ Line ◽

Global Gene Expression ◽

Branching Morphogenesis ◽

Conditional Allele ◽

Recognition Motifs ◽

Regulatory Effects ◽

Incomplete Closure ◽

Vascular Branching

ABSTRACT The infant leukemia-associated gene Ott1 (Rbm15) has broad regulatory effects within murine hematopoiesis. However, germ line Ott1 deletion results in fetal demise prior to embryonic day 10.5, indicating additional developmental requirements for Ott1. The spen gene family, to which Ott1 belongs, has a transcriptional activation/repression domain and RNA recognition motifs and has a significant role in the development of the head and thorax in Drosophila melanogaster. Early Ott1-deficient embryos show growth retardation and incomplete closure of the notochord. Further analysis demonstrated placental defects in the spongiotrophoblast and syncytiotrophoblast layers, resulting in an arrest of vascular branching morphogenesis. The rescue of the placental defect using a conditional allele with a trophoblast-sparing cre transgene allowed embryos to form a normal placenta and survive gestation. This outcome showed that the process of vascular branching morphogenesis in Ott1-deficient animals was regulated by the trophoblast compartment rather than the fetal vasculature. Mice surviving to term manifested hyposplenia and abnormal cardiac development. Analysis of global gene expression of Ott1-deficient embryonic hearts showed an enrichment of hypoxia-related genes and a significant alteration of several candidate genes critical for cardiac development. Thus, Ott1-dependent pathways, in addition to being implicated in leukemogenesis, may also be important for the pathogenesis of placental insufficiency and cardiac malformations.

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