Analysis of Coronary Vessels in Cleared Embryonic Hearts

Ivins, Sarah; Roberts, Catherine; Vernay, Bertrand; Scambler, Peter J.

doi:10.3791/54800

Cited by 8 publications

(13 citation statements)

References 23 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Confocal analysis was performed as described previously [ 17 , 47 ]. Immunolabelled embryos (E9.5–11.5) were dehydrated through a methanol series before clearing with 1 part Benzyl alcohol/ 2 parts benzyl benzoate solution (BABB, Sigma) and examined by epifluorescence on an inverted LSM710 confocal system mounted on an AxioObserver Z1 microscope (Carl Zeiss Ltd, United Kingdom) [ 17 , 47 ].…”

Section: Methodsmentioning

confidence: 99%

Loss of CXCL12/CXCR4 signalling impacts several aspects of cardiovascular development but does not exacerbate Tbx1 haploinsufficiency

Page¹,

Ridge²,

Diaz³

et al. 2018

PLoS ONE

Self Cite

View full text Add to dashboard Cite

The CXCL12-CXCR4 pathway has crucial roles in stem cell homing and maintenance, neuronal guidance, cancer progression, inflammation, remote-conditioning, cell migration and development. Recently, work in chick suggested that signalling via CXCR4 in neural crest cells (NCCs) has a role in the 22q11.2 deletion syndrome (22q11.2DS), a disorder where haploinsufficiency of the transcription factor TBX1 is responsible for the major structural defects. We tested this idea in mouse models. Our analysis of genes with altered expression in Tbx1 mutant mouse models showed down-regulation of Cxcl12 in pharyngeal surface ectoderm and rostral mesoderm, both tissues with the potential to signal to migrating NCCs. Conditional mutagenesis of Tbx1 in the pharyngeal surface ectoderm is associated with hypo/aplasia of the 4th pharyngeal arch artery (PAA) and interruption of the aortic arch type B (IAA-B), the cardiovascular defect most typical of 22q11.2DS. We therefore analysed constitutive mouse mutants of the ligand (CXCL12) and receptor (CXCR4) components of the pathway, in addition to ectodermal conditionals of Cxcl12 and NCC conditionals of Cxcr4. However, none of these typical 22q11.2DS features were detected in constitutively or conditionally mutant embryos. Instead, duplicated carotid arteries were observed, a phenotype recapitulated in Tie-2Cre (endothelial) conditional knock outs of Cxcr4. Previous studies have demonstrated genetic interaction between signalling pathways and Tbx1 haploinsufficiency e.g. FGF, WNT, SMAD-dependent. We therefore tested for possible epistasis between Tbx1 and the CXCL12 signalling axis by examining Tbx1 and Cxcl12 double heterozygotes as well as Tbx1/Cxcl12/Cxcr4 triple heterozygotes, but failed to identify any exacerbation of the Tbx1 haploinsufficient arch artery phenotype. We conclude that CXCL12 signalling via NCC/CXCR4 has no major role in the genesis of the Tbx1 loss of function phenotype. Instead, the pathway has a distinct effect on remodelling of head vessels and interventricular septation mediated via CXCL12 signalling from the pharyngeal surface ectoderm and second heart field to endothelial cells.

show abstract

Section: Methodsmentioning

confidence: 99%

Loss of CXCL12/CXCR4 signalling impacts several aspects of cardiovascular development but does not exacerbate Tbx1 haploinsufficiency

Page¹,

Ridge²,

Diaz³

et al. 2018

PLoS ONE

Self Cite

View full text Add to dashboard Cite

show abstract

“…Moreover, light-sheet microscopy is becoming more and more routinely-used equipment in histology 31 . The preparation of tissues for 3D histochemistry and imaging is relatively fast with a clearing procedure of 1–4 h and immunohistochemical staining for a week, whereas fluorescence signals are well-preserved, and their quenching is limited 9 , 17 . An unwanted effect of BABB clearing is tissue shrinkage.…”

Section: Discussionmentioning

confidence: 99%

“…Opacity of these tissues is mainly caused by lipid bilayers of cell membranes and not by ECM, because these tissues contain relatively small amounts of connective tissue. Clearing of ECM-rich tissues such as skin or gingiva needs an alternative approach by equalizing RIs of the different tissue compartments that are imaged 9 , 17 .…”

Section: Introductionmentioning

confidence: 99%

Three-dimensional histochemistry and imaging of human gingiva

Azaripour

Lagerweij

Scharfbillig

et al. 2018

Sci Rep

View full text Add to dashboard Cite

In the present study, 3D histochemistry and imaging methodology is described for human gingiva to analyze its vascular network. Fifteen human gingiva samples without signs of inflammation were cleared using a mixture of 2-parts benzyl benzoate and 1-part benzyl alcohol (BABB), after being immunofluorescently stained for CD31, marker of endothelial cells to visualize blood vessels in combination with fluorescent DNA dyes. Samples were imaged in 3D with the use of confocal microscopy and light-sheet microscopy and image processing. BABB clearing caused limited tissue shrinkage 13 ± 7% as surface area and 24 ± 1% as volume. Fluorescence remained intact in BABB-cleared gingiva samples and light-sheet microscopy was an excellent tool to image gingivae whereas confocal microscopy was not. Histochemistry on cryostat sections of gingiva samples after 3D imaging validated structures visualized in 3D. Three-dimensional images showed the vascular network in the stroma of gingiva with one capillary loop in each stromal papilla invading into the epithelium. The capillary loops were tortuous with structural irregularities that were not apparent in 2D images. It is concluded that 3D histochemistry and imaging methodology described here is a promising novel approach to study structural aspects of human gingiva in health and disease.

show abstract

“…Although this research was focused on answering key biological questions or development of LSFM setups, instead of method evaluation, a few conclusions from those studies can be drawn. First, BABB was mainly used to clear petite samples such as embryonic hearts . BABB might be also used to clear and image >2.0 mm thick regions of adult mouse heart (Figure c,g), as presented by Smith et al .…”

Section: Application Of Toc For Particular Peripheral Organsmentioning

confidence: 99%

Advances in Ex Situ Tissue Optical Clearing

Matryba

Kaczmarek

Gołąb

2019

Laser & Photonics Reviews

View full text Add to dashboard Cite

Examination of whole organs with subcellular resolution in health, disease, and during development is necessary to decipher their biological complexity. However, until recently, this has been virtually impossible due to the natural opacity of organ tissue. Recent progress in tissue optical clearing (TOC) has overcome this limitation by turning organs into transparent, light-permitting specimens. At least 20 original TOC methods have been developed in less than a decade, which were followed by hundreds of attempts that were aimed at their optimization and practical application. The majority of proof-of-concept studies have focused on the brain. However, it is apparent that TOC might be equally valuable when applied to peripheral organs or even the whole body. The progress in TOC for peripheral organs is delineated in an organ-by-organ fashion and whole-body clearing approaches are discussed. Additionally, physical and optical approaches for TOC affecting the optical properties of the samples and image quality are discussed to explore their advantages, limitations, and future possibilities.

show abstract

Analysis of Coronary Vessels in Cleared Embryonic Hearts

Cited by 8 publications

References 23 publications

Loss of CXCL12/CXCR4 signalling impacts several aspects of cardiovascular development but does not exacerbate Tbx1 haploinsufficiency

Loss of CXCL12/CXCR4 signalling impacts several aspects of cardiovascular development but does not exacerbate Tbx1 haploinsufficiency

Three-dimensional histochemistry and imaging of human gingiva

Advances in Ex Situ Tissue Optical Clearing

Contact Info

Product

Resources

About