En Face Endocardial Cushion Preparation for Planar Morphogenesis Analysis in Mouse Embryos

Summary

Classically, the endocardium of the mouse embryonic valve primordium has been analyzed using transversal, coronal, or sagittal sections. Our novel approach for en face, two-dimensional imaging of the endocardium at valvulogenic regions allows planar polarity and cell rearrangement analysis of the endocardium during valve development.

Abstract

The study of the cellular and molecular mechanisms underlying the development of the mammalian heart is essential to address human congenital heart disease. The development of the primitive cardiac valves involves the epithelial-to-mesenchymal transition (EMT) of endocardial cells from the atrioventricular canal (AVC) and outflow tract (OFT) regions of the heart in response to local inductive myocardial and endocardial signals. Once the cells delaminate and invade the extracellular matrix (cardiac jelly) located between the endocardium and the myocardium, the primitive endocardial cushions (EC) are formed. This process implies that the endocardium has to fill the gaps left by the delaminated cells and has to reorganize itself to converge (narrow) or extend (lengthen) along an axis. Current research has implicated the planar cell polarity (PCP) pathway in regulating the subcellular localization of the factors involved in this process. Classically, the initial phases of cardiac valve development have been studied in cross-sections of embryonic hearts or in ex vivo AVC or OFT explants cultured on collagen gels. These approaches allow the analysis of apico-basal polarity but do not allow the analysis of cell behavior within the plane of the epithelium or of the morphological changes of migrating cells. Here, we show an experimental approach that allows the visualization of the endocardium at valvulogenic regions as a planar field of cells. This experimental approach provides the opportunity to study PCP, planar topology, and intercellular communication within the endocardium of the OFT and AVC during valve development. Deciphering new cellular mechanisms involved in cardiac valve morphogenesis may contribute to understanding congenital heart disease associated with endocardial cushion defects.

Introduction

The heart is the first functional organ of a mammalian embryo. Around embryonic day (E) 7.5 in mice, bilateral precardiac mesoderm cells form the cardiac crescent in the ventral side¹.The cardiac crescent contains two populations of precardiac cells that include progenitors of the myocardium and the endocardium². Around E8.0, the cardiac precursors fuse in the midline, forming the primitive heart tube consisting of two epithelial tissues, the outer myocardium, and the inner endocardium, which is a specialized endothelium separated by an extracellular matrix named cardiac jelly. Later, at E8.5, the heart tube undergoes rightward looping. The looped heart has different anatomical regions with specific molecular signatures such as the outflow tract (OFT), the ventricles, and the atrio-ventricular canal (AVC)³. Although initially the heart tube expands at its inflow side through the addition of cells⁴, at E9.5, intensive cardiac proliferation results in ballooning of the chambers and establishment of the trabecular network⁵. Valve formation takes place in the AVC (future mitral and tricuspid valves) and in the OFT (future aortic and pulmonary valves).

The endocardium plays crucial roles in valve development. Endocardial cells undergo epithelial-mesenchymal transition (EMT) in the AVC and OFT to form the endocardial cushions, a structure that appears at the onset of valve development. Different signaling pathways activate this process; at E9.5 in mice, NOTCH activated in the endocardium in response to myocardial-derived BMP2 promotes invasive EMT of endocardial cells in the AVC and OFT regions through activation of TGFβ2 and SNAIL (SNAI1), which directly represses the expression of vascular endothelial cadherin (VE-cadherin), a transmembrane component of adherens junctions (AJs)^6,7,8. In the OFT, activation of the endocardium to initiate EMT is mediated by FGF8 and BMP4, whose expression is activated by NOTCH^9,10,11,12.

Progression of EMT involves cellular dynamics as cells change shape, break and remake junctions with their neighbors, delaminate, and begin to migrate¹³. These changes include AJ remodel and gradual disassembling^14,15, planar cell polarity (PCP) signaling, the loss of apico-basal polarity (ABP), apical constriction, and cytoskeletal organization^16,17. ABP refers to the distribution of proteins along the anterior-posterior axis of a cell. In the developing heart, ABP regulation in cardiomyocytes is required for ventricular development¹⁸. PCP refers to a polarized distribution of proteins within cells across the plane of a tissue and regulates cellular distribution; epithelia with a stable geometry are made up of hexagon-shaped cells, where only three cells converge at the vertices^19,20,21,22. Different cellular processes, such as cell division, neighbor exchange, or delamination occurring during epithelial morphogenesis, produce an increase in the number of cells that converge on a vertex and the number of neighboring cells that a given cell has²². These cellular behaviors related to PCP can be regulated by different signaling pathways, actin dynamics, or intracellular trafficking²³.

The data generated studying valve development in mice have been obtained from transversal, coronal, or sagittal sections of E8.5 and E9.5 embryonic hearts, where the endocardium is shown as a line of cells instead of as a field of cells-the endocardium covers the entire inner surface of the heart tube²⁴. Embryonic sections do not allow the analysis of PCP in the endocardium of mouse embryos. Our novel experimental method allows the analysis of endocardial cell distribution, AJ anisotropy, and single-cell shape analysis, as shown in the representative results. This type of data is required for PCP analysis, together with the description of other molecules related to PCP, not shown in this report. Whole-mount immunofluorescence, specific sample preparation and the use of genetically modified mice enable planar polarity analysis in the endocardium at the onset of valve development in mice.

Protocol

Animal studies were approved by the Centro Nacional de Investigaciones Cardiovasculares (CNIC) Animal Experimentation Ethics Committee and by the Community of Madrid (ref. PROEX 155.7/20). All animal procedures conformed to EU Directive 2010/63EU and Recommendation 2007/526/EC regarding the protection of animals used for experimental and other scientific purposes, enacted in Spanish law under Real Decreto 1201/2005.

1. Obtention of AVC and/or OFTs (adapted from Xiao et al.²⁵)

1. Set up crosses in the afternoon between female mTmG and male VE-Cadherin-Cre-ERT/+.
2. The next morning, check for a vaginal plug. If there is a vaginal plug, count it as half a day (E0.5). Depending on the experiment of interest, count 8 days or 9 days.
3. 24 h before dissection, gavage the pregnant female orally with 50 µL of 5 mg/mL 4-OH-Tamoxifen diluted in corn oil.
   ​NOTE: This dose will induce a limited amount of CRE-mediated recombination revealed by the presence of GFP-positive clones. The appropriate dose will have to be determined with each new batch of 4-OH-Tamoxifen.
4. Sacrifice the pregnant female at E8.5 or E9.5 by cervical dislocation.
5. Open the abdomen of the pregnant mice by using scissors and remove the uterus containing the embryos by using scissors and fine forceps.
6. Place the uterus in a Petri dish containing ice-cold 1x phosphate-buffered saline (PBS) with 0.1% Tween-20 (PBT).
7. Remove the individual deciduae from the uterus under a dissecting microscope and using fine forceps.
8. Using fine tweezers, make an incision in the white part of the decidua, which is where the embryo is, remove it gently, avoiding pulling, and transfer the embryos to a new Petri dish with fresh PBT using a P1000 with the tip cut off, leaving a diameter large enough for an embryo to pass through without breaking.
9. For genotyping, take a little piece of the yolk sac (0.5 mm²) or a piece of the tail (from the posterior end, counting 5 to 10 somites toward the head) and digest it in 100 µL of appropriate buffer.
10. Under the fume hood, transfer the embryos to ice-cold 4% paraformaldehyde (4% PFA) in sterile-filtered PBS in a microcentrifuge (2 mL) tube at 4 °C. Fix the embryos from 2 h to overnight (O/N) at 4 °C on a nutator.
11. Under the fume hood, remove the 4% PFA fixative from the microcentrifuge tubes without touching the embryos. Discard the PFA in an appropriate container.
12. Wash the embryos 5x in cold PBT for 10 min each at room temperature (RT).
13. Using fine forceps, remove the heart and transfer it to a new 1.5 mL microcentrifuge tube with fresh PBT.

2. Whole mount immunofluorescence of the mouse embryonic heart

1. Replace PBT with PBSTr (0.4% Triton X-100 in PBS) and wash the samples 3x, 5 min each, at RT on an orbital shaker.
2. Replace PBSTr with blocking solution (PBSTr with 10% heat-inactivated bovine serum). Incubate from 2 h to O/N at 4 °C on an orbital shaker.
3. Replace blocking solution with blocking solution containing primary antibody at the desired concentration (for anti-GFP or anti-VE-Cadherin antibody,use 1:1000 dilution). Incubate O/N on an orbital shaker at 4 °C.
4. After O/N incubation at 4 °C, incubate for 1.5 h at RT on an orbital shaker. This step is very important to increase the signal-to-noise ratio.
5. Remove and keep at 4 °C the primary antibody solution for up to three future experiments (add sodium azide to a final concentration of 0.02% for best conservation).
6. Wash the embryos 3x with PBSTr for 3 min each.
7. Wash the embryos with PBSTr 3x for 30 min each on an orbital shaker at 4 °C.
8. After the last wash, add 1 mL of blocking solution containing 1:1000 fluorescence-conjugated secondary antibody against the primary antibody host species. Add also 1:3000 DAPI to the solution and incubate the embryos O/N on an orbital shaker at 4 °C.
9. After O/N incubation at 4 °C, incubate for 1.5 h at RT on an orbital shaker. This step is very important to increase the signal-to-noise ratio.
10. Wash the embryos 3x with PBSTr for 3 min each.
11. Wash the embryos with PBSTr 3x-5x for 30 min each on an orbital shaker at RT.

3. Mounting the mouse embryonic AVC or OFTs

1. Put the hearts on a Petri dish (35 mm) containing PBS.
2. Using a tungsten wire and fine forceps, isolate the AVC or the OFT, and cut them longitudinally²⁶.
3. Prepare coverslips (22 mm x 22 mm), slides (60 mm x 24 mm), forceps, a P200 pipette with the appropriate tips, paper towel, and any aqueous glycerol-based mounting media for fluorescence, without DAPI.
4. Using a similar approach as in Xiao, C. et al.²⁵, stick two strips of tape on a slide, separated by 0.3-0.6 cm in order to create a 3D room space that will allow the samples to conserve it original shape without squashing them.
5. Carefully, and using the minimum volume of buffer, drop the samples onto the slide between the tape stripes using a cut P200 tip pipette. Use a dissecting microscope to increase the accuracy.
6. Using fine forceps, place the samples with the endocardium facing up (myocardium down on the slide).
7. Remove excess PBS with a paper towel (avoid touching the sample), and then dry the samples for 1-2 min at RT to make the samples stick to the slide.
8. Add 40 µL of aqueous-based mounting media on the tissue.
9. Place the coverslip over the two pieces of tape and lower it slowly onto the tissue with a needle or forceps. Avoid creating air bubbles.
10. Seal the coverslip using one drop of nail polish on each vertex of the coverslip.
11. Once the nail polish is dry, clean the excess mounting media from the sides of the coverslip by using an absorbent paper towel. Avoid moving the coverslip.
12. Add nail polish along the coverslip edges to fully seal it, preventing mounting media evaporation.
13. Using an upright or an inverted confocal microscope, take images at 10x for general view and at 63x with 3x zoom for detailed images.

Results

The data generated using this protocol show that it is possible to perform en face imaging of the endocardium of the AVC. The first objective was to analyze the cell shape of the endocardium during the formation of the valves at a cellular resolution (Figure 1). In order to highlight individual endocardial cells at E9.5, we used two transgenic mouse strains. (1) ROSA^mT/mG is a two-color fluorescent Cre reporter allele (tdTomato/mT and EGFP/mG) w...

Discussion

The endocardium is an epithelial monolayer that covers the entire inner surface of the embryonic heart tube. During valve development, endocardial cells at the prospective valvular regions undergo EMT, thus endocardial cells transform and rearrange their cytoskeleton to delaminate from the endocardium toward the cardiac jelly. We and others have obtained relevant data about valve development in mouse embryos by analyzing transversal sections of E8.5 and E9.5 embryonic hearts, where the endocardium is shown as a row of ce...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
4-OH-Tamoxifen	Sigma Aldrich	H-6278
16 % Paraformaldheyde	Electron Microscopy Sciences	157-10	Dilute to 4% in water
anti-GFP	Aves Labs	FGP-1010
anti-VECadherin	BD Biosciences	555289
Goat anti-Chicken, Alexa Fluor 488	Thermo Fisher Scientific	A-11039
Goat Anti-Mouse Alexa Fluor 647	Jackson ImmunoResearch	115-605-174
DAPI	AppliChem	A4099,0005
Slides Superfrost PLUS	VWR	631-0108	25 mm x 75 mm x 1.0 mm
Triton X-100	Sigma Aldrich	X100-100ML
Tween 20	A4974,0500	AppliChem
Vectashield Mounting Medium	Vector Laboratories	H-1000-10