Chapter 9 Development of Coronary Vessels

C H A P T E R

N I N E

Development of Coronary Vessels Xiu Rong Dong,*,† Colin T. Maguire,*,§ San-Pin Wu,} and Mark W. Majesky*,†,‡,§ Contents 1. Introduction 1.1. Origins of coronary vessels in the proepicardium 1.2. What is the PE? 1.3. Coronary vasculogenesis in the subepicardium 1.4. Coronary artery formation and smooth muscle cell recruitment 2. Microdissection and Explant Culture of the Proepicardium 2.1. Avian embryos 2.2. Mouse Embryos 3. Isolation of Total RNA from Individual PEs for Gene Expression Studies 3.1. Isolation of total RNA from single PEs 3.2. Analysis total RNA from individual PEs by RT-PCR 4. Explant Culture of the Epicardium 5. Analysis of PE and Epicardium In Vivo by Scanning Electron Microscopy 6. Methods to Study Coronary Vessel Development In Vivo 6.1. Whole-mount immunostaining of avian embryos 6.2. Whole-mount PECAM1 immunostaining of mouse embryos 6.3. Alternate antigen-retrieval methods 6.4. Confocal microscopy 6.5. Image processing 6.6. Whole-mount immunostaining for smooth muscle–marker proteins 6.7. Whole mount b-galactosidase staining 6.8. Preparation of coronary vascular casts Acknowledgments References * { { } }

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Carolina Cardiovascular Biology Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina Cardiovascular Sciences Graduate Program, Baylor College of Medicine, One Baylor Plaza, Houston, Texas Department of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, Texas

Methods in Enzymology, Volume 445 ISSN 0076-6879, DOI: 10.1016/S0076-6879(08)03009-7

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2008 Elsevier Inc. All rights reserved.

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Abstract This chapter summarizes experimental techniques used to study coronary vessel development from its origins in the proepicardium (PE) to the final assembled network of arteries, veins, and capillaries present in the mature heart. Methods are described for microdissection and culture of the PE and embryonic epicardial cells, isolation of total RNA from single PE primordia and analysis by RT-PCR, imaging of the epicardium and coronary vessels by whole-mount confocal microscopy and by scanning electron microscopy, and the preparation of coronary vascular corrosion casts to visualize the entire coronary artery network structure. These techniques form the basic tools to study the cellular and molecular pathways that guide development and remodeling of coronary vessels.

1. Introduction 1.1. Origins of coronary vessels in the proepicardium Coronary vessels arise from the proepicardium (PE), a primordium that contains precursors for the epicardium, coronary endothelial cells (CoECs), smooth muscle cells (CoSMCs), and interstitial fibroblasts (Mikawa and Fishman, 1992) (for review, see Majesky, 2004). Proepicardial cells arise outside the heart and extend villus-like projections that attach to the myocardium around Hamburger-Hamilton (HH) stage 17.5 in avian embryos (chick, quail) and E9.5 in the mouse (Hiruma and Hirakow, 1989; Manner et al., 2001; Viragh et al., 1993). PE cells then migrate over the surface of the heart to form an epicardial covering ( Manner, 1992; Viragh and Challice, 1981; Viragh et al., 1993). In response to signals from the myocardium, some epicardial cells undergo an epithelial to mesenchymal transition (EMT) and move into the subepicardium (Dettman et al., 1998; Gittenberger-de Groot et al., 1998; Perez-Pomares et al., 1998). From this position, coronary angioblasts form a subepicardial coronary plexus that surrounds the ventricles and encircles the aortic root (for review, see Tomanek, 2005). Capillary-like vessels then invade the wall of the aorta and make contact with the aortic lumen usually at two points within the aortic valve sinuses (Ando et al., 2004; Bogers et al., 1989; Eralp et al., 2005). These contacts initiate unidirectional blood flow through the coronary plexus, which promotes remodeling of the plexus into arteries, veins and capillaries, and stimulates recruitment of pericytes and CoSMCs.

1.2. What is the PE? In avian embryos, the PE arises from mesothelial cells lining the pericardial cavity where the sinus venosus (SV) joins the inflow tract of the heart. In mouse embryos, the PE arises from mesothelium covering the septum

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transversum beginning around E8.5. In both cases, the PE initiates development as bilateral primordia on either side of the ventral midline (Schulte et al., 2007). In mouse embryos, the bilateral primordia continue to develop as such until they fuse at the midline around E9.5, whereas in avian embryos the left side regresses while the right side continues to develop (Schulte et al., 2007). Formation of the PE is initiated by signals from the underlying liver bud that induce competent mesothelium to express PE markers and proliferate to form villus-like projections that extend into the pericardial cavity (Ishii et al., 2007). Each villus is composed of a simple squamous mesothelium covering a proteoglycan- and hyaluronic acid–rich extracellular matrix (ECM) core, which contains occasional clusters of mesenchymal cells and angioblasts embedded within ( Kalman et al., 1995; Kuhn and Liebherr, 1988; Munoz-Chapuli et al., 2002; Nahirney et al., 2003; Viragh and Challice, 1981). Together, the mesothelial and mesenchymal cells of the PE comprise the progenitor cells that are delivered to the heart to form the epicardium and coronary vessels (Dettman et al., 1998; Gittenberger-de Groot et al., 1998; Mikawa and Gourdie, 1996; Mikawa and Fishman, 1992; Perez-Pomares et al., 1998).

1.3. Coronary vasculogenesis in the subepicardium After contacting the heart, PE cells migrate as a continuous mesothelial sheet of cells in avian embryos to cover the surface of the myocardium and form the epicardium. In the mouse, aggregates of PE cells attach to the myocardium either as free-floating cell clusters or as fragments of villi that transfer to the myocardium after contact with the beating heart (Rodgers et al., 2008). This process is complete by E10.5 in the mouse embryo and HH21 in the chick embryo. The next critical step in coronary vessel formation is an epithelial to mesenchymal transition (EMT) that produces epicardiumderived mesenchymal cells (EPDCs) that enter the subepicardium and migrate into the myocardium. EPDCs are forerunners for CoSMCs, adventitial fibroblasts, and cardiac interstitial cells (Dettman et al., 1998; Landerholm et al., 1999; Mikawa and Gourdie, 1996). CoECs appear to arise from two sources. One source is from angioblasts formed in the region of the liver primordium and septum transversum, and carried to the heart by proepicardial villi (Kattan et al., 2004; Mikawa and Fishman, 1992; Poelmann et al., 1993; Vrancken Peeters et al., 1997). A second source is from the epicardium itself. A variety of studies suggest that at least some epicardial cells are initially multipotential, and that following EMT they are guided to various cell fates, including endothelium, by instructive signals present in the subepicardium or produced by myocardial cells. Coronary vasculogenesis occurs in a local environment that is rich in FGF2, FGF9, FGF16, VEGF-A, VEGF-B, and angiopoietin-1 (Lavine et al., 2005; Tomanek et al., 1999, 2006; Ward and Dumont, 2004). Genetic analysis

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suggests important roles for FGF receptor 1 (FGFR1)– and FGFR2-mediated signaling in the myocardium, which triggers a wave of sonic hedgehog (Shh) activation that is essential for VEGF-A, B, and C, and angiopoietin 2 expression during coronary vasculogenesis (Lavine et al., 2006). Furthermore, loss of function mutations for Friend of Gata-2 (FOG2) in the myocardium result in the absence of subepicardium formation and a failure of coronary vessels to form (Crispino et al., 2001; Tevosian et al., 2000).

1.4. Coronary artery formation and smooth muscle cell recruitment Around E13.5 in the mouse, or HH32 (day 7.5) in the chick, paired coronary ostia are formed by ‘‘controlled invasion of the aorta’’ by individual capillary-like vessels that surround the aortic root (Bogers et al., 1989); (Waldo et al., 1990). This remarkable process usually produces two stable connections with the aortic lumen, and these are almost always found in the opposed aortic valve sinuses that face away from the pulmonary artery. Contact with the aortic lumen initiates unidirectional blood flow through the coronary plexus. This directs a plexus remodeling process that involves the enlargement of main channels of blood flow into major distributing coronary arteries, pruning and regression of vascular channels that receive little or no flow, and the formation of coronary veins. Initiation of coronary blood flow also marks the onset of recruitment of CoSMCs and pericytes that will provide important survival and maturation signals for the endothelium, and will build a pressure-bearing artery wall. The first appearance of SMC differentiation markers in the coronary vasculature is around E16.0 in the rat at the level of the coronary stems (Ratajska et al., 2001). Smooth muscle a-actin was the first marker detected in the coronary vessel wall at E16.0, followed by SM-myosin heavy chain on E17, the 1E12 antigen (a smooth muscle–specific isoform of a-actinin) on E18, and finally smoothelin in the early postnatal period (Ratajska et al., 2001). This timing suggests that CoSMC differentiation is dependent on blood flow through the coronary vessels. CoSMC differentiation marker expression initiates in the coronary stems and proceeds in an orderly and continuous downstream sequence (Hellstrom et al., 1999; Hood and Rosenquist, 1992). An important role for PDGF signaling is suggested by a study from Tallquist et al. (2003), who used gene-targeting approaches to produce an allelic series of tyrosine to phenylalanine mutation in the intracellular domain of the PDGF receptor-b. Their analysis showed a strong correlation among the extent of CoSMC investment, the amount of PDGF-Rb expressed by CoSMCs, and the number of signal-transduction pathways that are activated by the receptor intracellular domain (Tallquist et al., 2003). A critical source of PDGFBB for CoSMC recruitment is from CoECs (Bjarnegard et al., 2004). Finally, there is a rapid expansion of the coronary vascular network shortly

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after birth triggered by rapid growth of the heart and increased demands for cardiac perfusion (Tomanek, 2005). In the mouse, capillary density increases three- to four-fold, and the number of coronary vessels that acquire a coating of SMCs increases at least 10-fold during the first 3 weeks after birth (Carmeliet et al., 1999).

2. Microdissection and Explant Culture of the Proepicardium 2.1. Avian embryos 2.1.1. Obtaining the embryo Fertilized quail (Cortunix cortunix) (Northwest Gamebirds, Kennewick, WA) or SPF chick (Charles River Laboratories, Wilmington, MA) eggs are incubated at 37
C in a moisture-controlled egg incubator (GQF Manufacturing, Inc., Savannah, GA) with rotation for 60 to 66 h. Under these conditions, the embryo should be at Hamburger-Hamilton (HH) stage 17.0 to 18.0 (Hamburger and Hamilton, 1951). In our experience, stage HH17.5 is optimal to visualize and microdissect the developing PE prior to its contact with the heart. To begin, clean the eggshell with 70% ethanol, and sterilize dissecting instruments in a 250-ml beaker containing water at a slow boil. To open the egg, use the blunt side of a pair of scissors and tap the small end of the egg to break the shell and then carefully make a radial cut in the shell and slide the yolk and egg contents out onto a 100-mm Petri dish. The embryo should be positioned on top of the yolk. Prepare a small piece of filter paper (1 cm  1 cm, size 3, 1003185, Whatman International Ltd., Maidstone, England) with a center window consisting of two overlapping holes made by a standard-sized, hand-held paper punch (7-mm diameter). Carefully position the autoclaved filter paper carrier over the embryo such that the entire embryo is visible within the central window of the paper carrier, and then lower the carrier onto the yolk surface. Using the edges of the filter paper as a guide, carefully cut the membrane covering the yolk, lift the filter paper carrying the embryo with sterile forceps, and place the embryo in cold 1  PBS, 2 antibiotic-antimycotic solution (AB/AM, Gibco/BRL) on ice until ready for further dissection. 2.1.2. Microdissection of avian PE Three steps to successful microdissection of the PE from avian embryos are identify, expose, and cut. The first step is to identify the PE in HH-stage17.5 embryos under a dissecting stereomicroscope (Leica MZ6, or equivalent). At this stage, the PE looks like a cauliflower-shaped cluster of villi located adjacent to the sinus venosus (SV) of the developing heart (Fig. 9.1).

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A

Quail PE HH17

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Figure 9.1 Coronary vessels are formed from progenitor cells that reach the heart via a transient structure called the proepicardium (PE). (A) A stage-HH17 quail embryo oriented with the ventral side up.The PE (arrow) is seen posterior to the looped heart tube (HT) and overlying the junction of the sinus venosus (SV) and the inflow tract of the heart. (B) Scanning electron micrograph of an HH17 quail embryo PE oriented similarly to that shown in A. Note the irregular surface of the PE, and the specialized cell^ cell junctions with numerous and interdigitated villi (inset). (C) An E9.5 mouse embryo with the PE (arrow) located just ventral to the heart tube (HT). (D) Scanning electron micrograph of mouse PE at E9.5. Note the less well-organized structure of the mouse PE (thick arrow) compared to the quail PE shown in (B). Note also the numerous small clusters of PE cells that have attached to the surface of the myocardium (thin arrow). The inset shows the numerous filopodial-type extensions that PE clusters exhibit when they initially attach to the myocardium.

To visualize the PE, use sterile forceps and a tungsten needle microscalpel (Conrad et al., 1993; Le Douarin et al., 1996), and expose the heart field by peeling off all membranes that cover the embryo body and the heart. In most cases, the PE will be found on the left side of the embryo. To microdissect the PE, make one cut between the PE and the SV then make a second half-moon–like cut on the bottom of the ‘‘cauliflower-like’’ PE cell cluster. Making the latter cut will free the PE from the embryo. After rinsing the dissected PE in PBS, the PE is drawn into a pipette tip using a volume of about 8 ml. Then transfer the PE in the pipette tip to 0.7 ml of culture medium (M199 medium containing 30 mM D-glucose, 5 mM L-glutamine, 1.25 mM putrescine, 1 AB/AM, and 10% fetal calf serum) in a 24-well plate. Position the PE in the center of the well with the pipette tip, and make sure it settles down on the bottom of the dish before

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placing into a 5% CO2/95% air–humidified, cell-culture incubator at 37
C as described (Landerholm et al., 1999; Lu et al., 2001).

2.2. Mouse Embryos The mouse PE differs from the avian PE in at least four ways (Figs. 9.1 and 9.2). First, the mouse PE spans the entire width of the embryo at the level of the looped heart owing to the continued growth of both the right and left sides of the PE primordia. In contrast, in avian embryos the left side regresses while only the right side continues to develop (Schulte et al., 2007). Therefore, the mouse PE can be seen on both sides of the embryo (Fig. 9.2). Second, the mouse PE has generally shorter and thinner villi than the avian PE. Third, the mouse PE arises from a mesothelial cell layer covering a protruding structure called the septum transversum (ST) (Fig. 9.2). Finally, the border of the mouse PE with the embryo body is not as clear as the avian A

Mouse embryo

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Figure 9.2 Whole mount b-galactosidase staining of a PE-specific reporter in transgenic mice. (A, B) Histochemical staining for LacZ activity in an E9.5 mouse embryo expressing a b-galactosidase reporter gene from the Tbx18 locus. Tbx18 is highly expressed in the mesothelial cells of the PE (arrow, A and B). Note in (A) that the PE appears over the junction of the sinus venosus (SV) with the inflow tract of the heart, and that it spans the entire width of the embryo at E9.5. HT, heart tube; PE, proepicardium; ST, septum transversum; SV, sinus venosus.

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PE (Fig. 9.2), and more attention is required in its microdissection. Three gentle cuts have to be made to free the mouse PE from the embryo. The first cut is between the PE and sinus venosus, the second cut is between the PE and the hind-limb bud and the third cut is horizontal to the ST. The same procedure for picking up the avian PE in a pipette tip and transferring it to culture medium applies to the dissected mouse PE. For explant culture, the dissected avian or mouse PE is placed in prewarmed M199 culture medium supplemented with 30 mM D-glucose, 5 mM glutamine, 1.25 mM putrescine, 2 antibiotic-antimycotic solution (Gibco-BRL, Gaithersburg, MD), and 10% fetal bovine serum in 24-well tissue culture trays (Becton-Dickinson, Lincoln Park, NJ), and incubated at 37
C in a 95% air/5% CO2 tissue culture incubator as described (Lu et al., 2001).

3. Isolation of Total RNA from Individual PEs for Gene Expression Studies 3.1. Isolation of total RNA from single PEs Obtain single avian or mouse PEs using the microdissection procedures described above, rinse with PBS, and drop PE into 200 ml of solution D (4 M guanidinium isothiocyanate, 25 mM sodium citrate, 0.5% (w/v) N-laurosarcosine (Sarkosyl), 0.1M 2-mercaptoethanol) (Chomczynski and Sacchi, 2006). At this step, the PE lysate can be stored at 4
C in solution D for RNA isolation at a later date. To proceed with RNA isolation, to 200 ml of RNA sample in solution D, add sequentially 20 ml of sodium acetate (2 M, pH 4.0), 200 ml of water-saturated phenol, and 40 ml of chloroform: isoamyl alcohol (49:1), mix well and place on ice for 15 min, and then centrifuge at 4
C for 20 min at 14,400 rpm in a bench-top centrifuge (Eppendorf ). Carefully transfer the aqueous phase (200 ml) into a 1.5ml microfuge tube, add 200 ml isopropanol and 20 mg of oyster glycogen (Boehringer, Inc.) as a carrier, mix well, and leave at –20
C overnight to precipitate. On the second day, collect the RNA pellet at 14,400 rpm for 30 min at 4
C in a bench-top centrifuge. The RNA-glycogen pellet is then washed with 95% ethanol once, air dried (not too long), and resuspended in 20 ml of DEPC-treated H2O. Residual genomic DNA carried down with the pellet is removed by the addition of 0.5 ml of RNase inhibitor (20 U/ml, Applied Biosystems), and 1.0 ml of RNase-free DNase (1 U/ml, Promega) to 20 ml of RNA sample, followed by incubation for 30 min at 37
C. After DNase digestion is complete, RNA is extracted by addition of 11 ml of phenol and 11 ml of chloroform, followed by vortex mixing and centrifugation at 4
C for 5 min at 14,400 rpm. The aqueous phase is transferred to a fresh 1.5-ml microfuge tube; an equal volume of chloroform:isoamyl

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alcohol (24:1) is added to the sample and vortexed well, and then centrifuged at 4
C for 5 min at 14,400 rpm. Transfer the aqueous phase to a fresh 1.5-ml microfuge tube, add 2.5 volumes of 100% ethanol and 20 mg of oyster glycogen to the RNA sample tube, mix well, and leave at –20
C overnight to precipitate. On the third day, spin down the purified RNA pellet at 14,400 rpm at 4
C for 30 min, wash the pellet once with 95% ethanol, carefully remove residual ethanol with a pipette tip, air dry the RNA pellet, and resuspend each RNA pellet from a single PE in 20 ml of TE buffer made with DEPC-treated H2O. For long-term storage, keep the RNA sample at –80
C until use.

3.2. Analysis total RNA from individual PEs by RT-PCR In our experience, 20 ml of total RNA obtained from a single PE (1500 cells) ( Jenkins et al., 2005) are sufficient for amplification of up to 10 individual gene products. The concentration of total RNA isolated from a single PE is generally too low to be reliably measured using a UV spectrophotometer. Therefore, a standard source of total RNA with a known RNA concentration (usually whole embryo) is serially diluted in 1:10 in TE buffer, and 2 ml of serially diluted samples are reverse transcribed using oligo-d(T) primers, and then amplified for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene expression for 20, 25, and 30 cycles of PCR following the manufacturer’s protocol exactly (Applied Biosystems). At the same time, 2 ml of single PE RNA sample is also reverse transcribed and amplified for GAPDH gene expression for 20, 25, and 30 cycles of PCR, and the intensity of gene product over the linear amplification range is matched with that of the known RNA standard. Further adjustments of the dilution series of RNA standard may be needed to obtain the best estimate of RNA concentration in PE samples. The RNA sample should be stored at –80
C until use.

4. Explant Culture of the Epicardium Prior to obtaining hearts for explant culture, 12-well or 24-well culture plates are coated with 0.1% gelatin at room temperature overnight, and fresh culture medium is prepared (DMEM containing 10% fetal bovine serum and 1 AB/AM). Embryonic mouse hearts are collected at E12.5 by aseptic technique, taking particular care not to damage the outer epicardial layer when using forceps or other dissecting instruments. Place in ice-cold PBS, trim off the outflow tract vessels and both atria, place the trimmed heart in the center of a gelatin-coated well (24-well tray) containing 200 ml of culture medium described above, and gently place in cell culture

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incubator a 37
C without bumping so as not to disturb attachment of the heart to the gelatin substrate. After 24 h, heart explants are examined, wells containing epicardial cell outgrowth are marked, and the tray gently returned to the incubator for another 24 h. After 48 h, remove the heart with forceps from the wells that exhibit epicardial outgrowth, return the plate to the incubator and repeat after another 24 h. Do not allow cultures to reach the point when fibroblasts appear in the cell layer surrounding the heart. In some cases, no epicardial cell outgrowth is visible around the explanted heart until the heart is removed from the well, and small patches of epicardial cells are evident underneath where the heart contacted the gelatin substrate. If no epicardial cells are present in the well after removing the heart explant, it probably means that the heart did not make good contact with the gelatin substrate. The procedure for obtaining epicardium from explanted avian embryo hearts at HH stage 25 is the same as described above, except that culture medium is M199 containing 10% FBS and AB/AM. Remove the culture medium from epicardial cell–containing wells, rinse wells with PBS, and place the 12- or 24-well tray on ice. Add 200 ml of solution D per well, and then scrape the cell layers with a pipette tip while pipetting up and down to dissolve the cultured epicardial cell monolayer. Collect the epicardial cell lysate into a 1.5-ml microfuge tube where it can be stored at 4
C for RNA isolation at a later time. The subsequent procedure is identical to that described in Section 3.1.

5. Analysis of PE and Epicardium In Vivo by Scanning Electron Microscopy Avian embryos (stage HH17.5) or mouse embryos (E9.5) are obtained, washed in PBS, and freed from any extraembryonic membranes. Because the looped heart tube obstructs the view of the PE, embryos can be stretched in the craniocaudal axis, thereby displacing the heart more cranially and away from the underlying PE. Following dissection and stretching, embryos are immediately fixed with a mixture of 2.5% glutaraldehyde, 2% paraformaldehyde, and 15 mM sodium phosphate pH 7.4 for 24 h. Embryos are then dehydrated through an ascending series of alcohol washes (10 min each wash): (1) 30% ethanol (2), (2) 50% ethanol (1), (3) 75% ethanol (1), and (4) 100% ethanol (3). When completely dehydrated in 100% ethanol, embryos are critical-point dried in CO2 using a Balzers Union CPD 020 (BAL-TEC AG) or equivalent. Subsequent to critical-point drying, embryos are adhered to 13-mm aluminum stubs with conductive carbon adhesive pads and sputter-coated with a 60/40 Au/Pd alloy to an approximate thickness of 10 to 15 nm using

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C

B

A

AV LV 880x

E5

D

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E11

20x LV RV

RV E7

E 40x

20x

40x

5x

E11

F 40x

40x

E13 10x

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Figure 9.3 Imaging of developing coronary vasculature. (A) Scanning electron micrograph of the epicardium in an HH25 (E5) quail heart. Note that the epicardial cells are characterized by dense microvilli ruffling at their lateral edges. (B to F) Confocal image stacks of whole-mount quail embryos stained for endothelial cell (EC) or smooth muscle cell (SMC) markers. (B) Low-power image of E7 (HH26) heart shows the early network of vessels (arrow) and clusters of QH1-positive angioblasts (arrowhead and inset). (C) Double immunostaining for ECs (QH1, red) and SMCs (SMaA, yellow) at E11 (HH37) shows a large septal artery (arrow) running along the interventricular septum. Inset: Small artery being covered by SMCs (green), while nearby vein (red) remains uncovered by SMCs (arrowhead). (D) SMCs (SMaActin, green) can be seen migrating (arrow) over growing arteries (QH1, red) at E11 (HH37). Inset shows SMCs covering the arteriole network in the myocardium. (E) Endothelial cells (green, ZO1, arrow) align parallel to the direction of blood flow, while SMCs (red, caldesmon, arrowheads) orient in a circumferential fashion. Inset shows SMCs organizing on a large septal artery costained for SM22 (green) and SMaActin (red) at E13 (HH39). (F) The right coronary artery (arrow, green, SM22) and its main branches distribute blood flow to the myocardium. Large veins (arrow, red, QH1) lack SMCs and return blood flow to the right atrium at E134 (HH39).

a Hummer X sputter coater (Anatech Ltd, Alexandria, VA) or equivalent. Sputter-coated PE and hearts are examined and imaged using a Zeiss Supra 25 FESEM (Thornwood, NY), or equivalent, set to an accelerating voltage of 5 kV (Fig. 9.1). Avian or murine embryos are obtained at the desired developmental stage, and hearts are carefully exposed by removing membranes or tissues that obscure it, taking care not to allow forceps to close around the heart, thus damaging the epicardial layer. Fixation, dehydration, critical-point drying, sputter coating, and viewing in the scanning electron microscope, use the methods described above (Section 5) (Fig. 9.3).

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6. Methods to Study Coronary Vessel Development In Vivo 6.1. Whole-mount immunostaining of avian embryos This protocol produces excellent results in avian embryos from E3 to E13, and in mouse embryos up to E12.5. On the first day, whole avian embryos or isolated hearts are fixed in 4% paraformaldehyde (PFA) for 15 min. Specimens are simultaneously permeabilized with PBT (PBS plus 0.2% Triton X-100) and blocked with 0.1% BSA and 2% normal goat serum for 1h at room temperature. Saponin (0.1%) can be substituted for 0.2% Triton X-100 for a less harsh antigen retrieval option, but then 0.1% saponin must be maintained during every subsequent step. Primary antibodies are added to a blocking buffer (0.1% BSA and 2% normal goat serum) and incubated overnight at 4
C. To identify endothelial cells in quail embryos, the QH1 monoclonal antibody (Developmental Studies Hybridoma Bank, University of Iowa) is used at a dilution of 1:50 from the supernatant. On the second day, embryos are rinsed in PBS, and incubated in a secondary block using PBT and normal goat serum at room temperature for 1 h. Matching secondary antibodies are added in 0.1% BSA and incubated for 1 hour at room temperature (1:400 goat anti-mouse IgG1 (g1)-AlexaFluor 594, Invitrogen, A21125). Nuclei are counterstained using either 1 mM DRAQ5 (Biostatus Limited, Leicestershire, UK), 5 nM Sytox Green (Invitrogen) or 5 nM TOPRO-3 (Invitrogen), rinsed with PBS, and cleared in a solution of 1:1 glycerol:PBS for at least 3 h at 4
C before imaging (Fig. 9.3).

6.2. Whole-mount PECAM1 immunostaining of mouse embryos This protocol is optimized for use in older specimens (E12.5-E18.5) when the heart tissue has become thicker and more pigmented. Isolated hearts are washed in ice-cold PBS and fixed with 4% PFA for 30 min, then permeabilized in methanol/DMSO (4:1) for 30 min at 4
C. Specimens are bleached in methanol/DMSO/30% H2O2 (4:1:1) for 1hr at room temperature. Smaller specimens can be bleached adequately for shorter durations. Specimens are subsequently permeabilized with PBT.3 (PBS plus 0.3% Triton X-100) and incubated for 1 h at room temperature. The primary antibody PECAM-1 (CD31, clone MEC13.3, BD Pharmingen, cat550247) is diluted to 1:100 with PBS, 0.1% BSA, and 2% blocking reagent (Roche, cat-1096176) and incubated overnight at 4
C. Specimens are washed with PBT.3 at 4
C for 1 h, followed by PBT.5 (PBS plus 0.5% Triton X-100) at 4
C for 1 h. After washing, a matching secondary antibody

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(donkey anti-rat IgG-Alexa 594, 1:400, Invitrogen, A21209) is added to PBS plus 0.1% BSA, and incubated for 1 h on a rocking table at room temperature. Nuclei are counterstained with DRAQ5, Sytox Green, or TOPRO-3 as described previously, and rinsed with PBS and cleared in a solution of 1:1 glycerol:PBS for 30-60 min at room temperature before imaging (Fig. 9.3).

6.3. Alternate antigen-retrieval methods The above-described protocols use Triton X-100, a nonionic surfactant, for antigen retrieval. However, some antigens are retrieved poorly, or not at all, by Triton X-100. Therefore, modified antigen retrieval methods are required. Many antibodies against components of the actin cytoskeleton produce better results if fixed with ice-cold methanol for 15 min followed by a quick 30-s permeablization step with acetone/methanol (1:3). Antibodies against SMaA (Sigma, A2547), SMgA (Seven Hills Bioreagents, cat#LMAB-b4), and calponin (Sigma, C6047) work very well using an acetone/methanol antigen–retrieval method. Except for omitting Triton X-100, every subsequent step of the above protocol remains unchanged. In order to identify which antigen retrieval method yields the best results, it is recommended that both methods described above are tested.

6.4. Confocal microscopy Fluorescent antibody staining results are best visualized using a confocal microscope, such as a Zeiss LSM5 Pascal confocal microscope (Carl Zeiss MicroImaging Inc., Thornwood, NY), an Olympus FV500 confocal microscope (Olympus America Inc., Center Valley, PA) or equivalent. Confocal microscopy gives superior spatial resolution and control over images in the Z-plane of the sample. Whole-mount stained specimens are gently laid inside of glass-bottom culture dishes (MatTek Corporation, Asland, MA) and scanned. A limitation of confocal microscopy is the shallow penetration depth of the laser light (100 mm). It is therefore important to place a glass cover-slide on top of the specimen and slowly press down to flatten the tissue sample. Images are acquired as single confocal images or confocal image stacks, which are presented as average Z-plane projections. Light sources and filter sets should be carefully matched to the fluorescent reporter group to be detected, usually conjugated to the secondary antibody.

6.5. Image processing To achieve accurate morphological representation, each confocal image scan is saved as either 8-bit TIFF files or 12-bit LSM files (depending on the confocal microscope used), and then converted into 8-bit Tiff files using

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ImageJ software (NIH, Bethesda, MD) and the LSM toolbox 4.0b plug-in. Each TIFF file is then opened in Adobe Photoshop 7.0.1 (Adobe Systems, San Jose, CA) and converted to RGB, which produces a properly pseudocolored image (original green channel images appear green, etc.). Images are combined using Photoshop layers and blending tools to produce a clear morphological image (Fig. 9.3). Alternative, and sometimes newer, methods do exist that can produce similar results.

6.6. Whole-mount immunostaining for smooth muscle–marker proteins The development of coronary smooth muscle throughout the vascular network is best studied by whole-mount immunostaining with SMC marker– specific antibodies, or by use of various transgenic mouse lines with lacZ or EGFP reporters driven by vascular SMC–specific promoter/enhancer cis regulatory elements (see below). For the whole-mount immunostaining, freshly dissected hearts are fixed and stained with a smooth muscle–marker antibody of interest. Commonly used antibodies for this purpose include SMaActin, SM22a, calponin, SMgA, caldesmon, and SM-myosin heavy chain (Owens et al., 2004). At early stages of heart development, the myocardium will express some of these smooth-muscle selective markers. Therefore, parallel immunostaining with MF20 (Developmental Studies Hybridoma Bank, University of Iowa), can be used to identify cardiac myocytes, and QH1 (avian) or PECAM1 (mouse) will identify endothelial cells. When antibodies to two or more cell types are combined during confocal imaging, patterns of smooth muscle formation in the developing coronary vascular network can be revealed in exquisite detail. When using low-power objectives (5 and 10) with the confocal microscope, it is important that confocal image stacks are presented as average Z-plane projections (Fig. 9.3).

6.7. Whole mount b-galactosidase staining To visualize coronary smooth muscle in transgenic mice, a number of different transgenic lines have been developed in which vascular SMC– specific cis regulatory elements drive expression of a bacterial b-galactosidase reporter gene in vivo (Kim et al., 1997; Li et al., 1996; Madsen et al., 1998; Mack and Owens, 1999). To visualize lacZ activity in coronary smooth muscle, whole embryos or isolated hearts are fixed in fresh 2% paraformaldehyde/0.2% glutaraldehyde (pH7.4) for 1 h on ice, washed three times with rinse buffer (100 mM sodium phosphate, 2 mM MgCl2, and 0.1% Triton X-100 at pH 7.9) at room temperature, and stained with a solution of rinse buffer containing 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, and 1 mg/ml X-gal substrate) at 37
C overnight. Postfixation is performed in 4% paraformaldehyde at 4
C overnight (Fig. 9.4).

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B

A

E18.5

E12.5 D

C

E18.5

E18.5

Figure 9.4 Whole-mount PECAM staining. Confocal image stacks of whole-mount mouse hearts stained for endothelial cells (PECAM). (A) E12.5 left ventricle free wall shows primary coronary vascular plexus prior to the onset of blood flow through the coronary vasculature (PECAM, red). (B) E18.5 left ventricle costained for ECs (PECAM, red lines, thick arrow), nuclei (TOPRO-3, red spheres), and red blood cells (green autofluorescence, thin arrow). (C) A dorsal view (10) of E18.5 heart shows branching pattern of a left main coronary artery (arrow) that penetrates into the myocardium and quickly ramifies into an arteriole bed (*)(PECAM, green; ZO1, red). (D) A higher-power image (20) of the left main coronary artery and branch vessels stained for PECAM (red) at E18.5.

6.8. Preparation of coronary vascular casts Vascular casting is an excellent method to visualize the architecture of the entire coronary vasculature down to the precapillary level. Once made, the cast is a permanent representation of the vascular network. We prepare murine embryo coronary vascular casts as described (Adamson et al., 2002) with the following minor modifications. In brief, uteri containing embryos at E18.5 are removed and immediately immersed in ice-cold PBS until further dissection. Individual embryos are perfused through the umbilical artery with heparinized lidocaine (1% lidocaine from VetTek, Inc., 1 U/ml

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A

B

800 mm Figure 9.5 Structure of coronary artery vasculature. (A) Whole mount b-galactosidae staining of coronary smooth muscle. Heart was obtained at P0 (shortly after birth) from a transgenic mouse pup expressing b-galactosidase under the control of muscle-specific regulatory elements of the SM22a promoter.The main coronary arteries are well formed at this time, and contain several layers of smooth muscle cells in the tunica media. Note also the strong expression of lacZ activity in the walls of the aorta and pulmonary trunk (top, dark blue). (B) Vascular corrosion cast of the coronary artery network at E18.5. Coronary casts were prepared as described in the text, mounted on a small piece of modeling clay made to fit onto an aluminum stub, and examined by scanning electron microscopy. Note the branching architecture of the main distributing coronary arteries, and the ability of vascular casting to capture small arterial vessels in the heart.

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heparin in 0.9% sodium chloride, Sigma) followed by 4% paraformaldehyde (pH 7.4) via a 32-gauge needle. A small incision is made in the umbilical vein to serve as a vent and to monitor completeness of perfusion. The methyl methylacrylate casting compound is prepared as follows: 1 ml monomer base, 0.3 ml catalyst, 0.02 ml promoter, a minimum quantity of pigment dye (Bateson’s #17 kit, Polysciences, Inc.) to monitor completeness of perfusion of the casting compound as empirically determined for each application, and 0.48 ml of Jet Acrylic liquid (Land Dental Mfg. Co., Ltd). Liquid casting compound is injected into embryos through the umbilical artery via a 30-gauge needle until dye-colored casting compound becomes visible in limb vessels, indicating successful perfusion with the casting compound. All perfusions on E18.5 embryos are carried out manually using a 1-ml syringe due to the small size of vessels involved and the need for careful control of perfusion pressure. Vascular casts are allowed to cure overnight at 4
C, followed by digestion of the surrounding tissues to completion with daily changes of 7 M potassium hydroxide. Digested coronary casts are mounted on a base of nonhardening modeling clay (Van Aken, Intl.) made to fit onto 13-mm aluminum SEM stubs (see above). For this purpose, the aortic cast is broken free of the systemic vascular cast, and the broken edge of the aortic cast is gently inserted into the modeling clay so that the coronary cast is elevated off the clay surface with the apex of the ventricles oriented upward. The clay base is then mounted on aluminum stubs with double-sided tape, sputter coated as described above, and visualized by SEM (Fig. 9.5). Casts of adult hearts are made by similar methods, using retroperfusion from the femoral artery.

ACKNOWLEDGMENTS The authors acknowledge Tom Landerholm, Jun Lu, Robert J. Tomanek, and Robert J. Schwartz Vicky Madden and Robert Bagnell for helpful discussions. Support for this work is from the National Institutes of Health (HL-19242, HL-07816), the American Heart Association, and the Carolina Cardiovascular Biology Center.

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