Temporal and Spatial Expression of Collagens During MurineAtrioventricular Heart Valve Development and Maintenance

Jacqueline D. Peacock1, Yinhui Lu2, Manuel Koch3, Karl E. Kadler2, and Joy Lincoln1,*

1Department of Molecular and Cellular Pharmacology, Leonard M. Miller School of Medicine, Universityof Miami, Miami, Florida

2Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences, University of Manchester,Manchester, United Kingdom

3Center for Biochemistry and Department of Dermatology, Medical Faculty, University of Cologne, Cologne,Germany

AbstractHeart valve function is achieved by organization of matrix components including collagens, yet thedistribution of collagens in valvular structures is not well defined. Therefore, we examined thetemporal and spatial expression of select fibril-, network-, beaded filament-forming, and FACITcollagens in endocardial cushions, remodeling, maturing, and adult murine atrioventricular heartvalves. Of the genes examined, col1a1, col2a1, and col3a1 transcripts are most highly expressed inendocardial cushions. Expression of col1a1, col1a2, col2a1, and col3a1 remain high, along withcol12a1 in remodeling valves. Maturing neonate valves predominantly express col1a1, col1a2,col3a1, col5a2, col11a1, and col12a1 within defined proximal and distal regions. In adult valves,collagen protein distribution is highly compartmentalized, with ColI and ColXII observed on theventricular surface and ColIII and ColVa1 detected throughout the leaflets. Together, theseexpression data identify patterning of collagen types in developing and maintained heart valves,which likely relate to valve structure and function.

Keywordscollagen; heart; valves; extracellular matrix

INTRODUCTIONCongenital heart disease, related to defects in heart valve structure and function, affect almost1% of live births (Rosamond et al., 2007). Efficient heart valve function is achieved bycompartmentalization of specialized extracellular matrix (ECM) layers arranged according toblood flow (Rabkin-Aikawa et al., 2005). These layers of connective tissue arise from valveprecursor cells of the endocardial cushions during development, and provide the mature valvewith the necessary strength, flexibility, and compliance in response to changes in hemodynamicpressures during the cardiac cycle (Gross and Kugel, 1931; Lincoln et al., 2004; Person et al.,2005; Rabkin-Aikawa et al., 2005). Previous studies have shown that each matrix layer offersa unique structural integrity within distinct compartments of normal valve structures, attributedto differential contributions of several ECM components including collagens (Lincoln et al.,

*Correspondence to: Joy Lincoln, Department of Molecular and Cellular Pharmacology, Leonard M. Miller School of Medicine,University of Miami, 1600 NW 10th Avenue, Miami, FL, 33136. E-mail: jlincoln@med.miami.edu

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Published in final edited form as:Dev Dyn. 2008 October ; 237(10): 3051–3058. doi:10.1002/dvdy.21719.

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2006b). In contrast, diseased or malfunctioning valves lose their layer compartmentalizationand show excessive deposition of disorganized matrix components and diminished structuralintegrity (Hinton et al., 2006). Consequently, the ability of the valve leaflets to open and closeduring the cardiac cycle is weakened leading to valvular insufficiency or regurgitation.Therefore, the establishment and maintenance of defined connective tissue architecture isessential for normal heart valve structure and function. Despite the clinical significance, thetemporal and spatial distribution of ECM components within developing and mature valves isnot well defined.

There are several function-based collagen classes including fibril-,network-, and beadedfilament-forming, as well as fibril-associated collagens with interrupted triple helices(FACITs). The structural integrities of different connective tissue systems are related to thecontribution and organization of each of the collagen fiber classes (Kadler et al., 2007). Fibril-forming collagens including colI, colIII, colV, and colXI, prominent in bone, skin, and tendons,provide tensile strength by forming parallel bundles of highly organized fibrils (Kadler et al.,2007), while FACITs, including colIX, colXII, and colXIV, are proteoglycans that serve tobridge and stabilize collagen fibrils (Shaw and Olsen 1991). In contrast, network-formingcolIV, colVIII, and colX form lattices, including that of the basement membrane, which providefiltration, or crosslink and stabilize collagen fibrils (Stephan et al., 2004). Previous expressionstudies in heart valves have identified a high abundance of fibril-forming collagens includingtypes I, II, III, V, XI (Swiderski et al., 1994; Lincoln et al., 2006a,b), as well as network- andbeaded filament-forming collagen types IV and VI, respectively (Liu et al., 1997; Klewer etal., 1998; Person et al., 2005; Lincoln et al., 2006a).

In connective tissue systems, collagen distribution and organization are indispensable fornormal tissue structure and function; disruptions in collagen genes are known to cause defectsin several connective tissues, including skin, bone and heart valves (Malfait and De Paepe,2005; Tinkle and Wenstrup, 2005; Grau et al., 2007). Human mutations in COL1A1, COL1A2,COL5A1, and COL11 genes can result in Ehlers-Danlos syndrome (EDS), primarily associatedwith skin and bone abnormalities, although mitral and aortic valve dysfunction have also beenreported (Malfait et al., 2006). The association of EDS with valve pathology is supported bystudies in col5a1+/-;col11a1-/- mouse models of the disease that show pathological heart valvestructures with increased colI and colIII deposition (Wenstrup et al., 2004; Lincoln et al.,2006b). Patients with Osteogenesis Imperfecta related to COL1 mutations, show a highprevalence of mitral valve insufficiency (Glesby and Pyeritz, 1989) and col1-/- mice die inutero with reported cardiac defects (Löhler et al., 1984). Valve dysfunction is also observed inover 50% of patients with Stickler Syndrome, a disease caused by mutations in COL11a1 andCOL2A1 (Liberfarb et al., 1986). Collectively, these previous studies from both human diseaseand mouse models highlight the importance of collagen organization and distribution fornormal heart valve structure and function. Despite the significance of collagen contribution toheart valve function, the relationships and role of differential collagens in normal heart valvestructures are not yet clear.

In this study, we examine the temporal and spatial expression of select fibril-, network-, beadedfilament-forming, and FACIT collagens in endocardial cushions (embryonic day (E) 12.5),remodeling (E17.5), maturing (neonate), and maintained (adult) murine atrioventricular heartvalves. TaqMan Low-Density Array (TLDA) analysis identified predominantly high transcriptlevels of col1a1, col1a2, and col3a1 at all stages. In addition to fibril-forming collagens,transcript levels of col12a1, a FACIT collagen, are significantly increased at the remodelingand maturation stages. At the neonatal stage, we observe the greatest number of increasedcollagen transcript levels compared to any other time point. Collagen proteins I, III, Va2, XIa1,XII, and XIV are predominantly expressed throughout the leaflet of neonate valves, withsomewhat diminished expression at the distal tip of the leaflets. By adult stages, collagen

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expression is highly compartmentalized within the valve structures. Collagen types I and XIIare enriched within the ventricular region of the valve, colVa1 within proximal valve regionsincluding the annulus, while colIII expression is observed throughout the valve leaflet. Thesedata show a collective array of collagen expression during important stages of valve formation,maturation, and maintenance that are likely essential for normal heart valve structure, integrity,and function.

RESULTSCollagen Genes Are Differentially Expressed During Atrioventricular Valve Development,Remodeling, and Maintenance

Collagens are building blocks required to maintain the structure and function of manyconnective tissue systems including heart valves (Lincoln et al., 2006b; Kadler et al., 2007).TLDA analysis was performed to detect quantitative changes in transcript levels of selectcollagen genes in atrioventricular canal regions from E12.5, E17.5, neonatal, and adult (2months) mouse hearts. Transcript levels of fibril-forming collagens (Fig. 1A) are higher thanthat of network-, beaded filament-forming, and FACIT collagens (Fig. 1B) at all time pointsexamined. At E12.5, representative of endocardial cushion stages, col1a1, col1a2, col2a1, andcol3a1 have the highest transcript levels. During valve remodeling (E17.5), col1a1 remainspredominant, and col12a1 transcript levels increase significantly compared to E12.5. Notably,col5a1 transcript is significantly decreased at E17.5. The transition from remodeling valves atE17.5 to maturing neonatal valves is associated with the greatest number of collagen geneswith significantly increased transcript abundance. Transcript levels of col1a1, col1a2, col3a1,col5a2, col6a1, col12a1, and col14a1 are notably high at this stage. Although col1a1, col1a2,col3a1, col5a1, col5a2, col12a1, and col14a1 transcript levels remain high in adult heartvalves, transcript levels of col1a1, col1a2, col3a1, col5a1, col10a1, col11a1, col9a2, andcol12a1 actually decrease in the adult compared to neonatal stages. Table 1 summarizes TLDAdata shown in Figure 1 and categorizes approximate transcript levels greater than 5,000transcripts (+++), 500-4,999 transcripts (++), 50-499 transcripts (+), and fewer than 50transcripts (empty).

Collagens I, III, and XII Are Differentially Localized in Remodeling Atrioventricular HeartValves at E17.5

Heart valve remodeling during late stages of embryonic development is associated withchanges in ECM distribution and organization (Lincoln et al., 2006b). TLDA analysis revealedapparently high transcript levels of col1a1, col1a2, and col3a1, and significantly increasedcol12a1 in remodeling valves at E17.5 (Fig. 1). Therefore, in situ hybridization andimmunohistochemistry were performed to support these findings and determine the spatialdistribution of these collagens in remodeling atrioventricular valve structures (Fig. 2). At thetranscript level, col1a1 (Fig. 2A) and col3a1 (Fig. 2B) expression is observed throughout themitral and tricuspid valve leaflets (arrows, Fig. 2A,B) and chordae tendineae (arrowheads, Fig.2A,B). Expression is also noted in the myocardium (#, Figs. 2A,B), likely associated withcapillary walls and the fibrous skeleton (Lincoln et al., 2006a). At the protein level, colI andcolIII are most highly expressed within proximal regions of the mitral valve leaflets (arrows,Fig. 2C,C’,D,D’), with diminished expression in distal regions (arrowheads, Fig. 2C,C’,D,D’).Additional expression was also detected in the ventricular myocardium (#, Fig. 2C,D) and greatvessels (*, Fig. 2C,D). The subtle variation in collagen expression observed by in situhybridization and immunohistochemistry may be attributed to differences in transcript stabilityand protein turnover. Compared to colI and colIII, collagen XII is more widely expressed invalve leaflets (arrow, Fig. 2E,E’), although expression was similarly undetected at the distaltip (arrowhead, Fig. 2E,E’). These findings demonstrate the differential expression of fibril-and network-forming collagens during stages of heart valve remodeling.

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Fibril- and Network-Forming Collagens Are Highly Expressed in Neonate Mitral ValvesBetween E17.5 and neonatal stages, heart valves undergo extensive maturation and furtherorganization of connective tissue (Hinton et al., 2006). TLDA analyses revealed that col1a1,col1a2, col3a1, col5a2, col12a1, and col14a1 transcript levels were amongst the mostpredominant at this time, and identified significantly increased col11a1 levels at neonatalstages compared to remodeling valves.

Immunohistochemistry and in situ hybridization were performed to determine their spatiallocalization in maturing, neonate valve structures. mRNA expression of fibril-forming collagentypes col1a1 (Fig. 3A) and col3a1 (Fig. 3B) is observed throughout the mitral valve leaflets(arrows), further supported at the protein level by immunofluorescence (Fig. 3C,D). Inaddition, protein expression of other fibril-forming collagens including Va2 (Fig. 3E), andXIa1 (Fig. 3F), and network-forming collagens XII (Fig. 3G) and XIV (Fig. 3H) are alsoobserved throughout maturing neonate valve leaflets at this stage. Interestingly, proteinexpression of colIII (Fig. 3D), colVa2 (Fig. 3E), colXIa1 (Fig. 3F), colXII (Fig. 3G), andcolXIV (Fig. 3H) is notably reduced at the distal tips of the leaflets (arrowhead). In additionto valve expression, colI and colIII are observed in the ventricular myocardium (*, Fig.3C,D,H). These data highlight the spatial expression of defined collagens in neonatal maturingheart valves.

Adult Mitral Valve Structures Show Differential Collagen Expression in Atrial and VentricularCompartments

Normal adult heart valves are characterized by compartmentalization of specified ECM withindistinct regions of the valve tissue (Hinton et al., 2006). TLDA analysis determined thattranscript levels of col1a1, col1a2, col3a1, col5a1 are comparatively high at this stage andtherefore to confirm these findings and determine spatial compartmentalization,immunofluorescence was performed. ColI expression is specifically enriched along theventricular surface of the valve leaflet (arrow, Fig. 4A), but is no longer detected on the atrialsurface (arrowhead, Fig. 4A). Similarly, colXII, a network-forming collagen, is detected alongthe ventricular portion of the valve leaflet (arrow, Fig. 4D). In contrast, colIII is presentthroughout the valve leaflet (arrow, Fig. 4B), while colVa1 is detected in the proximal portionof the valve leaflet (arrow, Fig. 4C). Interestingly, colI (Fig. 4A), colIII (Fig. 4B), and colVa1(Fig. 4C) are also highly expressed in the annular regions (*), with additional coII and coIIIIexpression detected in the ventricular myocardium (concave arrows, Fig. 4A,B). Collectively,these expression patterns demonstrate the high order of collagen matrix compartmentalizationin mature and maintained heart valve structures.

Collagen Fibrils Are Highly Organized in Tight Bundles in Neonate Mitral ValvesBundles of aligned collagen fibrils can be observed in mitral heart valves from neonate miceusing transmission electron microscopy (Fig. 5). In the maturing leaflet, large amounts of ECMcontaining collagen fibrils appear to surround the fibroblast-like valve interstitial cells (*, Fig.5B). Collagen fibrils are presented as highly organized, parallel longitudinal bundles (arrows,Fig. 5A), as well as fibril bundles that run perpendicular to the longitudinal axis of the heartvalve leaflet (arrowheads, Fig. 5B). The high abundance of collagen fibrils (Fig. 5B) arelocalized towards the ventricular surface of the valve leaflet (Fig. 5A), consistent with thefibrosa matrix layer.

DISCUSSIONConnective tissues of skin, bone, tendon, and cartilage are composed of highly organizedcollagen scaffolds that facilitate tissue function, and there is ever emerging data to suggest asimilar requirement for collagens in heart valve structures (Bosman and Stamenkovic, 2003;

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Lincoln et al., 2006a). However, the composition and localization of different collagen typesrequired to establish and maintain valve function has not been fully examined. To address this,we performed TLDA, in situ hybridization, and immunohistochemical studies to investigatethe temporal and spatial expression of collagens in murine atrioventricular heart valves atembryonic, neonatal, and adult stages. In this study, the transcript levels of 18 collagen geneswere examined and all were detectable during at least one stage of murine heart valvedevelopment or maintenance. Our findings show that endocardial cushions at E12.5 containhigh levels of col1a1, col2a1, and col3a1 transcripts. During valve matrix remodeling at E17.5,transcript levels of col1a1, col5a1, and col12a1 increase, although col1a1 and col3a1 levelsremain predominantly high. The greatest number of significantly increased collagen transcriptswas observed in maturing neonatal valves, when collagen proteins begin to showcompartmentalization within regions of the valve leaflets. In the adult valves, col1a1,col1a2, and col3a1 transcript levels are the most highly observed. However, col12a1 andcoI14a1 are also detected at high levels. At this stage, compartmentalization of connectivetissue layers is apparent and differential collagens are observed in annular regions. Collectively,these data identify the temporal and spatial localization of collagen types during importantstages of valve formation, maturation, and maintenance that likely contribute to normal heartvalve structure and function.

During endocardial cushion formation, the ECM supports proliferation and migration of newlytransformed mesenchyme cells into the cardiac jelly, and provides a repository for inductivemolecules (Potts and Runyan, 1989; Person et al., 2005). The lower abundance of collagengenes at this stage compared to later time points likely reflects the infiltrative and compliantarchitecture required for matrix function at this time (Swiderski et al., 1994; Lincoln et al.,2006b; Butcher et al., 2007). As valvulogenesis progresses, primitive valve structures becomeessential for maintaining unidirectional blood flow as hemodynamic forces increase (Sedmeraet al., 1999; Lincoln et al., 2006b; Butcher et al., 2007). This is reflected in the extensive ECMremodeling and significant increases in differential collagen transcript levels in the valves anddeveloping annular regions until the neonatal stages (Lincoln et al., 2004). Deposition ofcollagens during the remodeling stages appears most active in proximal regions while selectcollagens, particularly FACITs, appear decreased at the distal tips. The distal tip region, or“free edge” is associated with high levels of proteoglycans in porcine valves (Stephens et al.,2008). This defined architecture of low-level collagens and abundant proteoglycanspresumably contributes to a more pliable matrix at the distal tips for leaflet coaptation duringthe cardiac cycle (Sacks et al., 2002). Between neonatal and adult stages, organization ofspecific collagen proteins along the atrial and ventricular surfaces is very apparent. In line withother connective systems, it is suggested that the network-forming colXII may stabilize colIfibrils along the ventricular surface and provide a more elastic matrix adjacent to the greatesthemodynamic forces (Iimoto et al., 1988; Koch et al., 1995; Young et al., 2002; Butcher et al.,2007). The overall reduction in collagen gene expression in the adult is similar to thatpreviously described in human aortic valves and is likely due to the decrease in hemodynamicpressure observed after birth following separation into the pulmonary and systemic circulatorysystems (Aikawa et al., 2006). Collectively, these data suggest that distribution andorganization of multiple collagen types are highly adaptive to ensure a defined and homeoticmatrix required for function during stages of embryonic heart valve formation and adult valvemaintenance.

The requirement of collagen type, assembly, distribution, and organization for connectivetissue function is well established and is supported by observations of tissue dysfunction inmouse models and human patients harboring mutations in collagen genes (Malfait and DePaepe, 2005; Wenstrup et al., 2006; Grau et al., 2007). Mitral valve prolapse, or valvularinsufficiency, has been reported in many forms of collagen mutation diseases associated withprimary defects in bone, cartilage, tendon, and skin (Liberfarb et al., 1986; Glesby and Pyeritz,

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1989; Lincoln et al., 2006a). Several collagens are abundantly expressed in heart valvestructures, and despite the availability of mouse models carrying mutations in collagen genes,the roles played by different collagen classes during valvulogenesis in vivo warrant furtherinvestigation (Aszódi et al., 2000; Lincoln et al., 2006a). Insights into detrimental effects onheart valve structure and function following the loss of individual collagens in heart valvesmay be predicted from the discrete expression patterns revealed in this report. In conclusion,this study has examined the temporal and spatial relationships between fibril-, network-forming, and FACIT collagens during important stages of valve development and maturationand identified collagen genes that may be attributed to currently unappreciated forms of valvepathology.

EXPERIMENTAL PROCEDURESTissue Preparation

C57/BL6 mouse embryos were collected at E12.5 and E17.5, with detection of copulation plugconsidered embryonic day 0.5. E12.5 whole embryos and hearts from E17.5 embryos, 1-dayneonate mice, and 2-month-old mice were collected and fixed in 4% paraformaldehydeovernight at 4°C. Tissue was embedded for 5-μm-thick paraffin wax or 10-μm-thick frozensectioning. Paraffin sections were deparaffinized in xylene, re-hydrated through a gradedethanol series, and rinsed in PBS as previously described (Lincoln et al., 2004). Frozen sectionswere stored at -20°C and allowed to dry at 42°C for 20 min and rinsed in 1×PBS prior toimmunohistochemical staining or in situ hybridization (Lincoln et al., 2007). All animalprocedures were approved and performed in accordance with institutional guidelines.

Taqman Low Density Array (TLDA)Gene expression was investigated using TaqMan Low Density Array (TLDA). TLDA cardsconsist of 384 (4×96) wells pre-loaded with selected Taqman oligos for high-throughput, lowreaction volume, real-time PCR. Expression of 96 customized genes, including 2 endogenouscontrols were analyzed in 4 independent cDNA samples collected from wild-type mice atembryonic, postnatal, and adult time points. Following RNA extraction, 1 μg of RNA wassubject to cDNA synthesis according to the manufacturer’s instructions (Applied Biosystems).Ten microliters (10 μl) of each cDNA sample was diluted in 90 μl of distilled water and 100μl of Taqman master mix (no. 4304437, Applied Biosystems) for each experimental run. FourcDNA samples per taqman array card were loaded and 1 μl of reaction mixture was distributedonto each of the wells containing the specific taqman oligo by centrifugation. Standardrecommended PCR protocols were performed (50°C for 2 min, 94.5°C for 10 min, 97°C for30 sec, 59.7°C for 1 min, with steps 3 and 4 repeated for 40 cycles) using the ABI 7900 HTFast Real-Time PCR System. ΔCT values were determined by normalization to Gapdh usingthe RQ SDS manager software (Applied Biosystems). For more intuitive representation of thedata, approximate transcript copy number was estimated by assuming amplicons doubledduring each PCR cycle. Sample wells with a CT count of 40 were excluded from analysis asoutliers when more than two standard deviations above the mean of replicates. Statisticallysignificant differences in transcript levels were determined using Student’s t-test (P < 0.05).Collagen gene names are italicized and include corresponding arabic numerals.

Histology and ImmunostainingTissue sections were prepared as described above, subjected to necessary antigen retrieval (seeTable 2), and blocked in 10% heat inactivated goat serum/1×PBS:0.1% Tween 20 for 1 hr atroom temperature. Antigen retrieval with bovine testicular hyaluronidase (Sigma) was used at500 U/ml in phosphate buffer according to the manufacturer’s instructions for 30 min at 37°C. Alternatively, chondroitinase ABC (Sigma, 2 U/ml) was applied for 30 min at 37°Caccording to the manufacturer’s instructions. Primary antibody incubations were carried out

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overnight at 4°C. Alternatively, tissue sections were incubated in block solution and served asnegative controls. Following overnight incubation, sections were washed in 1×PBS andincubated with respective anti-rabbit or anti-mouse Alexa 488 or 568 secondary antibodies at1:400 dilution for 1 hr at room temperature (see Table 2).

In Situ HybridizationPrepared 10-μm-thick frozen sections were subjected to in situ hybridization as previouslydescribed (Lincoln et al., 2007). The plasmid used to generate the mouse col1a1-specificriboprobe was a kind gift from Dr. Katherine Yutzey. The col3a1 riboprobe based on the cDNAIMAGE clone no. 5705762 was a generous gift from Dr. Benoit De Crombrugghe.

Electron MicroscopyHearts subject to electron microscopy were collected at neonatal stages and fixed in 2%glutaraldehyde, 100 mM phosphate buffer (pH 7.0) for 30 min at room temperature. Heartswere bisected longitudinally and fixed for an additional 2 hr at 4°C, washed in 200 mMphosphate buffer with 0.1% sodium azide, and stored in 1×PBS until embedding. Transmissionelectron microscopy was performed as described previously (Humphries et al., 2008).

ACKNOWLEDGMENTSWe thank Drs. David Birk, Benoit De Crombrugghe, Stephen Henry, and Katherine Yutzey for their kind contributionsof antibodies and in situ hybridization probes, and Dr. Lina Shehadeh for her scientific expertise. This work wassupported by James and Esther King Biomedical Research Program (JL-07KN07), NHLBI Training Grant inCardiovascular Signaling (HL007188, Directed by Dr. James D. Potter), and The Florida Heart Research Institute.

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Fig. 1.TLDA analysis to determine transcript levels of select collagen genes in atrioventricular canalregions from E12.5, E17.5, neonatal, and adult mouse hearts. Average absolute transcriptnumbers of each chosen collagen were calculated from ΔCt values normalized to gadph levels.A: Transcript levels of fibril-forming collagens at endocardial cushion (E12.5), valveremodeling (E17.5), valve maturation (neonate), and maintained (2 months) stages. B:Transcript levels of network-, beaded filament-forming, and FACIT collagens at the same timepoints. Asterisks indicate significant changes in transcript levels from the previous time pointfor each collagen gene examined.

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Fig. 2.Collagens I, III, and XII are differentially localized in remodeling atrioventricular heart valvesat E17.5. In situ hybridization (A, B) and immunofluorescence (C-E) were used to detect spatialexpression of highly expressing collagens in remodeling valves at E17.5. Col1a1 (A) andcol3a1 (B) mRNA expression in atrioventricular heart valve leaflets (arrow) and chordaetendineae (arrowhead). #, indicates observed expression in the myocardium. ColI (C, C’),colIII (D, D’), and colXII (E, E’) protein expression in atrioventricular valve leaflets. Mitralvalve leaflets are outlined and shown at higher magnification in C’, D’, E’. Arrows indicatehigh levels of colI (C, C’), colIII (D, D’), and colXII (E, E’) expression in proximal regions ofthe valve leaflet, while arrowheads refer to reduced expression at the distal tips. Note additionalcolI and colIII expression in the ventricular myocardium (#, A, B) and vessels (*, A, B). mv,mitral valve; tv, tricuspid valve; ivs, intraventricular septum; smv, septal mitral valve. Scalebar in A-E = 500 μm; in C’-E’ = 200 μm.

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Fig. 3.Differential expression of collagens I, III, Va2, XIa1, XII, and XIV in maturing neonate mitralvalves. In situ hybridization (A, B) and immunofluorescence (C-H) were used to detect spatialexpression of highly expressed collagens in maturing mitral valves in neonatal mice. Col1a1(A) and col3a1 (B) are expressed throughout the mitral valve leaflets (arrows). Byimmunofluorescence, collagens I (C), III (D), Va2 (E), XIa1 (F), XII (G), and XIV (H) aredetected throughout the mitral valve leaflets (arrow), with reduced expression of colIII, colVa2,colXIa1, colXII, and colXIV observed at the distal tips (arrowhead). Note additional expressionof colI, colIII, and colXIV in the ventricular myocardium (*, C, D, H). #, non-specific auto-fluorescent staining of red blood cells. Blue DAPI staining indicates nuclei. mv, mitral valve;lvw, left ventricular wall. Scale bar = 200 μm.

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Fig. 4.Adult mitral valves show differential expression in atrial and ventricular compartments of thevalve leaflets. Immunofluorescence was performed to detect the spatial expression of collagensI, III, Va1, and XII in adult murine mitral valve leaflets. A: ColI expression is detected in theventricular surface of the mitral valve leaflet (arrow), the valve annulus (*), and the ventricularmyocardium (concave arrow). ColI expression was not observed on the atrial surface of thevalve leaflet (arrowhead). B: ColIII is detected throughout the valve leaflet (arrow), in theventricular myocardium (concave arrow), and the valve annulus (*). C: ColVa1 expression isobserved in the proximal portion of the valve leaflet (arrow) and in the annulus of the mitralvalve (*). D: Expression of colXII is detected along the ventricular surface of the valve leafletand almost absent on the atrial leaflet surface (arrowhead) and the annulus (*). Low levels ofexpression are also observed in the annulus (*). Nuclei are stained blue with DAPI. Scale bar= 200 μm. mmv, mural mitral valve; smv, septal mitral valve; LA, left atria; LV, left ventricle;lvw, left ventricular wall.

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Fig. 5.Collagen fibrils are highly organized in tight bundles in neonatal mitral valve leaflets.Transmission electron microscopy was used to observe collagen fibril organization at theultrastructural level in mitral valve leaflets from neonate mice. A: Low magnification of themitral valve leaflet respective to the atrial and ventricular surface. B: Higher magnification ofthe boxed area shown in A. Consistent with the fibrosa layer close to the ventricular surface,arrows indicate parallel collagen fibrils, and arrowheads show fibrils running perpendicular tothe plane of section.

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