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. Author manuscript; available in PMC: 2026 Sep 29.
Published before final editing as: Anat Rec (Hoboken). 2026 Jul 9:10.1002/ar.70285. doi: 10.1002/ar.70285

SOX6 is expressed in various cell lineages in the developing mouse heart and contributes to proper valvuloseptal development

Hannah G Tarolli 1, Andrew B Harvey 1, Inara Devji 1, Raymond N Deepe 1, Jenna R Drummond 1, Renélyn A Wolters 1, Andy Wessels 1
PMCID: PMC13617672  NIHMSID: NIHMS2209834  PMID: 42422969

Abstract

Cells derived from the endocardium, epicardium, cardiac neural crest, and second heart field play a critical role in the formation of the valvuloseptal structures of the heart. Previous studies have shown that the expression of the transcription factor SOX9 in these cell populations is essential in the regulation of this process. SOX9 interacts with other SOX family members including SOX5 and SOX6 to cooperatively regulate other developmental events. Although SOX6 has documented roles in the postnatal heart, its role in cardiac development has not yet been examined. For the current study we decided to investigate the importance of SOX6 in valvuloseptal morphogenesis. Expression studies revealed that SOX6 is expressed in many of the cell lineages that contribute to the formation of the heart. Given the pattern of SOX6 expression in the endocardium and endocardial-derived cells as well as in the coronary endothelium, we focused on the role of SOX6 in these cell populations by generating endothelial/endocardial-specific Sox6 knockout mice. These conditional knockout mice presented with ventricular septal defects, enlarged atrioventricular valves, and coronary abnormalities. Cardiovascular defects observed in the endothelial/endocardial-specific Sox6 knockout mouse demonstrate that SOX6 plays an important role in valvuloseptal development.

Keywords: development, endocardium, heart, SOX6, valves, vasculature, VSDs

1 |. INTRODUCTION

Proper development of the valvuloseptal structures is of crucial importance for the formation of the four-chambered heart. These structures sustain two separated, serially linked, blood flows throughout life. In the postnatal heart, the atrial septum (AS) physically separates the left and right atrium, the ventricular septum (VS) separates both ventricles, while the atrioventricular (AV) valves and semilunar valves (SLVs) are responsible for maintaining the unidirectional blood flow throughout the heart. AV septation relies on the proper development of several mesenchymal components including the mesenchymal cap, dorsal mesenchymal protrusion (DMP), the fused major AV cushions (AVCs), and the proximal outflow tract (OFT) cushions, together forming the AV mesenchymal complex (AVMC) (Briggs et al., 2012; Briggs et al., 2013; Burns et al., 2016; Deepe et al., 2020). Proper fusion of these components is required for complete AV septation.

Each individual valve is composed of several valve leaflets which during their development receive contribution from different cell lineages. The SLV leaflets that develop from the OFT cushions receive cells from the cardiac neural crest, anterior second heart field (aSHF), and SHF-derived endocardium, whereas the SLV leaflets that derive from the intercalated ridges (ICRs) are primarily derived from the SHF (Drummond et al., 2025; Dyer & Kirby, 2009; Eley et al., 2018; Kirby et al., 1983; Verzi et al., 2005). The AV valve leaflets that derive from the major AVCs mostly contain endocardial-derived cells (ENDCs) and the AV valve leaflets that derive from the lateral AVCs contain ENDCs and epicardial-derived cells (EPDCs) (Gittenberger-de Groot et al., 1998; Männer, 1999; Pérez-Pomares et al., 2002; Person et al., 2005; Wessels et al., 2012).

The SRY-type box family of transcription factors is involved in the regulation of various developmental processes and disease mechanisms. All family members show similarities within their high-mobility-group (HMG) DNA binding domain (Bowles et al., 2000). In the context of heart development, SOX9 is the most frequently investigated SOX family member and has been shown to play an important role in different aspects of valvuloseptal development. For instance, deletion of Sox9 from the endocardium leads to hypoplasia of the AVCs as a result of perturbation of endocardial-to-mesenchymal transformation (endMT), cell proliferation, and remodeling of the extracellular matrix (ECM) (Akiyama et al., 2004; Garside et al., 2015; Lincoln et al., 2007). In Tie2-Cre;Sox9fl/fl mice, specifically, the development of the AVCs is severely compromised and these embryos are embryonically lethal (Garside et al., 2015).

The deletion of Sox9 from the SHF results in atrioventricular septal defects (AVSDs) due to hypoplasia within the mesenchymal cap (Deepe et al., 2022) and in ventricular septal defects (VSDs) caused by perturbed development of the outlet septum (Drummond et al., 2025). Finally, the deletion of Sox9 from the epicardial cell lineage inhibits the migration of EPDCs into the AV valves and leads to myxomatous valve abnormalities (Harvey et al., 2024). The evidence that SOX9 and SOX6 interact in other developmental contexts (Ikeda et al., 2004; Lefebvre et al., 1998; Liu & Lefebvre, 2015), combined with the knowledge gap surrounding SOX6 in embryonic heart development, highlights the need to further investigate SOX6.

SOX6 is involved in the regulation of many biological processes, including chondrogenesis, cancer progression, skeletal muscle differentiation, and postnatal heart maturation (An et al., 2011; An et al., 2016; Jiang et al., 2018; Lefebvre et al., 1998; Li et al., 2013; Quiat et al., 2011; Smits et al., 2001; Wang et al., 2018). In chondrogenesis, SOX6 is required for activating transcription of type 2 collagen by interacting with SOX9 (Lefebvre et al., 1998). The importance of SOX6 in this context is seen when global Sox6 knockout specimens present with skeletal abnormalities that affect their ability to breathe and/or cause dwarfism, leading to neonatal lethality (Smits et al., 2001). However, the hearts of the Sox6 global knockout specimens were not thoroughly examined. In the postnatal heart, SOX6 represses transcription of Cyclin D1 to inhibit proliferation and promote heart maturation (An et al., 2016; Li et al., 2013). SOX6 also appears to play a role in cardiac conduction as indicated by the development of AV heart block in mice with a genetic disruption in Sox6 (Hagiwara et al., 2000). The structure of the cardiomyocytes in the hearts of these mice was consistent with myopathy phenotypes, which was revealed by an increased number of intracellular deposits (Hagiwara et al., 2000). While these studies demonstrate the significance of SOX6 in the postnatal heart, its role in cardiovascular development remains unexplored. The current study is the first to examine SOX6 in the early developing embryonic mouse heart.

In this manuscript we report the expression of SOX6 in the endocardium, epicardium, cardiac neural crest, and second heart field (SHF) during cardiac development in the mouse. We determined that SOX6 is widely expressed in the developing mesenchyme that derives from these cell lineages. The expression profile of SOX6 in the respective cell lineages that play an important role in valvuloseptal development suggested that SOX6, like SOX9, might play a significant role in heart development. To test this hypothesis, we created Sox6 knockout mice and found that the deletion of Sox6 indeed leads to a spectrum of cardiac abnormalities.

2 |. RESULTS

2.1 |. SOX6 expression and valvuloseptal development

The development of the valvuloseptal structures in the heart relies heavily on the contribution of cells that derive from various cell lineages. To gain insight into the expression of SOX6 in these respective cells at different developmental stages, we used the Wnt1-Cre, Tie2-Cre, and Mef2c-Cre mouse models, respectively as previously described, to identify cardiac neural crest-derived cells (CNCCs), ENDCs, and second heart field-derived cells (here referred to as SHFCs) (Kisanuki et al., 2001; Lewis et al., 2013; Verzi et al., 2005). We determined that SOX6 is expressed in each of these cell lineages within the developing OFT (Figures 1–3) and AV (Figures 4–6) cushions and valves.

FIGURE 1.

FIGURE 1

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SOX6 expression in the developing OFT at E10 (a–a’) and E10.5 (b–c’). Immunofluorescence reveals that SOX6 is expressed in some CNCCs (a–a’), SHFCs (b–b’), and ENDCs (c–c’) within the OFT cushions. CNCCs that are migrating proximally into the cushions begin expressing SOX6 (a’). SHFCs at the most proximal region also show SOX6 expression (b’). ENDCs have not significantly populated the OFT cushions at this stage, however, some cells within the endothelial lining express SOX6 (c’). a’, b’, and c’ are higher magnifications of the white rectangles in a, b, and c, respectively. White arrowheads point to cells from the respective cell lineages. SOX6, red; GFP, green; MF20, blue. OFT, outflow tract. myo, myocardium. endo, endocardium. Scalebars in (a, b, c) and (a’, b’, c’) are 100 and 50 μm, respectively.

FIGURE 3.

FIGURE 3

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SOX6 expression in the cells of the developing semilunar valves at E14.5 and E16.5. CNCCs (a), SHFCs (b), and ENDCs (c) within the leaflets of the pulmonary valve express SOX6. Scalebars = 50 μm. Cells in the aortic valve and CNCC mesenchyme express SOX6 at E14.5 (d–d”). An IHC image of a Wnt1-Cre;R26mG mouse heart reveals SOX6 is expressed in the CNCCs within the leaflets of the aortic valve (d′) and the condensed CNCCs in the developing ventricular septum (d”). (d’) and (d”) are higher magnifications of the rectangles outlined in (d). SOX6 expression in the valves of the pulmonary artery (e–g) and aorta (h–j) at E16.5. There are only a few CNCCs that remain in the leaflets of the semilunar valves and continue to express SOX6 (e, h). The cells within the condensed CNCC region just below the aorta stain strongly for SOX6 (h). SHFCs heavily populate the leaflets of the semilunar valves (f, i). Some ENDCs contribute to the semilunar valves, and only a handful of them express SOX6 (g, j). SOX6, red; GFP, green; SLV, semilunar valve. RV, right ventricle. CNCCs, cardiac neural crest cells. epi, epicardium. Scalebars in (a–c), (d’–d”), (e–j) = 50 μm. Scalebar in (d) = 100 μm.

FIGURE 4.

FIGURE 4

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The AV cushions and mesenchymal cap of the AV septum in a Tie2-Cre mouse express SOX6 at E11.5. Nearly every cell in the mesenchymal cap (b–b”) and AV cushions (c–c”) express SOX6. b–c” are higher magnifications of a–a” indicated by the white rectangles. SOX6, red. TNNT2, blue. DAPI, green. AVC, atrioventricular cushions. Scalebars in (a–a”) and (b–c”) are 100 and 50 μm, respectively.

FIGURE 6.

FIGURE 6

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SOX6 is expressed in the coronary vasculature at E14.5. The endothelial cells within the coronary arteries (indicated by white arrowheads), express SOX6. SOX6, red. GFP, green. MF20, blue. Scalebars = 50 μm.

2.1.1 |. SOX6 expression in the developing OFT cushions and ICRs (E10–12.5)

During the very first stages of OFT development, the OFT is a single tube consisting of three layers. The outermost layer is a cylinder of myocardial cells and the inner layer is a lining of endocardial cells. In between these cell layers is an initially acellular, ECM-rich space, generally referred to as the cardiac jelly. Soon after the initial formation, the cardiac jelly starts to expand on opposite sides within the OFT, a process that leads to the formation of two OFT cushions (Drummond et al., 2025). Around embryonic day 10 (E10), CNCCs migrate into the OFT cushions from the distal end of the OFT. At this early developmental stage, some expression of SOX6 is detected in the CNCCs (Figure 1a,a’). As the OFT cushions grow in size, the population of SOX6-expressing CNCCs expands. As reported previously, the endocardial lining of the OFT cushions is by and large SHF-derived (Drummond et al., 2025). Around E10.5, endMT of the SHF-derived endocardial lining of the proximal OFT cushions leads to the generation of SHF-derived ENDCs (SHF/ENDCs) that subsequently start to populate the proximal OFT cushions (Figure 1b,b’). Most of these ENDCs within the OFT cushions, as well as some of the SHF-derived cells within the SHF-derived endocardial lining (Drummond et al., 2025), express SOX6 (Figure 1b,b’). At this stage in development, apart from the small population of SHF-derived ENDCs in the proximal region of the OFT, there are very few ENDCs in the OFT cushions (Figure 1c,c’) (Drummond et al., 2025).

The distal OFT cushions give rise to four out of the six leaflets of the SLVs; the remaining two leaflets derive from the ICRs (Drummond et al., 2025; Eley et al., 2018). These ICRs will develop into the anterior leaflet of the pulmonary valve and the non-coronary leaflet of the aortic valve (Drummond et al., 2025; Eley et al., 2018). At E10.5, the development of the ICRs begins as an inward expansion of the SHF-derived OFT wall (Drummond et al., 2025; Eley et al., 2018). At E11, an early stage of ICR development, very little SOX6 expression is detected (Figure 2a,a’,a”). As development progresses, however, the level of expression for SOX6 increases in the cells that comprise the ICRs, and at E12.5 nearly all SHFCs express SOX6 (Figure 2b–g).

FIGURE 2.

FIGURE 2

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SOX6 expression in the developing OFT. Cells in the early-developing ICR of a Mef2c-Cre;R26nG mouse at E11 in the ICR are TNNT2-negative (a) and GFP-positive (a’) indicating that they are SHFCs. It appears that SOX6 is beginning to be expressed in the SHFCs in the early ICR (a”). The OFT cushions and intercalated ridges can be distinguished in frontal cross sections at E12.5 (b–g). The CNCCs that form the aorticopulmonary septum (APS) do not express SOX6 (b–c). Some CNCCs within the OFT cushions express SOX6 (b–c). ICRs are exclusively SHF-derived as indicated by GFP (d–e). SOX6 is expressed in SHFCs in both the ICRs and OFT cushions (d–e). The ENDCs present in the ICRs and OFT cushions when compared to CNCCs and SHFCs express SOX6 (f–g). At E12.5, SOX6 is expressed in various cell lineages of the OFT (h–j’). The OFT cushions at this stage are more densely populated with cells from the neural crest (h–h’), SHF (i–i’), and endocardium (j–j’). SOX6 is expressed in many of these cells. SOX6, red; GFP, green. ICR, intercalated ridge. OFTC, outflow tract cushions. APS, aorticopulmonary septum. dOFTC, distal outflow tract cushions. pOFTC, proximal outflow tract cushions. Scalebars = 50 μm.

By E12.5, the OFT cushions start to fuse in a distal-to-proximal direction. It is interesting to note that the condensed CNCCs that form a bridge over the most distal end of the fusing OFT cushions, thereby creating the base of the aorticopulmonary septum, do not express SOX6 (Figure 2b,c). Frontal and transverse views of the developing OFT at this stage demonstrate that, within the most distal OFT cushions, there is an expansion of CNCCs (Figure 2b,c,h,h’), SHFCs (Figure 2d,e,i,i’), and ENDCs (Figure 2f,g,j,j’) within the OFT cushions and ICRs, and many of these cells express SOX6. The OFT cushions will eventually contribute to a subset of leaflets of the SLVs of the aorta and pulmonary artery (Combs & Yutzey, 2009; Hinton et al., 2006; Hinton & Yutzey, 2011; Lockhart et al., 2011).

2.1.2 |. Expression of SOX6 in the developing SLVs (E14.5-E16.5)

By E14.5, the septation of the common OFT is complete and results in the creation of separate aortic and pulmonary sub-valvar outlet components. The leaflets that are derived from the OFT cushions still contain CNCCs (Figure 3a), SHFCs (Figure 3b), and ENDCs (Figure 3c), but the leaflets that have originated from the ICRs almost exclusively contain SHFCs (Figure 3b) (Drummond et al., 2025; Eley et al., 2018). At E14.5, a region of condensed CNCCs has formed in the outlet septum. The condensed CNCC-derived mesenchyme stains strongly for SOX6 (Figure 3d,d’,d”,h). OFT septation is heavily dependent on the contribution of CNCCs. Most, if not all, CNCCs in the OFT will eventually undergo apoptosis. It is believed that this process is critically important to allow for the myocardial cells to migrate into the cushions, a process that results in the complete closure and muscularization of the OFT septum (van den Hoff et al., 1999; van den Hoff & Wessels, 2020). At E16.5, the developing SLVs have gone through more remodeling and begin to resemble their mature morphology. Most CNCCs, SHFCs, and ENDCs within the leaflets of the pulmonary (Figure 3e–g) and aortic (Figure 3h–j) valves continue to express SOX6. The process of valve morphogenesis involves ECM reorganization (Hinton & Yutzey, 2011; Lockhart et al., 2011). Consistent with the staining pattern at E14.5, the cells within the condensed CNCC population just proximal to the aortic valve continue to stain intensely for SOX6 (Figure 3h).

2.1.3 |. SOX6 expression in the developing AV valves

The AV valves arise from the major and lateral AVCs (Wessels et al., 2012). Initially, these cushions are acellular ECM-rich swellings on the myocardial wall of the AV canal and are lined with a single layer of endocardial cells. The major AVCs are the first AVCs to develop and are located on the anterior and posterior wall of the AV canal. After their initial formation, endMT of a subset of cells in the endocardial lining leads to the formation of a population of ENDCs. After detaching from the endocardial lining, the ENDCs migrate and populate the cushions where further expansion occurs as a result of proliferation (Akiyama et al., 2004). Therefore, these major AVCs are often referred to as “endocardial cushions.” The lateral AVCs develop later and also initially become populated by ENDCs (Wessels et al., 2012). However, following an epicardial-to-mesenchymal transition (epiMT), EPDCs that have detached from the epicardial lining at the AV junction start to populate the AV sulcus, forming a distinct boundary between the atrium and ventricle called the annulus fibrosis (AF) (Kolditz et al., 2008; Wessels et al., 2012; Zhou et al., 2010) and eventually migrate into the AV valve leaflets that derive from the lateral AVCs (Gittenberger-de Groot et al., 1998; Harvey et al., 2024; Wessels et al., 2012).

To examine SOX6 expression in the ENDCs and EPDCs within the developing AVCs/valve leaflets, we stained Tie2-Cre;R26mG and Wt1-Cre;R26mG specimens with SOX6-specific antibodies at different developmental stages. At E11.5, the mesenchymal cap and the fused AVCs are developing, which is a crucial milestone for the proper formation of the AV septum. It is known that these structures are mostly comprised of ENDCs (Bolender & Markwald, 1979; Person et al., 2005; Wessels et al., 2012; Wessels & Sedmera, 2003). SOX6 is expressed in nearly every ENDC in both the mesenchymal cap and AVCs at this stage (Figure 4). By E12.5, most ENDCs within the AVCs express SOX6 (Figure 5a,a’). At this stage, the EPDCs have started to migrate into the AV sulcus and begin to form the AF. At E12.5, SOX6 expression is seen within some cells in the epicardial lining as well as in EPDCs within the AV sulcus (Figure 5b,b’). Throughout these expression studies, we noticed a visual overlap in the expression patterns of SOX6 and SOX9; however, closer observation revealed distinct differences as well. In particular, the expression of SOX6 in the EPDCs at the AV junction reveals unique expression. While SOX6 is expressed in the epicardial lining and migrating EPDCs at E12.5 (Figure 5b,b’), SOX9 is expressed heavily in the EPDCs of the AV sulcus (Figure 5c,c’), where there is significantly less SOX6 expression. By E14.5, the AVCs are in the process of remodeling and begin to resemble the leaflets of the AV valves in later stages. ENDCs within the developing AV valves still express SOX6 (Figure 5d,d’). SOX6 expression is also seen in EPDCs in the AF and those that have started to populate the parietal AV valve leaflets (Figure 5e,e’). Similar to the comparison of SOX6 and SOX9 expression at E12.5, there is little SOX6 expression in the EPDCs within the sulcus, whereas SOX9 is strongly expressed in the sulcus at E14.5 (Figure 5f,f’).

FIGURE 5.

FIGURE 5

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The expression pattern of SOX6 and SOX9 in the AV canal at E12.5 and E14.5. ENDCs populate the AV cushions at this stage and most, if not all, express SOX6 (a–a’). While SOX6 stains epicardial cells within the epicardial lining of the heart, only some cells within the epicardial-derived AV sulcus express SOX6 (b–b’). SOX9 is also expressed in the ENDCs of the AVCs and stains the EPDCs within the AV sulcus strongly at E12.5 (c’c’). SOX6 expression remains in virtually all of the ENDCs within the developing AV valves at E14.5 (d–d’). EPDCs are beginning to migrate into the parietal leaflets of the AV valves and express SOX6 as they enter the leaflet (e–e’). The AVCs continue to express SOX9 and the EPDCs within the AV sulcus still stain strongly for SOX9 (f–f’). a’, b′, c′ and d’, e’, f’ are higher magnifications of the white rectangles in a, b, c and d, e, f. respectively. GFP, green; MF20, blue. AVC, atrioventricular cushion. RV, right ventricle. LV, left ventricle. TriV, tricuspid valve. MiV, mitral valve. AL, anterior leaflet. PL, posterior leaflet. ML, mural leaflet. SL, septal leaflet. Scalebars in (a, b, c, d) and (a’, b′, c′, d’) are 100 and 50 μm, respectively.

2.1.4 |. SOX6 is expressed in the endothelial lining of the coronary vasculature

The coronary blood circulation functions to supply the heart with the necessary oxygen and nutrients. Development of the coronary vasculature begins at the sinus venosus and spreads in a ventral-to-dorsal direction to form the complete coronary plexus (Dyer et al., 2014; Wang et al., 2022). Coronary vasculature consists of an inner endothelial cell lining surrounded by smooth muscle cells (Tian et al., 2015). To determine if SOX6 is expressed in the endothelial cells of the coronary vasculature, we stained Tie2-Cre;R26mG mice for GFP and SOX6 and found that SOX6 is expressed in the coronary endothelium (Figure 6).

2.2 |. Cardiovascular defects in Tie2-Cre;Sox6fl/fl knockout mice

2.2.1 |. SOX6 expression in the endothelial/endocardial cell lineage is important for heart development

Given the expression of SOX6 observed throughout cardiovascular development, the established importance of SOX6 in other developmental and pathological conditions, and the observed overlap between SOX6 and SOX9 expression in the respective lineages studied, we focus in this study on the role of SOX6 in the endothelial/endocardial cell lineage during heart development. Although we examined SHF-specific (Mef2c-Cre;Sox6fl/fl) and neural crest-specific (Wnt1-Cre;Sox6fl/fl) embryos as well, the preliminary results from the endothelial/endocardial-specific Sox6 knockouts exhibited more severe phenotypes. To determine the importance of SOX6 in the cardiac endothelium/endocardium, we created endothelial/endocardial-specific Sox6 knockout specimens using the Tie2-Cre mouse in combination with the Sox6fl/fl mouse (Tie2-Cre;Sox6fl/fl). We analyzed Tie2-Cre;Sox6fl/fl offspring from E11.5 to P0 (Table 1). To determine whether this approach would efficiently delete Sox6 from the endocardial cell lineage, we examined the expression of SOX6 in control and Tie2-Cre;Sox6fl/fl embryos at E11.5 and compared it to the expression of SOX9. Both SOX9 and SOX6 are expressed in the mesenchyme of the AVCs and in the mesenchymal cap of control hearts at E11.5 (Figure 7a,c). While SOX9 expression is still observed in AVCs and cap of Tie2-Cre;Sox6fl/fl embryos (Figure 7b), the mesenchyme of these tissues in the Tie2-Cre;Sox6fl/fl embryos by and large no longer expresses SOX6 (Figure 7d). Upon observing the endothelial/endocardial-specific Sox6 knockout hearts, we noticed that the SLVs develop normally (Figure 8). The leaflets of both the pulmonary (Figure 8a,b) and aortic (Figure 8c,d) valves in Tie2-Cre;Sox6fl/fl specimens at E15.5 appear properly developed compared to controls.

TABLE 1.

Total number of control and Tie2-Cre;Sox6fl/fl specimens per embryonic stage.

Stage Total controls Total knockouts
E11.5 6 3
E12.5 9 1
E13.5 18 2
E14.5 41 7
E15.5 24 11
E16.5 7 0
E17.5 9 1
E18.5 4 6
P0 8 7
Total 126 38
Percentage 77% 23%
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FIGURE 7.

FIGURE 7

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SOX6 and SOX9 expression in control and Tie2-Cre;Sox6fl/fl specimens at E11.5. SOX9 and SOX6 are expressed in the cells of the mesenchymal cap and AV cushions (a, c). While SOX9 is still expressed in these structures in the Tie2-Cre;Sox6fl/fl mouse (b), SOX6 expression is by and large lost in those cells in the Tie2-Cre;Sox6fl/fl mouse (d). Scalebars = 100 μm.

FIGURE 8.

FIGURE 8

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The semilunar valves develop normally in Tie2-Cre;Sox6fl/fl mice. Representative H & E images of the pulmonary (a–b) and aortic (c–d) valves reveal that the leaflets develop normally in the endothelial/endocardial-specific knockouts compared to littermate controls. LV, left ventricle. RV, right ventricle. LA, left atrium. PuV, pulmonary valve. AoV, aortic valve. Scalebars = 100 μm.

2.2.2 |. Endothelial/endocardial-specific deletion of Sox6 leads to perturbation of ventricular septation

Histological and immunofluorescent analysis of E11.5-E13.5 Tie2-Cre;Sox6fl/fl offspring did not reveal any significant morphological defects. However, between E14.5–15.5, stages in which ventricular septation is typically completed, 7 out of 18 Tie2-Cre;Sox6fl/fl specimens showed gpersisting interventricular communication of varying degrees (Figure 9). At E14.5 and E15.5, Tie2-Cre;Sox6fl/fl specimens were found with an interventricular communication located between the fused major AVCs and the crest of the muscular VS (Figure 9a–d). Another Tie2-Cre;Sox6fl/fl specimen, when compared to a littermate control (Figure 9e), at E15.5 exhibited a muscular VSD, namely due to the presence of muscle on both sides of the VSD (Figure 9f).

FIGURE 9.

FIGURE 9

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VSDs in Tie2-Cre;Sox6fl/fl mice. H&E images of control (a) and Tie2-Cre;Sox6fl/fl (b) specimens at E14.5. H&E images of control (c) and Tie2-Cre;Sox6fl/fl (d) specimens at E15.5. Immunofluorescent stains of control (e) and Tie2-Cre;Sox6fl/fl (f) specimens at E15.5 reveal efficient deletion of Sox6. RV, right ventricle. LV, left ventricle. VSD, ventricular septal defect. AL, anterior leaflet. PL, parietal leaflet. EPDCs, epicardial-derived cells. Scalebars in (a, b) and (c–f) are 50 and 100 μm, respectively.

2.2.3 |. Abnormal valve development resulting from deletion of Sox6 from the endothelial/endocardial lineage

In addition to the septal abnormalities observed at E14.5 and E15.5, the Tie2-Cre;Sox6fl/fl mice presented with enlarged AVCs. Enlarged and dysmorphic AV valves were observed in 10 Tie2-Cre;Sox6fl/fl specimens at E15.5 (Figure 10a–c) and postnatal day zero (P0) (Figure 10d–f see also Table 2). Fifteen out of the 25 total Tie2-Cre;Sox6fl/fl specimens observed between E15.5 and P0 presented with visually enlarged valve leaflets (Table 2).

FIGURE 10.

FIGURE 10

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Endothelial/endocardial-specific Sox6 knockouts present with enlarged mitral valves. H&E images of control and Tie2-Cre;Sox6fl/fl specimens at E15.5 (a–b) and P0 (d–e). Quantification of the volume of the anterior leaflet of the mitral valve at E15.5 (c) and P0 (f). There is a significant increase in volume of the AL at E15.5 in the Tie2-Cre;Sox6fl/fl specimens (n = 10) compared to controls (n = 10). While the measured volume of the AL in Tie2-Cre;Sox6fl/fl specimens at P0 (n = 6) is larger than that in controls (n = 3), statistical analysis did not show a statistical difference. The enlarged leaflets of Tie2-Cre;Sox6fl/fl specimens show considerable levels of CD109 expression (g–h). Representative IHC images of a control (g) and Tie2-Cre;Sox6fl/fl specimen (h) at P0. Quantification of the cell count in the anterior leaflet of the mitral valve reveals that the cell numbers in the Tie2-Cre;Sox6fl/fl specimens analyzed (n = 2) are somewhat higher compared to controls (n = 2) (i). Versican expression in leaflets of control (j) and Tie2-Cre;Sox6fl/fl (k) specimens reveals expanded versican expression in knockouts compared to controls. AL, anterior leaflet. PL, posterior leaflet. Scalebars in (a–b, c–d) and (g–h, j–k) are = 50 and 100 μm, respectively.

TABLE 2.

Breakdown of Tie2-Cre;Sox6fl/fl specimens and abnormal cardiac phenotypes by stage.

T2Sox6fl/fl AGE VSD Enlarged valve Thin myocardium Dysmorphic coronaries
E11.5 (n = 3) N/A N/A N/A N/A
E12.5 (N = 1) N/A N/A N/A N/A
E13.5 (n = 2) N/A N/A N/A N/A
E14.5 (n = 7) 4 N/A 3 1
E15.5 (n = 11) 3 10 6 8
E17.5 (n = 1) 0 0 0 0
E18.5 (n = 6) 0 2 3 1
P0 (n = 7) 0 3 0 0
Total (n = 38) 7 15 12 10
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When comparing Tie2-Cre;Sox6fl/fl mice (n = 10) to littermate controls (n = 10) at E15.5 using AMIRA 3D analysis, the anterior leaflet of the mitral valve in the Tie2-Cre;Sox6fl/fl specimens exhibited about an 85% increase in size with a significant difference (p = 0.002) (Figure 10c). In a P0 litter of 11 specimens in total, three out of the six Tie2-Cre;Sox6fl/fl specimens presented with a notably enlarged anterior leaflet of the mitral valve when compared to littermates (Table 2). AMIRA 3D analysis of the anterior leaflet of the mitral valves of the Tie2-Cre;Sox6fl/fl mice (n = 6) showed that these leaflets were on average larger in volume compared to Cre-negative littermate controls (n = 3), although the statistical analysis of the data did not reveal statistical significance (Figure 10f).

A previous study showed CD109 is associated with myxomatous valve progression and is expressed in the coronary vasculature (Harvey et al., 2024). In the myxomatous-like valves of epicardial-specific Sox9 knockout mice (Wt1-Cre;Sox9fl/fl), an expansion of CD109 expression was observed (Harvey et al., 2024). Since the dysmorphic valves in the Tie2-Cre;Sox6fl/fl mice also have myxomatous-like features, we examined the expression of CD109 in these mice. Similar to what was observed in the Wt1-Cre;Sox9fl/fl mice, there also appeared to be more CD109 in the dysmorphic valves of the Tie2-Cre;Sox6fl/fl mice (Figure 10g,h, see also j,k).

To determine if the increase in size of the anterior leaflet of the mitral valve was due to an increase in cell number, we conducted a total cell count on the specimens at P0. When comparing two Tie2-Cre;Sox6fl/fl specimens to two littermate controls, an increase in cell number (>17%) in the anterior leaflet of the MV was found (Figure 10i). It is known from previous studies that ENDCs comprise the anterior leaflet of the mitral valve (Bolender & Markwald, 1979; Person et al., 2005; Wessels et al., 2012; Wessels & Sedmera, 2003). In the mitral valves of Tie2-Cre;Sox6fl/fl specimens, the anterior leaflet remains composed of ENDCs, indicated by GFP-staining, and suggests that the expanded mesenchymal cell population is from the endocardium.

A common characteristic of myxomatous valves is disorganized ECM (Harvey et al., 2024; Sauls et al., 2012). We stained the myxomatous-like valves of Tie2-Cre;Sox6fl/fl specimens with versican, a proteoglycan important for proper valvulogenesis, to investigate the ECM organization. In addition to the expression of CD109, we also observed disorganized expression of versican in the anterior leaflet of the mitral valve in Tie2-Cre;Sox6fl/fl hearts compared to littermate controls (Figure 10j,k).

The two main cellular events that determine the cell number in the AVCs and the valves that derive from these cushions after endMT has generated the first wave of ENDCs are proliferation and apoptosis. To examine if an increase in proliferation of ENDCs was causing the increase in cell numbers in the enlarged anterior leaflets, we stained serial sections of three Tie2-Cre;Sox6fl/fl specimens and three littermate controls at E11.5, and four Tie2-Cre;Sox6fl/fl specimens and four littermate controls at E14.5 with KI-67 to mark proliferating cells. MF20 was used to delineate the myocardium and DAPI nuclear staining was used to identify the ENDCs within the cushions/leaflets (Figure 11a–d’). Subsequent data analysis using CellProfiler and ImageJ revealed no significant differences in proliferation index in the Tie2-Cre;Sox6fl/fl specimens when compared to controls at E11.5 (Figure 11e) or E14.5 (Figure 11f). A qualitative analysis of apoptosis in these stages did not indicate a difference either (data not shown). Although we did not conduct proliferation/apoptosis assays at stages between E11.5 and E14.4, and hence it is possible that a temporary increase in proliferation activity (or reduction in apoptosis) in the E12.5-E13.5 window may have taken place, we believe that the contribution of these processes may have been relatively small. It is possible that endMT may have had an impact as well as SOX6 has an established role in inhibiting EMT in, for instance, cancer (Jiang et al., 2018; Wang et al., 2018).

FIGURE 11.

FIGURE 11

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KI67 stains and corresponding proliferation graphs on E11.5 and E14.5 Tie2-Cre;Sox6fl/fl hearts and littermate controls. Representative images of AVC mesenchyme stained for KI67 to mark proliferation at E11.5 (a–b’) and E14.5 (c–d’). Proliferation assay quantified via KI67 index (e–f). There is no significant difference in proliferation levels at E11.5 in Tie2-Cre;Sox6fl/fl specimens (n = 3) compared to controls (n = 3) (e), or at E14.5 in Tie2-Cre;Sox6fl/fl specimens (n = 4) compared to controls (n = 4) (f). AVC, atrioventricular cushions. AL, anterior leaflet. LA, left atrium. LV, left ventricle. VS, ventricular septum. Scalebars = 50 μm.

2.2.4 |. Vasculature and ventricular development is impaired in Tie2-Cre;Sox6fl/fl mice

The coronary vasculature is important for the blood supply to the cardiac tissues. Ventricular compaction requires sufficient nutrient support provided by the coronary vascular system in order for the cardiomyocytes to proliferate (Rhee et al., 2018; Wang et al., 2022). Because SOX6 expression was observed within the coronary vasculature endothelial cells, the impact of an endothelial/endocardial-specific Sox6 deletion on the coronary vasculature was investigated. Upon initial observation of the heart, the ventricular myocardial wall of Tie2-Cre;Sox6fl/fl mice appeared thinner compared to littermate controls (Figure 12a,b). Quantification of Tie2-Cre;Sox6fl/fl specimens (n = 6) and littermate controls (n = 6) at E15.5 revealed a significant decrease in the thickness of the right ventricular compact myocardium (p = 0.029) (Figure 12c). Some studies have shown that when the coronary vasculature does not fully or properly develop, the process of compaction is negatively affected, indicated by a thinner compact myocardium (Wang et al., 2022). To examine the possibility that the thinner myocardium in the Tie2-Cre;Sox6fl/fl specimens could result from abnormalities in the coronary vascular system, the vasculature was marked with CD109, which stains the coronary endothelium (Harvey et al., 2024). Inspection of Tie2-Cre;Sox6fl/fl specimens at E15.5 showed irregular and dilated coronary vasculature in the majority of the hearts examined compared to littermate controls (Figure 12d,e), indicating that SOX6 in the endothelium has a role in the development/patterning of the coronary vasculature and an indirect role on myocardial development.

FIGURE 12.

FIGURE 12

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Tie2-Cre;Sox6fl/fl specimens have thinner myocardial wall and abnormal vasculature at E15.5. H&E images of a control (a) and Tie2-Cre;Sox6fl/fl (b) heart reveal a thinner ventricular myocardium. Quantification of the compact myocardium reveals a significant decrease in the right ventricular myocardial thickness in Tie2-Cre;Sox6fl/fl specimens (n = 6) compared to controls (n = 6) (c). IHC images of a control (d) and Tie2-Cre;Sox6fl/fl specimen (e) with coronary vasculature marker, CD109, reveal abnormal vasculature in endothelial/endocardial-specific Sox6 knockouts. RV, right ventricle. LV, left ventricle. Scalebars in (a–b) and (d–e) = 200 and 50 μm, respectively.

3 |. DISCUSSION

3.1 |. Overview of SOX6 expression in the different cell lineages

Cardiac Neural Crest: CNCCs are important players in heart development. Their contribution is of particular significance to the development of the OFT and the associated aorticopulmonary septum. In our immunofluorescent SOX6 expression studies, we determined that, based on the intensity of staining when compared to the staining intensity of SOX6 immunofluorescence in other cell populations, SOX6 was expressed at a low-to-moderate level in CNCCs migrating into the OFT cushions during early developmental stages (see also, Drummond et al., 2025). The expression of SOX6 in the CNCCs becomes more prominent as the number of CNCCs increases in the OFT cushions at later stages of development; SOX6 expression is also observed in the CNCCs found in the OFT cushion-derived developing leaflets of the SLVs at later stages in development. Finally, particularly strong staining of SOX6 is seen in the condensed population of CNCCs at the base of the outlet septum (Anderson et al., 2023). As during normal development, CNCCs will eventually undergo apoptosis (van den Hoff et al., 1999; van den Hoff & Wessels, 2020), and given the fact that SOX6 is known to be involved in the regulation of apoptosis in other settings (An et al., 2016; Jia et al., 2016; Li et al., 2013), it is plausible that SOX6 might be involved in the regulation of this process.

Second Heart Field: The SHF contributes to the formation of the myocardial wall of the OFT, the endocardial lining of the OFT, and to a population of ENDCs that are primarily generated by an endMT from the SHF-derived endocardium (Drummond et al., 2025). The SHF is also the main source of cells that form the ICRs of the OFT (Drummond et al., 2025; Eley et al., 2018). We determined that, with the exception of the SHF-derived myocardial wall, all SHF-derived cell populations in the OFT express SOX6. The expression of SOX6 in the SHF-derived cells in the ICRs is similar to that of SOX9 in the ICRs (Drummond et al., 2025). In our earlier paper on the SHF-specific Sox9 knockout mice (Mef2c-Cre;Sox9fl/fl), we found that in the absence of SOX9 in the SHF, the development of the ICRs and the contribution of the SHF-derived cells to the ICRs were unaffected (Drummond et al., 2025), indicating that SOX9 by itself was not critically important for ICR development. As the expression of SOX6 in the ICRs was not affected in the SHF-specific Sox9 knockout mice, it is possible that SOX6 could have a compensatory or supplementary function to SOX9 in the SHF as it relates to the development of the ICRs. To explore this hypothesis, the generation of SHF-specific double Sox6/Sox9 knockouts could be considered. This approach, however, was out of the scope of this expression study. In addition, it is probably more likely that the development of the ICRs is not dependent on SOX6 signaling at all.

The epicardium: The significance of the epicardium to the development of the heart is well-established. At the AV junction, EPDCs, generated by an epiMT, initially populate the sub-epicardial space in the AV groove where they form the AV sulcus mesenchyme. Subsequently, they migrate into the AV myocardium, where they physically separate the atrial from the ventricular myocardium by forming the annulus fibrosis. From there, these AV-EPDCs migrate into the developing lateral AV valve leaflets. SOX6 expression was found in the epicardial lining as well as in the EPDCs that contribute to the development of the AV junction and in EPDCs that migrate into the ventricular myocardial walls. The expression profile of SOX6 in the epicardium and EPDCs significantly overlaps with that of SOX9. We have shown earlier that deletion of SOX9 from the epicardial lineage results in a reduction of the number of EPDCs that populate the AV sulcus and an inhibition of migration of the EPDCs into the lateral AVCs and that this eventually leads to abnormal valve development, in particular in the postnatal stages (Harvey et al., 2024). Whether SOX6 is equally important to AV valve development remains to be evaluated.

The endocardium: The endMT of cells in the endocardial lining of the AVCs at early stages of development results in the formation of ENDCs that migrate and expand into the AVCs (they are therefore often referred to as the endocardial cushions). Whereas the endocardium of the AVCs itself does not express SOX6, the population of ENDCs that populates the AVCs is characterized by robust expression of SOX6. It is also important to note that, with a few exceptions, SOX6 is not expressed in the endocardium lining the ventricular chambers. In this context, it is important to note that SOX6 expression was detected in the endothelial cells of the coronary vasculature.

3.2 |. Deletion of Sox6 from the endocardial cell lineage interferes with ventricular septation

Thorough analysis of the importance of SOX6 expression in all of the cell lineages described above will ultimately reveal the importance of SOX6 in the regulation of the behavior of these respective cell populations during valvuloseptal development. The overlap in expression patterns of SOX6 and SOX9 in the CNCCs, EPDCs, SHF, and ENDCs and the documented SOX6/SOX9 interaction in other biological systems emphasizes a need to pursue the role of SOX6 in these cell populations in more detail (Han & Lefebvre, 2008; Ikeda et al., 2004; Lefebvre et al., 1998; Liu & Lefebvre, 2015). For the current study, we opted to focus on the endocardial cell lineage. Studies on the potential role of SOX6 in the other cardiac cell lineages have been initiated and are ongoing.

To assess the importance of SOX6 in the endocardial cell lineage, we generated an endocardial-specific Sox6 knockout mouse using the endothelial/endocardial-specific Tie2-Cre mouse model in combination with a floxed Sox6 mouse to create Tie2-Cre;Sox6fl/fl specimens. To determine the impact of deleting Sox6 in the endocardial lineages, we analyzed the cardiac morphology of the Tie2-Cre;Sox6fl/fl embryonic offspring at different stages.

Loss of SOX6 from the endocardial lineage did not lead to noticeable abnormalities in the development of the OFT. Given the limited endocardial contribution to the OFT, when compared to the contribution of the endocardial cell lineage to the tissues at the AV junction, this was not totally unexpected. Structural defects were, however, observed at the level of the VS where persisting ventricular communication was observed in a number of Tie2-Cre;Sox6fl/fl mice. This included specimens with pronounced VSDs at E14.5 and E15.5. In addition, in some hearts a small interventricular communication was observed between the muscular VS and the fused AVCs at stages in which this small gap typically is closed. It is possible that these (small) communications could have been the result of delayed development with the possibility that the communication could have closed at later stages. The etiology of the observed VSDs has not been determined. AV septation largely depends on the proper formation of the major AVCs, the mesenchymal cap on the primary atrial septum (pAS), and the DMP: altogether, known as the AVMC (Deepe et al., 2020). SOX6-expressing ENDCs significantly contribute to the AVCs and the mesenchymal cap. The DMP does not contain ENDCs and is formed by the SHF. Impaired development of the mesenchymal cap can lead to primary atrial defects (pASDs) as seen in the SHF-specific Sox9 knockout mouse (Deepe et al., 2022). No pASDs were observed in the Tie2-Cre;Sox6fl/fl mice leading us to believe that the mesenchymal cap develops properly in this model. The loss of SOX6 from the endocardial cell lineage resulted in hypertrophy of the AVCs, a condition that has been associated with VSDs in, for instance, the neurofibromin-1 knockout mouse (Lakkis & Epstein, 1998). Given the fact that we observed that deletion of Sox6 led to a thinning of the ventricular wall in a subset of specimen inspected, it is also possible that a mild hypoplasia of the myocardial components of the VS (see below) may have contributed to the VSD phenotype.

3.3 |. Endocardial deletion of Sox6 in AV valve development

Several Tie2-Cre;Sox6fl/fl specimens at E15.5 presented with enlarged AV valve leaflets. This enlargement was particularly noticeable in the anterior leaflet of the mitral valve. Quantitative analysis revealed an increase in the cell number within the enlarged leaflets when compared to the leaflets in control specimens. The mesenchyme cell population that appears expanded within the anterior leaflets of Tie2-Cre;Sox6fl/fl specimens consists of ENDCs. In earlier studies, we have demonstrated that while mesenchyme of the anterior mitral valve leaflet is virtually entirely endocardial-derived, the posterior leaflet receives significant contribution of EPDCs (Bolender & Markwald, 1979; Harvey et al., 2024; Markwald et al., 1996; Wessels et al., 2012; Wessels & Pérez-Pomares, 2004; Wessels & Sedmera, 2003; Wolters et al., 2021). Given the fact that Sox6 was only deleted from the endocardial cell lineage, we infer that the increase in the number of ENDCs in the AV valve leaflets is a direct consequence of the absence of SOX6 in these cells, potentially due to an increase in proliferation, decrease in apoptosis, increase in endMT, or a combination of these processes.

The enlarged leaflets in Tie2-Cre;Sox6fl/fl specimens closely resemble myxomatous valves. Another model of myxomatous valve disease includes the published Wt1-Cre;Sox9fl/fl;R26mG model (Harvey et al., 2024). In this model, the posterior leaflet of the mitral valve is myxomatous-like and has expanded CD109 expression when compared to controls (Harvey et al., 2024). The myxomatous valve-like phenotypes in the Wt1-Cre;Sox9fl/fl;R26mG model occur in the posterior leaflet of the mitral valve because the contribution of EPDCs to the posterior leaflet is impaired. Contrastingly, the affected leaflet in Tie2-Cre;Sox6fl/fl specimens is the anterior leaflet. CD109 expression is also expanded in the enlarged leaflets of Tie2-Cre;Sox6fl/fl specimens. The expanded CD109 expression in each mouse model could suggest either a common mechanism for the enlarged/myxomatous leaflet or similar gene targets between SOX6 and SOX9. However, it is also possible that the cause of the enlarged leaflet in Tie2-Cre;Sox6fl/fl specimens is completely unrelated to that of the development of myxomatous valves in Wt1-Cre;Sox9fl/fl;R26mG specimens. Given that there are more ENDCs within the anterior leaflet of the Tie2-Cre;Sox6fl/fl mice, and that in Wt1-Cre;Sox9fl/fl;R26mG embryos, there is a compensation of ENDCs into the parietal leaflet in the absence of EPDC migration, it would make sense that the more ENDCs present in the developing valve, the more CD109 is expressed. While SOX6 has been shown to play a role in each of these events, further experimentation is required to determine which, if any, of these mechanisms is involved in the AV valve phenotype in the Tie2-Cre;Sox6fl/fl mouse (An et al., 2016; Jiang et al., 2018; Li et al., 2013; Wang et al., 2018).

3.4 |. Significance of SOX6 in the coronary vasculature

Along with VSDs and enlarged leaflets, several Tie2-Cre;Sox6fl/fl embryos showed abnormal patterning of the coronary vessels. In combination with this patterning defect, we established that the compact myocardium of the ventricular walls in these mice was significantly thinner than that of littermate controls. From this we infer that the loss of SOX6 from the endothelial/endocardial-derived cells might impair the development of the coronary vasculature, which may affect the oxygen and nutrient supply to the myocardium, resulting in (mild) myocardial hypoplasia (Rhee et al., 2018; Wang et al., 2022). It is also possible that the deletion of Sox6 interferes with the normal interaction between the coronary endothelium cells and myocardium (Chen et al., 2013; Grego-Bessa et al., 2007).

3.5 |. Conclusions

In this paper we have established the expression profile of SOX6 in various cell lineages significant in cardiovascular development. To determine the importance of SOX6 expression in the endothelial/endocardial cell lineage we created a lineage-specific Sox6 knockout mouse and demonstrated that SOX6 is critically important for proper valvuloseptal development. In addition, we have shown that SOX6 is involved in the development of the coronary vasculature and in the development of the myocardial structures in the heart. Unraveling the distinct mechanisms by which SOX6 contributes to these developmental processes and cardiovascular defects requires further experimentation.

4 |. EXPERIMENTAL PROCEDURES

4.1 |. Mice

All mice used for this study are on a C57BL/6 background. The lineage trace mouse models used in these studies include the Wnt1-Cre, Tie2-Cre, Mef2c-AHF-Cre, and mWt1/IRES/GFP-Cre (Wt1-Cre) lines, which are all previously described (Kisanuki et al., 2001; Lewis et al., 2013; Verzi et al., 2005; Wessels et al., 2012). The mouse lines that were purchased from Jackson Laboratory include the B6.129(Cg)-Gt(Rosa)26Sortm4(ACTB-tdTomato,EGFPLuo)/J (ROSA26mTmG dual fluorescence lineage trace reporter expressing membrane bound tdTomato or eGFP) mouse, and the B6N.129S6-Gt(ROSA)26Sortm1(CAG-tdTomato*,-EGFP*)Ees/J (ROSA26nTnG dual fluorescence lineage trace reporter expressing nuclear tdTomato or eGFP), abbreviated as R26mG and R26nG, respectively. To visualize the cell lineage in these transgenic lines, a Cre-positive male was mated with a female harboring the R26mG or R26nG reporter allele. To produce conditional knockout mice, the bona fide Sox6fl/fl mouse, provided by Dr. Veronique Lefebvre, was used in combination with the previously mentioned Cre-recombinase mouse models (Kisanuki et al., 2001; Lewis et al., 2013; Verzi et al., 2005; Wessels et al., 2012). Sox6fl/fl;R26nG or Sox6fl/+;R26nG females were crossed with Cre-positive males heterozygous at the Sox6 locus (Sox6fl/+) to create conditional knockouts and littermate controls. Mice harboring only one Sox6 floxed allele (Wnt1-Cre;Sox6fl/+;R26nG, Tie2-Cre;Sox6fl/+;R26nG, Mef2c-Cre;Sox6fl/+;R26nG, or Wt1-Cre;Sox6fl/+;R26nG animals) did not reveal abnormalities and were thus used alongside homozygous controls (Sox6+/+ animals). Embryonic mice were staged at 0.5 embryonic days at midday post-plug detection. The House Mouse: Atlas of Embryonic Development was used to observe features upon embryonic dissection and confirm the developmental stage. To extract the embryos, the pregnant female mouse was anesthetized with isoflurane and then sacrificed by cervical dislocation. For postnatal heart extraction, neonatal mice were anesthetized using isoflurane, followed by decapitation. Hearts were then extracted from the neonatal mice. These protocols follow the NIH guidelines and have been approved by the MUSC Institutional Animal Care and Use Committee (IACUC) under protocol ID number IACUC-2020-01140-1. Male and female embryos and neonates were used for these experiments.

4.2 |. Tissue fixation, immunofluorescence staining, and imaging

The tissue fixation, Hematoxylin and Eosin (H&E) staining, and immunofluorescence staining protocols are as previously described with the following primary antibodies: SOX6 (Novus; NBP1–86149; 1:500), SOX6 (Santa Cruz; sc-393,314; 1:250), SOX9 (Novus; NBP1–85551; 1:500), eGFP (Aves Labs; GFP-1020; 1:500), MF20 (DSHB; MF20; 1:100), TNNT2 (ABClonal; A4914; 1:200), CD109 (R&D; AF7717; 1:200) (Harvey et al., 2024). Secondary antibodies were purchased from Jackson Immunoresearch and include donkey-anti-rabbit Cy5, donkey-anti-sheep Cy5, goat-anti-mouse Cy5, donkey-anti-mouse TRITC, donkey-anti-rabbit TRITC, and donkey-anti-chicken FITC. All secondary antibodies were used at a 1:100 dilution. Images were taken with the Zeiss AxioImager II fluorescent microscope and edited using AxioVision SE64 and Adobe® Photoshop®.

4.3 |. Quantification of compact myocardial thickness

H&E images of the guide series were taken on an Olympus Bx40 microscope using the cellSens Standard software. Measurements were taken on 10 images for each specimen, ~20 microns apart. There were six measurements on each picture, 3 on the left ventricle (LV) and 3 on the right ventricle (RV), totaling 60 measurements per specimen. Separate averages were taken of the RV and LV measurements for the control and conditional knockout specimens.

4.4 |. Cell count

IHC, as described in the earlier section, was used first to visualize nuclei with DAPI staining on sections 5 μm apart. For each P0 specimen, staining throughout the entire mitral valve was performed to ensure accuracy. After imaging, Microsoft Paint was used to crop out the myocardium marked by MF20 or TNNT2, therefore leaving only cells within the mitral valve. CellProfiler was used for the cell count quantification.

4.5 |. Proliferation assay

To delineate proliferating cells, an anti-KI67 antibody was used. DAPI was used to mark all cells. For the six specimens at E11.5, five 5 μm thick sections at 10 μm intervals were used. For analysis of the four specimens at E14.5, six 5 μm thick sections at 10 μm intervals were used. CellProfiler was used to determine the total number of cells within the AVCs or valves, while ImageJ was used to manually count the KI67+ cells. To calculate the KI67 index, the number of KI67+ cells was divided by the total number of cells.

4.6 |. AMIRA 3D mitral valve volume quantification

Using both the IHC images from the cell count along with the H&E guide series from corresponding P0 specimens, we used AMIRA 3D software to outline the anterior leaflet of the mitral valve on sections 5 μm apart through the entire mitral valve. For the E15.5 specimens, we used H&E stains with sections 25 μm apart to outline the anterior leaflet. AMIRA 3D software then calculated the total volume of the outlined area.

4.7 |. Statistics

GraphPad Prism 10 was used for statistical analyses. For experiments with sufficient sample sizes, a Student’s unpaired t-test was used to analyze significance. If the p value was less than 0.05, the results were considered significant.

ACKNOWLEDGMENTS

We would like to thank Dr. Veronique Lefebvre from Children’s Hospital of Philadelphia for providing the Sox6fl/fl mouse model used in these studies.

Funding information

American Heart Association, Grant/Award Numbers: 25PRE1362464, POST-1369464; National Institutes of Health, Grant/Award Numbers: R01-HL122906, R01-HL122906-07S1, R01-HL162913, T32-GM132055

Footnotes

CONFLICT OF INTEREST STATEMENT

The authors declare no conflicts of interest.

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.

The MUSC Institutional Animal Care and Use Committee has approved all of the experiments according to both federal and institutional guidelines.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

The MUSC Institutional Animal Care and Use Committee has approved all of the experiments according to both federal and institutional guidelines.

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