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. Author manuscript; available in PMC: 2020 Jan 1.
Published in final edited form as: Anat Rec (Hoboken). 2018 Nov 9;302(1):108–116. doi: 10.1002/ar.23913

Dynamic Expression Profiles of Sox9 in Embryonic, Post Natal and Adult Heart Valve Cell Populations

Donika Gallina 1,2, Joy Lincoln 1,2,3,#
PMCID: PMC6312467  NIHMSID: NIHMS982171  PMID: 30412364

Abstract

Heart valves are dynamic structures and abnormalities during embryonic development can lead to premature lethality or congenital malformations present at birth. The transcription factor Sox9 has been shown to be critical for early and late stages of valve formation, but its defined expression pattern throughout embryonic, post natal and adult growth and maturation is incomplete. Here we show that in the developing embryo, Sox9 is not detected in valve endothelial cells lining the primitive valve structures, but is highly expressed in the endothelial-derived valve interstitial cell population following endothelial-to-mesenchymal transformation. Expression is maintained in this cell population after birth, but is additionally detected in valve endothelial cells from post natal day 1. Using a specific antibody to detect a phosphorylated form of Sox9 at Serine 181 (pSox9), we note enrichment of pSox9 in VECs at post natal days 1 and 10 and this pattern correlates with the known upstream kinase RockI, and downstream target, Aggrecan. The contribution of Sox9 to post natal growth and maturation of the valve is not known, but this study provides insights for future work examining the differential functions of Sox9 protein in valve cell populations.

INTRODUCTION

Valvular heart disease is a growing public health problem affecting approximately 2.5% of people in the US, with a wide age-related variation from 0.7–13.3% (Nkomo et al., 2006). The prevalence significantly increases with age, from <2% before 65 years, to 8.5% between 65–75 years, and 13.2% after 75 years, and these numbers are expected to increase as the median age rises (Iung and Vahanian, 2011). Due to its higher incidence in the aging population, heart valve disease was largely viewed as a passive disorder and the result of degenerative wear-and-tear over a lifetime. However, today it is seen as an active process involving phenotypic changes in valve cell populations and pathological remodeling of the extracellular matrix (ECM), leading to biomechanical failure (Lincoln and Yutzey, 2011). Acquired valve disease traditionally in the elderly, is likely the result of life-long exposure to known risk factors including elevated cholesterol, low-density lipoproteins (LDL) triglycerides, decreased high-density lipoproteins, male sex, tobacco-use, hypertension and diabetes mellitus (Stewart et al., 1997). In addition to pathogenesis manifested later in life, valvular disease can be present at birth (congenital) and largely determined by structural malformations. Such conditions include bicuspid aortic valve; whereby affected individuals are born with two, rather than three aortic valve cusps, and mitral valve prolapse characterized by leaflet thickening and abnormal bulging of the leaflets into the left atrium (reviewed (Lincoln and Yutzey, 2011)). These structural malformations accelerate disease pathogenesis, likely as a result of changes in biomechanical stress. The etiology of congenital valve disease is asymptomatic or attributed to defects during embryonic valve development and genetic components have been identified (Devereux et al., 1982; Cripe et al., 2004; Garg et al., 2005; Martin et al., 2007; Sutherell et al., 2007; Sauls et al., 2012). In addition, gene targeting studies in mice have highlighted key regulators that when misexpressed, lead to premature lethality, or valve malformations at birth (reviewed (Lincoln and Yutzey, 2011))(Sauls et al., 2012). At present, surgical valve replacement remains the standard treatment option for congenital or acquired valvular heart disease which comes with insuperable complications, financial burdens, and no guarantee of long-term success. Furthermore, there are no approved pharmacological treatments available to stop the progression or treat (reverse) valve pathogenesis. While there remains a critical need to develop new medical therapies, the field is restricted by the limited number of known molecular targets for generating mechanistic-based approaches.

Heart valves open and close over 100,000 times a day to regulate unidirectional blood flow, and this requires the coordinated movement of the valve leaflets or cusps, and supporting apparatus during the cardiac cycle. This is achieved by three highly organized layers of ECM (collagen, proteoglycans, elastin) that each provide a unique biomechanical property to withstand the complex hemodynamics experienced during systole and diastole (Hinton et al., 2006). Several molecular markers of the valve ECM components have been shown to be common with other connective tissue systems including cartilage; highlighting similarities in mechanical demand (Lincoln et al., 2006). Homeostasis of the valve ECM is maintained by a heterogeneous population of valve interstitial cells (VICs) that in healthy adults are phenotypically similar to fibroblasts (Horne et al., 2015). In addition to this cell population, the valve leaflet or cusp is encapsulated by a single cell layer of valve endothelial cells (VECs) that primarily functions as a barrier between the blood and the inner valve tissue, but have also been shown to molecularly communicate with underlying VICs to regulate their behavior (Bosse et al., 2013; Anstine et al., 2016; Huk et al., 2016). The mature valve structures are derived from mesenchyme precursors cells within the endocardial cushions during embryonic development (Lincoln et al., 2004b). Multiple cell lineages contribute to the valve precursor cell pool and proliferate to expand numbers. As development progress, the endocardial cushions undergo extensive remodeling and at this time, precursor cells downregulate mesenchyme markers and upregulate smooth muscle α-actin as they transition towards a VIC, and diversified ECM is laid down. Remodeling, growth and maturation continue after birth until approximately post natal day 10 when the stratified ECM layers are formed and smooth muscle α-actin is significantly downregulated in VICs (reviewed (Tao et al., 2012))(Horne et al., 2015). Many regulators of valve development have been implicated to be misexpressed or dysregulated in valve disease, including the transcription factor, Sox9.

In the skeletal system, Sox9 is required for cartilage formation. In mouse chimeras, Sox9−/− cells are excluded from all cartilage tissue and fail to express chondrocyte-specific markers (Bi et al., 1999). In addition, Sox9 haploinsufficiency leads to defective cartilage primorida and premature skeletal mineralization (Bi et al., 2001). It is well established that Sox9 promotes chondrogenic phenotypes through binding and transactivating several cartilaginous ECM proteins including Col2a1 and Aggrecan in the nuclei of cells (Ng et al., 1997; Sekiya et al., 2000; Kou and Ikegawa, 2004; Lefebvre and Smits, 2005). In addition, Sox9 attenuates mineralization by inhibiting RUNX2 function at the protein level, and directly repressing Runx2 and Spp1 transcripts (Zhou et al., 2006; Cheng and Genever, 2010; Peacock et al., 2011). In heart valves, Sox9 is required during endocardial cushion formation for proliferation of valve precursor cells, as targeted loss of function mice die before E14.5 with severely hypoplastic cushions (Lincoln et al., 2007). Consistent with this phenotype, ChIP-seq analysis observed enrichment of molecular interactions between Sox9 and proliferation associated genes, including key components of the AP-1 complex at E12.5 (Garside et al., 2015). Deletion of Sox9 at later stages of valvulogenesis (~E15.5) leads to reduced expression of chondrogenic ECM proteins found in healthy developing valves, including Col2a1 (Peacock et al., 2010). In addition, surviving heterozygote mice develop early onset valve calcification by 3 months of age (Peacock et al., 2010). These data suggest that Sox9 plays diverse roles in cell proliferation and ECM remodeling during differential stages of valve development. In addition, findings show that similar to the skeletal system, Sox9 plays pivotal roles in positively regulating cartilage-like and preventing mineralization in heart valves.

The goal of this current study was to examine the dynamic expression pattern of Sox9 during valve formation with a focus on post natal growth and maturation stages in mice, as little is known about Sox9 function during this time. In embryonic valve structures, Sox9 has a restricted expression pattern in the mesenchyme/VIC cell population that is maintained after birth following their maturation into VICs. Sox9 expression in the VEC population is first detected at post natal stages and is noted to be in a phosphorylated form. This expression pattern correlates with RockI, a known upstream kinase, and the Sox9 downstream target gene, Aggrecan. Together, these analyses provide insights into the temporal and spatial distribution of Sox9 during differential stages of valve development, growth and maturation.

MATERIALS and METHODS

Mice.

C57BL/6J mice were fed regular chow mix and housed in a controlled environment with 12-hour light/dark cycles at 21°C and 23% humidity and water ad libitum. Animals were euthanized by CO2 exposure followed by secondary euthanization by cervical dislocation (adult mice) or decapitation (pups). All procedures were approved by The Research Institute at Nationwide Children’s Hospital Institutional Animal Care and Use Committee (Protocol # AR11–00076).

Tissue Preparation.

C57/BL6 mouse embryos were collected at embryonic day (E) 11.5, E14.5 and E18.5, with detection of copulation plug considered 0.5. E11.5 and E14.5 whole embryos, and hearts from E18.5 embryos, post natal day (PND) 1, PND10 and 4 month old mice were collected and fixed in 4% paraformaldehyde/1×PBS overnight at 4°C. Tissue was embedded in paraffin wax and sectioned at 7μm. Paraffin was removed in xylene, and tissue sections were re-hydrated through a graded ethanol series, and rinsed in 1×PBS as previously described (Lincoln et al., 2004a). Tissue sections containing outflow tract cushions or aortic valves were then subject to immunohistochemistry.

Immunostaining and quantitation.

For antibody detection, fixed tissue sections were subjected to antigen retrieval by boiling for 10 minutes in unmasking solution (Vector Laboratories) prior to blocking for 1 hour at room temperature (1% BSA, 0.1% Cold water fish skin gelatin, 0.1% Tween 20 in PBS with 0.05% NaN3) as described (Peacock et al., 2008). Tissue sections were incubated overnight at 4°C with primary antibodies against Sox9 (1–100 amino acids (aa)) (Rabbit, Abcam, ab26414, 1:50), Sox9 (173–185aa) (Rabbit, Abcam, ab3697, 1:50), phospho(181) Sox9 (pSox9) (Rabbit, Abcam, ab59252, 1:50), CD31 (Rabbit, Abcam, ab28364, 1:20, and Goat, Santa Cruz, sc-1506, 1:100), Col2 (Rabbit, Abcam, ab34712, 1:100), Aggrecan (Mouse, Abcam, ab3778, 1:50), and RockI (Mouse, Santa Cruz, sc-17794, 1:100). For double staining using primary antibodies raised in different species (pSox9 (Rabbit) and Aggrecan (Mouse), pSox9 (Rabbit) and Rock1 (Mouse), CD31 (Rabbit) and Rock1 (Mouse), pSox9 (Rabbit) and CD31 (goat), Sox9 (Rabbit) and CD31 (goat)), tissue sections were incubated at 4°C overnight with both antibodies. For primary antibody detection, sections were incubated for 1 hour at room temperature with appropriate Alexa-Fluor IgG secondary antibodies (Goat anti-Rabbit, Donkey anti-Rabbit, Donkey anti-Mouse, Donkey anti-Goat) (Life technologies, 1:1000/PBS). For double staining using primary antibodies both raised in rabbit (Sox9 and CD31, pSox9 and CD31, pSox9 and Col2a1) modifications were made by incubating tissue sections at 4°C overnight with either Rabbit-Sox9 or Rabbit-pSox9 followed by an additional incubation step with goat anti-rabbit IgG (H+L) Fab fragments (Jackson Immunoresearch Laboratories, 10 μg/ml) for 60 minutes at room temperature. Donkey anti-goat IgG Alexa Fluor secondary antibody (Life technologies, 1:1000/1×PBS) was then applied for 60 minutes at room temperature. Tissue sections were further incubated with Rabbit-CD31 or Rabbit-Col2 at 4°C overnight, followed by incubation with Alexa Fluor 488 Donkey anti-Rabbit (1:1000) for 60 minutes at room temperature. All sections were mounted in Vectashield anti-fade medium with DAPI (Vector Laboratories) to detect cell nuclei. Images were visualized using an Olympus BX51 microscope and captured using an Olympus DP71 camera and CellSens software. Image brightness, contrast and removal of auto fluorescent red blood cells were edited using Adobe Photoshop CC. To determine the levels of Sox9 immunoreactivity, Image J software was used to calculate the average number of pixels within threshold regions from 5 aortic valve sections from 4 different animals at each time point. This was repeated in 3 distinct regions of the aortic valve structure relative to the annulus, namely proximal, medial and distal. Data was normalized for the cell number from each region. To determine the percentage of Sox9 or pSox9 immunoreactivity in VECs and VICs, the number of cells with positive reactivity for Sox9 or pSox9 (with or without CD31 reactivity) was recorded over the total number of DAPI-positive nuclei at each time point. To further quantitate the cellular localization of Sox9 or pSox9 in each cell type, the number of cells with nuclear, cytoplasmic, or nuclear and cytoplasmic staining was reported over the total number of immunoreactivie positive VECs or VICs at each time point. GraphPad Prism 7.0a was used to determine the errors bars as standard error over the mean, and two-tailed, unpaired t-test determined statistical significance as indicated in the figure legends.

RESULTS

Sox9 is expressed in embryonic mesenchymal cells and VICs of the developing valve, but undetected in VECs.

The requirement of Sox9 for valve development has been well described (Lincoln et al., 2007), however its temporal and spatial expression pattern during valvulogenesis in mice is not complete. To address this, immunohistochemistry was performed in tissue sections of wild type mice at embryonic stages E11.5 (during EMT), E14.5 and E18.5 (valve primorida elongation and remodeling) using an antibody to detect total Sox9 at 1–100aa. Immunohistochemistry shows that 97.5% of mesenchyme progenitor cells within the outflow tract endocardial cushions express total Sox9 at E11.5 (highlighted in Figure 1A, arrowheads Figure 1A’, 1D), however levels are not detected in CD31-positive endothelial cells lining the cushions (arrows, Figure 1A’). Within the immunoreactive cell population, 86.4% cells express Sox9 exclusively in the nucleus, and 13.6% express Sox9 in both the nuclear and cytoplasmic compartments (Figure 1E). At E14.5, Sox9 expression remains high (97%) in the non-endothelial, progenitor cell population (arrowheads Figure 1B, B’, 1D). However, nuclear Sox9 expression is reduced to 77.5%, while nuclear and cytoplasmic localization is unchanged, and cytoplasmic Sox9 localization is increased to 7.0% (Figure 1E). At E18.5 in the elongating aortic valve cusp, the majority of the non-endothelial VICs continue to express Sox9 (92%) (Figures 1C, C’, D), and Sox9 remains undetected in VECs (Figure 1C, arrowheads Figure 1C’). Sox9 nuclear expression continues to decrease at this time point, while nuclear and cytoplasmic (28.8%), and cytoplasmic (11.6%) localization increase. In data not shown, comparable patterns of Sox9 expression were observed in developing valve structures within the atrioventricular canal (mitral and tricuspid). Furthermore, an alternative total Sox9 (against 173–185aa) antibody showed similar enrichment in non-endothelial progenitor cells and was undetectable in VECs, and this immunoreactive pattern was not observed in no primary, or no secondary antibody controls (Supplementary Figure 1). Together, these descriptive and quantitative studies suggest that during valvulogenesis, Sox9 is predominantly expressed in mesenchyme and VIC cells, with undetectable levels observed in VECs.

Figure 1. Sox9 is detected in VIC, but not VEC populations in developing valve structures.

Figure 1.

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Immunohistochemistry (A-C’) to show total Sox9 expression (red) pattern in developing outflow tract valve structures at E11.5 (A, A’), E14.5 (B, B’) and E18.5 (C, C’). Sox9 is double labeled with CD31 (green) to show endothelial cells (A-C). A’-C’ represent high magnification of the boxed areas shown in A-C respectively. Endocardial cushion regions are outlined in A and B. Arrows indicate valve endothelial cells, arrowheads show mesenchyme cells (A’) and VICs (B’, C’). Nuclei are shown as DAPI-positive cells (blue). Scale bars in A-C indicate 50μm and 10μm in A’-C’. (D) % of VICs that express Sox9 over the total number of DAPI-positive VICs (CD31-negative). * indicates statistical significance p<0.05. (E) localization of Sox9 in VICs over the total number of immunoreactive VICs. * indicates statistical significance p<0.05 between different time points. x, #, + indicate statistical significance p<0.05 between VICs with differential cellular localization of Sox9 expression within the same time point (x = nuclear vs. cytoplasmic, # = nuclear vs. nuclear+ cytoplasmic, + = cytoplasmic vs. nuclear+ cytoplasmic. NCC, non-coronary cusp; LC, Left coronary cusp; RC, Right coronary cusp.

Sox9 is expressed in VICs and VECs at post natal stages of valve growth and maturation.

To determine expression patterns of total Sox9 after birth, immunohistochemistry was performed at PND1, PND10 and 4 months of age. At PND1, the aortic valve cusps continue to grow and remodel and at this time 98.1% of VICs are immunopositive for Sox9 (Figure 2A, 2D). Of these, Sox9 is exclusively nuclear in 60% of positive VICs, nuclear and cytoplasmic in 12.7% and exclusively cytoplasmic in 27.3% (Figure 2A’, 2E). At this time point, Sox9 is now detected in 100% of CD31-positive endothelial cells and is largely nuclear and cytoplasmic (Figure 2A, 2D, 2E). Similar endothelial expression was also observed using an independent antibody targeting 173–185aa of total Sox9 (Supplementary Figure 1). Growth and remodeling of the valve cusp is thought to decline by PND10, however Sox9 expression remains high in VICs (94.4%) (Figure 2B, arrowheads Figure 2B, B’, 2D) and the VEC (100%) (arrows Figure 2B, B’, 2D) populations. However, compared to PND1, exclusive nuclear localization of Sox9 is reduced in VICs to 28%, although nuclear and cytoplasmic, and cytoplasmic localization are increased to 49.9% and 22% respectively (Figure 2E). In VECs, cytoplasmic localization is increased (14.9%) at the expense of decreased nuclear and cytoplasmic localization (reduced to 85.1%) (Figure 2E). At 4 months of age, the aortic valve is considered mature and homeostatic and Sox9 is expressed in 89.9% of VICs and 100% of VECs (Figure 2C, C’, D) and this is consistent with older time points up to 15 months of age (data not shown). Compared to PND10, nuclear (2.9%), and nuclear and cytoplasmic (11.2%) Sox9 expression is reduced, but increased in the cytoplasmic compartment (85.9%) of the VIC population. To determine the pixel intensity of Sox9 immunoreactivity across the aortic valve cusp structure, quantitative analysis was performed. As shown in Figure 2F and graphically represented in Figure 2G, pixel intensity was significantly high in cells located within the distal (tip) region of the PND1 and PND10 aortic valve, suggesting enriched Sox9 expression at this location. Of note, no significant differences in pixel intensity were observed between atrial and ventricular sides of the cusp (data not shown). These observations show that post natal stages of valve growth and maturation are associated with detectable Sox9 expression, predominantly in the nucleus and cytoplasm in VICs and VECs, with enhanced immunoreactivity in VICs towards the distal tip. While expression remains high at 4 months of age, localization is predominantly cytoplasmic.

Figure 2. Sox9 is detected in VICs and VECs at post natal stages of valve growth and maturation.

Figure 2.

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Immunohistochemistry (A-C’) to show total Sox9 expression (red) pattern in maturing outflow tract valve structures at PND1 (A, A’), PND10 (B, B’) and 4 months (C, C’). Sox9 is double labeled with CD31 (green) to highlight endothelial cells (A-C’). A’-C’ represent high magnification of the boxed areas shown in A-C respectively. Arrows indicate valve endothelial cells, and arrowheads show valve interstitial cells. Nuclei are shown as DAPI-positive cells (blue). Scale bars in A-C indicate 50μm, and 10μm in A’-C’. (D) % of VICs and VECs that express pSox9 over the total number of DAPI-positive cells. * indicates statistical significance p<0.05. (E) localization of pSox9 in each cell type over the total number of immunoreactive cells * indicates statistical significance p<0.05 between different time points. x, #, + indicate statistical significance p<0.05 between each cell population with differential cellular localization of Sox9 expression within the same time point (x = nuclear vs. cytoplasmic, # = nuclear vs. nuclear+ cytoplasmic, + = cytoplasmic vs. nuclear+ cytoplasmic.(F) Representative images of Sox9 expression in VECs and VICs within proximal, medial and distal regions of the maturing aortic valve. (G) Quantitation of pixel number based on Sox9 immunoreactivity, normalized to cell number in regions shown in G. *, indicates statistical significance p<0.05.

pSox9 is enriched in VECs during post natal growth and maturation.

Post translational modifications of Sox9 have been shown to alter Sox9 nuclear/cytoplasmic shuttling (reviewed (Sim et al., 2008)). Further, phosphorylation of Sox9 at serine 181 directly affects gene transcription in non-valvular cell types (Huang et al., 2000; Liu et al., 2013). Using an antibody to specifically detect phosphorylation of Sox9 at Serine 181, we were not able to detect pSox9 expression within the developing valve structures at embryonic stages E11.5; which is in contrast to total Sox9 expression detected in mesenchyme cells and VICs (Figure 1A). At E14.5, low levels of pSox9 are observed outside the nucleus of mesenchyme cells within the aortic valve primordia (Figure 3B, arrowheads, Figure 3B’), and this continues at E18.5 (Figure 3C’). By PND1, pSox9 expression remains relatively low in VICs with only 41.7% of the cells showing immunoreactivity (arrowheads, Figure 3D’, 3G). Of these, 31.1% exclusively express pSox9 in the nucleus, 56.7% in the nuclear and cytoplasmic compartment, and 12.2% in the cytoplasm (Figure 3H). In contrast, pSox9 is detected in 100% of CD31-positive VECs in the nucleus and cytoplasm (arrows, Figure 3D’, 3G). At PND10, valve formation is largely complete as the final layer of elastin is laid down, (Hinton et al., 2006) and this is associated with an overall decrease in the number of VICs expressing pSox9 (23.3%) (Figure 3E, arrowheads Figure 3E’, G); including a decrease in nuclear (21.3%) localization, and an increase in nuclear and cytoplasmic (66.3%) localization (Figure 3H). In contrast, pSox9 continues to be expressed in 100% of the VEC population with a small, but significant shift in localization from the nucleus and cytoplasm (down to 85.1%), to the cytoplasm (increased to 14.9%) (Figure 3H). By 4 months, pSox9 expression is diffuse throughout the VIC and VEC cell populations, with a significant increase in the number of immunoreactive VICs, but the same number of immunoreactive VECs (100%) (Figure 3F, F’, G). Localization of pSox9 is predominantly cytoplasmic at the time (VICs; 90.5%, VECs; 84.3%) (Figure 3H), with reductions in the number of cells expressing pSox9 in nuclear, and nuclear and cytoplasmic compartments. Worthy of mention, nuclear pSox9 was not detected in VECs at this later time point. These data report pSox9 expression in growing and mature aortic valve structures, with notable enrichment in the VEC population, yet more predominant nuclear expression in VICs during post natal stages.

Figure 3. pSox9 expression is enriched in VECs during post natal stages of growth and maturation.

Figure 3.

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Immunohistochemistry to detect expression of Sox9 phosphorylated at Serine 181 (pSox9, red) with CD31 (endothelial cells, green) at E11.5 (A, A’), E14.5 (B, B’), E18.5 (C, C’), PND1 (D, D’), PND10 (E, E’) and 4 months of age (F, F’) in aortic valve structures. Nuclei are shown as DAPI-positive cells (blue). Endocardial cushion regions are outlined in Figures A and B. A’-F’ represent high magnification of the boxed areas shown in A-F respectively. Endocardial cushion regions are outlined in Figures D and E. Arrows indicate valve endothelial cells, arrowheads show mesenchyme cells (A’) and VICs (B’, F’). Scale bars in A-F indicate 50μm, and 10μm in A’-F’. (G) % of VICs and VECs that express pSox9 over the total number of DAPI-positive cells. * indicates statistical significance p<0.05. (H) Localization of pSox9 in each cell type over the total number of immunoreactive cells * indicates statistical significance p<0.05 between different time points. x, #, + indicate statistical significance p<0.05 between each cell population with differential cellular localization of Sox9 expression within the same time point (x = nuclear vs. cytoplasmic, # = nuclear vs. nuclear+ cytoplasmic, + = cytoplasmic vs. nuclear+ cytoplasmic. NCC, non-coronary cusp; LC, Left coronary cusp; RC, Right coronary cusp.

Enrichment of pSox9 in VECs during early post natal stages correlates with RockI and Aggrecan.

RhoA kinases RockI/II directly phosphorylate Sox9 at S181 in chondrocytes in vitro (Haudenschild et al., 2010), and studies have shown that phosphorylation increases Sox9 nuclear localization and promotes transactivation of target genes (Huang et al., 2000). Here, pSox9 expression correlates with CD31-positive RockI expression in VECs at PND10 (Figure 4A’-B’). To further determine a correlation between pSox9 and known target genes, we examined co-expression with Aggrecan and Col2. As shown, pSox9 is associated with Aggrecan immunoreactivity (Figure 4C, C’), but not Col2 (Figure 4D, D’), which is expressed within the annular region of the aortic valve (arrowhead Figure 4D). These results suggest that post translational modification of Sox9 may modulate its function in the valve cell populations through the differential regulation of diverse target genes.

Figure 4. pSox9 expression is associated RockI and Aggrecan in VECs at post natal stages.

Figure 4.

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Immunohistochemistry at PND10 to detect pSox9 (red) expression with RockI (green) (A, A’), Rock1 (red) with CD31 (green) (B, B’), pSox9 (red) with Aggrecan (green) (C, C’) and pSox9 (red) with Col2 (green) (D, D’). Nuclei are shown as DAPI-positive cells (blue). A’-D’ represent high magnification of the boxed areas shown in A-D respectively. Arrows indicate valve endothelial cells, arrowheads show VICs. Scale bars in A-D indicate 50μm, and 10μm in A’-D’.

DISCUSSION

This study highlights previously unappreciated dynamic expression patterns of Sox9 in heart valve cell populations of embryonic, post natal and adult aortic valve structures. Previous reports by our group and others have used the Tie2cre transgene to show a requirement of Sox9 in endothelial-derived cells during murine valvulogenesis (Akiyama et al., 2004; Lincoln et al., 2007). This current study did not detect Sox9 in endothelial cells of embryonic aortic valve structures by immunofluorescence (Figure 1), therefore suggesting that Sox9 functions in Tie2-cre derived mesenchyme cells. This conclusion is consistent with valve phenotypes observed in Sox9 mutant embryos; including normal transformation of endothelial cells, but decreased proliferation of newly transformed mesenchyme cells that leads to hypoplastic endocardial cushions and premature lethality by E14.5 (Akiyama et al., 2004; Lincoln et al., 2007). Further, Sox9 has been shown to molecularly interact with proliferation genes including Junb, Cops5, and Fos family members at E12.5, as determined by a ChIP-seq study by The Hoodless group (Garside et al., 2015). As shown in Figure 1, Sox9 expression remains high in mesenchyme cells and maturing VICs as the valve continues to grow, and reduced nuclear expression correlates with decreased cell proliferation (Hinton et al., 2006) (Anstine et al., 2016). Together, these studies support the proliferative role for Sox9 in the mesenchyme, but not endothelial cell population during valvulogenesis.

After birth, the valve continues to elongate and remodel until around PND10. At PND1, we show that Sox9 is detected in the majority of VICs, but in addition it is now detected in the majority of VECs. The regulation and function of Sox9 in these cell populations of the growing valve at PND1 are not known, but are likely diverse based on localization and post translational modification differences. In VICs, Sox9 is predominantly nuclear, whereas distribution in VECs is both nuclear and cytoplasmic. There is also a clear difference in the expression pattern of pSox9; highly enriched in the VEC population after birth. In general, the function of pSox9 is largely unexplored and few studies have been reported. In chick neural crest cells, Sox9 phosphorylation is required for delamination (Liu et al., 2013), however target genes are not known. Upstream, cyclic-AMP-dependent protein kinase A (PKA)-mediated phosphorylation enhances Sox9’s ability to transactivate Col2a1 (Huang et al., 2000). In this current study, PKA expression was not examined, but recent RNA-seq analysis of VECs from our lab detected transcript levels at post natal stages (Anstine et al., 2016). In addition to PKA, ROCK is also sufficient to directly phosphorylate Sox9 and enhance nuclear localization in response to Tgfβ1 treatment in chondrocytes. Here we show correlative expression between Sox9 and ROCK1 in post natal VECs, and this is associated with its known downstream target Aggrecan, but not Col2a1 expression (Figure 4). This might suggest that pSox9 functions in VECs to regulate proteoglycan synthesis particularly towards the peripheral edges of the valve leaflets, during stages of post natal remodeling. However, further work is required to test this. Interestingly, our previous work using the Col2a1-cre transgene to delete Sox9 in a sub-population of VICs localized adjacent to the endothelial layer from E15.5, revealed a requirement in these cells for expression of chondrogenic-related matrix proteins including Col2a1 and Cartilage Link Protein. (Lincoln et al., 2007). Together, these observations further highlight the diverse temporal and spatial requirements for Sox9 during development, growth and maturation.

From PND10, Sox9 and pSox9 remain high in VICs and VECs although there is a defined shift from nuclear (nuclear and cytoplasmic) towards cytoplasmic localization. Therefore, suggesting that its function as a transcription factor may decline during later stages of valve maintenance and its most critical window of requirement is during embryonic development in VICs, and post natal growth in both VICs and VECs. Beyond this descriptive report, future studies are needed to elucidate the contribution of Sox9 in these diverse cell populations within the valve, and explore the differential targets of pSox9 in the VECs, versus un-phosphorylated Sox9 in the VICs during the growth and maturation stages of post natal life. Aberrations during this stage could underlie valve defects manifested late in life or increase susceptibility to acquired disease or degenerative wear-and-tear in the adult population.

Supplementary Material

Supp figS1. Supplementary Figure 1. Sox9 (173–185 aa) expression, and negative controls for immunostaining of Sox9 and pSox9.

(A-J) Negative (A-H) and positive (I-J) controls for Sox9 immunoreactivity shown in Figures 1–4. Negative controls include no primary antibody (A, B), no secondary antibody for pSox9 (C, D) and Sox9 1–100aa (E, F). (G-H) Immunoreactivity of Sox9 detecting 173–185 aa. Note undetected expression in CD31-positive (green) (arrows) VECs at E14.5, expression in CD31-positive (green), and expression in mesenchyme cells (arrowheads). (I-J) No secondary antibody control for Sox9 173–185aa. Blue stain indicates DAPI-positive nuclei. Scale bars in A-F and I,J indicate 50μm, and 10μm in G and H.

ACKNOWLEDGMENTS

We thank Kaitlyn Thatcher for her technical support. This work was supported by The National Institute of Health, R01 HL127033 (JL), T32 HL098039 (VG, PM).

Grant Information:

National Institute of Health, R01 HL127033 (JL)

National Institute of Health, T32 HL098039 (DG, PIs: Drs. Vidu Garg and Peter Mohler)

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Associated Data

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

Supplementary Materials

Supp figS1. Supplementary Figure 1. Sox9 (173–185 aa) expression, and negative controls for immunostaining of Sox9 and pSox9.

(A-J) Negative (A-H) and positive (I-J) controls for Sox9 immunoreactivity shown in Figures 1–4. Negative controls include no primary antibody (A, B), no secondary antibody for pSox9 (C, D) and Sox9 1–100aa (E, F). (G-H) Immunoreactivity of Sox9 detecting 173–185 aa. Note undetected expression in CD31-positive (green) (arrows) VECs at E14.5, expression in CD31-positive (green), and expression in mesenchyme cells (arrowheads). (I-J) No secondary antibody control for Sox9 173–185aa. Blue stain indicates DAPI-positive nuclei. Scale bars in A-F and I,J indicate 50μm, and 10μm in G and H.

NIHMS982171-supplement-Supp_figS1.tif (65.5MB, tif)

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