Direct programming of human pluripotent stem cells into endothelial progenitors with SOX17 and FGF2.

Transcription factors (TFs) are pivotal in guiding stem cell behavior, including their maintenance and differentiation. Using single-cell RNA sequencing, we investigated TFs expressed in endothelial progenitors (EPs) derived from human pluripotent stem cells (hPSCs) and identified upregulated expression of SOXF factors SOX7, SOX17, and SOX18 in the EP population. To test whether overexpression of these factors increases differentiation efficiency, we established inducible hPSC lines for each SOXF factor and found only SOX17 overexpression robustly increased the percentage of cells expressing CD34 and vascular endothelial cadherin (VEC). Conversely, SOX17 knockdown via CRISPR-Cas13d significantly compromised EP differentiation. Intriguingly, we discovered SOX17 overexpression alone was sufficient to generate CD34+VEC+CD31- cells, and, when combined with FGF2 treatment, more than 90% of CD34+VEC+CD31+ EP was produced. These cells are capable of further differentiating into endothelial cells. These findings underscore an undiscovered role of SOX17 in programming hPSCs toward an EP lineage, illuminating pivotal mechanisms in EP differentiation.

SOX17/ETV2 improves the direct reprogramming of adult fibroblasts to endothelial cells.

An autologous source of vascular endothelial cells (ECs) is valuable for vascular regeneration and tissue engineering without the concern of immune rejection. The transcription factor ETS variant 2 (ETV2) has been shown to directly convert patient fibroblasts into vascular EC-like cells. However, reprogramming efficiency is low and there are limitations in EC functions, such as eNOS expression. In this study, we directly reprogram adult human dermal fibroblasts into reprogrammed ECs (rECs) by overexpressing SOX17 in conjunction with ETV2. We find several advantages to rEC generation using this approach, including improved reprogramming efficiency, increased enrichment of EC genes, formation of large blood vessels carrying blood from the host, and, most importantly, expression of eNOS in vivo. From these results, we present an improved method to reprogram adult fibroblasts into functional ECs and posit ideas for the future that could potentially further improve the reprogramming process.

SOX17 enables immune evasion of early colorectal adenomas and cancers.

A hallmark of cancer is the avoidance of immune destruction. This process has been primarily investigated in locally advanced or metastatic cancer1-3; however, much less is known about how pre-malignant or early invasive tumours evade immune detection. Here, to understand this process in early colorectal cancers (CRCs), we investigated how naive colon cancer organoids that were engineered in vitro to harbour Apc-null, KrasG12D and Trp53-null (AKP) mutations adapted to the in vivo native colonic environment. Comprehensive transcriptomic and chromatin analyses revealed that the endoderm-specifying transcription factor SOX17 became strongly upregulated in vivo. Notably, whereas SOX17 loss did not affect AKP organoid propagation in vitro, its loss markedly reduced the ability of AKP tumours to persist in vivo. The small fraction of SOX17-null tumours that grew displayed notable interferon-γ (IFNγ)-producing effector-like CD8+ T cell infiltrates in contrast to the immune-suppressive microenvironment in wild-type counterparts. Mechanistically, in both endogenous Apc-null pre-malignant adenomas and transplanted organoid-derived AKP CRCs, SOX17 suppresses the ability of tumour cells to sense and respond to IFNγ, preventing anti-tumour T cell responses. Finally, SOX17 engages a fetal intestinal programme that drives differentiation away from LGR5+ tumour cells to produce immune-evasive LGR5- tumour cells with lower expression of major histocompatibility complex class I (MHC-I). We propose that SOX17 is a transcription factor that is engaged during the early steps of colon cancer to orchestrate an immune-evasive programme that permits CRC initiation and progression.

Mechanistic study of transcription factor Sox18 during heart development.

Heart development is a delicate and complex process regulated by coordination of various signaling pathways. In this study, we investigated the role of sox18 in heart development by modulating Wnt/ß-Catenin signaling pathways. Our spatiotemporal expression analysis revealed that sox18 is mainly expressed in the heart, branchial arch, pharyngeal arch, spinal cord, and intersegmental vessels at the tailbud stage of Xenopus tropicalis embryo. Overexpression of sox18 in the X. tropicalis embryos causes heart edema, while loss-of-function of sox18 can change the signal of developmental heart marker gata4 at different stages, suggesting that sox18 plays an essential role in the development of the heart. Knockdown of SOX18 in human umbilical vein endothelial cells suggests a link between Sox18 and ß-CATENIN, a key regulator of the Wnt signaling pathway. Sox18 negatively regulates islet1 and tbx3, the downstream factors of Wnt/ß-Catenin signaling, during the linear heart tube formation and the heart looping stage. Taken together, our findings highlight the crucial role of Sox18 in the development of the heart via inhibiting Wnt/ß-Catenin signaling.

Human Primordial Germ Cell-Like Cell Induction from Pluripotent Stem Cells by SOX17 and PRDM1 Expression.

Human primordial germ cell (PGC) development initiates about 2 weeks after fertilization during embryogenesis. Unique molecular events follow, including epigenetic resetting, to establish functional gametes (egg and sperm). Due to the inaccessibility of human embryos, it is essential to have an amenable experimental platform to investigate the mechanisms and potential dysfunctions of the events. We previously established a PGC-like cell (PGCLC) differentiation method using human pluripotent stem cells (PSCs) via induction of precursor cells followed by stimulation with a cytokine cocktail including BMP. We also revealed that the expression of PGC specifiers, SOX17 and PRDM1, can robustly induce PGCLCs from PSCs without the cytokines. The balance of SOX17 and PRDM1 is critical for germ cell fate since the two factors also regulate endoderm differentiation. Here we describe a detailed procedure for PGCLC differentiation with the balanced induction of SOX17 and PRDM1. The protocol can be used for PGC induction in other mammalian species exhibiting PGCs with SOX17 expression. Together, these studies will advance the understanding of germ cell biology and its applications in reproductive technology and medicine.

SOX18 Promotes the Proliferation of Dermal Papilla Cells via the Wnt/ß-Catenin Signaling Pathway.

SRY-box transcription factor 18 (SOX18) is known to play a crucial role in the growth and development of hair follicles (HF) in both humans and mice. However, the specific effect of SOX18 on sheep hair follicles remains largely unknown. In our previous study, we observed that SOX18 was specifically expressed within dermal papilla cells (DPCs) in ovine hair follicles, leading us to investigate its potential role in the growth of hair follicles in sheep. In the present study, we aimed to examine the effect of SOX18 in DPCs and preliminarily study its regulatory mechanism through RNA-seq. We initially found that the overexpression of SOX18 promoted the proliferation of DPCs compared to the negative control group, while the interference of SOX18 had the opposite effect. To gain further insight into the regulatory mechanism of SOX18, we conducted RNA-seq analysis after knocking down SOX18 in Hu sheep DPCs. The result showed that the Wnt/ß-Catenin signaling pathway was involved in the growth process of DPC after SOX18 knockdown. Subsequently, we investigated the effect of SOX18 on the Wnt/ß-Catenin signaling pathway in DPCs using TOP/FOP-flash, qRT-PCR, and Western blot (WB) analysis. Our data demonstrated that SOX18 could activate the Wnt/ß-Catenin signaling pathway in DPCs. Additionally, we observed that SOX18 could rescue the proliferation of DPCs after inhibiting the Wnt/ß-Catenin signaling pathway. These findings underscore the essential role of SOX18 as a functional molecule governing the proliferation of DPCs. Additionally, these findings also greatly enhance our understanding of the role of SOX18 in the proliferation of DPCs and the growth of wool in Hu sheep.

Compound AC1Q3QWB upregulates CDKN1A and SOX17 by interrupting the HOTAIR-EZH2 interaction and enhances the efficacy of tazemetostat in endometrial cancer.

Endometrial cancer (EC) is a common malignancy of the female reproductive system, with an escalating incidence. Recurrent/metastatic EC presents a poor prognosis. The interaction between the long non-coding RNA (lncRNA) HOTAIR and the polycomb repressive complex 2 (PRC2) induces abnormal silencing of tumor suppressor genes, exerting a pivotal role in tumorigenesis. We have previously discovered AC1Q3QWB (AQB), a small-molecule compound targeting HOTAIR-EZH2 interaction. In the present study, we unveil that AQB selectively hampers the interaction between HOTAIR and EZH2 within EC cells, thus reversing the epigenetic suppression of tumor suppressor genes. Furthermore, our findings demonstrate AQB's synergistic effect with tazemetostat (TAZ), an EZH2 inhibitor, significantly boosting the expression of CDKN1A and SOX17. This, in turn, induces cell cycle arrest and impedes EC cell proliferation, migration, and invasion. In vivo experiments further validate AQB's potential by enhancing TAZ's anti-tumor efficacy at lower doses. Our results advocate AQB, a recently discovered small-molecule inhibitor, as a promising agent against EC cells. When combined with TAZ, it offers a novel therapeutic strategy for EC treatment.

Lymphatic Defects in Zebrafish sox18 Mutants Are Exacerbated by Perturbed VEGFC Signaling, While Masked by Elevated sox7 Expression.

Mutations in the transcription factor-coding gene SOX18, the growth factor-coding gene VEGFC and its receptor-coding gene VEGFR3/FLT4 cause primary lymphedema in humans. In mammals, SOX18, together with COUP-TFII/NR2F2, activates the expression of Prox1, a master regulator in lymphatic identity and development. Knockdown studies have also suggested an involvement of Sox18, Coup-tfII/Nr2f2, and Prox1 in zebrafish lymphatic development. Mutants in the corresponding genes initially failed to recapitulate the lymphatic defects observed in morphants. In this paper, we describe a novel zebrafish sox18 mutant allele, sa12315, which behaves as a null. The formation of the lymphatic thoracic duct is affected in sox18 homozygous mutants, but defects are milder in both zygotic and maternal-zygotic sox18 mutants than in sox18 morphants. Remarkably, in sox18 mutants, the expression of the closely related sox7 gene is elevated where lymphatic precursors arise. Sox7 could thus mask the absence of a functional Sox18 protein and account for the mild lymphatic phenotype in sox18 mutants, as shown in mice. Partial knockdown of vegfc exacerbates lymphatic defects in sox18 mutants, making them visible in heterozygotes. Our data thus reinforce the genetic interaction between Sox18 and Vegfc in lymphatic development, previously suggested by knockdown studies, and highlight the ability of Sox7 to compensate for Sox18 lymphatic dysfunction.

E2F1 Mediates SOX17 Deficiency-Induced Pulmonary Hypertension.

BACKGROUND: Rare genetic variants and genetic variation at loci in an enhancer in SOX17 (SRY-box transcription factor 17) are identified in patients with idiopathic pulmonary arterial hypertension (PAH) and PAH with congenital heart disease. However, the exact role of genetic variants or mutations in SOX17 in PAH pathogenesis has not been reported. METHODS: SOX17 expression was evaluated in the lungs and pulmonary endothelial cells (ECs) of patients with idiopathic PAH. Mice with Tie2Cre-mediated Sox17 knockdown and EC-specific Sox17 deletion were generated to determine the role of SOX17 deficiency in the pathogenesis of PAH. Human pulmonary ECs were cultured to understand the role of SOX17 deficiency. Single-cell RNA sequencing, RNA-sequencing analysis, and luciferase assay were performed to understand the underlying molecular mechanisms of SOX17 deficiency-induced PAH. E2F1 (E2F transcription factor 1) inhibitor HLM006474 was used in EC-specific Sox17 mice. RESULTS: SOX17 expression was downregulated in the lung and pulmonary ECs from patients with idiopathic PAH. Mice with Tie2Cre-mediated Sox17 knockdown and EC-specific Sox17 deletion induced spontaneously mild pulmonary hypertension. Loss of endothelial Sox17 in EC exacerbated hypoxia-induced pulmonary hypertension in mice. Loss of SOX17 in lung ECs induced endothelial dysfunctions including upregulation of cell cycle programming, proliferative and antiapoptotic phenotypes, augmentation of paracrine effect on pulmonary arterial smooth muscle cells, impaired cellular junction, and BMP (bone morphogenetic protein) signaling. E2F1 signaling was shown to mediate the SOX17 deficiency-induced EC dysfunction. Pharmacological inhibition of E2F1 in Sox17 EC-deficient mice attenuated pulmonary hypertension development. CONCLUSIONS: Our study demonstrated that endothelial SOX17 deficiency induces pulmonary hypertension through E2F1. Thus, targeting E2F1 signaling represents a promising approach in patients with PAH.

The dynamic expression of SOX17 in germ cells from human female foetus and adult ovaries after specification.

Background: SOX17 has been identified as a critical factor in specification of human primordial germ cells, but whether SOX17 regulates development of germ cells after sex differentiation is poorly understood. Methods: We collected specimens of gonadal ridge from an embryo (n=1), and ovaries of foetuses (n=23) and adults (n=3). Germ cells were labelled with SOX17, VASA (classic germ cells marker), phosphohistone H3 (PHH3, mitosis marker) and synaptonemal complex protein 3 (SCP3, meiosis marker). Results: SOX17 was detected in both cytoplasm and nucleus of oogonia and oocytes of primordial and primary follicles from 15 to 28 gestational weeks (GW). However, it was exclusively expressed in cytoplasm of oogonia at 7 GW, and in nucleus of oocytes in secondary follicles. Co-expression rates of SOX17 in VASA+ germ cells ranged from 81.29% to 97.81% in foetuses. Co-staining rates of SOX17 and PHH3 or SCP3 were 0%-34% and 0%-57%, respectively. Interestingly, we distinguished a subpopulation of SOX17+VASA- germ cells in fetal ovaries. These cells clustered in the cortex and could be co-stained with the mitosis marker PHH3 but not the meiosis marker SCP3. Conclusions: The dynamic expression of SOX17 was detected in human female germ cells. We discovered a population of SOX17+ VASA- germ cells clustering at the cortex of ovaries. We could not find a relationship between mitosis or meiosis and SOX17 or VASA staining in germ cells. Our findings provide insight into the potential role of SOX17 involving germ cells maturation after specification, although the mechanism is unclear and needs further investigation.

Sox7-positive endothelial progenitors establish coronary arteries and govern ventricular compaction.

The cardiac endothelium influences ventricular chamber development by coordinating trabeculation and compaction. However, the endothelial-specific molecular mechanisms mediating this coordination are not fully understood. Here, we identify the Sox7 transcription factor as a critical cue instructing cardiac endothelium identity during ventricular chamber development. Endothelial-specific loss of Sox7 function in mice results in cardiac ventricular defects similar to non-compaction cardiomyopathy, with a change in the proportions of trabecular and compact cardiomyocytes in the mutant hearts. This phenotype is paralleled by abnormal coronary artery formation. Loss of Sox7 function disrupts the transcriptional regulation of the Notch pathway and connexins 37 and 40, which govern coronary arterial specification. Upon Sox7 endothelial-specific deletion, single-nuclei transcriptomics analysis identifies the depletion of a subset of Sox9/Gpc3-positive endocardial progenitor cells and an increase in erythro-myeloid cell lineages. Fate mapping analysis reveals that a subset of Sox7-null endothelial cells transdifferentiate into hematopoietic but not cardiomyocyte lineages. Our findings determine that Sox7 maintains cardiac endothelial cell identity, which is crucial to the cellular cross-talk that drives ventricular compaction and coronary artery development.

Pan-cancer Analysis Reveals Cancer-dependent Expression of SOX17 and Associated Clinical Outcomes.

BACKGROUND/AIM: SRY-box containing gene 17 (SOX17) plays a pivotal role in cancer onset and progression and is considered a potential target for cancer diagnosis and treatment. However, the expression pattern of SOX17 in cancer and its clinical relevance remains unknown. Here, we explored the relationship between the expression of SOX17 and drug response by examining SOX17 expression patterns across multiple cancer types. MATERIALS AND METHODS: Single-cell and bulk RNA-seq analyses were used to explore the expression profile of SOX17. Analysis results were verified with qPCR and immunohistochemistry. Survival, drug response, and co-expression analyses were performed to illustrate its correlation with clinical outcomes. RESULTS: The results revealed that abnormal expression of SOX17 is highly heterogenous across multiple cancer types, indicating that SOX17 manifests as a cancer type-dependent feature. Furthermore, the expression pattern of SOX17 is also associated with cancer prognosis in certain cancer types. Strong SOX17 expression correlates with the potency of small molecule drugs that affect PI3K/mTOR signaling. FGF18, a gene highly relevant to SOX17, is involved in the PI3K-AKT signaling pathway. Single-cell RNA-seq analysis demonstrated that SOX17 is mainly expressed in endothelial cells and barely expressed in other cells but spreads to other cell types during the development of ovarian cancer. CONCLUSION: Our study revealed the expression pattern of SOX17 in pan-cancer through bulk and single-cell RNA-seq analyses and determined that SOX17 is related to the diagnosis, staging, and prognosis of some tumors. These findings have clinical implications and may help identify mechanistic pathways amenable to pharmacological interventions.

Evaluation of the determinants for improved pluripotency induction and maintenance by engineered SOX17.

An engineered SOX17 variant with point mutations within its DNA binding domain termed SOX17FNV is a more potent pluripotency inducer than SOX2, yet the underlying mechanism remains unclear. Although wild-type SOX17 was incapable of inducing pluripotency, SOX17FNV outperformed SOX2 in mouse and human pluripotency reprogramming. In embryonic stem cells, SOX17FNV could replace SOX2 to maintain pluripotency despite considerable sequence differences and upregulated genes expressed in cleavage-stage embryos. Mechanistically, SOX17FNV co-bound OCT4 more cooperatively than SOX2 in the context of the canonical SoxOct DNA element. SOX2, SOX17, and SOX17FNV were all able to bind nucleosome core particles in vitro, which is a prerequisite for pioneer transcription factors. Experiments using purified proteins and in cellular contexts showed that SOX17 variants phase-separated more efficiently than SOX2, suggesting an enhanced ability to self-organise. Systematic deletion analyses showed that the N-terminus of SOX17FNV was dispensable for its reprogramming activity. However, the C-terminus encodes essential domains indicating multivalent interactions that drive transactivation and reprogramming. We defined a minimal SOX17FNV (miniSOX) that can support reprogramming with high activity, reducing the payload of reprogramming cassettes. This study uncovers the mechanisms behind SOX17FNV-induced pluripotency and establishes engineered SOX factors as powerful cell engineering tools.

SOX17-mediated LPAR4 expression plays a pivotal role in cardiac development and regeneration after myocardial infarction.

Lysophosphatidic acid receptor 4 (LPAR4) exhibits transient expression at the cardiac progenitor stage during pluripotent stem cell (PSC)-derived cardiac differentiation. Using RNA sequencing, promoter analyses, and a loss-of-function study in human PSCs, we discovered that SRY-box transcription factor 17 (SOX17) is an essential upstream factor of LPAR4 during cardiac differentiation. We conducted mouse embryo analyses to further verify our human PSC in vitro findings and confirmed the transient and sequential expression of SOX17 and LPAR4 during in vivo cardiac development. In an adult bone marrow transplantation model using LPAR4 promoter-driven GFP cells, we observed two LPAR4+ cell types in the heart following myocardial infarction (MI). Cardiac differentiation potential was shown in heart-resident LPAR4+ cells, which are SOX17+, but not bone marrow-derived infiltrated LPAR4+ cells. Furthermore, we tested various strategies to enhance cardiac repair through the regulation of downstream signals of LPAR4. During the early stages following MI, the downstream inhibition of LPAR4 by a p38 mitogen-activated protein kinase (p38 MAPK) blocker improved cardiac function and reduced fibrotic scarring compared to that observed following LPAR4 stimulation. These findings improve our understanding of heart development and suggest novel therapeutic strategies that enhance repair and regeneration after injury by modulating LPAR4 signaling.

Targeting SOX18 Transcription Factor Activity by Small-Molecule Inhibitor Sm4 in Non-Small Lung Cancer Cell Lines.

The transcription factor SOX18 has been shown to play a crucial role in lung cancer progression and metastasis. In this study, we investigated the effect of Sm4, a SOX18 inhibitor, on cell cycle regulation in non-small cell lung cancer (NSCLC) cell lines LXF-289 and SK-MES-1, as well as normal human lung fibroblast cell line IMR-90. Our results demonstrated that Sm4 treatment induced cytotoxic effects on all three cell lines, with a greater effect observed in NSCLC adenocarcinoma cells. Sm4 treatment led to S-phase cell accumulation and upregulation of p21, a key regulator of the S-to-G2/M phase transition. While no significant changes in SOX7 or SOX17 protein expression were observed, Sm4 treatment resulted in a significant upregulation of SOX17 gene expression. Furthermore, our findings suggest a complex interplay between SOX18 and p21 in the context of lung cancer, with a positive correlation observed between SOX18 expression and p21 nuclear presence in clinical tissue samples obtained from lung cancer patients. These results suggest that Sm4 has the potential to disrupt the cell cycle and target cancer cell growth by modulating SOX18 activity and p21 expression. Further investigation is necessary to fully understand the relationship between SOX18 and p21 in lung cancer and to explore the therapeutic potential of SOX18 inhibition in lung cancer.

SOX17 Enhancer Variants Disrupt Transcription Factor Binding And Enhancer Inactivity Drives Pulmonary Hypertension.

BACKGROUND: Pulmonary arterial hypertension (PAH) is a rare disease characterized by remodeling of the pulmonary arteries, increased vascular resistance, and right-sided heart failure. Genome-wide association studies of idiopathic/heritable PAH established novel genetic risk variants, including conserved enhancers upstream of transcription factor (TF) SOX17 containing 2 independent signals. SOX17 is an important TF in embryonic development and in the homeostasis of pulmonary artery endothelial cells (hPAEC) in the adult. Rare pathogenic mutations in SOX17 cause heritable PAH. We hypothesized that PAH risk alleles in an enhancer region impair TF-binding upstream of SOX17, which in turn reduces SOX17 expression and contributes to disturbed endothelial cell function and PAH development. METHODS: CRISPR manipulation and siRNA were used to modulate SOX17 expression. Electromobility shift assays were used to confirm in silico-predicted TF differential binding to the SOX17 variants. Functional assays in hPAECs were used to establish the biological consequences of SOX17 loss. In silico analysis with the connectivity map was used to predict compounds that rescue disturbed SOX17 signaling. Mice with deletion of the SOX17-signal 1 enhancer region (SOX17-4593/enhKO) were phenotyped in response to chronic hypoxia and SU5416/hypoxia. RESULTS: CRISPR inhibition of SOX17-signal 2 and deletion of SOX17-signal 1 specifically decreased SOX17 expression. Electromobility shift assays demonstrated differential binding of hPAEC nuclear proteins to the risk and nonrisk alleles from both SOX17 signals. Candidate TFs HOXA5 and ROR-α were identified through in silico analysis and antibody electromobility shift assays. Analysis of the hPAEC transcriptomes revealed alteration of PAH-relevant pathways on SOX17 silencing, including extracellular matrix regulation. SOX17 silencing in hPAECs resulted in increased apoptosis, proliferation, and disturbance of barrier function. With the use of the connectivity map, compounds were identified that reversed the SOX17-dysfunction transcriptomic signatures in hPAECs. SOX17 enhancer knockout in mice reduced lung SOX17 expression, resulting in more severe pulmonary vascular leak and hypoxia or SU5416/hypoxia-induced pulmonary hypertension. CONCLUSIONS: Common PAH risk variants upstream of the SOX17 promoter reduce endothelial SOX17 expression, at least in part, through differential binding of HOXA5 and ROR-α. Reduced SOX17 expression results in disturbed hPAEC function and PAH. Existing drug compounds can reverse the disturbed SOX17 pulmonary endothelial transcriptomic signature.

Single-Cell RNA Sequencing of Sox17-Expressing Lineages Reveals Distinct Gene Regulatory Networks and Dynamic Developmental Trajectories.

During early embryogenesis, the transcription factor SOX17 contributes to hepato-pancreato-biliary system formation and vascular-hematopoietic emergence. To better understand Sox17 function in the developing endoderm and endothelium, we developed a dual-color temporal lineage-tracing strategy in mice combined with single-cell RNA sequencing to analyze 6934 cells from Sox17-expressing lineages at embryonic days 9.0-9.5. Our analyses showed 19 distinct cellular clusters combined from all 3 germ layers. Differential gene expression, trajectory and RNA-velocity analyses of endothelial cells revealed a heterogenous population of uncommitted and specialized endothelial subtypes, including 2 hemogenic populations that arise from different origins. Similarly, analyses of posterior foregut endoderm revealed subsets of hepatic, pancreatic, and biliary progenitors with overlapping developmental potency. Calculated gene-regulatory networks predict gene regulons that are dominated by cell type-specific transcription factors unique to each lineage. Vastly different Sox17 regulons found in endoderm versus endothelial cells support the differential interactions of SOX17 with other regulatory factors thereby enabling lineage-specific regulatory actions.

SOX7 deficiency causes ventricular septal defects through its effects on endocardial-to-mesenchymal transition and the expression of Wnt4 and Bmp2.

SOX7 is a transcription factor-encoding gene located in a region on chromosome 8p23.1 that is recurrently deleted in individuals with ventricular septal defects (VSDs). We have previously shown that Sox7-/- embryos die of heart failure around E11.5. Here, we demonstrate that these embryos have hypocellular endocardial cushions with severely reduced numbers of mesenchymal cells. Ablation of Sox7 in the endocardium also resulted in hypocellular endocardial cushions, and we observed VSDs in rare E15.5 Sox7flox/-;Tie2-Cre and Sox7flox/flox;Tie2-Cre embryos that survived to E15.5. In atrioventricular explant studies, we showed that SOX7 deficiency leads to a severe reduction in endocardial-to-mesenchymal transition (EndMT). RNA-seq studies performed on E9.5 Sox7-/- heart tubes revealed severely reduced Wnt4 transcript levels. Wnt4 is expressed in the endocardium and promotes EndMT by acting in a paracrine manner to increase the expression of Bmp2 in the myocardium. Both WNT4 and BMP2 have been previously implicated in the development of VSDs in individuals with 46,XX sex reversal with dysgenesis of kidney, adrenals and lungs (SERKAL) syndrome and in individuals with short stature, facial dysmorphism and skeletal anomalies with or without cardiac anomalies 1 (SSFSC1) syndrome, respectively. We now show that Sox7 and Wnt4 interact genetically in the development of VSDs through their additive effects on endocardial cushion development with Sox7+/-;Wnt4+/- double heterozygous embryos having hypocellular endocardial cushions and perimembranous and muscular VSDs not seen in their Sox7+/- and Wnt4+/- littermates. These results provide additional evidence that SOX7, WNT4 and BMP2 function in the same pathway during mammalian septal development and that their deficiency can contribute to the development of VSDs in humans.