SOX9 is an important antagonist of the canonical Wnt signalling pathway (Figure 4). Nevertheless, the underlying mechanism has not been clearly addressed. Its molecular mechanism is possibly involved in three aspects: promoting the degradation of β-catenin, inhibiting the formation of a β-catenin-TCF/LEF complex and prohibiting its transcriptional activity, and transcriptionally activating Wnt-related antagonists.

Many studies suggest that SOX9 has the potential to activate the canonical Wnt/β-catenin signalling pathway (Figure 5) in stem/progenitor cells or cancer cells. It has been proven that SOX9 activates the canonical Wnt signalling pathway predominantly by promoting the stability and nucleus translocation of β-catenin (Figures 5A, B) (Kormish et al., 2010; Kawai et al., 2016; Zhang et al., 2019; Chen et al., 2022; Li et al., 2023), in which the inactivation of GSK3β by SOX9 via the phosphorylation of Ser9, an increased level of β-catenin translocation into cell nuclei, and the enhanced transcriptional activity of the β-catenin/TCF complex are involved (Huang et al., 2019). Moreover, SOX9 could directly bind to the enhancer of Wnt target genes in conjugation with TCF and promote their transcription (Figure 5C) (Ramakrishnan et al., 2023).

It is also notable that SOX9 plays a critical role in promoting the transcription of receptors and co-receptors of the Wnt signalling pathway (Figure 5D), including FZDs, LRP family members and TCF4 (Wang et al., 2013; Leung et al., 2016; Ma et al., 2016). The findings of ChIP-sequencing demonstrated that SOX9 bound to the enhancers of Fzd8 and Sox4 and promoted their transcription in intestinal stem cells (ISCs) (Huang et al., 2017). In lung tip progenitors, SOX9 directly upregulated the transcription of LGR5 and CD44, which are the amplifiers of the Wnt signalling pathway (Sun et al., 2022). Moreover, SOX9 was also reported to bind the promoter of ubiquitin-specific peptidase 22 (USP22), a deubiquitinating enzyme, promote its transcription, and activate the Wnt/β-catenin pathway (Miao et al., 2022).

The expression of SOX9 is regulated and maintained by the Wnt/β-catenin signalling pathway, as supported by many studies (Blache et al., 2004; Snowball et al., 2015; Liang et al., 2022; Liu et al., 2022; Sun et al., 2022). In tracheal cartilage and human lung bud tip progenitors, the elevation of either the WNT protein or Wnt signalling pathway amplifier enhanced the expression of SOX9 and maintained its level in the cell nucleus (Nasr et al., 2021; Hein et al., 2022). β-catenin participated in this process because it could partially rescue the loss of the SOX9 transcription and protein level that was caused by the absence of Wnt signalling activators (Sun et al., 2022). Meanwhile, the β-catenin/TCF4 complex could directly bind to the promoter of the Sox9 gene and activate its transcription (Figure 5C) (Blache et al., 2004). Notably, post-transcriptional modifications are also involved in the regulation of the Wnt signalling pathway on SOX9. During neural crest delamination, the canonical Wnt signalling indirectly induces the phosphorylation of SOX9 on S64 and S181 and facilitates the modification of SOX9 by small ubiquitin-like modifier (SUMO) (Liu et al., 2013).

The Wnt/β-catenin pathway also plays an important role in inhibiting SOX9. It not only silences the Sox9 gene via DNA methylation in limb bud mesenchymal cells (Kumar and Lassar, 2014), but also promotes the degradation of SOX9, which is predominantly in a ubiquitin/26S proteasome-dependent way (Akiyama et al., 2004; Jin et al., 2006). Microtubule-associated serine/threonine kinase 4 (Mast4), the stability of which is maintained by Wnt signals, might participate in this degradation (Kim et al., 2022). Moreover, β-catenin inhibits the transcriptional activity of SOX9 in a dose-dependent manner (Akiyama et al., 2004). However, the molecular mechanism of this inhibition remains unclear.

In summary, many parts of signalling transduction are involved in the cross-regulation between SOX9 and the canonical Wnt/β-catenin signalling pathway, including the expression and stability maintenance of key proteins, the nuclear translocation of signal molecules, the formation and activity of transcription complex, and selective downstream target genes, all of which form a complicated signal network. The effect of SOX9-Wnt cross-regulation and underlying mechanisms are tissue- and cell type-specific, and might have changes during the development or under pathological conditions, which will be addressed in details in the next section.

The effects of SOX9-Wnt interactions on airway progenitors are space-specific. For instance, the canonical Wnt/β-catenin signalling pathway is required to upregulate the expression of SOX9 and maintain the homeostasis of distal lung tip epithelial progenitors during cell differentiation and tissue development (Ustiyan et al., 2016; Ostrin et al., 2018; Hein et al., 2022; Sun et al., 2022). In contrast, β-catenin inhibits the expression of SOX9 in the progenitors of proximal airway epithelial cells (Ustiyan et al., 2016). The absence of β-catenin further leads to the expanded expression of SOX9 in the proximal airway (Rockich et al., 2013). These findings support the hypothesis that the space-specific control of SOX9 by β-catenin maintains a normal proximal–peripheral patterning of lung tubules during pulmonary branching morphogenesis (Ustiyan et al., 2016).

Moreover, during the development of the fetal lung, SOX9 amplifies Wnt signalling, promotes lung tip epithelial progenitor cell proliferation, and prohibits precocious airway differentiation (Sun et al., 2022). In the proximal airway, the reciprocal crosstalk between Wnt/Lef1 signalling pathway and SOX9 also dynamically controls the healing of superficial airway epithelium after injuries by regulating the asymmetrical division of submucosal gland progenitors, with one SOX9⁺Wnt⁻ daughter cell maintaining stemness and quiescence and the other SOX9⁻Wnt⁺ cell maintaining proliferation (Ievlev et al., 2022). Apart from its activity in epithelial cells, Wnt signalling is also required to activate SOX9 during the differentiation of tracheal mesenchymal stem cells and tracheal cartilage development (Snowball et al., 2015).

It was found that SOX9 phosphorylates GSK3β, upregulates the level of nuclear β-catenin, and promotes the proliferation and differentiation of pancreatic progenitors (McDonald et al., 2012). Another study indicated that miR-690 induces Sox9 silencing and inactivates Wnt signalling, which leads to the arrested differentiation of β-cells from iPSC-derived insulin-producing cells (Xu et al., 2019). Nevertheless, the activation of Wnt signalling arrests the generation of β-cells by attenuating SOX9-dependent multipotent pancreatic progenitor cells (Dettmer et al., 2021). These findings collectively suggest that during SOX9-dependent pancreatic development and β-cell differentiation, the cross-regulation between SOX9 and Wnt signalling prevents the over-activation of SOX9. Intriguingly, the inactivation of SOX9 and a relatively low Wnt signalling level are crucial for the maintenance of functional adult β-cells, as evidenced by a study that found that aberrant activation of Wnt signalling and SOX9 in adult β-cells resulted in diabetes mellitus (Puri et al., 2013).

It has been demonstrated that the expression of SOX9 in the intestinal crypts, which is regulated by the Wnt signalling pathway, is indispensable for the development of Paneth cells, a type of highly specialized secretory epithelial cell that constructs the niches for ISCs (Blache et al., 2004; Bastide et al., 2007; Mori-Akiyama et al., 2007). Moreover, high-mobility group A1 (Hmga1) chromatin remodeling proteins upregulate Wnt agonist receptors and SOX9 and maintain ISC niches by expanding the Paneth cell compartments (Xian et al., 2017). Apart from its involvement with Paneth cells, SOX9 is also involved in the maintenance of an undifferentiated phenotype of intestinal epithelial progenitors in a Wnt-dependent manner (Blache et al., 2004). The molecular mechanism of the SOX9-Wnt axis in regulating the proliferation of ISCs was also explored. In diabetic mice, SOX9 transcriptionally activated the repressors of the canonical Wnt signalling pathway, such as Wnt4 and Tab2 (Huang et al., 2017). The absence of SOX9 led to the overactivation of the Wnt signalling pathway and abnormal proliferation and differentiation of ISCs. A bimodal role of SOX9 was proposed: A low level of Wnt-dependent SOX9 promoted ISC proliferation in the stem/progenitor cell compartment, whereas a high level of Wnt-independent SOX9 prohibited cell proliferation and induced terminal maturation of enteroendocrine precursors (Formeister et al., 2009).

The SOX9-Wnt signalling axis controls the lineage decisions of osteoblasts and chondrocytes (Akiyama et al., 2004; Hill et al., 2005). On one hand, the inactivation of Wnt/β-catenin signalling by SOX9 promotes cell differentiation from mesenchymal stem/progenitor cells (MSCs) into chondrocytes and the formation of cartilage nodules at the expense of osteogenesis (Hill et al., 2005; Topol et al., 2009; Dy et al., 2012; Venkatesan et al., 2012). On the other hand, the canonical Wnt/β-catenin signalling pathway represses the expression of SOX9, prohibits the chondrogenic potential of osteochondroprogenitors, and stimulates differentiation towards the osteoblast lineage (Hill et al., 2005; Kumar and Lassar, 2014; Lefebvre and Bhattaram, 2016). In addition, phase-specific mutual antagonism between SOX9 and β-catenin was found to be involved in the lineage decisions of osteochondroprogenitors. It was found that in D0 osteochondroprogenitors, SOX9 combined with the promoter of the target gene Ccn2, while β-catenin/TCF complex competed with SOX9 and bound to Ccn2 in D26 hypertrophic chondrocytes (Huang et al., 2010). Such phase-specific regulations coordinate osteoblast recruitment, cartilage renewal, and bone formation.

However, reciprocal inhibition between SOX9 and Wnt/β-catenin signalling is disturbed in osteoblasts and chondrocytes under pathological conditions. Hydrostatic pressure induces the activation of Wnt/β-catenin signalling and, consequently, elevates the expression of SOX9 in MSCs, leading to a higher level of chondrogenic differentiation (Cheng et al., 2022). Nevertheless, during the induction of osteonecrosis of the femoral head (ONFH), the inhibition of SOX9 downregulates the Wnt/β-catenin signalling pathway and suppresses osteogenic differentiation (Meng and Zhu, 2023). It was also reported that the perturbation of mutual SOX9-Wnt inhibition contributed to dysplasia. In developing limbs, a truncated SOX9 mutant interfered with SOX9-mediated Wnt inhibition, resulting in campomelia, a kind of genetic disorder with skeletal malformation (Au et al., 2023). In addition, Wnt11, which instigates the noncanonical Wnt signalling pathway, was reported to promote the transcription of SOX9 and support the chondrogenic differentiation of MSCs (Liu et al., 2014).

Neural crest cells (NCCs) are a transient population of embryonic multipotent stem cells that give rise to a wide variety of cell and tissue types, including cartilage and bone, most neurons, and all glia of the peripheral nervous system (Achilleos and Trainor, 2012; Liu et al., 2013). Similarly to MSC-derived osteochondrogenesis, during NCC-derived craniofacial osteochondrogenesis, Wnt/β-catenin signalling enhances osteogenic potential by counteracting SOX9, and arrests cell differentiation towards chondrocytes in which Yap/Taz is involved (Dash and Trainor, 2020; Zhao et al., 2022). In addition, the absence of Med23 induces abnormally elevated expression of SOX9, which leads to the inhibition of Wnt signalling and the perturbation of NCC-derived mesenchymal proliferation in the palatal shelf (Dash et al., 2021).

Apart from their involvement in osteochondrogenesis, NCCs play a more important role in neural crest induction, which is mediated by Wnt signals, with SOX9 serving as a crucial downstream transcriptional activator (Lee et al., 2004). Yardley and Garcia-Castro (Yardley and Garcia-Castro, 2012) confirmed that the upregulation of WNT molecules is prior to the elevated expression of the neural crest marker, SOX9, during the transformation from non-neural ectoderm to the neural crest. Moreover, during the initiation of neural crest delamination, the canonical Wnt signalling promotes SOX9 phosphorylation and SUMOylation (Liu et al., 2013). SOX9 was also reported as a biomarker of adult neural-crest-derived stem cells, and it interacted with the canonical Wnt signalling pathway to maintain stemness (Hoving et al., 2021).

It was proposed that in the limbal niches, SOX9 and Wnt/β-catenin signalling mutually antagonize to achieve a balance among quiescence, proliferation, and differentiation of limbal epithelial stem/progenitor cells (LEPCs). In cultured LEPCs, Sox9 knockdown caused a decreased level of GSK3β and increased expression of canonical-Wnt-signalling-related genes, such as CTNNB1 (encoding β-catenin) and WNT4, which repressed cell proliferation and promoted differentiation (Menzel-Severing et al., 2018). In turn, the expression of SOX9 was significantly suppressed with the treatment of exogenous GSK3β inhibitors and was enhanced by C59, a small-molecule Wnt inhibitor. Likewise, an elevated level of Wnt3a, a Wnt ligand that only activates the canonical Wnt signalling pathway, downregulated the expression of SOX9 and impaired the stem cell phenotype (Peng et al., 2015). Our ongoing study also found out that Wnt16b, a ligand of both the canonical and the non-canonical Wnt signalling pathway, promoted the proliferation of LEPCs via downregulating the expression of SOX9 and upregulating SOX11 (unpublished data). Although it was reported that the nucleocytoplasmic shuttling of SOX9 and β-catenin might be crucial in the regulation of LEPC proliferation and differentiation (Menzel-Severing et al., 2018), its mechanism has not been thoroughly elucidated. This balance might be controlled by the ligands expressed in the mesenchymal cells, such as Dickkopf-2 (Walker et al., 2020), or the activation of ΔNp63 (Gouveia et al., 2019).

SOX9 is the marker of hair follicle stem cells. The cross-regulation between SOX9 and WNT signalling determines the specification and cell fate commitment of hair follicle stem cells (Xu et al., 2015). During hair formation, a Pcad^high placode cell undergoes asymmetric cell division in a WNT^high environment and generates two daughter cells: one WNT^lowSOX9⁺ and the other WNT^highSOX9^-. WNT^lowSOX9⁺ cells migrate towards the suprabasal layer and maintain stemness, while WNT^highSOX9^- cells remain in the basal layer and undergo terminal differentiation (Ouspenskaia et al., 2016). The underlying mechanism of this cell specification lies in a WNT signalling gradient, in which WNT^lowSOX9⁺ cells respond to paracrine SHH expressed by WNT^highSOX9^- cells. Moreover, the knockdown of SOX9 led to the dampened expression of Wnt signalling pathway genes, such as LEF1, TCF1, and c-Myc in goat hair follicle stem cells (He et al., 2018).

It was reported that in the salivary glands, the population of SOX9⁺ progenitor cells increases after radiation damage via the activation of the Wnt/β-catenin pathway (Xu et al., 2022). During the development of teeth, SOX9 and Wnt signalling regulate mesenchymal and epithelial interactions and control the expansion and differentiation of apical stem/progenitor cells (Lav et al., 2023). In addition, adipose mesenchymal-stem-cell-derived exosomes activate the Wnt/β-catenin pathway by upregulating the expression of SOX9, leading to accelerated proliferation and migration of human skin fibroblast cells and promoting skin wound healing (Qian et al., 2021).

In the embryonic neural stem cells (NSCs) of mice, NF-α1 inhibited the Wnt/β-catenin signalling pathway and repressed cell proliferation, while it activated the MAPK/MEK/Sox9 signalling pathway and promoted the differentiation of NSCs into astrocytes (Selvaraj et al., 2017). Notably, the promotive effect of NF-α1 on SOX9 preceded its inhibition on β-catenin. Nevertheless, whether NF-α1 indirectly inhibits β-catenin via SOX9 remains unclear.