Introduction

The lung is a structurally and functionally intricate organ, with over 40 different, known cell types1. The advent of single-cell sequencing and other technologies continues to increase this number and contributes to the striking complexity of the lung. At its most proximal portion, the cartilaginous conducting airways harbor a pseudostratified mucociliary epithelium that plays a vital role in host defense. Inhaled harmful particles, pathogenic organisms, and debris are expectorated from the airways via a process known as mucociliary clearance (MCC). Inhaled contents are first entrapped by a layer of mucus, produced by goblet cells, that are then transported proximally by the unidirectional beating of cilia from terminal bronchioles to the trachea2. The coordinated removal of debris by ciliated cells and mucus-producing goblet cells is facilitated by lubrication from a periciliary water layer. The concerted undertaken by these multiple cell types together comprise the mucociliary escalator2. The bronchioles, also referred to as the conducting airways, are additionally important for moving gases to and from the distal lung, which contain the alveolar sacs necessary for gas exchange. Atmospheric oxygen undergoes exchange for blood carbon dioxide, a process that promotes cellular respiration for all tissues of the body.

The wingless related-integration site (Wnt)/β-catenin signaling pathway plays an instrumental role in stem cell self-renewal across several tissue epithelia3. R-spondin ligands are cysteine-rich glycoproteins that bind to their cognate leucine-rich repeat-containing G-protein coupled receptor (LGR) LGR4/5/6 receptors and E3 ubiquitin ligases ring finger protein (RNF)43/ZNRF3 via their furin-like domains4,5. In the absence of R-spondins, RNF43/ZNR43 ubiquitinate the Frizzled receptors that targets them for degradation (Fig. 1a, b). As such, Wnt signaling is dampened. Under canonical conditions in the absence of Wnt ligand, β-catenin is in complex with several other proteins including Adenomatous polyposis coli (APC), Glycogen synthase kinase-3a, Glycogen synthase kinase-3β (GSK3β), casein kinase I (CKI), Axin, and Disheveled, among others (Fig. 1a)3. These proteins together comprise a destruction complex. CKIγ first phosphorylates β-catenin at residue serine 45, a priming event that allows for recognition by GSK3β, which phosphorylates β-catenin at serine 33, serine 37, and threonine 413. These N-terminal post-translational modifications mark β-catenin for ubiquitination and subsequent proteasomal degradation by the E3 ubiquitin ligase β-transducin repeat-containing protein (Fig. 1a).

Fig. 1: Overview of canonical Wnt signaling.
figure 1

a In the absence of RSPO binding to LGR4/5/6, ubiquitin ligase ZNRF43/RNF43 ubiquitinates the Frizzled receptor which leads to receptor complex endocytosis, β-catenin degradation and subsequent inhibition of Wnt-driven transcriptional activity5. b The binding of RSPO to LGR4/5/6 potentiates Wnt signaling by removing ZNR43/RNF43 ubiquitin ligase from the cell membrane, which would otherwise mark Frizzled receptor for ubiquitination. Frizzled receptors are then able to interact with both Wnt ligand and LRP5/6 co-receptor to drive Wnt signaling cascade. β-catenin then escapes cytoplasmic proteasomal degradation, resulting in its nuclear translocation, interactions with transcription factors. TCF/LEF, and subsequent transactivation of Wnt target genes like c-MYC, CyclinD1, and Axin25.

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However, upon Wnt transcription and translation, the ligand enters to the endoplasmic reticulum (ER) and encounters Porcupine, an ER-resident protein that palmitoylates N-terminal cysteine residues of Wnt proteins. This lipid tail modification has been shown to be necessary for Wnt ligand secretion6,7. Wnt ligand then binds to LRP5/6 and appropriate Frizzled co-receptor on the same or neighboring cell. Moreover, R-spondins can potentiate and amplify Wnt signaling by forming complexes with their cognate LGR pair and binding to RNF43/ZNR43 to prevent Frizzled receptor ubiquitination. Together, these events allow for a cascade of molecular events that results in disassembly of the cytoplasmic destruction complex (Fig. 1b). β-catenin then accumulates in the cytoplasm, subsequently translocates to the nucleus, and interacts with transcription factors T-cell factor/Lymphoid enhancer factor 1 (TCF/LEF) (Fig. 1b). In this way, β-catenin drives transactivation of downstream target genes such as c-MYC, AXIN2, and CYCLIN D1 among others (Fig. 1b). The canonical Wnt signaling described above is important in a variety of cellular processes including proliferation, self-renewal, epithelial to mesenchymal transitions, and migration and motility.

In contrast, non-canonical Wnt signaling is often thought of as the β-catenin-independent pathway. One arm of this pathway regulates planar cell polarity (PCP), during which Frizzled receptors trigger downstream activation of RhoA and Rac GTPases that promote cytoskeletal remodeling8. A second arm of non-canonical Wnt signaling lies in Wnt-Frizzled binding that triggers Phospholipase C and downstream Ca2+ activity for regulation of cell migration and fate decisions8. It is important to note that Porcupine palmitoylates all 19 mammalian Wnt ligands and is therefore necessary for their secretion, including those that partake in non-canonical signaling.

Over the past several years, much research has been put forth toward carefully dissecting the nuanced role of Wnt signaling across several disciplines pertaining to lung biology. This review aims to highlight the major contributions made to our current understanding of the Wnt signaling pathway in lung and airway development, its role in proximal and distal airway homeostasis and relevant niche biology, as well as its role in directed differentiation efforts of induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs) to the lung lineage, in organoid culture models, and its perturbations in disease states.

Development

Detailed staging and mechanisms of both human and murine lung development have been well reviewed by others9,10,11,12. To briefly summarize murine lung development, during the embryonic stage, the ventral anterior foregut endoderm (AFE) expresses the transcription factor Nkx2.1 at mouse embryonic day (E) 9.0 as a sign of the specification to promote initial lung budding. From E9.5–E12.5, two lung buds with high Nkx2.1 expression and a proximal portion with low Nkx2.1 that later forms the trachea emerge concomitantly with tracheo-esophageal septation. From E12.5–E16.5 during the pseudoglandular stage, the lung buds undergo a period of branching morphogenesis to form the lung tree and terminal bronchioles. Upon completion of the canalicular and saccular stages of development (E16.5-Postnatal (P) day 4), the terminal bronchioles narrow and begin to form epithelial sacs. These structures later form fully mature alveolar structures for gas exchange by P21 during the alveolarization phase9. In contrast, alveolarization begins pre-partum during human lung development and continues postnatally into childhood13.

Coordinated development of the conducting and distal airways is a vital process during which both the epithelial and mesenchymal compartments play integral roles. The develo** lung endodermal buds penetrate the splanchnic mesoderm and mesothelium around E9.5. The develo** distal lung then acquires four distinct layers, each with its own unique anatomical, cellular, morphologic, and molecular profiles: endoderm (epithelium), subepithelial mesoderm (mesenchyme), submesothelial mesoderm (mesenchyme), and mesothelium. Transient amplifying submesothelial cells give rise to a parabronchial smooth muscle cell (PSMC) progenitor population. This cell population then migrates more proximally around the bronchi and differentiates into smooth muscle cells (SMCs)14,15. Studies assessing the expression levels of various Wnt/β-catenin signaling members and activity reporters in these aforementioned compartments during lung development have identified contrasting findings16,17. However, a myriad of studies collectively demonstrates that the develo** lung mesenchyme displays several highly regulated interactions with the lung endoderm in both mouse and human that together coordinate normal lung organogenesis9, many of which are Wnt-mediated.

Embryonic stage (E9.5–E12.5)

Wnt signaling plays a role in some of the earliest stages of cardiopulmonary specification. Wnt2+ Gli1+ Isl1+ cells comprise the multipotent cardiopulmonary mesoderm progenitors (CPPs) that orchestrate heart and lung development. Lineage tracing of Wnt2+ CPPs at E8.5 demonstrates their capacity to generate the cardiac inflow tract and pulmonary mesoderm cell lineage by E17.518. These cells are important for the vital epithelial–mesenchymal interactions that occur during lung development.

Crosstalk from develo** mesenchyme to endoderm

Wnt-driven mesenchymal-to-endodermal crosstalk is critically important from the earliest stages of lung development. From E9.5–12.5, there is active Wnt/β-catenin signaling in both the epithelium and the mesenchyme adjacent to the future proximal airway as measured by TOPGAL and AXIN2-LacZ Wnt activity reporters19,20. Canonical Wnt2/2b ligands are spatiotemporally regulated by Hox5 genes during this stage, with notable mesodermal expression near the ventral aspect of the anterior foregut between E9.0 and E10.521 (Fig. 2a). Together, Wnt2/2b cooperate to promote Nkx2.1+ lung endodermal specification (Fig. 2a), as mice without them display lung agenesis22,23,24. Wnt2/2b converges on canonical signaling in the develo** endoderm, as deletion of β-catenin in the anterior foregut also display lung agenesis, resulting in the upregulation of a Sox2+ digestive progenitor identity19,22. In contrast, constitutive activation of β-catenin prevents tracheo-esophageal septation and instead drives Nkx2.1+ lung endodermal progenitor expansion19,22.

Fig. 2: Wnt signals mediate epithelial–mesenchymal interactions during lung development.
figure 2

a During the embryonic stage (E9.0–12.5) of development, lung bud emerges from tracheal-esophageal septation occurs9. Mesenchymal (brown) HOX5 spatiotemporally regulates endodermal (pink) Wnt2/2b to establish NKX2.1 lung progenitor via downstream β-catenin signaling21,22,23,24. Endodermal Wnt ligands also promote mesenchymal FGF10 and β-catenin, which then allow for SMC differentiation and cartilage and basal cell development27. b During the pseudoglandular stage (E12.5–E16.5), lung buds undergo branching morphogenesis to develop terminal bronchioles9. Mesenchymal Wnt5a promotes tracheal and cartilage formation via ROR2-dependent mechanisms33. Wntless (Wls)-regulated Notum suppresses mesenchymal Wnt and is necessary for tracheal development and branching morphogenesis39,40. A Wnt7b-BMP4 signaling axis also promotes epithelial proliferation and mesenchymal vascular SMC (VSMC) differentiation and SMC proliferation35,36,37,38. Further, epithelial Wnt5a expression is highest in distal tips25. Similar to constitutive β-catenin activation, deletion of Barx1 results in the loss of tracheo-esophageal septation as evidenced by single, contiguous luminal layer of Nkx2.1+ tracheal and Sox2+ esophageal epithelium by E10.519,22,25.

Crosstalk from develo** endoderm to mesenchyme

As early as E11.5, canonical Wnt2 ligand signaling promotes fibroblast growth factor 10 (Fgf10) signaling that, in turn, facilitates differentiation of immature platelet-derived growth factor receptor (Pdgfr)α/β+ smooth muscle cells by regulating the expression of myocardin and Mrtf-B, critical transcription factors in myogenesis26 (Fig. 2a). Mesenchymal Wnt2 also promotes endodermal Wnt7b expression that later signals back to the subepithelial mesenchyme to further drive SMC differentiation26. More recently, epithelial Wnt ligands were shown to activate mesenchymal β-catenin, which alongside Fgf10/Fgfr2, regulate both cartilage progenitors and basal cell development27 (Fig. 2a).

Pseudoglandular stage (E12.5–E16.5)

Constitutive activation of β-catenin in surfactant protein C (Sftpc)+ cells drives distal conducting airway dilatation concomitant with no differentiation to either the secretory or ciliated cell fate28. Consistent with this, R-spondin 2 (Rspo2) facilitates normal embryonic lung growth and the start of branching morphogenesis by potentiating Wnt/β-catenin signaling29. A recent study identified, however, that Rspo2 antagonizes RNF43 and zinc and ring finger (ZNRF)3 to regulate limb and lung formation in xenopus4. These efforts resulted in a paradigm shift, demonstrating that Rspo2 can potentiate Wnt signaling in the absence of LGR4,30. Interestingly, while Rspo2 drives branching morphogenesis earlier in development, it has no role in the differentiation of epithelial or mesenchymal cell types29. This suggests functional redundancy in the varying upstream regulators of Wnt signaling in embryonic lung development.

Several Wnt ligands have been well characterized in the pseudoglandular stage. Wnt5a is diffusely expressed in both the epithelium and mesenchyme as early as E12.067,68,69. The bronchoalveolar duct junction (BADJ) houses the Scgb1a1+ Sftpc+ Sca1+ bronchoalveolar stem cell (BASC)70,71. Further, Sftpc+ ATII cells in the alveolar region undergo self-renewal and give rise to ATI cells72. Most recently, a family of stem/progenitor cells referred to as the lineage-negative epithelial progenitors (LNEPs) have been characterized as quiescent at homeostasis but are mobilized under injurious conditions to regenerate the lungs73,74. Although the intricate complexity of the identity of these stem cell progenitors continues to be an area of current investigation, Wnt signaling plays critical roles in several of these regional compartments in regeneration.

From a cell biology perspective, several of the aforementioned identified progenitor populations or non-stem supporting niche cells employ Wnt signaling in bronchiolar, bronchioalveolar, or alveolar regeneration. Although an early study reported that β-catenin in club cells is dispensable for bronchiolar epithelial repair75, several subsequent findings indicate a highly important and intricately regulated role for this pathway at the cellular level in repair.

In the bronchioles and BADJ following naphthalene injury, ciliated cells induce Wnt7b expression that signals to the PSMCs to induce Fgf10 expression76,77 (Fig. 3c). Mesenchymal Fgf10 then induces Ak strain transforming (AKT)-mediated phosphorylation of β-catenin at S552 to promote BASC expansion and subsequent epithelial regeneration78 (Fig. 3c). Constitutive activation of β-catenin increases bronchiolar stem cell expansion and attenuates differentiation79. β-catenin stabilization also skews sex-determining region Y-box 2 (Sox2)+ LNEPs toward an ATII-like rather than K5+ cell fate by inhibiting Notch and hypoxia signaling following influenza infection139. BPD also shares a common downstream activated, phosphorylated form of β-catenin at Y489 that is also observed in IPF127,140, though much remains to be explored in this disease context. COPD also displays perturbed non-canonical Wnt signaling biology, as pulmonary fibroblasts secrete Wnt5a to inhibit alveolar canonical Wnt/β-catenin signaling, thereby preventing epithelial repair141. Recent work has also begun to think about the role of Wnt signaling in the context of aging as well. Lehmann et al. reported that aged ATII cells exhibit increase senescence that is driven by activation of Wnt/β-catenin signaling and is associated with profibrotic changes142.

Prospective/discussion of Wnt signaling in disease biology

The work of Nabhan et al. has paved the way for understanding the role of single-cell niches within distal lung regeneration83. The success of this lies, in part, because its generated understanding of the Wnt-producing (Wnt-expressing, Porcupine-positive) cells and the Wnt-responsive (Axin2+) cells via single-cell sequencing studies. This approach should be adopted in the context of disease processes, as it could hold particular promise with disease processes with known intermediates such as the malignant transformation underpinning lung squamous cell carcinoma. There also exists a plethora of outstanding questions largely underexplored within the sphere of aging. For example, how do stem cell niches throughout the airway and lung change with aging, both at the cellular and molecular levels? And how, if at all, do the alterations in Wnt signaling that occur in aging relate to distinct pathophysiology of disease processes?

Summary

Taken together, it is clear that Wnt signaling plays a major role in lung development, lung repair and regeneration, and the progression of many lung diseases. The rapid technological advances in the fields of molecular and cellular biology are greatly facilitating the study of Wnt signaling in lung biology. Advancing our knowledge on the exact mechanisms of Wnt signaling in the lung will allow for the development of more Wnt pathway targeted therapies that will hopefully lead to a therapeutic benefit for patients with lung diseases.