Congenital disorders are medical conditions that are present from birth. Although many congenital disorders are rare, they can have a severe impact on the quality of life of those affected.

For example, congenital pulmonary airway malformation (CPAM) is a rare congenital disorder that occurs in around 1 out of every 25,000 pregnancies. In CPAM, abnormal, fluid-filled sac-like pockets of tissue, known as cysts, form within the lungs of unborn babies. After birth, these cysts become air-filled and do not behave like normal lung tissue and stop a baby’s lungs from working properly. In severe cases, babies with CPAM need surgery immediately after birth.

We still do not understand exactly what the underlying causes of CPAM might be. CPAM is not considered to be hereditary – that is, it does not appear to be passed down in families – nor is it obviously linked to any environmental factors. CPAM is also very difficult to study, because researchers cannot access tissue samples during the critical early stages of the disease.

To overcome these difficulties, Luo et al. wanted to find a way to study CPAM in the laboratory. First, they developed a non-human animal ‘model’ that naturally forms CPAM-like lung cysts, using genetically modified mice where the gene for the signaling molecule Bmpr1a had been deleted in lung cells.

Normally, Bmpr1a is part of a set of the molecular instructions, collectively termed BMP signaling, which guide healthy lung development early in life. However, mouse embryos lacking Bmpr1a developed abnormal lung cysts that were similar to those found in CPAM patients, suggesting that problems with BMP signalling might also trigger CPAM in humans.

Luo et al. also identified several other genes in the Bmpr1a-deficient mouse lungs that had abnormal patterns of activity. All these genes were known to be controlled by BMP signaling, and to play a role in the development and organisation of lung tissue. This suggests that when these genes are not controlled properly, they could drive formation of CPAM cysts when BMP signaling is compromised.

This work is a significant advance in the tools available to study CPAM. Luo et al.’s results also shed new light on the molecular mechanisms underpinning this rare disorder. In the future, Luo et al. hope this knowledge will help us develop better treatments for CPAM, or even help to prevent it altogether.

Congenital pulmonary cysts, resulting from abnormal fetal lung development, cause respiratory distress, infection, and pneumothorax in neonates. The dynamic pathogenic process and mechanisms are difficult to study in humans and few animal models are available. It is known that lung development begins with the specification of the respiratory domain in the ventral wall of the anterior foregut endoderm, as indicated by the expression of Nkx2-1, and the separation of the respiratory tract from the dorsal esophagus. The primary lung epithelial buds then undergo reiterated elongation and division to form the conducting airways (branching morphogenesis), followed by distal saccular formation and peripheral alveolarization to give rise to millions of gas exchange units (McCulley et al., 2015; Morrisey and Hogan, 2010). This well-known process literally describes the growth of respiratory epithelium based on the extensive studies done in the past decades. It is notable that lung morphogenesis is a complex process relying on the highly coordinated development of both lung epithelium and lung mesenchyme. Evidence has increasingly suggested that lung mesenchymal lineages, including airway and vascular smooth muscle cells (SMCs), pericytes, and stromal fibroblasts, are as important as epithelial cells in proper lung development (Noe et al., 2019; Ren et al., 2016; Luo et al., 2015). In addition to acting as a mechanical framework that supports the formation of the bronchi, bronchioles, and the distal alveoli, the lung mesenchyme provides a microenvironment that regulates the growth of epithelium through a variety of morphogenic signals, such as Wnts, fibroblast growth factors (Fgfs), and bone morphogenetic proteins (BMPs) (McCulley et al., 2015; Nikolić et al., 2018; Morrisey et al., 2013; Luo et al., 2018).

BMPs are a family of growth factors that direct many biological processes, including organogenesis (Wang et al., 2014). BMPs bind to the receptor complex of BMP receptor II (Bmpr2) and BMP receptor I (Bmpr1a or Bmpr1b), which in turn activates the intracellular Smad-dependent and Smad-independent pathways (Shi and Massagué, 2003). Mice with conventional Bmpr1a gene deletions are early embryonic lethal (E7.5–9.5) before lung organogenesis (Mishina et al., 1995). As reported by us and other groups, both hyperactivation and inhibition of BMP signaling in fetal lung epithelial cells result in lung malformation, which is primarily due to the defects in distal lung epithelial cell proliferation and differentiation/maturation (Luo et al., 2016; Sun et al., 2008; Weaver et al., 1999; Bellusci et al., 1996). However, the role of mesenchymal BMP signaling in regulating fetal lung development has not been studied due to the lack of lung mesenchyme-specific targeting tools in the past. We recently generated a lung mesenchyme-specific Tbx4 lung enhancer-driven loxP/Cre mouse driver line (Zhang et al., 2013) which enables us to manipulate BMP signaling specifically in lung mesenchymal cells in vivo and to study its role in the lung development.

Although Bmpr1a is expressed predominantly in fetal mouse lung airway epithelial cells at the early gestation stage (embryonic day [E]12.5 to E14.5), its expression in fetal lung mesenchyme is also evident during mid-gestation (Sun et al., 2008). Herein, we specifically deleted Bmpr1a in fetal lung mesenchymal cells, which resulted in abnormal airway development and subsequent prenatal cystic malformation. This pathological phenotype resembles the features observed in pediatric patients diagnosed with congenital pulmonary airway malformation (CPAM). Therefore, investigating the abnormal lung phenotypes caused by lung mesenchyme-specific Bmpr1a knockout and revealing the underlying molecular and cellular mechanisms will significantly enhance our understanding of congenital lung diseases.

Our current study provides solid in vivo evidence for the first time that Bmpr1a-mediated BMP signaling in lung mesenchymal cells is indispensable in lung development. Lung mesenchymal deletion of Bmpr1a at an early developmental stage causes abnormal branching morphogenesis and then severe lung cysts at late gestation in mice. Interestingly, congenital pulmonary cysts are reported in human CPAM patients as a result of abnormal prenatal lung development (Stocker, 2009). Reduction of airway SMCs and decreased subepithelial elastin fibers are also found to be the common pathological changes in the cystic airways of human CPAM samples (Jiang et al., 2019). Although the congenital pulmonary cysts in CPAM patients can be identified by a prenatal ultrasound examination, the tissue specimens for pathological analysis are only available from postnatal surgery, when advanced airway cysts have already developed. This makes it difficult to study the pathogenic molecular mechanisms that trigger early cyst formation during fetal lung development in human subjects. Studies in mouse models suggest that altered growth factor signaling in a prenatal time window is sufficient to cause cyst formation (Miao et al., 2021). The abnormal BMP signaling pathway may be involved in the initiation of CPAM, but not in the later stage when cystic lesions have formed. Interestingly, integrated suppression of BMP signaling pathway was recently found in human CPAM specimens by an RNA-seq approach (Zhang et al., 2022). Therefore, the lung mesenchymal Bmpr1a knockout model developed here is a potentially useful tool for studying the pathogenic process of CPAM.

It has been reported that epithelial overexpression of Bmp4 appears to induce extensive mesenchymal SMC differentiation in vivo (Badri et al., 2008), and that BMP4 promotes myocyte differentiation and inhibits cell proliferation in cultured human fetal lung fibroblasts via the Smad1 pathway (Jeffery et al., 2005). Smad1 and Smad5 are critical downstream effectors of BMP type I receptors, mediating BMP canonical signal pathway (Wang et al., 2014; Katagiri and Watabe, 2016). However, our in vivo study indicates that simultaneous deletion of Smad1 and Smad5 in mouse lung mesenchyme does not affect the differentiation of airway SMCs although it disrupts early embryonic lung morphogenesis. This suggests that the decrease in phosphorylated Smad1 and Smad5 in the Bmpr1a CKO lungs is not responsible for the defective airway SMC growth. In addition to the Smad-dependent BMP signal pathway, BMP-activated Bmp receptors also function through MAPK (Wang et al., 2014; Katagiri and Watabe, 2016; von Bubnoff and Cho, 2001). Many studies have shown that p38 MAPK regulates cell proliferation in a variety of SMC types, including airway SMCs (Chen and Khalil, 2006; Pelaia et al., 2008). The p38 MAPK is also involved in myogenic differentiation and myofibroblast trans-differentiation (Choi et al., 2011; Meyer-Ter-Vehn et al., 2006; Sebe et al., 2008). In the present study, reduced phosphorylation of p38 was detected in mesenchymal Bmpr1a CKO lungs while addition of BMP4 activated the p38 signal pathway in mouse primary fetal lung mesenchymal cells. Moreover, the myogenic response to BMP4 stimulation in the cultured fetal lung mesenchymal cells could be blocked by treating the cells with a p38 inhibitor. This suggests that p38 is the primary intracellular signaling component to mediate BMP’s regulatory effects on airway myogenesis.

Despite being commonly characterized by the expression of a variety of myogenesis-associated genes, airway and vascular SMCs seem to originate from distinct mesenchymal progenitor populations (Zhang et al., 2013), and are regulated by distinct molecular mechanisms. Our study reveals that Myocd is a prominent target gene that links Bmpr1a to the genes associated with airway SMC development. However, Bmpr1a, p38, and Myocd are not critical in fetal pulmonary vascular SMC development, and inhibition of these molecules in vivo and in vitro does not have any observable effect on vascular SMCs in fetal lungs. Our previous study has demonstrated that Myocd is abundant in airway SMCs but barely expressed in vascular SMCs (Young et al., 2020), and that cardiovascular SMC development is mediated by myocardin-related transcription factors, the other members of Myocd family (Parmacek, 2007). Distinct regulation between airway and vascular SMCs is also evidenced by the phenotypes of mice with various mutations (Badri et al., 2008; Murphy et al., 2008). For instance, hypomorphic FGF-10 mice only display decreased expression of airway smooth muscle actin (Mailleux et al., 2005), while deletion of Wnt-7b selectively disrupts the development of vascular SMCs (Shu et al., 2002). Although reduced expression of Bmpr1a is associated with various forms of acquired as well as primary nonfamilial pulmonary hypertension (Du et al., 2003), the role of Bmpr1a-mediated signaling in regulating prenatal lung vascular SMC development has not been studied. Our current data suggest that Bmpr1a is dispensable in fetal vascular SMC development.

Previous studies have suggested that airway SMCs initially develop from local mesenchymal cells around the tips of epithelial buds undergoing bifurcation. These cells wrap around the bifurcating cleft and neck of the terminal buds, and then grow rigidly alongside the elongating bronchial tree (Kim et al., 2015; Badri et al., 2008; Kumar et al., 2014). Inhibition of SMC differentiation in embryonic lung explant culture, such as by blocking L-type Ca²⁺ channels using nifedipine, or by inhibiting FGF and SHH signaling using SU5402SHH and cyclopamine, prevented airway terminal bifurcation (Kim et al., 2015). However, our current and a recently published study demonstrate that abrogation of airway SMCs by deleting mesenchymal Myocd does not cause any significant abnormalities in airway branching morphogenesis. This suggests that loss of SMCs is neither the primary nor sole mechanism accounting for the reduction of airway branching, dilation of terminal airways, and development of airway cysts in our Bmpr1a CKO lungs. Notably, the airway subepithelial elastin defects were uniquely found in the Bmpr1a knockout lungs with extensive cystic lesions but not in the Myocd CKO lungs. Interestingly, elastin expression in the developing lung parenchyma prior to alveologenesis is predominantly localized to the mesenchyme in the developing airways, as indicated by our elastin immunostaining data, suggesting that elastin plays a role in airway branching. Previous study has revealed that the perinatal development of terminal airway branches is arrested in mice lacking elastin (Eln -/-), resulting in dilated distal air sacs that form abnormally large cavities (Wendel et al., 2000). It is worthy to note that airway smooth muscles are normal in these elastin null mice, implying that loss of elastic fiber alone is not sufficient to cause airway cystic pathology, but could serve as an additional factor contributing to the lesions in vivo. This assumption is also supported by the fact that congenital airway cysts in the lungs of human CPAM patients are characterized by compromised smooth muscle and elastin fiber (Jiang et al., 2019). Future studies are needed to investigate whether the combined defects of airway SMCs and elastin fibers are the pathogenic mechanisms behind congenital airway cyst formation.

The upregulation of multiple Bmp ligands, particularly Bmp4, in the Bmpr1a CKO lung tissues as compared with the WT lungs is a complex scenario. The isolated fetal lung mesenchymal cells of Bmpr1a CKO mice also showed increased Bmp4 expression as compared to the WT lung cells. This might be due to an autoregulatory feedback loop between Bmp ligand expression and intracellular BMP signaling. Previous studies have shown that disruption of BMP signaling by LDN-193189 can substantially improve Bmp4 production (Kim and Lee, 2019), and that deletion of Bmpr1a and Acvr1 in the lens-forming ectoderm increases Bmp2, -4, and -7 transcripts (Huang et al., 2015). These in vitro and in vivo studies clearly suggest the inverse relationship between mesenchymal BMP signaling activity and Bmp ligand expression. Misexpression of BMP4 results in a decrease of Sox2⁺ proximal progenitors and an expansion of Sox9⁺/Id2⁺ distal progenitors in fetal mouse lungs, leading to malformed airways (Wang et al., 2013). Consistently, overexpression of Bmp antagonist Xnoggin or gremlin in fetal distal lung epithelium leads to a severe reduction of distal epithelial cell types and a concurrent expanded expression of the proximal epithelial cell markers CC10 and Foxj1 (Weaver et al., 1999; Lu et al., 2001). In our Bmpr1a CKO lungs, the proximal-distal patterning of lung airways is also substantially disturbed, as indicated by the aberrant distribution of proximal and distal epithelial cells. Evidence increasingly suggests that the lung mesenchyme is closely involved in respiratory lineage specification and epithelial differentiation by producing many critical signaling molecules, such as Bmp ligands, that direct endodermal expression of specific cell markers in a temporal and spatial fashion (McCulley et al., 2015; Morrisey and Hogan, 2010). Therefore, the decrease in proximal cell types and ectopic expression of distal cell markers (Sox9) in the Bmpr1a CKO lungs may be subsequently caused by abnormal increase of Bmp ligands. This aberrant epithelial growth may also contribute to airway cyst formation, as multiple mouse models have suggested that loss of Sox2⁺ or expansion of Sox9⁺ in airway epithelia are associated with altered epithelial differentiation and branching, as well as cystic pathology in fetal lungs (Wang et al., 2013; Liberti et al., 2019; Rockich et al., 2013).

In summary, mesenchymal Bmpr1a is essential for lung development, particularly airway branching morphogenesis. Disruption of Bmpr1a-mediated mesenchymal signaling causes prenatal airway malformation and cystic lesions. Multiple mechanisms, including deficiency of airway smooth muscle, airway elastin fiber defect, and perturbation of the Sox2-Sox9 epithelial progenitor axis, may contribute to the congenital airway cystic pathogenesis. In addition, the downstream Smad-independent p38 pathway appears to be critical in mediating BMP-regulated airway smooth muscle development. Our study helps to understand both normal lung development and the mechanisms of related airway malformation and congenital pulmonary diseases.

The sequencing data have been deposited to the NCBI GEO repository with accession number GSE97946. All data generated and analyzed during this study are included in the manuscript and supporting files.

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Author details

1. Yongfeng Luo

   Department of Surgery, Children’s Hospital Los Angeles, Keck School of Medicine, University of Southern California, Los Angeles, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Supervision, Validation, Investigation, Visualization, Methodology, Writing – original draft, Project administration, Writing – review and editing

   For correspondence

   yluo@chla.usc.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-8765-0273

2. Ke Cao

   Division of Pulmonary, Critical Care & Sleep Medicine, Department of Internal Medicine, University of Cincinnati College of Medicine, Cincinnati, United States

   Contribution

   Data curation, Formal analysis, Validation, Investigation, Methodology

   Competing interests

   No competing interests declared

3. Joanne Chiu

   Department of Surgery, Children’s Hospital Los Angeles, Keck School of Medicine, University of Southern California, Los Angeles, United States

   Contribution

   Investigation, Methodology

   Competing interests

   No competing interests declared

4. Hui Chen

   Division of Pulmonary, Critical Care & Sleep Medicine, Department of Internal Medicine, University of Cincinnati College of Medicine, Cincinnati, United States

   Contribution

   Investigation, Methodology

   Competing interests

   No competing interests declared

5. Hong-Jun Wang

   Division of Pulmonary, Critical Care & Sleep Medicine, Department of Internal Medicine, University of Cincinnati College of Medicine, Cincinnati, United States

   Contribution

   Investigation

   Competing interests

   No competing interests declared

6. Matthew E Thornton

   Division of Maternal Fetal Medicine, Department of Obstetrics and Gynecology, Keck School of Medicine, University of Southern California, Los Angeles, United States

   Contribution

   Formal analysis, Investigation, Visualization, Methodology

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-1083-2703

7. Brendan H Grubbs

   Division of Maternal Fetal Medicine, Department of Obstetrics and Gynecology, Keck School of Medicine, University of Southern California, Los Angeles, United States

   Contribution

   Resources, Formal analysis

   Competing interests

   No competing interests declared

8. Martin Kolb

   Department of Medicine, McMaster University, Hamilton, Canada

   Contribution

   Formal analysis, Writing – review and editing

   Competing interests

   No competing interests declared

9. Michael S Parmacek

   Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, United States

   Contribution

   Resources, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-1449-4665

10. Yuji Mishina

    Department of Biologic and Material Sciences, University of Michigan-Ann Arbor, Ann Arbor, United States

    Contribution

    Conceptualization, Resources, Writing – review and editing

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-6268-4204

11. Wei Shi

    Division of Pulmonary, Critical Care & Sleep Medicine, Department of Internal Medicine, University of Cincinnati College of Medicine, Cincinnati, United States

    Contribution

    Conceptualization, Resources, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing – original draft, Project administration, Writing – review and editing

    For correspondence

    shiwe@ucmail.uc.edu

    Competing interests

    No competing interests declared

    Additional information

    Lead contact

    "This ORCID iD identifies the author of this article:" 0000-0001-6499-2473

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