Defective mesenchymal Bmpr1a-mediated BMP signaling causes congenital pulmonary cysts

Reviewer #1 (Public Review):

The goal of this paper is to characterize the molecular mechanisms that lead to lung cyst formation in a murine model wherein the Bmpr1a receptor gene has been inactivated in the fetal lung mesenchyme. In this context, it is important to note that very little is known regarding how lung cysts form, and generally the presumption has been that these pathological structures result from dysregulated events in the epithelium. Thus, the emphasis in this paper on derangements in a fundamental developmental signaling pathway in the lung mesenchyme that results in cyst formation is both novel and significant.

In this manuscript, the authors seek to understand how abnormal lung development leads to the formation of cysts in the lung. Cysts are enlarged pathological balloon-like structures that interfere with normal gas exchange and characterize a variety of pediatric and adult lung diseases. To date, the molecular signals underlying cyst formation are poorly understood. Using genetically modified mice, the focus herein is on how inactivation of a specific gene known to transduce key developmental signals (Bmpr1a) leads to the development of cysts. One novelty of this work is that the gene inactivation has been targeted to a set of primitive fetal lung cells that give rise to structural and contractile cells supporting bronchial airways. Alterations in the function of this particular cell type has not previously been examined in the context of lung cyst pathogenesis.

Notably, the experiments and models are state-of-art and the authors are careful in their interpretations. It should be noted, however, that there are also several concerns that limit enthusiasm at this time. These include a lack of data evaluating relative histological similarities and differences between cysts generated in their murine model and human lung cysts, and whether there is information implicating a role for the gene studied in this paper in human cysts. Secondly, despite an abundance of data, at the end of the day, the key molecular signals are not clearly identified.

Additional Feedback

Overall, this is a well-executed paper that addresses how derangements in signals emanating from the fetal lung mesenchyme in embryonic life lead to cyst formation. This work, therefore, seeks to fill in basic deficiencies in knowledge since the pathogenesis of lung cyst formation is poorly understood and the role of altered mesenchymal cell activity in this process has not been carefully addressed. For the most part, the experiments are clearly presented, and the models are relevant and state-of-the art. Although enthusiasm is high, there are several overriding concerns, which the authors should consider.

While the paper seeks to understand the key molecular events leading to cyst formation and a plethora of data is provided, this goal is largely not clearly met. As a result, the paper ends up being descriptive. Further, without these data, a so-called definitive rescue experiment is not possible at this time. In addition, the experiments, particularly toward the end of the manuscript are not well integrated with the overall body of the Results. This is particularly true for figure 7. While interesting, the results in some of these latter figures are insufficiently linked to the primary observations. This issue further contributes to concerns that the manuscript is largely descriptive.

Importantly, it would be useful to have provided more detailed information on the structure and histological properties of the murine cysts and how such findings relate to human lung cysts. Also, the authors should examine whether there is any information on Bmpr1a in human cyst formation (i.e GWAS data).

Throughout the paper, there is a lack of quantification for the histological findings. Littermate controls should also be clearly defined genetically,

Specific Concerns by figure

Figure 1 suppl: "Doxycycline" is misspelled.

Figure1c Suppl: Hard to discern clear-cut expression of Bmpr1a protein in mesenchyme in WT. Comparable images with similar sizes of airways should be used.

Figure 2a: Expression of several genes studied and altered should be identified on scatter plot.

Figure 2c: Authors should also consider staining for other smooth muscle markers.

Figure 3: ELN expression should be defined in a clear quantitative manner.

Figure 4: Additional information on p38 dependent signaling (? Including in vivo studies) would potentially help to understand key molecular events and perhaps could help to address key mechanistic events, including their location and identity.

Figure 6: Would be helpful to know whether Bmpr1a receptor is expressed in Myocd KO.

Figure 7: Not clear how these findings, though interesting, relate to the body of studies and the pathogenesis of cyst formation. Other points: 1) The authors should re-examine/repeat co-staining in the KO mouse lung (right 2 images in the top group of 4) for Foxj1, Sox2, and CDH (right 2 images, Figure 7A). For one thing, the cadherin stain in the 2 KO images seems localized to the lumen. Secondly, the pattern of cadherin staining looks exactly the same in both KO images, suggesting an error and/or duplication 2) authors should place arrows on the heat map showing the location of SPC, Sox2, Sox9, and FoxJ1 bands 3) figure 7D graph needs numbers on y axis.

Reviewer #2 (Public Review):

Congenital cystic airway abnormalities (CPAM) are a common disorder in airway lung development whose etiology is poorly understood and can be fatal if not effectively treated at birth. This study by Luo and colleagues provides compelling new evidence in mice and cultured fetal mesenchymal cells that loss of mesenchymal Bmpr1a signaling disrupts branching morphogenesis, leading to the formation of numerous pulmonary cysts. Their airways were deficient in underlying smooth muscle and subepithelial elastin fibers along with perturbations in Sox2-Sox9 proximal-distal epithelial development. Interestingly, these changes were independent of canonical Smad1/5 signaling and suggestive of non-canonical signaling perhaps through p38 signaling. They were also independent of simply ablating non-vascular mesenchymal cells using Myocd-ko mice. Although the study does not define how loss of Bmpr1a causes cystic formation or whether this pathway is related to human CPAM disease, the findings are considered highly significant because they provide new evidence for BMP signaling in branching morphogenesis. The knowledge may pave the way for future studies designed to understand and prevent or treat newborn infants with CPAM. There are only a few weaknesses.

Major Weaknesses:
1. The authors may be aware that a recent paper (https://doi.org/10.1038/s41598-022-24858-3) reported on transcriptional changes seen in human CPAM. It would seem that some of the molecular changes seen in human CPAM move in the opposite direction of what is reported in mice lacking mesenchymal Bmrp1a. Perhaps the authors could comment on these differences in the discussion and whether they potentially explain the etiology of CPAM or branching morphogenesis in general.
2. Figure 4 shows that BMP4 increases SMADs, p38, and several muscle genes in mesenchymal cells. Figure 5 extends this finding with a clever strategy to label airway and vascular smooth muscle with different fluorescent molecules used to isolate different types of mesenchymal cells. It shows that non-vascular smooth muscle cells but not perivascular smooth muscles are responsive to BMP4 signaling as defined by increased expression of Myh11. Are there cell-restricted responses to the other genes shown in Figure 4? Given the lack of SMAD signaling and the increase seen in p38 signaling, would blocking p38 signaling influence the BMP responsiveness of these nonvascular smooth muscle cells?
3. Figure 6 shows that mesenchymal loss of Myocd causes a deficiency of airway smooth muscle cells, but this was not sufficient to create cysts. Did the authors ever check to see if it changed Sox2-Sox9 staining in the airway epithelium?
4. Figure 7 shows that mesenchymal loss of Bmpr1a proximalizes the distal airway as defined by loss of Sox2 and FoxJ1 (a ciliated marker) and gain in (Sox9 and SP-C) staining. But Club cells expressing Scgb1a1 and Cyp2F2 are the predominant epithelial cells in the distal airway. The transcriptomics data in panel B shows expression of these genes is less in the mutant mice. Does this mean they fail to generate Club cells or there is just less expression per cell? In other words, what are the primary epithelial cells present in the airways of mice with loss of mesenchymal Bmpr1a?