Decoding the IGF1 signaling gene regulatory network behind alveologenesis from a mouse model of bronchopulmonary dysplasia

  1. Feng Gao
  2. Changgong Li
  3. Susan M Smith
  4. Neil Peinado
  5. Golenaz Kohbodi
  6. Evelyn Tran
  7. Yong-Hwee Eddie Loh
  8. Wei Li
  9. Zea Borok
  10. Parviz Minoo

  • Curated by eLife

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    Evaluation Summary:

    This paper will be of interest to lung biologists, developmental biologists, and neonatologists interested in lung injury. In this manuscript, the authors used gene expression signatures to construct a gene regulatory network to identify genes associated with alveologenesis. While reviewers were impressed with the novelty of the approach, questions were raised about the robustness of the results in mice and the validation in human samples.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

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Abstract

Lung development is precisely controlled by underlying gene regulatory networks (GRN). Disruption of genes in the network can interrupt normal development and cause diseases such as bronchopulmonary dysplasia (BPD) – a chronic lung disease in preterm infants with morbid and sometimes lethal consequences characterized by lung immaturity and reduced alveolarization. Here, we generated a transgenic mouse exhibiting a moderate severity BPD phenotype by blocking IGF1 signaling in secondary crest myofibroblasts (SCMF) at the onset of alveologenesis. Using approaches mirroring the construction of the model GRN in sea urchin’s development, we constructed the IGF1 signaling network underlying alveologenesis using this mouse model that phenocopies BPD. The constructed GRN, consisting of 43 genes, provides a bird’s eye view of how the genes downstream of IGF1 are regulatorily connected. The GRN also reveals a mechanistic interpretation of how the effects of IGF1 signaling are transduced within SCMF from its specification genes to its effector genes and then from SCMF to its neighboring alveolar epithelial cells with WNT5A and FGF10 signaling as the bridge. Consistently, blocking WNT5A signaling in mice phenocopies BPD as inferred by the network. A comparative study on human samples suggests that a GRN of similar components and wiring underlies human BPD. Our network view of alveologenesis is transforming our perspective to understand and treat BPD. This new perspective calls for the construction of the full signaling GRN underlying alveologenesis, upon which targeted therapies for this neonatal chronic lung disease can be viably developed.

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  1. Version published to 10.7554/elife.77522 on eLife
  2. Version published to 10.1101/2022.01.24.477613v4 on bioRxiv
  3. eLife

    Author Response

    Reviewer 2

    In this manuscript, Gao et al claim that they have constructed a gene regulatory network underlying alveologenesis and its significance to bronchopulmonary dysplasia (BPD). Using RTPCR and in situ hybridization, the authors claim that Igf1 and Igf1r are expressed in secondary crest myofibroblasts (SCMFs) and their loss of function using Gli1-creER results in alveolar simplification, a tissue level disorganization of alveoli that phenocopies BPD. Further, the authors investigate transcriptomic changes in mesenchymal and epithelial populations from control and Igf1r mutant lungs. For this, the authors developed a 47-gene panel that they claim to represent signaling modules within SCMFs and used this panel for RT-PCR analysis. These data are used to generate an interaction network to evaluate signaling partners, co-effectors mediated by IGF1 signaling in SCMFs, other fibroblasts and alveolar epithelial cells. Using this GRN, the authors concluded that Wnt5a is a key signaling molecule downstream of IGF1 signaling that regulates alveologenesis.

    While the authors' claims are salient, some of the conclusions were previously shown by others. For example, a role for Wnt5a driven Ror/Vangl2 has already been shown to be a key mediator of alveologenesis, by virtue of the same signaling effectors identified in this study (Zhang 2020 eLife).

    Response: For the network construction, we not only used our own data, but also considered perturbation analyses and gene regulation data contributed from the work reported by others in the lung research community. The goal was to decipher the genetic regulations among these genes, connect them together, and build the GRN so the lung research community can employ it as a tool to study lung development and its disease in a network-type context.

    We are aware of the excellent study by Zhang et al. 2020 and appreciate their findings related to WNT5a/Ror/Vangl2 functions in lung development. With due respect, we would like to point out that Zhang and colleagues inactivated WNT5a by Tbx4Cre which is highly expressed during the embryonic stage (i.e. Cebra-Thomas et al., 2003). This approach cannot exclude the indirect effects of the mutation that originate during the embryonic phase, on alveologenesis, which is an entirely postnatal process. In contrast, our study used a conditional CreER model to inactivate WNT5a specifically during alveologenesis (i.e. PN2). More importantly, our work identified the upstream and downstream regulatory connections of this signaling pathway and further elucidated its role and function from the network ground.

    Additionally, the genetic loss of function studies performed here are not specific to SCMFs and instead they target broader alveolar and airway fibroblasts.

    Response: Please see our Response to General Comment #3 above for the specificity of Gli1 to SCMFs.

    The construction of a gene regulatory network is a potentially exciting tool, but this requires additional perturbations to distinct nodes identified in this work. It would be of particular interest to determine whether there is any redundancy among these nodes and what are the downstream effectors that are specific to each node. While I recognize that this is outside the scope of this work, the authors need to demonstrate the significance of at least one such node.

    Response: We agree with the general validity of the reviewer’s comment that much more can be done that is not in this first report of the alveologenesis GRN. There is always much more.

    The reviewer has raised some critically important and interesting points. We appreciate the reviewer’s acknowledgement that these very key studies are not within the scope of what is presented in this initial report on GRN construction. Each of such studies will likely require a separate report. The content of the present manuscript was chosen with the goal of disseminating a highly cogent and focused set of data that introduces the utility of GRN in alveologenesis. As noted above by the Editors, this is a novel approach in “translating publicly available data into meaningful biological insights”. We hope this clarifies the main purpose of our study and this manuscript.

  4. eLife

    Evaluation Summary:

    This paper will be of interest to lung biologists, developmental biologists, and neonatologists interested in lung injury. In this manuscript, the authors used gene expression signatures to construct a gene regulatory network to identify genes associated with alveologenesis. While reviewers were impressed with the novelty of the approach, questions were raised about the robustness of the results in mice and the validation in human samples.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

  5. eLife

    Reviewer #1 (Public Review):

    In this manuscript, Gao and colleagues use existing comprehensive RNA expression datasets and generate additional data from targeted gene deletion or pathway manipulation in secondary crest myofibroblasts (MFBs) during mouse lung alveolar septation. They integrate the results and use software tools to infer potential active signaling between MFBs and nearby alveolar epithelial type I (AT1) and AT2 cells. By examining which genes are increased and decreased upon deletion of IGF1 or IGF1R in MFBs at postnatal day 2, they generate a gene regulatory network (GNR) for the MFB during alveolar septation. They subsequently identify WNT5a as putatively signaling in an autocrine manner (via Ror2) and to AT2 cells (via Ror1), and FGF10 signaling to both AT1 and AT2 cells (via FGFR2) and in an autocrine manner (via FGFR1/3/4). They flesh out the GRN within the MFB by pharmacologic inhibition of FGFR2 and silencing of Wnt5a expression followed by qRT-PCR expression analysis of genes within the GRN, by which they build upon their initial GRN derived from modulation of IGF1 signaling. They also examine the expression of AT1 and AT2 marker genes in bulk cell RNA of all non-MFB cells by qRT-PCR, adding these connections onto their map. Overall this manuscript represents a cutting-edge and novel approach to extracting useful information from publicly available gene expression datasets. The work should be of high interest to lung developmental biologists and researchers interested in understanding BPD pathophysiology.

  6. eLife

    Reviewer #2 (Public Review):

    In this manuscript, Gao et al claim that they have constructed a gene regulatory network underlying alveologenesis and its significance to bronchopulmonary dysplasia (BPD). Using RT-PCR and in situ hybridization, the authors claim that Igf1 and Igf1r are expressed in secondary crest myofibroblasts (SCMFs) and their loss of function using Gli1-creER results in alveolar simplification, a tissue level disorganization of alveoli that phenocopies BPD. Further, the authors investigate transcriptomic changes in mesenchymal and epithelial populations from control and Igf1r mutant lungs. For this, the authors developed a 47-gene panel that they claim to represent signaling modules within SCMFs and used this panel for RT-PCR analysis. These data are used to generate an interaction network to evaluate signaling partners, co-effectors mediated by IGF1 signaling in SCMFs, other fibroblasts and alveolar epithelial cells. Using this GRN, the authors concluded that Wnt5a is a key signaling molecule downstream of IGF1 signaling that regulates alveologenesis.

    While the authors' claims are salient, some of the conclusions were previously shown by others. For example, a role for Wnt5a driven Ror/Vangl2 has already been shown to be a key mediator of alveologenesis, by virtue of the same signaling effectors identified in this study (Zhang 2020 eLife). Additionally, the genetic loss of function studies performed here are not specific to SCMFs and instead they target broader alveolar and airway fibroblasts. The construction of a gene regulatory network is a potentially exciting tool, but this requires additional perturbations to distinct nodes identified in this work. It would be of particular interest to determine whether there is any redundancy among these nodes and what are the downstream effectors that are specific to each node. While I recognize that this is outside the scope of this work, the authors need to demonstrate the significance of at least one such node.

  7. eLife

    Reviewer #3 (Public Review):

    This paper uses a unique approach to untangle the molecular mechanisms driving the pathogenesis of BPD. Strengths of the study include using a new genetic mouse model and the approach of applying gene regulatory networks to this model. There are, however, significant weaknesses in the study which make the conclusions weakly supported by the data. It is not clear that the markers the authors use to define "secondary crest myofibroblasts" are truly labeling a distinct cell population. The authors identify Pdgfra+/Igf+ or Pdgfra+/Igfr+ cells as defining this population by RNA ISH, but the images presented are not convincing and not rigorously quantified. At least two large single-cell transcriptomes of the developing mouse lung have been published in the last 12 months (Zepp et al, Cell Stem Cell 2021 and Negretti et al, Development 2021) and the examination of the expression of Igf1 and Igfr in specific myofibroblast populations over time should be explored, a method perhaps more specific and sophisticated than the whole-lung RTqpCR used in this paper. The use of Dermo1-Cre will drive mutant allele expression in all mesenchymal cells, not just SCMF. While Gli1 is a previously published marker of SCMF, does this marker have specificity in the context of newer single-cell transcriptomic datasets? In addition, there exist significant concerns about the rigor of this study, including a lack of information about the number of technical and biological replicates used. Moreover, whole lung qPCR is used on human lung as an attempt to validate these methods, however, there is no significant clinical data given about the patients from who this RNA was obtained (e.g., at what age did they die? from what cause? what gestational age were they born?). Given these significant concerns about the strength of the data to support these conclusions, the impact of this work is difficult to assess and diminished in scope.

  8. Version published to 10.1101/2022.01.24.477613v3 on bioRxiv
  9. Version published to 10.1101/2022.01.24.477613v2 on bioRxiv
  10. Version published to 10.1101/2022.01.24.477613v1 on bioRxiv