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Chromatin remodeler Znhit1 controls bone morphogenetic protein signaling in embryonic lung tissue branching

  • Wei Wei
    Affiliations
    State Key Laboratory of Genetic Engineering, School of Life Sciences, Greater Bay Area Institute of Precision Medicine (Guangzhou), Zhongshan Hospital, Fudan University, Shanghai, China

    The Joint Laboratory for Lung Development and Related Diseases of West China Second University Hospital, Sichuan University and School of Life Sciences of Fudan University, Chengdu, China
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  • Xiaofang Tang
    Affiliations
    State Key Laboratory of Genetic Engineering, School of Life Sciences, Greater Bay Area Institute of Precision Medicine (Guangzhou), Zhongshan Hospital, Fudan University, Shanghai, China
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  • Ning Jiang
    Affiliations
    State Key Laboratory of Genetic Engineering, School of Life Sciences, Greater Bay Area Institute of Precision Medicine (Guangzhou), Zhongshan Hospital, Fudan University, Shanghai, China
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  • Chao Ni
    Affiliations
    State Key Laboratory of Genetic Engineering, School of Life Sciences, Greater Bay Area Institute of Precision Medicine (Guangzhou), Zhongshan Hospital, Fudan University, Shanghai, China
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  • Hua He
    Affiliations
    The Joint Laboratory for Lung Development and Related Diseases of West China Second University Hospital, Sichuan University and School of Life Sciences of Fudan University, Chengdu, China
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  • Shenfei Sun
    Affiliations
    State Key Laboratory of Genetic Engineering, School of Life Sciences, Greater Bay Area Institute of Precision Medicine (Guangzhou), Zhongshan Hospital, Fudan University, Shanghai, China
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  • Meng Yu
    Affiliations
    State Key Laboratory of Genetic Engineering, School of Life Sciences, Greater Bay Area Institute of Precision Medicine (Guangzhou), Zhongshan Hospital, Fudan University, Shanghai, China
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  • Chuyue Yu
    Affiliations
    State Key Laboratory of Genetic Engineering, School of Life Sciences, Greater Bay Area Institute of Precision Medicine (Guangzhou), Zhongshan Hospital, Fudan University, Shanghai, China
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  • Mengdi Qiu
    Affiliations
    State Key Laboratory of Genetic Engineering, School of Life Sciences, Greater Bay Area Institute of Precision Medicine (Guangzhou), Zhongshan Hospital, Fudan University, Shanghai, China
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  • Dong Yan
    Affiliations
    State Key Laboratory of Genetic Engineering, School of Life Sciences, Greater Bay Area Institute of Precision Medicine (Guangzhou), Zhongshan Hospital, Fudan University, Shanghai, China
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  • Zhaocai Zhou
    Affiliations
    State Key Laboratory of Genetic Engineering, School of Life Sciences, Greater Bay Area Institute of Precision Medicine (Guangzhou), Zhongshan Hospital, Fudan University, Shanghai, China
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  • Yuanlin Song
    Affiliations
    Department of Pulmonary Medicine, Zhongshan Hospital, Fudan University, Shanghai, China

    Shanghai Key Laboratory of Lung Inflammation and Injury, Shanghai, China
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  • Hanmin Liu
    Correspondence
    For correspondence: Xinhua Lin; Bing Zhao; Hanmin Liu
    Affiliations
    The Joint Laboratory for Lung Development and Related Diseases of West China Second University Hospital, Sichuan University and School of Life Sciences of Fudan University, Chengdu, China
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  • Bing Zhao
    Correspondence
    For correspondence: Xinhua Lin; Bing Zhao; Hanmin Liu
    Affiliations
    State Key Laboratory of Genetic Engineering, School of Life Sciences, Greater Bay Area Institute of Precision Medicine (Guangzhou), Zhongshan Hospital, Fudan University, Shanghai, China
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  • Xinhua Lin
    Correspondence
    For correspondence: Xinhua Lin; Bing Zhao; Hanmin Liu
    Affiliations
    State Key Laboratory of Genetic Engineering, School of Life Sciences, Greater Bay Area Institute of Precision Medicine (Guangzhou), Zhongshan Hospital, Fudan University, Shanghai, China

    The Joint Laboratory for Lung Development and Related Diseases of West China Second University Hospital, Sichuan University and School of Life Sciences of Fudan University, Chengdu, China

    Shanghai Key Laboratory of Lung Inflammation and Injury, Shanghai, China
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Open AccessPublished:October 05, 2022DOI:https://doi.org/10.1016/j.jbc.2022.102490
      Branching morphogenesis is a key process essential for lung and other organ development in which cellular and tissue architecture branch out to maximize surface area. While this process is known to be regulated by differential gene expression of ligands and receptors, how chromatin remodeling regulates this process remains unclear. Znhit1 (zinc finger HIT-type containing 1), acting as a chromatin remodeler, has previously been shown to control the deposition of the histone variant H2A.Z. Here, we demonstrate that Znhit1 also plays an important role in regulating lung branching. Using Znhit1 conditional KO mice, we show that Znhit1 deficiency in the embryonic lung epithelium leads to failure of branching morphogenesis and neonatal lethality, which is accompanied by reduced cell proliferation and increased cell apoptosis of the epithelium. The results from the transcriptome and the chromatin immunoprecipitation assay reveal that this is partially regulated by the derepression of Bmp4, encoding bone morphogenetic protein (BMP) 4, which is a direct target of H2A.Z. Furthermore, we show that inhibition of BMP signaling by the protein inhibitor Noggin rescues the lung branching defects of Znhit1 mutants ex vivo. Taken together, our study identifies the critical role of Znhit1/H2A.Z in embryonic lung morphogenesis via the regulation of BMP signaling.

      Keywords

      Abbreviations:

      BMP (bone morphogenetic protein), CCSP (club cell secretory protein), ChIP (chromatin immunoprecipitation), COPD (chronic obstructive pulmonary disease), DMEM (Dulbecco's modified Eagle's medium), E9 (embryonic day 9), FGF (fibroblast growth factor), PH3 (phosphorylated histone H3), pSMAD1/5/9 (phosphor-SMAD1/5/9), qRT–PCR (quantitative RT–PCR), Znhit1 (zinc finger HIT-type containing 1)
      The lung is one of the essential organs that terrestrial vertebrates have evolved to adapt to life on land. The complex architecture of the lung enables the vital function of gas exchange, and lung development entails a series of remarkably elaborate branching events from the primary lung buds (
      • Burri P.H.
      Fetal and postnatal development of the lung.
      ). In mice, the lung is derived from the ventral anterior foregut endoderm and appears at embryonic day 9 (E9) (
      • Wells J.M.
      • Melton D.A.
      Vertebrate endoderm development.
      ). The primary lung buds are formed at the embryonic stage E9.5–E12.5. During the subsequent pseudoglandular stage (E12.5–E16.5), the lung forms the primary airway pattern. SOX9-expressing proximal epithelial progenitors give rise to bronchioles and gas exchange units, and SOX2-expressing proximal regions gradually develop into the conductive airways in the mature lung. The functional adult lung is progressively formed after canalicular, saccular, and final alveolar stages.
      The pattern formation of the lung is controlled by a series of important signaling molecules, including bone morphogenetic protein 4 (BMP4), SHH, fibroblast growth factor 10 (FGF10), and wingless (Wnt). Interactions between FGFs and BMPs are crucial to the formation of secondary lung buds. BMP4-deficient mouse embryos die before E10, whereas the excessive activation of BMP4 antagonizes the FGF10-induced epithelial bud growth (
      • Weaver M.
      • Dunn N.R.
      • Hogan B.L.
      Bmp4 and Fgf10 play opposing roles during lung bud morphogenesis.
      ,
      • Hogan B.L.
      • Yingling J.M.
      Epithelial/mesenchymal interactions and branching morphogenesis of the lung.
      ,
      • Wang Y.
      • Tian Y.
      • Morley M.P.
      • Lu M.M.
      • DeMayo F.J.
      • Olson E.N.
      • et al.
      Development and regeneration of Sox2+ endoderm progenitors are regulated by a HDAC1/2-Bmp4/Rb1 regulatory pathway.
      ). These signaling molecules working together with their downstream target genes regulate lung morphological development. In addition, epigenetic regulations, including DNA methylation, histone modification, and chromatin remodeling, also play vital roles in the regulation of gene expression during lung development.
      Chromatin remodeling is tightly coupled to lung branching. Loss of histone deacetylase 1/2 (
      • Wang Y.
      • Tian Y.
      • Morley M.P.
      • Lu M.M.
      • DeMayo F.J.
      • Olson E.N.
      • et al.
      Development and regeneration of Sox2+ endoderm progenitors are regulated by a HDAC1/2-Bmp4/Rb1 regulatory pathway.
      ) or Sin3a (
      • Yao C.
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      • Konda B.
      • Guan X.
      • Mizuno T.
      • Chiba N.
      • et al.
      Sin3a regulates epithelial progenitor cell fate during lung development.
      ), two components of the NuRD chromatin remodeling complex, impairs proximal airway epithelial differentiation partially through the activation of Bmp4 during mouse lung endoderm development. These findings suggest that chromatin remodeling is indispensable for proximal progenitor differentiation during lung development.
      The replacement of canonical histone H2A with histone variant H2A.Z leads to chromatin remodeling and subsequent gene expression changes (
      • Weber C.M.
      • Henikoff S.
      Histone variants: Dynamic punctuation in transcription.
      ,
      • Creyghton M.P.
      • Markoulaki S.
      • Levine S.S.
      • Hanna J.
      • Lodato M.A.
      • Sha K.
      • et al.
      H2AZ is enriched at polycomb complex target genes in ES cells and is necessary for lineage commitment.
      ,
      • Hu G.
      • Cui K.
      • Northrup D.
      • Liu C.
      • Wang C.
      • Tang Q.
      • et al.
      H2A.Z facilitates access of active and repressive complexes to chromatin in embryonic stem cell self-renewal and differentiation.
      ,
      • Subramanian V.
      • Mazumder A.
      • Surface L.E.
      • Butty V.L.
      • Fields P.A.
      • Alwan A.
      • et al.
      H2A.Z acidic patch couples chromatin dynamics to regulation of gene expression programs during ESC differentiation.
      ,
      • Cuadrado A.
      • Corrado N.
      • Perdiguero E.
      • Lafarga V.
      • Munoz-Canoves P.
      • Nebreda A.R.
      Essential role of p18Hamlet/SRCAP-mediated histone H2A.Z chromatin incorporation in muscle differentiation.
      ). As a key subunit of the SNF-2 related CBP activator protein complex, the zinc finger HIT-type containing 1 (Znhit1) mediates the exchange of histone variant H2A.Z for H2A. Our recent studies have demonstrated that Znhit1 incorporates H2A.Z for transcriptional regulation of genes in Lgr5-positive stem cell maintenance (
      • Zhao B.
      • Chen Y.
      • Jiang N.
      • Yang L.
      • Sun S.
      • Zhang Y.
      • et al.
      Znhit1 controls intestinal stem cell maintenance by regulating H2A.Z incorporation.
      ), hematopoietic homeostasis (
      • Sun S.
      • Jiang N.
      • Jiang Y.
      • He Q.
      • He H.
      • Wang X.
      • et al.
      Chromatin remodeler Znhit1 preserves hematopoietic stem cell quiescence by determining the accessibility of distal enhancers.
      ), and meiotic initiation (
      • Sun S.
      • Jiang Y.
      • Zhang Q.
      • Pan H.
      • Li X.
      • Yang L.
      • et al.
      Znhit1 controls meiotic initiation in male germ cells by coordinating with Stra8 to activate meiotic gene expression.
      ). However, the function of Znhit1/H2A.Z in mammalian pulmonary development remains unknown.
      In this study, we show that Znhit1 deficiency in embryonic respiratory epithelial cells results in severe branching defects, accompanied by reduced proliferation and increased apoptosis of the entire lung epithelia. The deletion of Znhit1 leads to increased expression of Bmp4 in lung epithelium and overactivation of its downstream target SMAD; also the incorporation of H2A.Z in Bmp4 promoter region is Znhit1 dependent. Inhibition of BMP4 signaling in 3D lung epithelial organoids with the BMP antagonist Noggin can partially rescue the branching defects as shown by enhanced bud formation. Taken together, our work demonstrates that the epigenetic factor Znhit1 acts as a repressor of Bmp4 expression through H2A.Z in the embryonic epithelium during lung branching morphogenesis.

      Results

      Znhit1 deletion impairs embryonic lung epithelium development

      To determine the functions of Znhit1 in lung development, we first examined the expression pattern of Znhit1 during fetal lung development by immunochemistry histological staining. As shown in Fig. 1A, Znhit1 was widely expressed throughout the lung from E12.5 to E18.5 with nuclear localization.
      Figure thumbnail gr1
      Figure 1Znhit1 deletion in embryonic lung epithelium impairs prenatal development. A, the expression pattern of Znhit1 is showing revealing dynamic changes of Znhit1 expression during lung prenatal development in mice (embryonic day; E12.5, E14.5, E16.5, and E18.5). The scale bars represent 200 μm. B, the high efficiency of Znhit1 KO driven by Shh-Cre was proved by immunohistochemical staining at E12.5. Quantitative RT–PCR (qRT–PCR) of RNA extracted from E13.5 control and Znhit1-deficient EpCAM+ sorted lung epithelial cells confirmed a significant decrease of Znhit1 expression in homozygous Znhit1 conditional KO (cKO) mice. Means ± SEM; ∗∗p < 0.01 using unpaired two-tailed Student’s t test. n = 4/group. The scale bars represent 100 μm. C, statistic table of postnatal death in Znhit1 cKO group. D, lungs from control and Znhit1 cKO mice (E18.5) are shown demonstrating cystic defects after deletion of Znhit1. The scale bars represent 200 μm (E12.5) and 1000 μm (E18.5). E, hematoxylin/eosin-stained sections of control and Znhit1 mutant lungs at E18.5. The scale bars represent 50 μm. Znhit1, zinc finger HIT-type containing 1.
      To assess the potential role(s) of Znhit1 in epithelium development, we used Shh-Cre (
      • Harfe B.D.
      • Scherz P.J.
      • Nissim S.
      • Tian H.
      • McMahon A.P.
      • Tabin C.J.
      Evidence for an expansion-based temporal Shh gradient in specifying vertebrate digit identities.
      ) to delete Znhit1 in the respiratory epithelium before birth. The efficiency of Cre-mediated epithelial deletion of Znhit1 was examined through quantitative RT–PCR (qRT–PCR) and immunostaining. The expression of Znhit1 was absent in the mutant lung epithelium at E12.5 but remained normal in the rest of the lung (Fig. 1B). The survival of Shh-Cre; Znhit1fl/fl (referred to as “Znhit1 cKO” [conditional KO] hereafter) mice during embryogenesis was as good as those of Znhit1fl/fl and Shh-Cre; Znhit1fl/+ (referred to as “control” hereafter) mice, with no significant weight/length changes (Figs. 1C; S1). While Znhit1fl/fl and control mice both survived normally without any observable malformations, Znhit1-mutant neonatal mice died of respiratory failure immediately after birth. Further analysis of fetal lung development revealed that Znhit1 cKO embryos did form primary lung buds at E12.5 but exhibited multiple distal cysts instead of well-formed lung tissue by E18.5 (Figs. 1, D and E; S2), indicating an important function of Znhit1 in epithelial pattern formation.

      Znhit1 is essential for embryonic lung distal epithelium branching

      To determine whether Znhit1 regulates lung branching, we performed hematoxylin and eosin staining with lung sections of E12.5, E14.5, and E16.5. The observable branching defects at E14.5 developed into cystic morphology by E16.5 (Fig. 2A). Along the proximodistal axis during branching morphogenesis, the lung endoderm with SOX2 expression marks the proximal airway progenitors (
      • Que J.
      • Luo X.
      • Schwartz R.J.
      • Hogan B.L.
      Multiple roles for Sox2 in the developing and adult mouse trachea.
      ,
      • Que J.
      • Okubo T.
      • Goldenring J.R.
      • Nam K.T.
      • Kurotani R.
      • Morrisey E.E.
      • et al.
      Multiple dose-dependent roles for Sox2 in the patterning and differentiation of anterior foregut endoderm.
      ), whereas SOX9 marks the distal progenitors (
      • Perl A.K.
      • Kist R.
      • Shan Z.
      • Scherer G.
      • Whitsett J.A.
      Normal lung development and function after Sox9 inactivation in the respiratory epithelium.
      ,
      • Rockich B.E.
      • Hrycaj S.M.
      • Shih H.P.
      • Nagy M.S.
      • Ferguson M.A.
      • Kopp J.L.
      • et al.
      Sox9 plays multiple roles in the lung epithelium during branching morphogenesis.
      ). In E14.5 sections, the SOX2+ region aggressively extended and the SOX9+ region was restricted in the distal epithelial tips accompanied by branching morphogenesis in control mice. But the SOX9+ area showed an extended distal pattern and fewer branches in Znhit1-deficient epithelium while the patterning of the SOX2+ region remained largely unchanged (Fig. 2B).
      Figure thumbnail gr2
      Figure 2Znhit1 is essential for embryonic lung distal epithelium branching. A, hematoxylin/eosin-stained sections reveal normal lung morphogenesis at embryonic day 12.5. (E12.5) but show branching defects at E14.5 and cystic lung structure at E16.5. The scale bars represent 100 μm. B, E14.5 control and Znhit1 conditional KO (cKO) lung sections were stained with SOX2 and SOX9. SOX9-positive cells were showing an extended distal pattern and fewer branches in Znhit1-deficient epithelium. The scale bars represent 500 μm. Quantification of SOX9-positive branches at E14.5. Means ± SEM; ∗∗∗∗p < 0.0001 using unpaired two-tailed Student’s t test. n = 4/group. C, E18.5 control and mutant lung sections were stained with SOX2 (proximal lung epithelial progenitor marker), proSPC (AT2 marker), HOPX (AT1 marker), and CCSP (club cell marker). Znhit1-deficient lungs show differentiation of distal epithelial cells but less SOX2-positive progenitors the day before birth. The scale bars represent 50 μm. Znhit1, zinc finger HIT-type containing 1.
      The abnormal branching patterns of Znhit1 mutant mice suggest the possible functions of Znhit1 in regulating cell differentiation, proliferation, and cell death. Cell differentiation events during lung cell lineage specifications have been shown to regulate branching morphogenesis (
      • Hogan B.L.
      • Barkauskas C.E.
      • Chapman H.A.
      • Epstein J.A.
      • Jain R.
      • Hsia C.C.
      • et al.
      Repair and regeneration of the respiratory system: Complexity, plasticity, and mechanisms of lung stem cell function.
      ). To test whether the branching defects of Znhit1 mutant in the epithelium are due to its role in cell differentiation, we examined markers for major cell types of lung epithelium at E18.5. Airway club cell differentiation indicated by club cell secretory protein (CCSP), as well as AT1 cells and AT2 cells labeled by HOPX and proSPC staining, was undisturbed (Fig. 2C). The SOX2+ cells were lost in Znhit1-deficient lungs at E18.5 but remained unchanged in the esophagus (Fig. S3A), indicating a specific function of Znhit1 in lung epithelium development. The endothelial and smooth muscle cells also differentiated normally without Znhit1 (Fig. S3, B and C). These results suggest that Znhit1 in lung epithelium is dispensable for epithelial cell differentiation but exerts a regulatory function in lung branching.

      Branching failure caused by Znhit1 deletion leads to decreased proliferation and increased apoptosis of lung epithelium

      Previous studies have shown that Znhit1 plays a role in p53-mediated cell apoptosis and cell cycle arrest in vitro (
      • Lafarga V.
      • Cuadrado A.
      • Nebreda A.R.
      p18(Hamlet) mediates different p53-dependent responses to DNA-damage inducing agents.
      ,
      • Yang Z.
      • Cao Y.
      • Zhu X.
      • Huang Y.
      • Ding Y.
      • Liu X.
      Znhit1 causes cell cycle arrest and down-regulates CDK6 expression.
      ). At the cellular level, the formation of new branches is also driven by the proliferation and cytoskeleton of lung epithelial cells. To examine whether cell proliferation is affected by the deletion of Znhit1, we performed immunofluorescence staining and quantification analyses. Phosphorylated histone H3 (PH3) is a marker of cell proliferation and labels cells in the G2 and M phases (
      • Hans F.
      • Dimitrov S.
      Histone H3 phosphorylation and cell division.
      ), whereas Ki67 can be found throughout the cell cycle except G0 phase (
      • Gerdes J.
      • Lemke H.
      • Baisch H.
      • Wacker H.H.
      • Schwab U.
      • Stein H.
      Cell cycle analysis of a cell proliferation-associated human nuclear antigen defined by the monoclonal antibody Ki-67.
      ,
      • McCormick D.
      • Chong H.
      • Hobbs C.
      • Datta C.
      • Hall P.A.
      Detection of the Ki-67 antigen in fixed and wax-embedded sections with the monoclonal antibody MIB1.
      ). NKX2.1 was used to label fetal lung epithelial cells. Both cell number and proliferation of fetal lung epithelial and mesenchymal cells were analyzed and counted by costaining NKX2.1 and PH3 on the E14.5 fetal lung tissue sections. We found that the proliferation of epithelial cells was reduced in Znhit1 cKO lungs (p < 0.05) with unchanged cell polarity and structure (Figs. 3A; S4A). Loss of Znhit1 seemed to cause cell cycle arrest in lung epithelium as no significant differences were found in Ki67 staining (Fig. S4, B and C).
      Figure thumbnail gr3
      Figure 3Branching failure caused by Znhit1 deletion leads to decreased proliferation and increased apoptosis of lung epithelium. A, region of interest (ROI) of phosphorylated histone H3 (PH3) staining was used for quantification. DAPI-labeled cell number quantification of epithelium and mesenchyme revealed a decreased proportion of epithelial cells in Znhit1 conditional KO (cKO) lungs at embryonic day 14.5 (E14.5). Loss of Znhit1 also impairs proliferation of epithelium into late G2 and mitosis stages. Means ± SEM; ∗p < 0.05, ∗∗p < 0.01 using unpaired two-tailed Student’s t test. n = 4/group. The scale bars represent 500 μm. B, apoptotic cell staining by cleaved caspase-3 at both E12.5 and E13.5. Apoptosis of epithelium raises at E13.5 in Znhit1 cKO lungs. Quantification of cleaved caspase-3-positive cells at E13.5. Means ± SEM; ∗∗∗∗p < 0.0001 using unpaired two-tailed Student’s t test. n = 5/group. The scale bars represent 50 μm. C, 0 h and 36 h of lung explant culture with/without pan-caspase inhibitor Z-VAD-FMK and quantification of lung tips with or without Z-VAD-FMK at 0 and 36 h culture. Inhibition of apoptosis did not rescue the branching defects in Znhit1-deficient lungs. Means ± SEM; ∗∗p < 0.01 using unpaired two-tailed Student’s t test. n = 5/group (without inhibitor), n = 4/group (with inhibitor). The scale bars represent 1000 μm. DAPI, 4′,6-diamidino-2-phenylindole; Znhit1, zinc finger HIT-type containing 1.
      Next, we examined whether the level of cell apoptosis was changed in Znhit1-deficient lungs. By cleaved caspase-3 immunostaining, significantly increased apoptosis in epithelium was observed in Znhit1-deficient lungs (Fig. 3B). To test whether increased cell apoptosis affects lung branching, we dissected E12.5 lungs and performed an explant culture with pan-caspase inhibitor Z-VAD-FMK to inhibit apoptosis (Fig. S5A). Quantification of lung tips showed that lung branching is reduced in the Znhit1-deficient group, despite inhibition of apoptosis (Figs. 3C; S5B). These results suggest that reduced proliferation and increased apoptosis of lung epithelial cells induced by cKO of Znhit1 cannot explain branching defects of lung epithelium.

      Znhit1 deficiency abolishes epithelium branching through activating BMP–SMADs signaling

      To assess the molecular consequences of Znhit1 deficiency in early lung endoderm, we isolated epithelial cells from Znhit1fl/fl and Znhit1 cKO lungs at E13.5 to perform RNA-Seq analysis. We found that 550 genes were significantly upregulated and 192 genes were significantly downregulated by Znhit1 deletion. Differently expressed genes were annotated to GO terms, including apoptosis, oxidative stress, and cell cycle–related clustering (Fig. S6). Consistent with increased apoptosis of lung epithelial cells in the phenotypic analysis, we found increased expression of proapoptotic Bcl family genes BOK and Bax (p < 0.0001) and decreased expression of Bcl2L2 (p < 0.0001), which can inhibit the activity of Bax and promote cell survival (Fig. 4A). Moreover, Cdkn1a and Ccng1, related to cell cycle arrest, were also upregulated in the mutant epithelium, accounting for the cell proliferation disorder (Fig. 4A). Interestingly, Znhit1 deletion significantly upregulated the expression of Bmp4 in lung epithelium (Fig. 4A). Upregulation of Bmp4 expression in the epithelium is further confirmed by qRT–PCR (Fig. 4B). As reported previously (
      • Weaver M.
      • Dunn N.R.
      • Hogan B.L.
      Bmp4 and Fgf10 play opposing roles during lung bud morphogenesis.
      ,
      • Weaver M.
      • Yingling J.M.
      • Dunn N.R.
      • Bellusci S.
      • Hogan B.L.
      Bmp signaling regulates proximal-distal differentiation of endoderm in mouse lung development.
      ), expression of Bmp4 is confined to the distal tips of the airway branches as observed in control lungs by in situ hybridization, but the expression of Bmp4 was elevated and pervaded throughout the epithelium of Znhit1 mutant lungs (Fig. 4C). Fgf10 signaling was not significantly altered in Znhit1 mutants (Fig. S7A), as were other critical genes involved in lung branching morphogenesis (Fig. S7B). To evaluate whether Bmp4 is directly regulated by H2A.Z, we performed anti-H2A.Z chromatin immunoprecipitation (ChIP) assay on embryonic lung samples. H2A.Z enrichment in the promoter region of Bmp4 significantly decreased in Znhit1-deficient lungs (Fig. 4D), supporting our hypothesis that Znhit1 promotes the incorporation of H2A.Z into the promoter region, thereby controlling Bmp4 transcription.
      Figure thumbnail gr4
      Figure 4Znhit1 deficiency abolishes epithelium branching through activating BMP–SMAD signaling. A, heatmap of differentially expressed genes from RNA-Seq data of EpCAM-sorted cells at embryonic day 13.5 (E13.5). B, quantitative RT–PCR (qRT–PCR) of Bmp4 with RNA extracted from E13.5 control and Znhit1-deficient EpCAM+ sorted lung epithelial cells. Means ± SEM; ∗∗p < 0.01 using unpaired two-tailed Student’s t test. n = 3/group. C, RNA in situ of Bmp4 in lung sections at E13.5. Znhit1 conditional KO (cKO) lungs showed increased Bmp4 expression around distal tips of epithelium. The scale bars represent 500 μm (tile scans) or 50 μm (zooms). D, chromatin immunoprecipitation (ChIP)–qPCR showing the fold enrichment of H2A.Z in the promoter region of Bmp4 in control and Znhit1 cKO lung samples at E13.5. Means ± SEM; ∗∗∗∗p < 0.0001 using unpaired two-tailed Student’s t test. n = 3/group. BMP, bone morphogenetic protein; Znhit1, zinc finger HIT-type containing 1.

      Canonical BMP–SMAD1/5/9 signaling is activated in Znhit1 cKO mice, and noggin treatment rescues lung budding formation in Znhit1 cKO lung epithelial organoids

      To examine the activation of BMP–SMAD1/5/9 signaling in Znhit1 cKO lungs, we used E13.5 lung sections to stain phosphor-SMAD1/5/9 (pSMAD1/5/9), a well-established indicator of BMP signaling activity (
      • Moustakas A.
      • Heldin C.H.
      The regulation of TGFbeta signal transduction.
      ). Expression of pSMAD1/5/9 exhibited an expanded pattern around the dilated epithelium with much stronger fluorescence intensity than that of controls (Fig. 5A), which is consistent with the expanded pattern of Bmp4. BMP signaling is an important regulator of anterior foregut endoderm development as well as a regulator of the proximal–distal patterning of the developing lung.
      Figure thumbnail gr5
      Figure 5Canonical BMP–SMAD1/5/9 signaling is activated in Znhit1 conditional KO (cKO) mice and Noggin treatment rescues lung budding formation in mutant lung epithelial organoids. A, phosphor-SMAD1/5/9 (pSMAD1/5/9) staining on embryonic day 13.5 (E13.5) lung sections. The scale bars represent 50 μm. Quantification of pSMAD1/5/9-positive cells. Means ± SEM; ∗p < 0.05 using unpaired two-tailed Student’s t test. n = 5/group. B, wildtype lungs of E12.5 were dissected and cultured in vitro with or without BMP4. After a culture of 48 h, lung explant with BMP4 showed expanded tips and fewer branches. The scale bars represent 200 μm. Quantification of lung branches at 0 and 48 h. Means ± SEM; ∗p < 0.05 using unpaired two-tailed Student’s t test. n = 3/group. C, lung organoids from E12.5 lung epithelial cells were cultured with Noggin for 7 days. Bright field scale bars represent 200 μm. Noggin treatment promoted budding formation in organoids derived from Znhit1 cKO epithelium. The budding number of mutant organoids was significantly increased with Noggin treatment. Means ± SEM; ∗p < 0.05 using unpaired two-tailed Student’s t test. n = 3/group. 3D lung organoids were stained SOX2 and SOX9 revealing cells at budding tips were double-positive cells. Immunofluorescence staining scale bars represent 50 μm. Quantitative RT–PCR (qRT–PCR) of Sox2 and Sox9 with RNA from lung epithelial organoids. Noggin treatment enhanced relative expression of Sox2/Sox9, whereas BMP4 treatment leads to decreased Sox2/Sox9 expression ratio. Means ± SEM; ∗p < 0.05 using unpaired one-way ANOVA test. n = 3/group. BMP, bone morphogenetic protein.
      Previous studies have shown that activation of BMP signaling through epigenetic changes in epithelium inhibits lung branching and proximal lung development (
      • Wang Y.
      • Tian Y.
      • Morley M.P.
      • Lu M.M.
      • DeMayo F.J.
      • Olson E.N.
      • et al.
      Development and regeneration of Sox2+ endoderm progenitors are regulated by a HDAC1/2-Bmp4/Rb1 regulatory pathway.
      ,
      • Li Q.
      • Jiao J.
      • Li H.
      • Wan H.
      • Zheng C.
      • Cai J.
      • et al.
      Histone arginine methylation by Prmt5 is required for lung branching morphogenesis through repression of BMP signaling.
      ). Our data in lung explant culture did show a similar branching defect with BMP4 treatment (Fig. 5B). To determine whether the activation of BMP–SMAD1/5/9 signaling in the Znhit1-deficient lungs contributes to branching defects described previously, we inhibited BMP signaling by using the BMP antagonist Noggin in an ex vivo lung organoid culture system. Lung epithelial organoids were generated from E12.5 lung epithelial cells in a 3D culture system, and the organoids’ budding sites were identified by SOX2 and SOX9 immunofluorescent wholemount staining (Fig. 5C). Inhibition of BMP signaling with Noggin treatment improved budding formation of lung epithelial organoids, with loss of Znhit1 (p < 0.05). Consistent with SOX2+ cell loss and SOX9+ cell expansion in vivo, activation of BMP signaling also caused a decreased expression ratio of Sox2/Sox9, whereas BMP signaling inhibition increased the Sox2/Sox9 expression ratio significantly. The organoid culture from fetal lung epithelium demonstrated that the activation of intraepithelial Bmp4 caused by Znhit1 ablation hindered the formation of lung buds during branching development.

      Discussion

      One important issue in lung development is how epigenetic regulation is involved in cell fate determination during branching morphogenesis. Recent studies have shown that Znhit1 acts as a critical regulator of chromatin organization (
      • Zhao B.
      • Chen Y.
      • Jiang N.
      • Yang L.
      • Sun S.
      • Zhang Y.
      • et al.
      Znhit1 controls intestinal stem cell maintenance by regulating H2A.Z incorporation.
      ,
      • Sun S.
      • Jiang N.
      • Jiang Y.
      • He Q.
      • He H.
      • Wang X.
      • et al.
      Chromatin remodeler Znhit1 preserves hematopoietic stem cell quiescence by determining the accessibility of distal enhancers.
      ,
      • Sun S.
      • Jiang Y.
      • Zhang Q.
      • Pan H.
      • Li X.
      • Yang L.
      • et al.
      Znhit1 controls meiotic initiation in male germ cells by coordinating with Stra8 to activate meiotic gene expression.
      ,
      • Xu M.
      • Yao J.
      • Shi Y.
      • Yi H.
      • Zhao W.
      • Lin X.
      • et al.
      The SRCAP chromatin remodeling complex promotes oxidative metabolism during prenatal heart development.
      ). In this study, we have provided convincing evidence that Znhit1 is essential for lung branching morphogenesis by coordinating with the histone variant H2A.Z to regulate Bmp4 expression (Fig. 6). Using Znhit1 cKO mice, we found the disrupted lung branching accompanied by decreased proliferation and increased apoptosis of lung epithelium in Znhit1 mutants. We demonstrate that Znhit1 can repress intraepithelial Bmp4 expression during embryonic development. Our results help to understand the mechanism by which lung development is tightly regulated under the context of chromatin.
      Figure thumbnail gr6
      Figure 6Working model of Znhit1 regulation of embryonic lung development. Znhit1 mediates the exchange of histone variant H2A.Z for H2A leading to repression of Bmp4 and permitting embryonic lung branching. Loss of Znhit1 in lung epithelial increases Bmp4 expression and causes abnormal lung branching. The scale bars represent 200 μm. Znhit1, zinc finger HIT-type containing 1.
      The process of lung development, including specification, branching, and patterning, is fine tuned by key signaling pathways, such as the Wnt, BMP, and FGF pathways (
      • Herriges M.
      • Morrisey E.E.
      Lung development: Orchestrating the generation and regeneration of a complex organ.
      ). Among these morphogen molecules, the moderate activity of BMP4 signaling plays a crucial role in regulating branching morphogenesis, and the derepression of Bmp4 expression in lung distal epithelium would lead to lung branching defects (
      • Wang Y.
      • Tian Y.
      • Morley M.P.
      • Lu M.M.
      • DeMayo F.J.
      • Olson E.N.
      • et al.
      Development and regeneration of Sox2+ endoderm progenitors are regulated by a HDAC1/2-Bmp4/Rb1 regulatory pathway.
      ). Therefore, it is important to clarify the regulatory mechanism of Bmp4 transcription. Previous studies have shown that histone deacetylase 1/2-mediated histone deacetylation or Prmt5-mediated histone methylation is required for repressing Bmp4 transcription (
      • Wang Y.
      • Tian Y.
      • Morley M.P.
      • Lu M.M.
      • DeMayo F.J.
      • Olson E.N.
      • et al.
      Development and regeneration of Sox2+ endoderm progenitors are regulated by a HDAC1/2-Bmp4/Rb1 regulatory pathway.
      ,
      • Li Q.
      • Jiao J.
      • Li H.
      • Wan H.
      • Zheng C.
      • Cai J.
      • et al.
      Histone arginine methylation by Prmt5 is required for lung branching morphogenesis through repression of BMP signaling.
      ). Our work showed that excessive BMP signaling achieved by loss of Znhit1 in lung epithelium resulted in abnormal fetal lung formation, and inhibition of BMP signaling rescued budding formation in Znhit1 cKO lung epithelial organoids, partially. Compared with the control group, H2A.Z enrichment in the promoter region of Bmp4 decreased dramatically in Znhit1 cKO embryonic lungs (Fig. 4D), indicating that Znhit1-dependent H2A.Z enrichment in the promoter region directly controls Bmp4 transcription. Thus, we identify the essential role of the chromatin remodeler Znhit1 in branching morphogenesis by regulating Bmp4 expression.
      Bmp4 is not only critical for lung development but also associated with chronic obstructive pulmonary disease (COPD) (
      • Zuo W.L.
      • Yang J.
      • Strulovici-Barel Y.
      • Salit J.
      • Rostami M.
      • Mezey J.G.
      • et al.
      Exaggerated BMP4 signalling alters human airway basal progenitor cell differentiation to cigarette smoking-related phenotypes.
      ). Inhalational exposure to tobacco smoke is one of the major risk factors for COPD (
      • Labaki W.W.
      • Rosenberg S.R.
      Chronic obstructive pulmonary disease.
      ). And previous studies reported that the induced BMP4 signaling in human airway basal cells promotes cigarette smoking–related phenotypes (
      • Zuo W.L.
      • Yang J.
      • Strulovici-Barel Y.
      • Salit J.
      • Rostami M.
      • Mezey J.G.
      • et al.
      Exaggerated BMP4 signalling alters human airway basal progenitor cell differentiation to cigarette smoking-related phenotypes.
      ). In consideration of the crucial role of Znhit1/H2A.Z in regulating Bmp4 expression, we speculate that Znhit1/H2A.Z may also get involved in the pathogenesis of COPD.
      In summary, our studies demonstrate that Znhit1 in lung epithelium represses Bmp4 expression through mediating H2A.Z incorporation into the promoter region during branching morphogenesis. Whether the regulatory mechanism of Znhit1/H2A.Z on BMP signaling is conserved during chronic injury and the repair process of airways needs to be addressed in further studies.

      Experimental procedures

      Mice

      Znhit1fl/fl mice were generated by the Model Animal Research Center of Nanjing University (MARC) (
      • Zhao B.
      • Chen Y.
      • Jiang N.
      • Yang L.
      • Sun S.
      • Zhang Y.
      • et al.
      Znhit1 controls intestinal stem cell maintenance by regulating H2A.Z incorporation.
      ). Shh-Cre (The Jackson Laboratory, stock number 005622) mice were obtained from the Jackson Laboratory. For timed mating, embryonic day (E) 0.5 was determined by the presence of a copulation plug in the morning. Pregnant dams were sacrificed by CO2 inhalation, and embryos were harvested at different embryonic stages. All strains were maintained in C57BL/6 background. Mice were housed in pathogen-free conditions according to the protocols approved by the Institutional Animal Care and Use Committee of Fudan University.

      qRT–PCR

      Total RNA was extracted with RNeasy Mini Kit (Qiagen), and the reverse transcription reaction was performed using GoScript Reverse Transcription System (Promega). qRT–PCRs were performed with GoTaq qPCR Master Mix (Promega) in triplicates on CFX96 Touch System (Bio-Rad). Primer pairs are listed in Table S1.

      Lung immunohistochemistry, immunofluorescence, and biochemistry

      Embryos and embryonic lungs were fixed by 4% paraformaldehyde followed by paraffin embedding. Histological staining, immunohistochemistry, and immunofluorescence were performed on 5 μm paraffin sections. Brightfield images were obtained using a Zeiss Axio ImagerA2 microscope equipped with AxioVision Software (Carl Zeiss Microscopy, LLC). Fluorescent images were obtained using a Nikon A1Rsi inverted laser confocal microscope and Olympus FV3000 Confocal Laser Scanning Microscope. The following antibodies were used in this work: rabbit anti-ZNHIT1 (catalog no.: HPA019043; Sigma–Aldrich); mouse anti-SOX2 (catalog no.: sc-365823; Santa Cruz); rabbit anti-SOX9 (catalog no.: AB5535; Millipore); rabbit anti-CCSP (catalog no.: WRAB-3950; Seven Hills Bioreagents); guinea pig anti-proSPC (generated in the Whitsett laboratory, raised against the N terminus of proSPC); rabbit anti-HOPX (catalog no.: sc-30216; Santa Cruz); mouse anti-Alpha smooth muscle actin (catalog no.: A2547; Sigma); rabbit anti-CD31 (catalog no.: 28083-1-AP; Proteintech); rabbit anti-ZO-1 (catalog no.: 21773-1-AP; Proteintech); mouse anti-E-cadherin (catalog no.: 610182; BD Transduction); rabbit anti-PH3 (catalog no.: sc-8656-R; Santa Cruz); mouse anti-NKX2.1 (catalog no.: sc-53136; Santa Cruz); rat anti-TROMA-I (KRT8) (catalog no.: AB 531826; DSHB); mouse anti-Ki67 (catalog no.: 556003; BD Pharmingen); rabbit anti-E-cadherin (catalog no.: 3195S; Cell Signaling); rabbit anti-Active caspase-3 (catalog no.: AF835; R&D Systems); rabbit anti-pSMAD1/5/9 (catalog no.: 13820S; Cell Signaling); rabbit anti-Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (catalog no.: 4370S; Cell Signaling).

      Isolation of epithelial cells by magnetic cell sorting

      E13.5 lung lobes were harvested from control and Znhit1 cKO embryos to isolate epithelial cells. Dispase (Corning) was used for lobe digestion. After 15 min of digestion at 37 °C, samples were transferred to 5 ml of Dulbecco's modified Eagle's medium (DMEM) containing 25 mM Hepes (Gibco) and 120 units of DNAse I (Sigma–Aldrich). Sample dissociation was performed on GentleMACS Dissociator (Miltenyi Biotec), and 40 μm nylon cell strainers (Corning) were used to get single cells. Cell suspensions that passed through the strainers were pelleted by centrifugation at 1500 rpm (433g) for 6 min at 4 °C and resuspended in 10 ml DMEM (with Hepes and PenStrep) and centrifuged once again. Cells were resuspended in 90 μl of autoMACS Running Buffer (Miltenyi Biotec) and 10 μl FcR Blocking Reagent and incubated at 4 °C for 10 min. Then, 10 μl of CD326 MicroBeads (Miltenyi Biotec) was added to bind epithelial cells. After incubation for 15 min at 4 °C, cells were washed twice with autoMACS Running Buffer. Cell pellets were resuspended in 500 μl of Running Buffer to pass through a 40 μm nylon filter before sorting using an AutoMACS Pro Separator (Miltenyi Biotec). CD326-positive (epithelial) and -negative (nonepithelial) cells were separated. Sorting efficiency was determined by the qRT–PCR of Epcam (epithelial) and Twist2 (mesenchymal).

      RNA-Seq and analysis

      RNA-Seq was performed on samples extracted from isolated lung epithelium at E13.5. The reverse transcription reaction was performed using the Ovation RNA-Seq System V2 kit (NuGEN). Sequencing and alignment were done by Berry Genomics. Znhit1 cKO and control samples (n = 2 each) were analyzed. The raw data were uniquely mapped to the mm10 genome by TopHat, version 1.4.1 (
      • Trapnell C.
      • Pachter L.
      • Salzberg S.L.
      TopHat: Discovering splice junctions with RNA-seq.
      ). Expression values were assigned to gene level by Cufflinks, version 1.3.0 (
      • Trapnell C.
      • Roberts A.
      • Goff L.
      • Pertea G.
      • Kim D.
      • Kelley D.R.
      • et al.
      Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks.
      ). All differentially expressed genes had a log2-transformed fold change >1.2 and a threshold of p < 0.05.

      In situ hybridization

      Mice embryos were washed with diethyl pyrocarbonate–treated PBS and fixed in 4% paraformaldehyde overnight at 4 °C. Samples went through dehydration and paraffin embedding. In situ hybridization was performed on 6 μm paraffin sections with the RNA scope 2.0 kit (Advanced Cell Diagnostics). The probes used were as below: RNAscope Probe-Mm-Bmp4 (REF #401301).

      ChIP–qPCR

      Samples used for ChIP were freshly harvested embryonic lungs at E13.5 (n = 3). Samples were crosslinked with 1% formaldehyde for 10 min at room temperature, then quenched with glycine, and washed with PBS. ChIP protocol was done as previously described (
      • Zhao B.
      • Chen Y.
      • Jiang N.
      • Yang L.
      • Sun S.
      • Zhang Y.
      • et al.
      Znhit1 controls intestinal stem cell maintenance by regulating H2A.Z incorporation.
      ). The anti-H2A.Z antibody (ab4174; Abcam) and protein A/G magnetic beads (Millipore) were used for ChIP. ChIP–qPCR was performed with two pairs of designed primers in the promoter region of Bmp4 (Bmp4 #1 and Bmp4 #2 in Table S1).

      Lung explant culture

      E11.5 lung buds were dissected from the embryos and then cultured as previously described (
      • He H.
      • Huang M.
      • Sun S.
      • Wu Y.
      • Lin X.
      Epithelial heparan sulfate regulates Sonic Hedgehog signaling in lung development.
      ) in the presence of either 250 ng/ml BMP4 (OrganRegen) or bovine serum albumin medium for 48 h.

      Lung organoid culture

      E12.5 lungs were dissected from embryos and digested with prewarmed digestion buffer, containing collagenase I (Sigma) 200 U/ml and dispase II (Sigma) 6 U/ml for 15 min on a shaker at 37 °C. The cells were centrifuged at 300g for 4 min and washed with wash medium (DMEM supplemented with 1% penicillin/streptomycin and 1% fetal bovine serum) and cold PBS. The harvested cells were collected and embedded in Matrigel (R&D Systems) and then seeded on a 48-well plate.
      Lung organoid culture medium consists of advanced DMEM/F12 (Invitrogen) (supplemented with penicillin/streptomycin, Hepes, GlutaMAX, B27, and N-acetylcysteine) plus 5 mM nicotinamide (Sigma), 500 ng/ml R-spondin1 (OrganRegen), 50 ng/ml epidermal growth factor(OrganRegen), 100 ng/ml FGF10 (OrganRegen), 5 μM Y27632 (Tocris), 500 nM A83-01 (Tocris), and 500 nM SB202190 (Sigma). About 100 ng/ml Noggin (OrganRegen) was added for Noggin treatment, and 200 ng/ml BMP4 was added for BMP4 treatment. Culture medium was refreshed every 3 days.

      Statistical analysis

      For qRT–PCR results and cell quantification, values are expressed as the mean ± SEM. Data were analyzed using a two-tailed unpaired Student’s t test or one-way ANOVA test. p Values of 0.05 or less were considered statistically significant. Statistical analysis was performed with the GraphPad Prism 8 software (GraphPad Software, Inc).

      Data availability

      RNA-Seq RAW data have been deposited to the National Center for Biotechnology Information SRA database under accession number SRP325667. All other data described in the article are contained within the main text or supporting information.

      Supporting information

      This article contains supporting information.

      Conflict of interest

      The authors declare that they have no conflicts of interest with the contents of this article.

      Acknowledgments

      This work was supported by grants from the National Natural Science Foundation of China (grant nos.: 31730044 , 32192403 , 32022022 , and 31900581 ), the National Key Research and Development Program of China (grant no.: 2018YFC1003500 ), and the Science and Technology Commission of Shanghai Municipality (grant no.: 20DZ2261200 ).

      Author contributions

      W. W., B. Z., and X. L. conceptualization; W. W., X. T., and C. N. methodology; W. W. validation; W. W., N. J., and S. S. formal analysis; X. T., H. H., S. S., M. Y., C. Y., and M. Q. investigation; N. J. resources; N. J. data curation; W. W., B. Z., and X. L. writing–original draft; D. Y., Z. Z., and Y. S. writing–review & editing; W. W. visualization; H. L., B. Z., and X. L. supervision; C. N., Y. S., B. Z., and X. L. funding acquisition.

      Supporting information

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