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Mark1 Regulates Distal Airspace Expansion Through Type I Pneumocyte Flattening in Lung Development Katsumi Fumoto1, Hisako Takigawa-Imamura2, Kenta Sumiyama3, Shige H

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Mark1 Regulates Distal Airspace Expansion Through Type I Pneumocyte Flattening in Lung Development Katsumi Fumoto1, Hisako Takigawa-Imamura2, Kenta Sumiyama3, Shige H © 2019. Published by The Company of Biologists Ltd | Journal of Cell Science (2019) 132, jcs235556. doi:10.1242/jcs.235556 RESEARCH ARTICLE Mark1 regulates distal airspace expansion through type I pneumocyte flattening in lung development Katsumi Fumoto1, Hisako Takigawa-Imamura2, Kenta Sumiyama3, Shige H. Yoshimura4, Natsumi Maehara1 and Akira Kikuchi1,* ABSTRACT Nelson, 2017). However, mechanistic insights into distal lung During the later stages of lung development, two types of sacculation have been less clear. It has recently been reported pneumocytes, cuboidal type II (AECII) and flattened type I (AECI) that a loss of Wnt signaling results in the reduction of apical alveolar epithelial cells, form distal lung saccules. Here, we highlight constriction, and thereby contributes to distal lung sacculation how fibroblasts expressing MAP-microtubule affinity regulating (Fumoto et al., 2017). The regulation of capillary density by kinase 1 (Mark1) are required for the terminal stages of pulmonary trinucleotide repeats-containing 6c (TNRC6C), an effector for development, called lung sacculation. In Mark1-knockout (KO) mice, micro (mi)RNA-mediated mRNA repression, is involved in distal sacculation and AECI flattening are significantly impaired. Fetal distal lung sacculation through modulating transforming growth β epithelial cells generate alveolar organoids and differentiate into factor (TGF- ) signaling (Guo et al., 2017). Epigenetic changes pneumocytes when co-cultured with fibroblasts. However, the size of mediated by histone deacetylase 3 (HDAC3) are required for organoids decreased and AECI flattening was impaired in the alveolar cell remodeling through the regulation of miRNA and β presence of Mark1 KO fibroblasts. In Mark1 KO fibroblasts TGF- signaling (Wang et al., 2016). Follistatin-like 1 (Fstl1), themselves, cilia formation and the Hedgehog pathway were an antagonist against bone morphogenetic protein, is required suppressed, resulting in the loss of type I collagen expression. for elastin deposition in the mesenchyme during lung The addition of type I collagen restored AECI flattening in organoids sacculation and alveologenesis (Geng et al., 2013). Thus, co-cultured with Mark1 KO fibroblasts and rescued the decreased although genetic, epigenetic and miRNA pathways for distal size of organoids. Mathematical modeling of distal lung sacculation lung sacculation have been described, to date it is unclear how supports the view that AECI flattening is necessary for the proper changes in the cell shape of AECI are regulated and involved in formation of saccule-like structures. These results suggest that distal lung sacculation. Mark1-mediated fibroblast activation induces AECI flattening and Map-microtubule affinity regulating kinase (Mark) family thereby regulates distal lung sacculation. kinases are AMP-activated protein kinase (AMPK)-related kinases, also known as Par-1 family kinases, that regulate KEY WORDS: Mark1, Lung, Pneumocyte, Sacculation, Hedgehog microtubule dynamics and cellular polarity (Bright et al., 2009). Mark family members share catalytic and variable C-terminal INTRODUCTION spacer domains (Timm et al., 2003). Microtubule-associated In the early stages of fetal lung development [embryonic day proteins (MAPs), doublecortin and PSD-95 (also known as (E)9.5–E16.5], the epithelium forms a branched bronchial tree, DLG4), which are substrates of Mark family kinases, are involved while the distal ends of bronchioles transform into saccular in neuronal functions (Hayashi et al., 2012; Wu et al., 2012). structures in the later stages (E17.5–E18.5) (Morrisey and Hogan, Recently, Mark family kinases have also been recognized as 2010). Alveolar epithelial cells consist mainly of two types of regulators of the Hippo pathway. Mark1 acts as a downstream pneumocytes: flattened type I alveolar epithelial cells (AECI), molecule of the tumor suppressor LKB1 (also known as STK11), which mediate gas exchange, and cuboidal type II epithelial cells forms a complex with components of the Hippo pathway, and (AECII), which secrete surfactant proteins to prevent alveoli mediates Yap activity through LKB1 (Lizcano et al., 2004; Mohseni collapsing. In fetal lung development, they are derived from et al., 2014). Mark3 forms a complex with MST1 or MST2 (MST1/ common progenitors that show a bi-potency to generate both types 2; also known as STK4 and STK3, respectively) and DLG5, and of pneumocytes (Desai et al., 2014; Treutlein et al., 2014). inhibits MST1/2 kinase activity (Kwan et al., 2016). Mark4 Mechanisms of branching morphogenesis by growth factor attenuates complex formation between MST1/2–SAV complex signaling and physical cues have been well studied (Varner and and LATS1, thereby repressing cell proliferation in breast cancer cells (Heidary Arash et al., 2017). Thus, Mark family kinases may be involved in various signaling pathways but their specific roles 1Department of Molecular Biology and Biochemistry, Graduate School of Medicine, in vivo are unclear. Osaka University, 2-2 Yamadaoka, Suita 565-0871, Japan. 2Department of Anatomy and Cell Biology, Graduate School of Medical Sciences, Kyushu Recently, Mark1 was found to be involved in lung branching University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. 3Laboratory for morphogenesis in vitro; however Mark1-knockout (KO) mice Mouse Genetic Engineering, RIKEN Center for Biosystems Dynamics Research did not show a phenotype during the pseudoglandular stage in Center, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan. 4Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan. vivo (Fumoto et al., 2017). However, Mark1 is also expressed in the mesenchyme but its physiological role has not been *Author for correspondence ([email protected]) addressed in the later stages of lung development. Here, we A.K., 0000-0003-3378-9522 showed that Mark1 is expressed in fetal lung fibroblasts and is involved in distal lung sacculation through type I collagen Received 27 June 2019; Accepted 5 November 2019 assembly. Journal of Cell Science 1 RESEARCH ARTICLE Journal of Cell Science (2019) 132, jcs235556. doi:10.1242/jcs.235556 RESULTS Rodríguez-Fraticelli et al., 2015). Therefore, to obtain novel Mark1 is expressed in the alveolar mesenchyme during the insights into how Mark1-expressing fibroblasts are involved in later stages of lung development lumen expansion and AECI flattening during development, we It was recently shown that Mark1 is induced by Wnt signaling in the developed in vitro distal lung sacculation models by using a culture of epithelium during the early stages of lung development (Fumoto fetal lung alveolar organoids in the presence of embryonic fibroblasts. et al., 2017). However, at E14.5, an in vivo phenotype of the Mark1 EpCAM-positive (EpCAM+) epithelial cells and Pdgfrα-positive KO lung was not obvious probably due to a redundancy of function (Pdgfrα+) fibroblasts were independently collected by magnet- among Mark family members (Fumoto et al., 2017). In the later assisted cell sorting (MACS). Although co-cultures of organoids stages of lung development, the expression of Mark1 mRNA was and fibroblasts have been reported previously (Barkauskas et al., reduced in the epithelium and increased in the mesenchyme 2013), the viability of fetal fibroblasts in Matrigel, which contains (Fig. 1A). In our previous study with immunostaining, Mark1 laminin, entactin and type IV collagen, was poor in our hands (data protein levels were indeed decreased in the epithelium of the E17.5 not shown). Therefore, Pdgfrα+ fibroblasts were first seeded on type lung compared with what was found in the E14.5 lung (Fumoto I collagen-coated dishes. Matrigel was then solidified on fibroblasts et al., 2017). In contrast, Mark1 was induced by Wnt activation in and finally single epithelial cells suspended in culture medium were cultured fibroblasts isolated from an E18.5 lung (Fig. S1A). The seeded on the solidified Matrigel (Fig. 2A,B). When co-cultured specific expression of Mark1 was confirmed in data from the with fibroblasts, epithelial cells formed single-layered alveolar LungGENS database (Du et al., 2015), revealing that Mark1, but not organoids, in which they expressed either one of AECI (Pdpn) or Mark2 and Mark3, is expressed in myofibroblasts at E18.5 AECII (proSP-C) markers, or both (Fig. 2C). Organoids co-cultured (Fig. 1B). The expression of Mark4 is not shown in the same with fibroblasts expressed alveolar markers more markedly than database. We also isolated E18.5 embryonic lung cells by epithelial cells cultured alone (Fig. 2D). This is consistent with fluorescence activated cell sorting (FACS), and found that Mark1 previous reports showing that Pdgfrα+ fibroblasts are required for was highly expressed in platelet-derived growth factor receptor α alveolar differentiation (Zepp et al., 2017). In contrast, the level of the (Pdgfrα)-high (Pdgfrαhigh) and -low (Pdgfrαlow) cells (Fig. S1B). club cell marker Scgb1a1 was decreased, suggesting that alveolar The expression of Mark1 in these fibroblasts was also confirmed organoids including both AECI and AECII were predominantly by immunohistochemistry (Fig. 1C), suggesting that multiple grown in this condition. The culture medium was supplemented with fibroblast subsets express Mark1 .Acta2, a myofibroblast marker, epidermal growth factor (EGF), noggin, R-spondin1 (Rspo1) and was found to be more highly expressed in Pdgfrahigh cells (Fig. S1B). fibroblast growth factor 10 (FGF10). FGF10
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