Bud formation precedes the appearance of differential cell proliferation during branching morphogenesis of mouse lung epithelium in vitro

Lung mesenchyme is able to support budding and cytodifferentiation of salivary epithelial rudiments in vitro. No difference in response was found between submandibular and parotid epithelium from mouse or rat. There are several further features of this result, which is contradictory to previous findings. (1) Lung mesenchyme is quantitatively less effective than submandibular mesenchyme for supporting submandibular morphogenesis. At least part of this difference is attributed to the inability of submandibular epithelium to replace lung epithelium in supporting the growth of lung mesenchyme. (2) Rat lung mesenchyme is quantitatively more effective than mouse lung mesenchyme when recombined with mouse submandibular epithelium. This may be at least partly due to mouse lung being more easily damaged by the procedures used. (3) Whereas the response of submandibular epithelium to submandibular mesenchyme is equally good on an agar or Millipore filter (MF) substratum, the response to lung mesenchyme is severely reduced or eliminated on MF. This difference is interpreted in terms of different mesenchymal cell densities necessary for submandibular or lung mesenchyme to support branching morphogenesis. Salivary buds formed in lung mesenchyme after 6 days are smaller and more closely packed than in salivary mesenchyme. In these heterotypic recombinates, the accumulation of amylase-resistant, PAS-positive material in the buds is initially accelerated and the tubular epithelium accumulates glycogen.

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Human embryonic lung epithelial tips are multipotent progenitors that can be expanded in vitro as long-term self-renewing organoids

eLife ◽

10.7554/elife.26575 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 84

Author(s):

Marko Z Nikolić ◽

Oriol Caritg ◽

Quitz Jeng ◽

Jo-Anne Johnson ◽

Dawei Sun ◽

...

Keyword(s):

Lung Development ◽

Mouse Lung ◽

Lung Epithelium ◽

Human Embryonic Lung ◽

Self Renewal ◽

Embryonic Mouse ◽

Multipotent Progenitors ◽

Lung Epithelial

The embryonic mouse lung is a widely used substitute for human lung development. For example, attempts to differentiate human pluripotent stem cells to lung epithelium rely on passing through progenitor states that have only been described in mouse. The tip epithelium of the branching mouse lung is a multipotent progenitor pool that self-renews and produces differentiating descendants. We hypothesized that the human distal tip epithelium is an analogous progenitor population and tested this by examining morphology, gene expression and in vitro self-renewal and differentiation capacity of human tips. These experiments confirm that human and mouse tips are analogous and identify signalling pathways that are sufficient for long-term self-renewal of human tips as differentiation-competent organoids. Moreover, we identify mouse-human differences, including markers that define progenitor states and signalling requirements for long-term self-renewal. Our organoid system provides a genetically-tractable tool that will allow these human-specific features of lung development to be investigated.

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Stage specificity in the mesenchyme requirement of rodent lung epithelium in vitro: a matter of growth control?

Development ◽

10.1242/dev.74.1.183 ◽

1983 ◽

Vol 74 (1) ◽

pp. 183-206

Author(s):

Kirstie A. Lawson

Keyword(s):

Cell Cycle ◽

Bronchial Epithelium ◽

Branching Morphogenesis ◽

S Phase ◽

Growth Fraction ◽

Early Event ◽

Rat Lung ◽

Lung Epithelium ◽

Dividing Cells

Epithelia from lung rudiments in which secondary bronchial buds are already established (14th and 13th gestational day for rat and mouse respectively) are able to undergo branching morphogenesis and cytodifferentiation in submandibular mesenchyme in vitro, whereas lung epithelium from one day younger foetuses rarely gives a morphogenetic response to submandibular mesenchyme and usually differentiates into primary (non-budding) bronchial epithelium. The failure of 13-day rat lung epithelium to respond to submandibular mesenchyme can be prevented by peeling off the submandibular mesenchyme from the lung epithelium after 2½ days culture and replacing the same mesenchyme, or renewing it with fresh salivary mesenchyme ex vivo. Changes in the epithelial contour are visible by 10 h and buds form within 24 h; this is followed by branching morphogenesis in more than 66% of the samples. The number of cells in S-phase in the epithelium is doubled within 3 to 5 h after the operation and the number of mitotic cells (colchicine block) is increased during an 11 to 19 h period after the operation. Substituting stomach mesenchyme for submandibular mesenchyme after the operation failed to elicit morphogenesis or an increase in the number of S-phase cells in the epithelium. The proportion of epithelial cells in S-phase in unoperated recombinates does not differ from the proportion in the primary bronchial epithelium (non-budding) of homotypic lung recombinates, whereas the proportion of S-phase cells in operated recombinates approaches that found in the buds of homotypic lung recombinates. The distribution of S-phase cells in visibly responding recombinates 15 to 17 h after operation shows the same heterogeneity as in homotypic lung recombinates, newly formed buds having twice as many cells labelled with [3H]thymidine as the non-budding area. Cell cycle parameters of intact rat lung growing in vitro were estimated using the labelled mitoses method. Primary bronchial epithelium and bronchial buds both had a total cell cycle time of about 13 h and an S-phase of about 10 h. The growth fraction was 0·54 in the primary bronchus and 0·95 in the buds. It is suggested that, also in the recombinates, differences in the proportion of S-phase cells at any one time in morphogenetically active and inactive areas of the epithelium are due to differences in the growth fraction. It is concluded that an early event in the morphogenetic response of lung epithelium to submandibular mesenchyme after removing and restoring the mesenchyme is an increase in the size of the population of dividing cells and it is suggested that a high proportion of dividing cells in an epithelial population is a prerequisite for further interaction of epithelium and mesenchyme leading to branching morphogenesis.

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Fibroblast growth factor 10 (FGF10) and branching morphogenesis in the embryonic mouse lung

Development ◽

10.1242/dev.124.23.4867 ◽

1997 ◽

Vol 124 (23) ◽

pp. 4867-4878 ◽

Cited By ~ 74

Author(s):

S. Bellusci ◽

J. Grindley ◽

H. Emoto ◽

N. Itoh ◽

B.L. Hogan

Keyword(s):

Growth Factor ◽

Fibroblast Growth Factor ◽

Lung Development ◽

Expression Patterns ◽

Branching Morphogenesis ◽

Mouse Lung ◽

Fibroblast Growth ◽

Embryonic Mouse ◽

Fibroblast Growth Factor 10

During mouse lung morphogenesis, the distal mesenchyme regulates the growth and branching of adjacent endoderm. We report here that fibroblast growth factor 10 (Fgf10) is expressed dynamically in the mesenchyme adjacent to the distal buds from the earliest stages of lung development. The temporal and spatial pattern of gene expression suggests that Fgf10 plays a role in directional outgrowth and possibly induction of epithelial buds, and that positive and negative regulators of Fgf10 are produced by the endoderm. In transgenic lungs overexpressing Shh in the endoderm, Fgf10 transcription is reduced, suggesting that high levels of SHH downregulate Fgf10. Addition of FGF10 to embryonic day 11.5 lung tissue (endoderm plus mesenchyme) in Matrigel or collagen gel culture elicits a cyst-like expansion of the endoderm after 24 hours. In Matrigel, but not collagen, this is followed by extensive budding after 48–60 hours. This response involves an increase in the rate of endodermal cell proliferation. The activity of FGF1, FGF7 and FGF10 was also tested directly on isolated endoderm in Matrigel culture. Under these conditions, FGF1 elicits immediate endodermal budding, while FGF7 and FGF10 initially induce expansion of the endoderm. However, within 24 hours, samples treated with FGF10 give rise to multiple buds, while FGF7-treated endoderm never progresses to bud formation, at all concentrations of factor tested. Although exogenous FGF1, FGF7 and FGF10 have overlapping activities in vitro, their in vivo expression patterns are quite distinct in relation to early branching events. We conclude that, during early lung development, localized sources of FGF10 in the mesoderm regulate endoderm proliferation and bud outgrowth.

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A Wearable Photobiomodulation Patch Using a Flexible Red-Wavelength OLED and Its In Vitro Differential Cell Proliferation Effects

Advanced Materials Technologies ◽

10.1002/admt.201700391 ◽

2018 ◽

Vol 3 (5) ◽

pp. 1700391 ◽

Cited By ~ 21

Author(s):

Yongmin Jeon ◽

Hye-Ryung Choi ◽

Myungsub Lim ◽

Seungyeop Choi ◽

Hyuncheol Kim ◽

...

Keyword(s):

Cell Proliferation ◽

Differential Cell

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Rac1 modulates mammalian lung branching morphogenesis in part through canonical Wnt signaling

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00274.2016 ◽

2016 ◽

Vol 311 (6) ◽

pp. L1036-L1049 ◽

Cited By ~ 9

Author(s):

Soula Danopoulos ◽

Michael Krainock ◽

Omar Toubat ◽

Matthew Thornton ◽

Brendan Grubbs ◽

...

Keyword(s):

Cell Proliferation ◽

Wnt Signaling ◽

Branching Morphogenesis ◽

Mouse Lung ◽

Canonical Wnt Signaling ◽

Embryonic Mouse ◽

Cell Proliferation And Differentiation ◽

Proliferation And Differentiation ◽

Canonical Wnt ◽

Lung Branching

Lung branching morphogenesis relies on a number of factors, including proper epithelial cell proliferation and differentiation, cell polarity, and migration. Rac1, a small Rho GTPase, orchestrates a number of these cellular processes, including cell proliferation and differentiation, cellular alignment, and polarization. Furthermore, Rac1 modulates both noncanonical and canonical Wnt signaling, important pathways in lung branching morphogenesis. Culture of embryonic mouse lung explants in the presence of the Rac1 inhibitor (NSC23766) resulted in a dose-dependent decrease in branching. Increased cell death and BrdU uptake were notably seen in the mesenchyme, while no direct effect on the epithelium was observed. Moreover, vasculogenesis was impaired following Rac1 inhibition as shown by decreased Vegfa expression and impaired LacZ staining in Flk1-Lacz reporter mice. Rac1 inhibition decreased Fgf10 expression in conjunction with many of its associated factors. Moreover, using the reporter lines TOPGAL and Axin2-LacZ, there was an evident decrease in canonical Wnt signaling in the explants treated with the Rac1 inhibitor. Activation of canonical Wnt pathway using WNT3a or WNT7b only partially rescued the branching inhibition. Moreover, these results were validated on human explants, where Rac1 inhibition resulted in impaired branching and decreased AXIN2 and FGFR2b expression. We therefore conclude that Rac1 regulates lung branching morphogenesis, in part through canonical Wnt signaling. However, the exact mechanisms by which Rac1 interacts with canonical Wnt in human and mouse lung requires further investigation.

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Long Noncoding RNA SNHG4 Remits Lipopolysaccharide‐Engendered Inflammatory Lung Damage by Inhibiting METTL3 - Mediated m6A Level of STAT2 mRNA

10.21203/rs.3.rs-573716/v1 ◽

2021 ◽

Author(s):

Si-Xiu Li ◽

Wen Yan ◽

Jian-Ping Liu ◽

Yu-Juan Zhao ◽

Lu Chen

Keyword(s):

Cell Proliferation ◽

Noncoding Rna ◽

Lung Damage ◽

Lung Fibroblasts ◽

Mouse Lung ◽

Treated Cell ◽

Cell Functions ◽

Tnf Α

Abstract Background: Emerging evidence suggests that long non coding RNA (lncRNA) small nucleolar RNA host gene 4 (SNHG4) has become a new insight into lipopolysaccharide (LPS) - induced microglia inflammation, its role in neonatal pneumonia (NP) remains to be largely unrevealed.Methods: RT-qPCR was used to determine SNHG4 and METTL3 expression in the serum from NP patients and normal volunteers, as well as in WI-38 cells treated with LPS. The SNHG4 overexpression vector (pcDNA-SNHG4) was transfected into LPS - treated cells. CCK-8, Transwell, annexin V-FITC/PI and ELISA assays were used to determine cell proliferation, migration, apoptosis and contents of IL-6, TNF-α, SOD and MDA, respectively. The level of SNHG4 in the promoter region of METTL3 was assessed with RIP assay. m6A quantitative analysis illustrated the m6A level with or without SNHG4 overexpression or METTL3 silencing. Bioinformatics analysis and RIP-PCR were used to predict and validate YTHDF1 - mediated m6A levels on signal transducer and activator of transcription 2 (STAT2) mRNA in METTL3 inhibited cells. Then rescue experiments were performed to explore effects of SNHG4 and METTL3 or STAT2 on LPS-treated cell functions. Subsequently, in vivo functional experiments were performed to investigate the role of SNHG4 in LPS induced pneumonia in mice. Results: SNHG4 was downregulated and METTL3 was upregulated in NP patients and LPS-treated cells. SNHG4 overexpression facilitated cell proliferation, migration and SOD concentration, and inhibited apoptosis and IL-6, TNF-α and MDA contents. Mechanistically, SNHG4 bound with METTL3 and downregulated METTL3 expression. Besides, total m6A modification level was lower in the SNHG4 overexpressed or METTL3 inhibited cells. METTL3 interference reduced m6A levels of STAT2 mRNA, decreased STAT2 mRNA stability and promoted STAT2 translation level. METTL3 or STAT2 upregulated reversed the effects of SNHG4 overexpression on LPS - treated cell functions. Conclusions: This study reveals that SNHG4 promotes LPS induced inflammation in human lung fibroblasts and mouse lung tissues in vitro and in vivo by inhibiting METTL3 - mediated m6A level of STAT2 mRNA, which may provide a potential therapeutic mechanism for NP.

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YAP is essential for mechanical force production and epithelial cell proliferation during lung branching morphogenesis

eLife ◽

10.7554/elife.21130 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 47

Author(s):

Chuwen Lin ◽

Erica Yao ◽

Kuan Zhang ◽

Xuan Jiang ◽

Stacey Croll ◽

...

Keyword(s):

Cell Proliferation ◽

Epithelial Cell ◽

Force Production ◽

Genomic Analysis ◽

Branching Morphogenesis ◽

Cell Number ◽

Mechanical Force ◽

Hippo Signaling ◽

Lung Epithelium ◽

Direct Induction

Branching morphogenesis is a fundamental program for tissue patterning. We show that active YAP, a key mediator of Hippo signaling, is distributed throughout the murine lung epithelium and loss of epithelial YAP severely disrupts branching. Failure to branch is restricted to regions where YAP activity is removed. This suggests that YAP controls local epithelial cell properties. In support of this model, mechanical force production is compromised and cell proliferation is reduced in Yap mutant lungs. We propose that defective force generation and insufficient epithelial cell number underlie the branching defects. Through genomic analysis, we also uncovered a feedback control of pMLC levels, which is critical for mechanical force production, likely through the direct induction of multiple regulators by YAP. Our work provides a molecular pathway that could control epithelial cell properties required for proper morphogenetic movement and pattern formation.

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Amphiregulin in lung branching morphogenesis: interaction with heparan sulfate proteoglycan modulates cell proliferation

Development ◽

10.1242/dev.122.6.1759 ◽

1996 ◽

Vol 122 (6) ◽

pp. 1759-1767 ◽

Cited By ~ 1

Author(s):

L. Schuger ◽

G.R. Johnson ◽

K. Gilbride ◽

G.D. Plowman ◽

R. Mandel

Keyword(s):

Cell Proliferation ◽

Epithelial Cells ◽

Heparan Sulfate ◽

Branching Morphogenesis ◽

Mesenchymal Cells ◽

Heparan Sulfate Proteoglycan ◽

Sulfate Proteoglycan ◽

Epithelial Proliferation ◽

Differential Cell ◽

Embryonic Lungs

Epithelial and mesenchymal cells isolated from mouse embryonic lungs synthesized and responded to amphiregulin (AR) in a different fashion. Mesenchymal cells produced and deposited 3- to 4-fold more AR than epithelial cells, proliferated in the presence of exogenous AR, and their spontaneous growth was blocked by up to 85% by anti-AR antibodies. In contrast, epithelial cells exhibited a broad response to this growth regulator factor depending on whether they were supplemented with extracellular matrix (ECM) and whether this ECM was of epithelial or mesenchymal origin. AR-treated epithelial cells proliferated by up to 3-fold in the presence of mesenchymal-deposited ECM, remained unchanged in the presence of epithelial-deposited ECM, and decreased in their proliferation rate below controls in the absence of ECM supplementation. This effect was abolished by treatment with the glycosaminoglycan-degrading enzymes heparinase and heparitinase suggesting the specific involvement of heparan sulfate proteoglycan (HSPG) in AR-mediated cell proliferation. In whole lung explants, branching morphogenesis was inhibited by antibodies against the AR heparan sulfate binding site and stimulated by exogenous AR. Since during development, epithelial cells are in contact with mesenchymal ECM at the tips of the growing buds and alongside the basement membrane, focal variations in the proportion of epithelial and mesenchymal HSPG will focally affect epithelial proliferation rates. Therefore, AR-HSPG interaction may underlie the process of branching morphogenesis by inducing differential cell proliferation.

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Role of oxygen and vascular development in epithelial branching morphogenesis of the developing mouse lung

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00185.2004 ◽

2005 ◽

Vol 288 (1) ◽

pp. L167-L178 ◽

Cited By ~ 101

Author(s):

Minke van Tuyl ◽

Jason Liu ◽

Jinxia Wang ◽

Maciek Kuliszewski ◽

Dick Tibboel ◽

...

Keyword(s):

Molecular Mechanisms ◽

Vascular Tissue ◽

Vascular Development ◽

Branching Morphogenesis ◽

Mouse Lung ◽

Vegf Receptor ◽

Lacz Gene ◽

Low Oxygen ◽

Pulmonary Vascular Development

Recent investigations have suggested an active role for endothelial cells in organ development, including the lung. Herein, we investigated some of the molecular mechanisms underlying normal pulmonary vascular development and their influence on epithelial branching morphogenesis. Because the lung in utero develops in a relative hypoxic environment, we first investigated the influence of low oxygen on epithelial and vascular branching morphogenesis. Two transgenic mouse models, the C101-LacZ (epithelial-LacZ marker) and the Tie2-LacZ (endothelial-LacZ marker), were used. At embryonic day 11.5, primitive lung buds were dissected and cultured at either 20 or 3% oxygen. At 24-h intervals, epithelial and endothelial LacZ gene expression was visualized by X-galactosidase staining. The rate of branching of both tissue elements was increased in explants cultured at 3% oxygen compared with 20% oxygen. Low oxygen increased expression of VEGF, but not that of the VEGF receptor (Flk-1). Expression of two crucial epithelial branching factors, fibroblast growth factor-10 and bone morphogenetic protein-4, were not affected by low oxygen. Epithelial differentiation was maintained at low oxygen as shown by surfactant protein C in situ hybridization. To explore epithelial-vascular interactions, we inhibited vascular development with antisense oligonucleotides targeted against either hypoxia inducible factor-1α or VEGF. Epithelial branching morphogenesis in vitro was dramatically abrogated when pulmonary vascular development was inhibited. Collectively, the in vitro data show that a low-oxygen environment enhances branching of both distal lung epithelium and vascular tissue and that pulmonary vascular development appears to be rate limiting for epithelial branching morphogenesis.

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