Budding epithelial morphogenesis driven by cell-matrix versus cell-cell adhesion

Wang, Shaohe; Matsumoto, Kazue; Lish, Samantha R.; Cartagena‐Rivera, Alexander X.; Yamada, Kenneth M.

doi:10.1016/j.cell.2021.05.015

Cited by 90 publications

(100 citation statements)

References 61 publications

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“…Additional environmental signals are likely required to support the further differentiation of mSG-PAC1 cells. During development, both morphogenesis and differentiation of the salivary endothelial networks 53,54 . Signals from the mesenchyme have recently been shown to promote acinar differentiation 19 .…”

Section: Discussionmentioning

confidence: 99%

Regulation of Myoepithelial Differentiation in 3-Dimensional Culture

Varney

Larsen

et al. 2021

Preprint

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Myoepithelial cells serve as contractile units in exocrine glands, including mammary, salivary, and lacrimal glands, and their dysfunction negatively impacts secretory function. In spite of their importance, mechanisms that regulate myoepithelial differentiation are poorly defined. To study this process, we employed an established murine salivary gland epithelial cell line, mSG-PAC-1, that we previously demonstrated recapitulates aspects of acinar cell differentiation when cultured as spheroids. Here, we report that mSG-PAC1 spheroids can be induced towards a myoepithelial phenotype by manipulating culture conditions, resulting in the inhibition of expression of the acinar marker, AQP5, the upregulation of myoepithelial markers alpha-SMA, calponin, and the integrin beta4 subunit, as well as the acquisition of myoepithelial morphology. Transcriptome analysis indicated that YAP/TAZ target genes are also upregulated. We demonstrate that the transcriptional co-activator TAZ is expressed by the same cells that express alpha-SMA in the epithelial compartment of the differentiating murine salivary gland and by mSG-PAC1 cells under conditions that promote a myoepithelial phenotype. Furthermore, siRNA targeting TAZ expression in mSG-PAC1 spheroids diminishes expression of myoepithelial markers, implicating TAZ in their regulation. Thus, we have demonstrated mSG-PAC1 cells are a reliable tool to complement in vivo approaches to understand mechanisms that promote myoepithelial differentiation.

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Section: Discussionmentioning

confidence: 99%

Regulation of Myoepithelial Differentiation in 3-Dimensional Culture

Varney

Larsen

et al. 2021

Preprint

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“…An extra G was added to the 5'-end if the sequence did not begin with G to facilitate transcription by the U6 promoter. The parental lentiviral vector (pW212; Addgene, 170810) co-expressing sgRNA with an red nuclear fluorescent reporter (NLS-mScarlet-I) and a blasticidin resistance gene was previously generated by our group (Wang et al 2021). To make the parental vector (pW299; Addgene, TBD) with a blue nuclear fluorescent reporter (NLS-tagBFP), mScarlet-I in pW212 was replaced with tagBFP using Gibson Assembly (Gibson et al 2009).…”

Section: Sgrna Designmentioning

confidence: 99%

“…Lentivirus packaging was performed as previously described (Wang et al 2021). Briefly, lenti-sgRNA plasmids were co-transfected with psPAX2 (Addgene, 12260) and pMD2.G (Addgene, 12259) into HEK293T cells by calcium co-precipitation to produce infectious lentiviral particles.…”

Section: Lentivirus Packagingmentioning

confidence: 99%

“…On the other hand, the ex vivo organotypic culture of mouse embryonic salivary glands enabled characterizations and manipulations that are only possible outside embryos (Borghese 1950). For example, live imaging of ex vivo cultured salivary glands revealed extensive dynamics of epithelial cells and the basement membrane matrix, which catalyzed the discovery that a combination of strong cell-matrix and weak cell-cell adhesions in surface epithelial cells drives branching morphogenesis of salivary glands (Larsen et al 2006; J.C. Hsu et al 2013; Harunaga et al 2014; Wang et al 2021). Furthermore, the mesenchyme encapsulating the salivary epithelium can be surgically removed to allow mesenchyme-free culture of the salivary epithelium in the presence of basement membrane extract and certain growth factors (Nogawa and Takahashi 1991).…”

Section: Introductionmentioning

confidence: 99%

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Efficient Gene Knockout in Salivary Gland Epithelial Explant Cultures

Sekiguchi

Mehlferber

Matsumoto

et al. 2022

Preprint

Self Cite

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We have developed methods to achieve efficient CRISPR-Cas9 mediated gene knockout in ex vivo mouse embryonic salivary epithelial organoids. Salivary epithelial organoids provide a valuable model for characterizing cell signaling, differentiation, and epithelial morphogenesis, but research has been limited by a paucity of efficient gene perturbation methods. Here, we demonstrate highly efficient gene perturbation by transient transduction of guide RNA-expressing lentiviruses into Cas9-expressing salivary epithelial buds isolated from Cas9 transgenic mice. We first show that salivary epithelial buds can be cultured in low-concentration, non-solidified Matrigel suspensions in 96-well plates, which greatly increases sample throughput compared to conventional cultures embedded in solidified Matrigel. We then describe an efficient workflow to produce experiment-ready, high-titer lentiviruses within 1 week after molecular cloning. To track transduced cells, we designed the lentiviral vector to co-express a nuclear fluorescent reporter with the guide RNA. We routinely achieve 50-80% transduction efficiency as indicated by the fluorescent reporter. Importantly, we detected robust loss of targeted protein products when testing 8 guide RNAs for 3 different genes. Moreover, targeting the β1 integrin gene (Itgb1) inhibited branching morphogenesis, which supports the importance of cell-matrix adhesion in driving branching morphogenesis. In summary, we have established a lentivirus-based method that can efficiently perturb genes of interest in salivary epithelial organoids, which will greatly facilitate studies of specific gene functions using this system.

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“…Moreover, downregulation of E-cadherin, together with increased cell-matrix interactions, is essential for budding epithelial morphogenesis. In this process, cells expressing low levels of E-cadherin engage with the basement membrane rather than with other cells, thus allowing epithelia to protrude and develop [ 56 ]. AJs remodeling occurs also in pathogenic processes, such as cancer dissemination.…”

Section: Cell-to-cell Contacts Adherens Junctions and Emtmentioning

confidence: 99%

The Autophagic Route of E-Cadherin and Cell Adhesion Molecules in Cancer Progression

Santarosa

Maestro

2021

Cancers

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Cell-to-cell adhesion is a key element in epithelial tissue integrity and homeostasis during embryogenesis, response to damage, and differentiation. Loss of cell adhesion and gain of mesenchymal features, a phenomenon known as epithelial to mesenchymal transition (EMT), are essential steps in cancer progression. Interestingly, downregulation or degradation by endocytosis of epithelial adhesion molecules (e.g., E-cadherin) associates with EMT and promotes cell migration. Autophagy is a physiological intracellular degradation and recycling process. In cancer, it is thought to exert a tumor suppressive role in the early phases of cell transformation but, once cells have gained a fully transformed phenotype, autophagy may fuel malignant progression by promoting EMT and conferring drug resistance. In this review, we discuss the crosstalk between autophagy, EMT, and turnover of epithelial cell adhesion molecules, with particular attention to E-cadherin.

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Budding epithelial morphogenesis driven by cell-matrix versus cell-cell adhesion

Cited by 90 publications

References 61 publications

Regulation of Myoepithelial Differentiation in 3-Dimensional Culture

Regulation of Myoepithelial Differentiation in 3-Dimensional Culture

Efficient Gene Knockout in Salivary Gland Epithelial Explant Cultures

The Autophagic Route of E-Cadherin and Cell Adhesion Molecules in Cancer Progression

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