isolation-myoepithelial-cells-from-adult-murine-lacrimal

Isolation of Myoepithelial Cells from Adult Murine Lacrimal and Submandibular Glands

The lacrimal gland (LG) is an exocrine tubuloacinar gland that secretes an aqueous layer of tear film. The LG epithelial tree is comprised of acinar, ...

The Scripps Research Institute

The developmental activities of morphogens depend on the gradients that they form in the extracellular matrix. Here, we show that differences in the binding of fibroblast growth factor 7 (FGF7) and FGF10 to heparan sulfate (HS) underlie the formation of different gradients that dictate distinct activities during branching morphogenesis. Reducing the binding affinity of FGF10 for HS by mutating a single residue in its HS-binding pocket converted FGF10 into a functional mimic of FGF7 with respect to gradient formation and regulation of branching morphogenesis. In particular, the mutant form of FGF10 caused lacrimal and salivary gland epithelium buds to branch rather than to elongate. In contrast, mutations that reduced the affinity of the FGF10 for its receptor affected the extent, but not the nature, of the response. Our data may provide a general model for understanding how binding to HS regulates other morphogenetic gradients.

Differential interactions of FGFs with heparan sulfate control gradient formation and branching morphogenesis.

Canonical Wnts promote myoblast differentiation; however, the role of β-catenin in adult myogenesis has been contentious, and its mechanism(s) unclear. Using CRISPR-generated β-catenin-null primary adult mouse myoblasts, we found that β-catenin was essential for morphological differentiation and timely deployment of the myogenic gene program. Alignment, elongation and fusion were grossly impaired in null cells, and myogenic gene expression was not coordinated with cytoskeletal and membrane remodeling events. Rescue studies and genome-wide analyses extended previous findings that a β-catenin-TCF/LEF interaction is not required for differentiation, and that β-catenin enhances MyoD binding to myogenic loci. We mapped cellular pathways controlled by β-catenin and defined novel targets in myoblasts, including the fusogenic genes myomaker and myomixer. We also showed that interaction of β-catenin with α-catenin was important for efficient differentiation. Overall the study suggests dual roles for β-catenin: a TCF/LEF-independent nuclear function that coordinates an extensive network of myogenic genes in cooperation with MyoD; and an α-catenin-dependent membrane function that helps control cell-cell interactions. β-Catenin-TCF/LEF complexes may function primarily in feedback regulation to control levels of β-catenin and thus prevent precocious/excessive myoblast fusion.

β-Catenin is essential for differentiation of primary myoblasts via cooperation with MyoD and α-catenin.

We present a protocol for isolation of putative epithelial progenitor cells from mouse lacrimal gland (LG) by fluorescence-activated cell sorting (FACS). Isolated LG epithelial progenitor cells can be cultured as 3D reaggregates within extracellular matrix gel or plated as a monolayer. 3D cultures could be maintained for several days and then dissociated with trypsin and plated as monolayer cultures, processed for analysis (e.g., mRNA/protein expression) and/or used for transplantations. Our goal is to provide researchers with a method that can be used as is or modified if isolation of other LG epithelial cell types is required.

Helen Makarenkova

Department of Molecular Medicine

The Scripps Research Institute

Isolation of Myoepithelial Cells from Adult Murine Lacrimal and Submandibular Glands

Helen Makarenkova

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Molecular Medicine

The Scripps Research Institute

Isolation of Myoepithelial Cells from Adult Murine Lacrimal and Submandibular Glands

Department of Molecular Medicine