genetic-engineering-primary-mouse-intestinal-organoids-using-magnetic

Genetic Engineering of Primary Mouse Intestinal Organoids Using Magnetic Nanoparticle Transduction Viral Vectors for Frozen Sectioning

Intestinal organoid cultures provide a unique opportunity to investigate intestinal stem cell and crypt biology in vitro, although efficient approaches to ...

3d-analysis-of-multi-cellular-responses-to-chemoattractant-gradients

3D Analysis of Multi-cellular Responses to Chemoattractant Gradients

Various limitations of 2D cell culture systems have sparked interest in 3D cell culture and analysis platforms, which would better mimic the spatial and ...

Johns Hopkins University School of Medicine

Johns Hopkins University

The mammary epithelium elaborates through hormonally regulated changes in proliferation, migration and differentiation. Non-muscle myosin II (NMII) functions at the interface between contractility, adhesion and signal transduction. It is therefore a plausible regulator of mammary morphogenesis. We tested the genetic requirement for NMIIA and NMIIB in mammary morphogenesis through deletion of the three NMII heavy chain-encoding genes (,  and ; also known as ,  and , respectively) that confer specificity to the complex. Surprisingly, mosaic loss, but not ubiquitous loss, of  and  induced high levels of proliferation in 3D culture. This phenotype was observed even when cells were cultured in basal medium, which does not support tissue level growth of wild-type epithelium. Mosaic loss of NMIIA and NMIIB combined with FGF signaling to induce hyperplasia. Mosaic analysis revealed that the cells that were null for both NMIIA and NMIIB, as well as wild-type cells, proliferated, indicating that the regulation of proliferation is both cell autonomous and non-autonomous within epithelial tissues. This phenotype appears to be mediated by cell-cell contact, as co-culture did not induce proliferation. Mosaic loss of NMIIA and NMIIB also induced excess proliferation  Our data therefore reveal a role for NMIIA and NMIIB as negative regulators of proliferation in the mammary epithelium.

Mosaic loss of non-muscle myosin IIA and IIB is sufficient to induce mammary epithelial proliferation.

We sought to understand how cells collectively elongate epithelial tubes. We first used 3D culture and biosensor imaging to demonstrate that epithelial cells enrich Ras activity, phosphatidylinositol (3,4,5)-trisphosphate (PIP), and F-actin to their leading edges during migration within tissues. PIP enrichment coincided with, and could enrich despite inhibition of, F-actin dynamics, revealing a conserved migratory logic compared with single cells. We discovered that migratory cells can intercalate into the basal tissue surface and contribute to tube elongation. We then connected molecular activities to subcellular mechanics using force inference analysis. Migration and transient intercalation required specific and similar anterior-posterior ratios of interfacial tension. Permanent intercalations were distinguished by their capture at the boundary through time-varying tension dynamics. Finally, we integrated our experimental and computational data to generate a finite element model of tube elongation. Our model revealed that intercalation, interfacial tension dynamics, and high basal stress are together sufficient for mammary morphogenesis.

Coordination of Receptor Tyrosine Kinase Signaling and Interfacial Tension Dynamics Drives Radial Intercalation and Tube Elongation.

The mammary epithelium is composed of an inner luminal and surrounding myoepithelial cell layer. The presence of cancer cells beyond the myoepithelium defines invasive breast cancer, yet the role of the myoepithelium during invasion remains unclear. We developed a 3D organotypic culture assay to model this process through lineage-specific expression of the prometastatic transcription factor  We sought to distinguish the functional role of the myoepithelium in regulating invasion and local dissemination. Myoepithelial-specific  expression induced cell-autonomous myoepithelial cell escape. Remarkably, luminal-specific  expression was rarely sufficient for escape. Time-lapse microscopy revealed that myoepithelial cells collectively restrain and reinternalize invading Twist1 luminal cells. Barrier function correlated with myoepithelial abundance and required the expression of α-smooth muscle actin and P-cadherin. We next demonstrated that myoepithelial cells can restrain and recapture invasive cancer cells. Our data establish the concept of the myoepithelium as a dynamic barrier to luminal dissemination and implicate both smooth muscle contractility and intercellular adhesion in barrier function.

ArticleTotal : 2	Year
Publication title Cited by 12	2019
Publication title Cited by 4	2019

Article	Year
Mosaic loss of non-muscle myosin IIA and IIB is sufficient to induce mammary epithelial proliferation. Journal of cell science\| PubMed ID: 28821574	2017
Coordination of Receptor Tyrosine Kinase Signaling and Interfacial Tension Dynamics Drives Radial Intercalation and Tube Elongation. Developmental cell\| PubMed ID: 29634937	2018
Myoepithelial cells are a dynamic barrier to epithelial dissemination. The Journal of cell biology\| PubMed ID: 30061105	2018

Andrew J. Ewald

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