FGF signaling dynamics regulates epithelial patterning and morphogenesis

Abstract

SummarySingle cell assays revealed that growth factor signaling dynamics is actively sensed by a cell and ultimately controls cell fate. However, the effects of growth factor signaling dynamics at the tissue level have been unknown. We used mammary epithelial organoids, time-lapse imaging, fibroblast growth factor 2 (FGF2) variants of different stabilities, mathematical modeling, and perturbation analysis to study the role of FGF2 signaling dynamics in epithelial morphogenesis. We found that fluctuant and sustained FGF signaling dynamics induced distinct morphological and functional states of mammary epithelium through differential employment of intracellular effectors ERK and AKT. ERK activity domains determined epithelial branch size, while AKT activity drove epithelial stratification. Furthermore, FGF signaling dynamics affected epithelial tissue mechanoresponsiveness to extracellular matrix, thereby impinging upon branch elongation. Our study provides new insights into regulation of epithelial patterning and branching morphogenesis by FGF signaling dynamics and into downstream signaling effectors that regulate cellular outcomes.

Fibroblast-induced mammary epithelial branching depends on fibroblast contractility

“…The classic mammary organoid model cultured in Matrigel with FGF2 [ 21 ] mimics epithelial stratification to some extent (reaching 3–4 layers of luminal cells in TEB-like ends of the branches) but does not support full myoepithelial coverage of branches [ 31 ]. We revealed that a stabilized form of FGF2 (FGF2-STAB; [ 32 , 33 ]) induces several TEB-like features in the organoids, including highly proliferative phenotype, multiple layers of luminal cells, and full myoepithelial cell coverage [ 34 ]. When we exposed the dispersed organoid-fibroblast co-cultures to FGF2-STAB, the organoids developed large branches with a set of features typical of TEBs in vivo, including stratified luminal cells, full myoepithelial coverage, and presence of basal-in-luminal cells (similar to cap-in-body cells in vivo [ 4 ]) ( Fig 5A–5G ).…”

A Mammary Organoid Model to Study Branching Morphogenesis

“…The need of a timed addition of growth factor supplementation to obtain proper elongation and branching events suggests that the signaling events regulating branching morphogenesis may work in waves of activation of downstream signaling pathways. Indeed, such models have been previously proposed in other organs such as the lung, the submandibular gland, and in mammary acini and spherical organoids ( Nitta et al, 2009 ; Ender et al, 2020 ; Rabata et al, 2020 ; Sumbal et al, 2020 ; Gagliardi et al, 2021 ). Further experiments using refined models for branching morphogenesis, including branched mammary organoid models, will be necessary to fully dissect the signaling events driving branching, bifurcation, and elongation in the mammary gland.…”

“…Although multiple organoid models derived from different organs have achieved high levels of complexity and morphological reproducibility of in vivo structures, the complexity of murine mammary organoids is still limited ( Clevers, 2016 ; Kim et al, 2021 ; Vazquez-Armendariz and Herold, 2021 ). Current mammary organoid culture protocols result in either bi-layered sphere-shaped organoids (referred to as cysts) or budding structures upon supplementation with different growth factors, such as FGF2, FGF7, FGF10, and Neuregulin1 ( Fata et al, 2007 ; Nguyen-Ngoc et al, 2015 ; Jardé et al, 2016 ; Sumbal et al, 2020 ). Organoid culture methods using Matrigel with culture medium supplemented with FGF2 have provided valuable insights into the differentiation potential of mammary organoids ( Jamieson et al, 2016 ).…”