Mammary gland development occurs mainly after birth and is composed of three successive stages: puberty, pregnancy and lactation, and involution. These developmental stages are associated with major tissue remodeling, including extensive changes in mammary epithelium as well as surrounding stroma. Three-dimensional (3D) mammary organoid culture has become an important tool in mammary gland biology and enabled invaluable discoveries on pubertal mammary branching morphogenesis and breast cancer. However, a suitable 3D organoid model recapitulating key aspects of lactation and involution has been missing. Here, we describe a robust and straightforward mouse mammary organoid system modeling lactation and involution-like process, which can be applied to study mechanisms of physiological mammary gland lactation and involution as well as pregnancy-associated breast cancer.

FGF signaling is essential for mammary gland development, yet the mechanisms by which different members of the FGF family control stem cell function and epithelial morphogenesis in this tissue are not well understood. Here, we have examined the requirement of Fgfr2 in mouse mammary gland morphogenesis using a postnatal organ regeneration model. We found that tissue regeneration from basal stem cells is a multistep event, including luminal differentiation and subsequent epithelial branching morphogenesis. Basal cells lacking Fgfr2 did not generate an epithelial network owing to a failure in luminal differentiation. Moreover, Fgfr2 null epithelium was unable to undergo ductal branch initiation and elongation due to a deficiency in directional migration. We identified FGF10 and FGF2 as stromal ligands that control distinct aspects of mammary ductal branching. FGF10 regulates branch initiation, which depends on directional epithelial migration. By contrast, FGF2 controls ductal elongation, requiring cell proliferation and epithelial expansion. Together, our data highlight a pleiotropic role of Fgfr2 in stem cell differentiation and branch initiation, and reveal that different FGF ligands regulate distinct aspects of epithelial behavior.

Fibroblast growth factor 2 (FGF2) plays important roles in tissue development and repair. Using heparan sulfates (HS)/heparin as a cofactor, FGF2 binds to FGF receptor (FGFR) and induces downstream signaling pathways, such as ERK pathway, that regulate cellular behavior. In most cell lines, FGF2 signaling displays biphasic dose-response profile, reaching maximal response to intermediate concentrations, but weak response to high levels of FGF2. Recent reports demonstrated that the biphasic cellular response results from competition between binding of FGF2 to HS and FGFR that impinge upon ERK signaling dynamics. However, the role of HS/heparin in FGF signaling has been controversial. Several studies suggested that heparin is not required for FGF-FGFR complex formation and that the main role of heparin is to protect FGF from degradation. In this study, we investigated the relationship between FGF2 stability, heparin dependence and ERK signaling dynamics using FGF2 variants with increased thermal stability (FGF2-STABs). FGF2-STABs showed higher efficiency in induction of FGFR-mediated proliferation, lower affinity to heparin and were less dependent on heparin than wild-type FGF2 (FGF2-wt) for induction of FGFR-mediated mitogenic response. Interestingly, in primary mammary fibroblasts, FGF2-wt displayed a sigmoidal dose-response profile, while FGF2-STABs showed a biphasic response. Moreover, at low concentrations, FGF2-STABs induced ERK signaling more potently and displayed a faster dynamics of full ERK activation and higher amplitudes of ERK signaling than FGF2-wt. Our results suggest that FGF2 stability and heparin dependence are important factors in FGF-FGFR signaling complex assembly and ERK signaling dynamics.

Fibroblast growth factor (FGF) signaling is crucial for mammary gland development. While multiple roles for FGF signaling in the epithelium were described, the function of FGF signaling in mammary stroma has not been elucidated. In this study, we investigated FGF signaling in mammary fibroblasts. We found that mammary fibroblasts express FGF receptors FGFR1 and FGFR2 and respond to FGF ligands. In particular, FGF2 and FGF9 induce sustained ERK1/2 signaling and promote fibroblast proliferation and migration in 2D. Intriguingly, only FGF2 induces fibroblast migration in 3D extracellular matrix (ECM) through regulation of actomyosin cytoskeleton and promotes force-mediated collagen remodeling by mammary fibroblasts. Moreover, FGF2 regulates production of ECM proteins by mammary fibroblasts, including collagens, fibronectin, osteopontin, and matrix metalloproteinases. Finally, using organotypic 3D co-cultures we show that FGF2 and FGF9 signaling in mammary fibroblasts enhances fibroblast-induced branching of mammary epithelium by modulating paracrine signaling and that knockdown of Fgfr1 and Fgfr2 in mammary fibroblasts reduces branching of mammary epithelium. Our results demonstrate a pleiotropic role for FGF signaling in mammary fibroblasts with implications for regulation of mammary stromal functions and epithelial branching morphogenesis.

Significance: Despite its importance in epithelial development and in breast cancer, the role of EGF receptor (EGFR) signaling and its regulation in the postnatal mammary stroma has remained unclear. Here, we show that sprouty 1 ( Spry1 ) modulates epithelial–stromal interactions by inhibiting EGFR-dependent stromal paracrine signaling and ECM remodeling. Spry1 loss leads to upregulated EGFR signaling and enhanced stromal activities, including ECM remodeling and growth factor production, and ultimately increased epithelial branching. By contrast, EGFR signaling reduction in fibroblasts leads to stunted epithelial branching. We show that EGFR and FGF receptor (FGFR) signaling constitute a critical part of stromal–epithelial interactions during postnatal development and that these signaling pathways are fine-tuned by different members of the Sprouty gene family to ensure normal epithelial morphogenesis of the mammary gland.

FGF signaling plays an essential role in lung development, homeostasis, and regeneration. We employed mouse 3D cell culture models and imaging to study ex vivo the role of FGF ligands and the interplay of FGF signaling with epithelial growth factor (EGF) and WNT signaling pathways in lung epithelial morphogenesis and differentiation. In non-adherent conditions, FGF signaling promoted formation of lungospheres from lung epithelial stem/progenitor cells (LSPCs). Ultrastructural and immunohistochemical analyses showed that LSPCs produced more differentiated lung cell progeny. In a 3D extracellular matrix, FGF2, FGF7, FGF9, and FGF10 promoted lung organoid formation. FGF9 showed reduced capacity to promote lung organoid formation, suggesting that FGF9 has a reduced ability to sustain LSPC survival and/or initial divisions. FGF7 and FGF10 produced bigger organoids and induced organoid branching with higher frequency than FGF2 or FGF9. Higher FGF concentration and/or the use of FGF2 with increased stability and affinity to FGF receptors both increased lung organoid and lungosphere formation efficiency, respectively, suggesting that the level of FGF signaling is a crucial driver of LSPC survival and differentiation, and also lung epithelial morphogenesis. EGF signaling played a supportive but non-essential role in FGF-induced lung organoid formation. Analysis of tissue architecture and cell type composition confirmed that the lung organoids contained alveolar-like regions with cells expressing alveolar type I and type II cell markers, as well as airway-like structures with club cells and ciliated cells. FGF ligands showed differences in promoting distinct lung epithelial cell types. FGF9 was a potent inducer of more proximal cell types, including ciliated and basal cells. FGF7 and FGF10 directed the differentiation toward distal lung lineages. WNT signaling enhanced the efficiency of lung organoid formation, but in the absence of FGF10 signaling, the organoids displayed limited branching and less differentiated phenotype. In summary, we present lung 3D cell culture models as useful tools to study the role and interplay of signaling pathways in postnatal lung development and homeostasis, and we reveal distinct roles for FGF ligands in regulation of mouse lung morphogenesis and differentiation ex vivo.