The mammary gland consists of numerous tissue compartments, including mammary epithelium, an array of stromal cells, and the extracellular matrix (ECM). Bidirectional interactions between the epithelium and its surrounding stroma are essential for proper mammary gland development and homeostasis, whereas their deregulation leads to developmental abnormalities and cancer. To study the relationships between the epithelium and the stroma, development of models that could recapitulate essential aspects of these interacting systems in vitro has become necessary. Here we describe a three-dimensional (3D) co-culture assay and show that the addition of fibroblasts to mammary organoid cultures promotes the epithelium to undergo branching morphogenesis, thus allowing the role of the stromal microenvironment to be examined in this essential developmental process.
- MeSH
- Cellular Microenvironment physiology MeSH
- Stromal Cells cytology MeSH
- Epithelium physiology MeSH
- Epithelial Cells cytology MeSH
- Extracellular Matrix physiology MeSH
- Fibroblasts cytology MeSH
- Coculture Techniques methods MeSH
- Cells, Cultured MeSH
- Mammary Glands, Animal cytology MeSH
- Morphogenesis physiology MeSH
- Mice MeSH
- Signal Transduction physiology MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
Angiogenesis is the process of new blood vessels growing from existing vasculature. Visualizing them as a three-dimensional (3D) model is a challenging, yet relevant, task as it would be of great help to researchers, pathologists, and medical doctors. A branching analysis on the 3D model would further facilitate research and diagnostic purposes. In this paper, a pipeline of vision algorithms is elaborated to visualize and analyze blood vessels in 3D from formalin-fixed paraffin-embedded (FFPE) granulation tissue sections with two different staining methods. First, a U-net neural network is used to segment blood vessels from the tissues. Second, image registration is used to align the consecutive images. Coarse registration using an image-intensity optimization technique, followed by finetuning using a neural network based on Spatial Transformers, results in an excellent alignment of images. Lastly, the corresponding segmented masks depicting the blood vessels are aligned and interpolated using the results of the image registration, resulting in a visualized 3D model. Additionally, a skeletonization algorithm is used to analyze the branching characteristics of the 3D vascular model. In summary, computer vision and deep learning is used to reconstruct, visualize and analyze a 3D vascular model from a set of parallel tissue samples. Our technique opens innovative perspectives in the pathophysiological understanding of vascular morphogenesis under different pathophysiological conditions and its potential diagnostic role.
Epithelial branching morphogenesis is an essential process in living organisms, through which organ-specific epithelial shapes are created. Interactions between epithelial cells and their stromal microenvironment instruct branching morphogenesis but remain incompletely understood. Here, we employed fibroblast-organoid or fibroblast-spheroid co-culture systems and time-lapse imaging to reveal that physical contact between fibroblasts and epithelial cells and fibroblast contractility are required to induce mammary epithelial branching. Pharmacological inhibition of ROCK or non-muscle myosin II, or fibroblast-specific knock-out of Myh9 abrogate fibroblast-induced epithelial branching. The process of fibroblast-induced branching requires epithelial proliferation and is associated with distinctive epithelial patterning of yes associated protein (YAP) activity along organoid branches, which is dependent on fibroblast contractility. Moreover, we provide evidence for the in vivo existence of contractile fibroblasts specifically surrounding terminal end buds (TEBs) of pubertal murine mammary glands, advocating for an important role of fibroblast contractility in branching in vivo. Together, we identify fibroblast contractility as a novel stromal factor driving mammary epithelial morphogenesis. Our study contributes to comprehensive understanding of overlapping but divergent employment of mechanically active fibroblasts in developmental versus tumorigenic programs.
- MeSH
- Epithelial Cells * metabolism MeSH
- Fibroblasts metabolism MeSH
- Coculture Techniques MeSH
- Mammary Glands, Animal * metabolism MeSH
- Morphogenesis physiology MeSH
- Mice MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
The role of the local microenvironment in influencing cell behavior is central to both normal development and cancer formation. Here, we show that sprouty 1 (SPRY1) modulates the microenvironment to enable proper mammary branching morphogenesis. This process occurs through negative regulation of epidermal growth factor receptor (EGFR) signaling in mammary stroma. Loss of SPRY1 resulted in up-regulation of EGFR-extracellular signal-regulated kinase (ERK) signaling in response to amphiregulin and transforming growth factor alpha stimulation. Consequently, stromal paracrine signaling and ECM remodeling is augmented, leading to increased epithelial branching in the mutant gland. By contrast, down-regulation of EGFR-ERK signaling due to gain of Sprouty function in the stroma led to stunted epithelial branching. Taken together, our results show that modulation of stromal paracrine signaling and ECM remodeling by SPRY1 regulates mammary epithelial morphogenesis during postnatal development.
- MeSH
- Adaptor Proteins, Signal Transducing deficiency metabolism MeSH
- Amphiregulin pharmacology MeSH
- Stromal Cells drug effects metabolism MeSH
- Time-Lapse Imaging MeSH
- Epithelium growth & development metabolism MeSH
- Epithelial Cells cytology drug effects MeSH
- ErbB Receptors metabolism MeSH
- Extracellular Matrix metabolism MeSH
- Extracellular Signal-Regulated MAP Kinases metabolism MeSH
- Fibroblasts drug effects metabolism MeSH
- Phosphoproteins deficiency metabolism MeSH
- Phosphorylation drug effects MeSH
- Collagen metabolism MeSH
- Ligands MeSH
- Membrane Proteins deficiency metabolism MeSH
- Mammary Glands, Animal drug effects metabolism MeSH
- Morphogenesis * drug effects MeSH
- Mutation genetics MeSH
- Mice, Knockout MeSH
- Mice, Nude MeSH
- Paracrine Communication * drug effects MeSH
- Cell Movement drug effects MeSH
- Proto-Oncogene Proteins c-akt metabolism MeSH
- Signal Transduction * drug effects MeSH
- Transforming Growth Factor alpha pharmacology MeSH
- Gene Expression Regulation, Developmental drug effects MeSH
- Animals MeSH
- Check Tag
- Male MeSH
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Research Support, N.I.H., Extramural MeSH
Mammary gland is composed of branched epithelial structure embedded within a complex stroma formed by several stromal cell types, including fibroblasts, and extracellular matrix (ECM). Development of mammary gland is tightly regulated by bidirectional epithelial-stromal interactions that include paracrine signaling, ECM remodeling and mechanosensing. Importantly, these interactions play crucial role in mammary gland homeostasis and when deregulated they contribute to tumorigenesis. Therefore, understanding the mechanisms underlying epithelial-stromal interactions is critical for elucidating regulation of normal mammary gland development and homeostasis and revealing novel strategies for breast cancer therapy. To this end, several three-dimensional (3D) cell culture models have been developed to study these interactions in vitro. In this chapter, a novel 3D organoid-fibrosphere coculture model of mammary gland is described with the capacity for studying not only the qualitative and quantitative aspects of interactions between mammary fibroblasts and epithelial organoids but also their radius and directionality.
- MeSH
- Cell Differentiation MeSH
- Spheroids, Cellular cytology MeSH
- Stromal Cells cytology MeSH
- Epithelial Cells cytology MeSH
- Fibroblasts cytology MeSH
- Coculture Techniques methods MeSH
- Cells, Cultured MeSH
- Mammary Glands, Animal cytology MeSH
- Mice MeSH
- Organoids cytology MeSH
- Paracrine Communication MeSH
- Cell Proliferation MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
Atopie zasahuje do etiopatogeneze profesionálních kontaktních dermatitid ruky (KDR) klinickými projevy (atopickou kožní diatézou – AKDI, atopickým ekzémem ruky – AER) nebo existencí sebe samotné. Kožní atopie přinejmenším zdvojnásobuje účinky iritancií a rizikových vlivů v profesích, u nichž je ER/DR běžným problémem a interferuje kauzálně se vznikem a průběhem profe- sionálních KDR. Nadprůměrně vysoký počet atopiků byl nalezen ve speciálních ambulancích klinik pro kožní nemoce z povolání (kolem 30–40 %), prevalence ER/DR u atopiků je udávána v letech 1970–1994 v rozsahu 14–78 % a atopie u postižených s ER/DR se v letech 1981–1995 pohybovala mezi 27–54 %. AER nejeví žádnou specifickou patognomickou morfu, k zamezení větším diagnostickým problémům je proto nezbytné spojení velké erudice, pečlivého morfologického popisu (včetně minor znaků), topografické analýzy, anamnézy, pracovního i domácího prostředí. Hanifin-Rajkova kritéria jsou nadále bez výhrad uznávána, diagnostické skóre AKDI v erlangenském pojetí využívá jen minor znaky a napomáhá včas preventivně odhalit silentní atopiky. Je snížený práh iritační reaktivity, iritancia vyvolávají snáze iritační dermatitidu ruky (IDR) a spouštějí nebo zhoršují stávající AER. Atopici jsou přinejmenším kontaktně senzibilizovatelní (IV. typ) tak jako neatopici a nasedající alergie zhoršuje prognózou. Mukózní atopie bez kožních atopických projevů není sdružena se zvýšeným rizikem IDR, frekvence ER/DR nebývá vyšší než u neatopiků. Zaměstnanci různých průmyslových odvětví včetně domácích aktivit s anamnézou AE jeví vyšší prevalence ER/DR než neatopici. Mezi silnými prediktory ER/DR byly popsány: přítomnost AE před 15. rokem věku, současný ekzém kdekoliv na kůži a přetrvá- vající suchá/svědivá kůže; k slabším prediktorům náleží ženské pohlaví, AE v rodinné anamnéze a přidružená respirační alergie. Důležitými všeobecnými riziky jsou xeróza, voda, práce ve vlhku, detergenty, organická rozpustidla, minerální oleje, pohonné hmoty, konzervancia, dezinficiencia, řezné a chladící kapaliny a četné další (ne)průmyslové chemikálie. Výrazné diagnosticko-posudkové problémy vykazuje rozlišení kontaktní IDR nasedlé na AER nebo primární IDR u atopika. Kontaktní ER lze přiznat, pokud AE je delší dobu zhojený a jsou-li splněna všechna kritéria profesionality; přiznání akutní ID za stejných podmínek je problematické, nikoliv však při velké míře expozice vyloučené. Sdělení zdůrazňuje potřebu zabývat se soustavně odhalováním atopiků, sledovat při volbě manuálního povolání, pracovním zařazování a domácích aktivitách všechna ekzémová onemocnění rukou, stejně jako věnovat pozornost následné preventivní péči o atopiky v zaměstnání. Závěrem je podán přehled možných příčin etiopatogeneze chronického průběhu a prognózy (kontaktní) ekzémové dermatitidy ruky.
Atopy interferes with the etiopathogenesis of contact dermatitis of the hand (CDH) by clinical manifestations (atopic skin diathesis – ASDI, atopic eczema of the hand – AEH) or by its mere existence. Skin atopy at least doubles the effects of irritants and occupational risk factors in occupational where HE/HD is a common problem and interferes causally with the development and course of occupational CDH. An above average number of atopic subjects was found in special out-patient clinics for occupational skin diseases (some 30–40%), the reported prevalence of HE/HD in atopic subjects was in 1970–1994 14–78% and atopies in subjects with HE/HD in subjects with HE/HD in 1981–1995 27–54%. AEH has no specific pathognomic morphea, however a combination of major knowledge, careful morphological descript (inc. minor signs), topographic analysis, case-history of the working and domestic environment are essential to prevent major diagnostic problems. Hanifin-Rajk ’ s criteria are accepted without reservations, the diagnostic ASDI score in the Erlangen concept uses not only minor signs and helps to discover in time preventively silent atopic subjects. The threshold of irritational reactivity is reduced, irritants produce more easily irritational dermatitis of the hand (IDH) and trigger or cause deterioration of existing AEH. Atopic subjects have at least contact sensitization (IVth type) similary as non-atopic subjects and subsequent allergy causes deterioration of the prognosis. Mucous atopy without atopic skin manifestations is not associated with in increased risk of IDH, frequency of HE/HD is usually not higher than in non-atopic subjects. Employees of various industrial branches incl. domestic activities with a history of AE have a higher prevalence of HE/HD than non-atopic subjects. Strong predictors of HE/HD include the presence of AE before the age of 15 years, the concomitant occurrence of eczema anywhere on the skin and persisting dry/itching skin, weaker predictors include female sex, AE in the family-history and associated respiratory allergies. Important general risks include xerosis, water, work in humid environment, detergents, organic solvents, mineral oils, fuels, preservatives, disinfectants, cutting lubricants, cooling liquids and many other (non)industrial chemical subs- tances. Major diagnostic problems arise from differentiation of contact IDH following AEH or primary IDH atopic subjects. Contact HE can be accepted if AE has been cured for some time and if all criteria of an occupational character are met. Acceptance of acute ID under the same conditions is problematic and can be ruled out unless there is major exposure. The paper emphasizes the necessity of systematic detection of atopic subjects during selection of a manual occupations, working enlistment, domestic activities, and in all eczematous diseases of the hands, similarly as subsequent preventive care of atopic subjects at their workplace. Finally the authors review possible causes of the etiopathogenesis of a chronic course and prognosis of (contact) eczematous dermatitis of the hand.
Fibroblast growth factor (FGF) signaling is crucial for mammary gland development. Although multiple roles for FGF signaling in the epithelium have been 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 murine 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 cultures. 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.
- MeSH
- Fibroblast Growth Factor 2 metabolism MeSH
- Fibroblast Growth Factor 9 metabolism MeSH
- Fibroblasts cytology metabolism MeSH
- MAP Kinase Signaling System * MeSH
- Mammary Glands, Animal cytology embryology MeSH
- Mice, Inbred ICR MeSH
- Mice MeSH
- Paracrine Communication * MeSH
- Receptor, Fibroblast Growth Factor, Type 1 metabolism MeSH
- Receptor, Fibroblast Growth Factor, Type 2 metabolism MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Shoot branching is a primary contributor to plant architecture, evolving independently in flowering plant sporophytes and moss gametophytes. Mechanistic understanding of branching is largely limited to flowering plants such as Arabidopsis, which have a recent evolutionary origin. We show that in gametophytic shoots of Physcomitrella, lateral branches arise by re-specification of epidermal cells into branch initials. A simple model co-ordinating the activity of leafy shoot tips can account for branching patterns, and three known and ancient hormonal regulators of sporophytic branching interact to generate the branching pattern- auxin, cytokinin and strigolactone. The mode of auxin transport required in branch patterning is a key divergence point from known sporophytic pathways. Although PIN-mediated basipetal auxin transport regulates branching patterns in flowering plants, this is not so in Physcomitrella, where bi-directional transport is required to generate realistic branching patterns. Experiments with callose synthesis inhibitors suggest plasmodesmal connectivity as a potential mechanism for transport.
- MeSH
- Models, Biological MeSH
- Biological Transport drug effects MeSH
- Cytokinins biosynthesis MeSH
- Plant Epidermis cytology growth & development MeSH
- Plants, Genetically Modified MeSH
- Indoleacetic Acids metabolism pharmacology MeSH
- Lactones pharmacology MeSH
- Bryopsida drug effects growth & development MeSH
- Morphogenesis drug effects MeSH
- Mutation genetics MeSH
- Gene Expression Regulation, Plant drug effects MeSH
- Plant Growth Regulators pharmacology MeSH
- Plant Proteins metabolism MeSH
- Body Patterning drug effects MeSH
- Plant Shoots drug effects growth & development MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
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.
- Publication type
- Journal Article MeSH
Natural cytokinins as well as the majority of their synthetic derivatives show negative effects on root growth and development. Changes in morphology, primarily linked to the inhibition of the cell division in the meristematic zone, are manifested as thickening and shortening of the primary root and impaired lateral root branching. Rational design of cytokinin derivatives can partially overcome these drawbacks and reduce the negative effects. Using our database of cytokinin derivatives, we selected several aromatic cytokinin analogs with modifications at the N9 atom of the adenine moiety. We found that tetrahydropyranyl and tetrahydrofuranyl substitutions at the N9 atom led to enhanced acropetal transport of the modified cytokinin, and both derivatives also showed weak anticytokinin activity. Consequently, changes in the distribution of the active cytokinin pool together with gradual metabolic conversion of the modified cytokinin to its free form prevent root growth inhibition that limits cytokinin utilization in micropropagation techniques.
