Katja Närhi

Sustained epithelial β-catenin activity induces precocious hair development but disrupts hair follicle down-growth and hair shaft formation

During embryonic and postnatal development, Wnt/β-catenin signaling is involved in several stages of hair morphogenesis from placode formation to hair shaft differentiation. Using a transgenic approach, we have investigated further the role of β-catenin signaling in embryonic hair development. Forced epithelial stabilization of β-catenin resulted in precocious and excessive induction of hair follicles even in the absence of Eda/Edar signaling, a pathway essential for primary hair placode formation. In addition, the spacing and size of the placodes was randomized. Surprisingly, the down-growth of follicles was suppressed and hair shaft production was severely impaired. Gene and reporter expression analyses revealed elevated mesenchymal Wnt activity, as well as increased BMP signaling, throughout the skin that was accompanied by upregulation of Sostdc1 (Wise, ectodin) expression. Our data suggest that BMPs are downstream of Wnt/β-catenin and that their interplay may be a critical component in establishing correct patterning of hair follicles through the reaction-diffusion mechanism.

Fgf20 governs formation of primary and secondary dermal condensations in developing hair follicles

In hair follicle development, a placode-derived signal is believed to induce formation of the dermal condensation, an essential component of ectodermal organs. However, the identity of this signal is unknown. Furthermore, although induction and patterning of hair follicles are intimately linked, it is not known whether the mesenchymal condensation is necessary for inducing the initial epithelial pattern. Here, we show that fibroblast growth factor 20 (Fgf20) is expressed in hair placodes and is induced by and functions downstream from epithelial ectodysplasin (Eda)/Edar and Wnt/β-Catenin signaling to initiate formation of the underlying dermal condensation. Fgf20 governs formation of primary and secondary dermal condensations in developing hair follicles and subsequent formation of guard, awl, and auchene hairs. Although primary dermal condensations are absent in Fgf20 mutant mice, a regular array of hair placodes is formed, demonstrating that the epithelial patterning process is independent of known histological and molecular markers of underlying mesenchymal patterns during the initial stages of hair follicle development.

Capturing complex tumour biology in vitro: histological and molecular characterisation of precision cut slices

Precision-cut slices of in vivo tumours permit interrogation in vitro of heterogeneous cells from solid tumours together with their native microenvironment. They offer a low throughput but high content in vitro experimental platform. Using mouse models as surrogates for three common human solid tumours, we describe a standardised workflow for systematic comparison of tumour slice cultivation methods and a tissue microarray-based method to archive them. Cultivated slices were compared to their in vivo source tissue using immunohistochemical and transcriptional biomarkers, particularly of cellular stress. Mechanical slicing induced minimal stress. Cultivation of tumour slices required organotypic support materials and atmospheric oxygen for maintenance of integrity and was associated with significant temporal and loco-regional changes in protein expression, for example HIF-1α. We recommend adherence to the robust workflow described, with recognition of temporal-spatial changes in protein expression before interrogation of tumour slices by pharmacological or other means.

Explant Culture of Embryonic Craniofacial Tissues: Analyzing Effects of Signaling Molecules on Gene Expression

The in vitro culture of embryonic tissue explants allows the continuous monitoring of growth and morphogenesis at specific embryonic stages. The functions of soluble regulatory molecules can be examined by adding them into culture medium or by introducing them with beads to specific locations in the tissue. Gene expression analysis using in situ hybridization, quantitative PCR, and reporter constructs can be combined with organ culture to examine the functions of the regulatory molecules.

The putative tumor suppressor gene EphA3 fails to demonstrate a crucial role in murine lung tumorigenesis or morphogenesis.

Treatment of non-small cell lung cancer (NSCLC) is based on histological analysis and molecular profiling of targetable driver oncogenes. Therapeutic responses are further defined by the landscape of passenger mutations, or loss of tumor suppressor genes. We report here a thorough study to address the physiological role of the putative lung cancer tumor suppressor EPH receptor A3 (EPHA3), a gene that is frequently mutated in human lung adenocarcinomas. Our data shows that homozygous or heterozygous loss of EphA3 does not alter the progression of murine adenocarcinomas that result from Kras mutation or loss of Trp53, and we detected negligible postnatal expression of EphA3 in adult wild-type lungs. Yet, EphA3 was expressed in the distal mesenchyme of developing mouse lungs, neighboring the epithelial expression of its Efna1 ligand; this is consistent with the known roles of EPH receptors in embryonic development. However, the partial loss of EphA3 leads only to subtle changes in epithelial Nkx2-1, endothelial Cd31 and mesenchymal Fgf10 RNA expression levels, and no macroscopic phenotypic effects on lung epithelial branching, mesenchymal cell proliferation, or abundance and localization of CD31-positive endothelia. The lack of a discernible lung phenotype in EphA3-null mice might indicate lack of an overt role for EPHA3 in the murine lung, or imply functional redundancy between EPHA receptors. Our study shows how biological complexity can challenge in vivo functional validation of mutations identified in sequencing efforts, and provides an incentive for the design of knock-in or conditional models to assign the role of EPHA3 mutation during lung tumorigenesis.

Analysis of tissue interactions in ectodermal organ culture.

The morphogenesis of ectodermal organs is regulated by epithelial mesenchymal interactions mediated by conserved signaling molecules. Analyzing the roles of these molecules will increase our understanding of mechanisms regulating organogenesis, and organ culture methods provide powerful tools in this context. Here we present two organ culture methods for skin and tooth development: the hanging drop method for the short-term culture of small explants and the Trowell-type method for the long-term cultures of variable size explants. The latter allows manipulations such as combining separated epithelial and mesenchymal tissues and the use of signal-releasing beads. The effects of signaling molecules on morphogenesis can be observed during culture by using tissues from GFP-reporter mice. After culture, the effects of signals on gene expression can be analyzed by in situ hybridization or quantitative RT-PCR.

Embryonic Explant Culture: Studying Effects of Regulatory Molecules on Gene Expression in Craniofacial Tissues

The ex vivo culture of embryonic tissue explants permits the continuous monitoring of growth and morphogenesis at specific embryonic stages. The functions of soluble regulatory molecules can be analyzed by introducing them into culture medium or locally with beads to the tissue. Gene expression in the manipulated tissue explants can be analyzed using in situ hybridization, quantitative PCR, and reporter constructs combined to organ culture to examine the functions of the signaling molecules.

Protocols and characterization data for 2D, 3D, and slice-based tumor models from the PREDECT project

Two-dimensional (2D) culture of cancer cells in vitro does not recapitulate the three-dimensional (3D) architecture, heterogeneity and complexity of human tumors. More representative models are required that better reflect key aspects of tumor biology. These are essential studies of cancer biology and immunology as well as for target validation and drug discovery. The Innovative Medicines Initiative (IMI) consortium PREDECT (www.predect.eu) characterized in vitro models of three solid tumor types with the goal to capture elements of tumor complexity and heterogeneity. 2D culture and 3D mono- and stromal co-cultures of increasing complexity, and precision-cut tumor slice models were established. Robust protocols for the generation of these platforms are described. Tissue microarrays were prepared from all the models, permitting immunohistochemical analysis of individual cells, capturing heterogeneity. 3D cultures were also characterized using image analysis. Detailed step-by-step protocols, exemplary datasets from the 2D, 3D, and slice models, and refined analytical methods were established and are presented.

Spatial aspects of oncogenic signalling determine the response to combination therapy in slice explants from Kras-driven lung tumours: Spatial response to combination therapy in tumour slices

A key question in precision medicine is how functional heterogeneity in solid tumours informs therapeutic sensitivity. We demonstrate that spatial characteristics of oncogenic signalling and therapy response can be modelled in precision‐cut slices from Kras‐driven non‐small‐cell lung cancer with varying histopathologies. Unexpectedly, profiling of in situ tumours demonstrated that signalling stratifies mostly according to histopathology, showing enhanced AKT and SRC activity in adenosquamous carcinoma, and mitogen‐activated protein kinase (MAPK) activity in adenocarcinoma. In addition, high intertumour and intratumour variability was detected, particularly of MAPK and mammalian target of rapamycin (mTOR) complex 1 activity. Using short‐term treatment of slice explants, we showed that cytotoxic responses to combination MAPK and phosphoinositide 3‐kinase–mTOR inhibition correlate with the spatially defined activities of both pathways. Thus, whereas genetic drivers determine histopathology spectra, histopathology‐associated and spatially variable signalling activities determine drug sensitivity. Our study is in support of spatial aspects of signalling heterogeneity being considered in clinical diagnostic settings, particularly to guide the selection of drug combinations.