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Background and Purpose: Wnt binding to Frizzleds (FZD) is a crucial step that leads to the initiation of signalling cascades governing multiple processes during embryonic development, stem cell regulation and adult tissue homeostasis. Recent efforts have enabled us to shed light on Wnt–FZD pharmacology using overexpressed HEK293 cells. However, assessing ligand binding at endogenous receptor expression levels is important due to differential binding behaviour in a native environment. Here, we study FZD paralogue, FZD7, and analyse its interactions with Wnt‐3a in live CRISPR‐Cas9‐edited SW480 cells typifying colorectal cancer. Experimental Approach: SW480 cells were CRISPR‐Cas9‐edited to insert a HiBiT tag on the N‐terminus of FZD7, preserving the native signal peptide. These cells were used to study eGFP‐Wnt‐3a association with endogenous and overexpressed HiBiT‐FZD7 using NanoBiT/bioluminescence resonance energy transfer (BRET) and NanoBiT to measure ligand binding and receptor internalization. Key Results: With this new assay the binding of eGFP‐Wnt‐3a to endogenous HiBiT‐FZD7 was compared with overexpressed receptors. Receptor overexpression results in increased membrane dynamics, leading to an apparent decrease in binding on‐rate and consequently in higher, up to 10 times, calculated Kd. Thus, measurements of binding affinities to FZD7 obtained in overexpressed cells are suboptimal compared with the measurements from endogenously expressing cells. Conclusions and Implications: Binding affinity measurements in the overexpressing cells fail to replicate ligand binding affinities assessed in a (patho)physiologically relevant context where receptor expression is lower. Therefore, future studies on Wnt–FZD7 binding should be performed using receptors expressed under endogenous promotion. [ABSTRACT FROM AUTHOR]

Most of the biological processes, including cell signaling, cancer invasion, embryogenesis, or neural development, are dependent on and guided by the complex architecture and composition of cellular microenvironments. Mimicking such microenvironments in cell coculture models is crucial for fundamental and applied biology investigations. The ability to combine different cell types grown as both two‐dimensional (2D) monolayers and three‐dimensional (3D) spheroids in specific defined location inside a microculture environments is a key towards in vitro tissue modeling and towards mimicking complex in vivo cellular processes. In this study, we introduce and investigate a method to create in vitro models of 2D cell monolayers cocultured with 3D spheroids in defined preorganization. We demonstrate the possibility of creating such complex cellular microenvironments in a high‐throughput and automated manner by creating arrays of such droplets containing prearranged 2D and 3D cellular microcolonies. Furthermore, we demonstrate an application of this approach to study paracrine propagation of Wnt signaling between 2D and 3D cellular colonies. This method provides a general approach for the miniaturized, high‐throughput, and automated formation of complex coculture cellular microarchitectures that will be useful for mimicking various in vivo complex cellular structures and for studying complex biological processes in vitro. [ABSTRACT FROM AUTHOR]

Author(s): Gary Davidson (corresponding author) [1, 2]; Wei Wu [1, 2]; Jinlong Shen [1]; Josipa Bilic [1]; Ursula Fenger [1]; Peter Stannek [1]; Andrei Glinka [1]; Christof Niehrs (corresponding author) [...]

Author(s): Bingyu Mao [1]; Wei Wu [1]; Gary Davidson [1]; Joachim Marhold [2]; Mingfa Li [2]; Bernard M. Mechler [2]; Hajo Delius [3]; Dana Hoppe [1]; Peter Stannek [1]; Carmen [...]

The superimposition of Barrovian-type metamorphism on low-pressure contact aureoles in magmatic arcs is subject to the kinetic constraints of temperature and influx of [H.sub.2]O and may be cryptically developed or not at all. The spatial distribution of detectable overprint will reflect these kinetic factors rather than the geometry of the imposed load. Many of the features believed indicative of magma loading, including apparent steep baric gradients, may be produced in this way. On the basis of adding areas of cryptic metamorphism to the area of obvious Barrovian overprint on the northeast side of the Mount Stuart batholith, we conclude that loading 2-3 m.y. after emplacement was probably much more extensive (sheetlike) than believed previously.

Abstract Background A fundamental requirement for development of diverse brain regions is the function of local organizers at morphological boundaries. These organizers are restricted groups of cells that secrete signaling molecules, which in turn regulate the fate of the adjacent neural tissue. The thalamus is located in the caudal diencephalon and is the central relay station between the sense organs and higher brain areas. The mid-diencephalic organizer (MDO) orchestrates the development of the thalamus by releasing secreted signaling molecules such as Shh. Results Here we show that canonical Wnt signaling in the caudal forebrain is required for the formation of the Shh-secreting MD organizer in zebrafish. Wnt signaling induces the MDO in a narrow time window of 4 hours - between 10 and 14 hours post fertilization. Loss of Wnt3 and Wnt3a prevents induction of the MDO, a phenotype also observed upon blockage of canonical Wnt signaling per se. Pharmaceutical activation of the canonical Wnt pathways in Wnt3/Wnt3a compound morphant embryos is able to restore the lack of the MDO. After blockage of Wnt signaling or knock-down of Wnt3/Wnt3a we find an increase of apoptotic cells specifically within the organizer primordium. Consistently, blockage of apoptosis restores the thalamus organizer MDO in Wnt deficient embryos. Conclusion We have identified canonical Wnt signaling as a novel pathway, that is required for proper formation of the MDO and consequently for the development of the major relay station of the brain - the thalamus. We propose that Wnt ligands are necessary to maintain the primordial tissue of the organizer during somitogenesis by suppressing Tp53-mediated apoptosis.

Research has been conducted on growth factor antagonists which are important for mediating the inductive effects of Spemann organized in amphibian embryos. The authors have tested the hypothesis that the dual inhibition of Wnt and BMP signals could confer head organizer activity, and they report the results.