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Summary: Smooth muscle guides the morphogenesis of several epithelia during organogenesis, including the mammalian airways. However, it remains unclear how airway smooth muscle differentiation is spatiotemporally patterned and whether it originates from transcriptionally distinct mesenchymal progenitors. Using single-cell RNA-sequencing of embryonic mouse lungs, we show that the pulmonary mesenchyme contains a continuum of cell identities, but no transcriptionally distinct progenitors. Transcriptional variability correlates with spatially distinct sub-epithelial and sub-mesothelial mesenchymal compartments that are regulated by Wnt signaling. Live-imaging and tension-sensors reveal compartment-specific migratory behaviors and cortical forces and show that sub-epithelial mesenchyme contributes to airway smooth muscle. Reconstructing differentiation trajectories reveals early activation of cytoskeletal and Wnt signaling genes. Consistently, Wnt activation induces the earliest stages of smooth muscle differentiation and local accumulation of mesenchymal F-actin, which influences epithelial morphology. Our single-cell approach uncovers the principles of pulmonary mesenchymal patterning and identifies a morphogenetically active mesenchymal layer that sculpts the airway epithelium.

Morphogenesis of tissue sheets is well studied, but mechanisms that shape bulk tissues are unclear. Here, the authors show that mesenchymal cells intercalate in 3D to shape the mouse branchial arch, with cortical forces driving intercalations in a Wnt5a-, Yap/Taz- and Piezo1-dependent manner.

The mechanical properties of tissues are pivotal for morphogenesis and disease progression. Recent approaches have enabled measurements of the spatial distributions of viscoelastic properties among embryonic and pathological model systems and facilitated the generation of important hypotheses such as durotaxis and tissue-scale phase transition. There likely are many unexpected aspects of embryo biomechanics we have yet to discover which will change our views of mechanisms that govern development and disease. One area in the blind spot of even the most recent approaches to measuring tissue stiffness is the potentially anisotropic nature of that parameter. Here, we report a magnetic micromanipulation device that generates a uniform magnetic field gradient within a large workspace and permits measurement of the variation of tissue stiffness along three orthogonal axes. By applying the device to the organ-stage mouse embryo, we identify spatially heterogenous and directionally anisotropic stiffness within the mandibular arch. Those properties correspond to the domain of expression and the angular distribution of fibronectin and have potential implications for mechanisms that orient collective cell movements and shape tissues during development. Assessment of anisotropic properties extends the repertoire of current methods and will enable the generation and testing of hypotheses.

The frequent comorbidity of Autism Spectrum Disorders (ASDs) with epilepsy suggests a shared underlying genetic susceptibility; several genes, when mutated, can contribute to both disorders. Recently, PRICKLE1 missense mutations were found to segregate with ASD. However, the mechanism by which mutations in this gene might contribute to ASD is unknown. To elucidate the role of PRICKLE1 in ASDs, we carried out studies in Prickle1(+/-) mice and Drosophila, yeast, and neuronal cell lines. We show that mice with Prickle1 mutations exhibit ASD-like behaviors. To find proteins that interact with PRICKLE1 in the central nervous system, we performed a yeast two-hybrid screen with a human brain cDNA library and isolated a peptide with homology to SYNAPSIN I (SYN1), a protein involved in synaptogenesis, synaptic vesicle formation, and regulation of neurotransmitter release. Endogenous Prickle1 and Syn1 co-localize in neurons and physically interact via the SYN1 region mutated in ASD and epilepsy. Finally, a mutation in PRICKLE1 disrupts its ability to increase the size of dense-core vesicles in PC12 cells. Taken together, these findings suggest PRICKLE1 mutations contribute to ASD by disrupting the interaction with SYN1 and regulation of synaptic vesicles.

Pattern formation is influenced by transcriptional regulation as well as by morphogenetic mechanisms that shape organ primordia, although factors that link these processes remain under-appreciated. Here we show that, apart from their established transcriptional roles in pattern formation, IRX3/5 help to shape the limb bud primordium by promoting the separation and intercalation of dividing mesodermal cells. Surprisingly, IRX3/5 are required for appropriate cell cycle progression and chromatid segregation during mitosis, possibly in a nontranscriptional manner. IRX3/5 associate with, promote the abundance of, and share overlapping functions with co-regulators of cell division such as the cohesin subunits SMC1, SMC3, NIPBL and CUX1. The findings imply that IRX3/5 coordinate early limb bud morphogenesis with skeletal pattern formation. [ABSTRACT FROM AUTHOR]

Numerous hypotheses invoke tissue stiffness as a key parameter that regulates morphogenesis and disease progression. However, current methods are insufficient to test hypotheses that concern physical properties deep in living tissues. Here we introduce, validate, and apply a magnetic device that generates a uniform magnetic field gradient within a space that is sufficient to accommodate an organ-stage mouse embryo under live conditions. The method allows rapid, nontoxic measurement of the three-dimensional (3D) spatial distribution of viscoelastic properties within mesenchyme and epithelia. Using the device, we identify an anteriorly biased mesodermal stiffness gradient along which cells move to shape the early limb bud. The stiffness gradient corresponds to a Wnt5adependent domain of fibronectin expression, raising the possibility that durotaxis underlies cell movements. Three-dimensional stiffness mapping enables the generation of hypotheses and potentially the rigorous testing of mechanisms of development and disease. [ABSTRACT FROM AUTHOR]

The movements of single actin filaments along a myosin-fixed glass surface were observed under a conventional fluorescence microscope. Although random at a low concentration, moving directions of filaments were aligned by the presence of over 1.0 mg/mL of unlabeled filaments. We found that actin filaments when at the intermediate concentrations ranging from 0.1 to 1.0 mg/mL, formed winding belt-like patterns and moved in a two-directional manner along the belts. These patterns were spread over a millimeter range and found to have bulged on the glass in a three-dimensional manner. Filaments did not get closer than about 37.5 nm to each other within each belt-pattern. The average width and the curvature radius of the pattern did not apparently change even when the range of actin concentrations was between 0.05 and 1.0 mg/mL or the sliding velocity between 1.2 and 3.2 μm/sec. However, when the length of filaments was shortened by ultrasonic treatments or the addition of gelsolin molecules, the curvature radius became small from 100 to 60 μm. These results indicate that this belt-forming nature of actin filaments may be due to some inter-filament interactions.

Physical processes ultimately shape tissue during development. Two emerging proposals are that cells migrate toward stiffer tissue (durotaxis) and that the extent of cell rearrangements reflects tissue phase, but it is unclear whether and how these concepts are related. Here, we identify fibronectin-dependent tissue stiffness as a control variable that underlies and unifies these phenomena in vivo. In murine limb bud mesoderm, cells are either caged, move directionally, or intercalate as a function of their location along a stiffness gradient. A modified Landau phase equation that incorporates tissue stiffness accurately predicts cell diffusivity upon loss or gain of fibronectin. Fibronectin is regulated by WNT5A-YAP feedback that controls cell movements, tissue shape, and skeletal pattern. The results identify a key determinant of phase transition and show how fibronectin-dependent directional cell movement emerges in a mixed-phase environment in vivo. [ABSTRACT FROM AUTHOR]