Biology subjects

Hopyan, S.

Publications and source records attributed to Hopyan, S..

10 recordsLinked to original sources

bioRxiv2023

Tissue stiffness mapping by light sheet elastography

Tissue stiffness plays a crucial role in regulating morphogenesis. The ability to measure and monitor the dynamic progression of tissue stiffness is important for generating and testing mechanistic hypotheses. Methods to measure tissue properties in vivo have been emerging but present challenges with spatial and temporal resolution especially in 3D, by their reliance on highly specialised equipment, and/or due to their invasive nature. Here, introduce light sheet elastography, a noninvasive method that couples low frequency shear waves with light sheet fluorescence microscopy by adapting commercially available instruments. With this method, we achieved in toto stiffness mapping of organ-stage mouse and zebrafish embryos at cellular resolution. Versatility of the method enabled time-lapse stiffness mapping during tissue remodelling and of the beating embryonic heart. This method expands the spectrum of tools available to biologists and presents new opportunities for uncovering the mechanical basis of morphogenesis.

bioengineering↗

bioRxiv2023

Mesenchymal Wnts are required for morphogenetic movements of calvarial osteoblasts during apical expansion

Apical expansion of calvarial osteoblast progenitors from the cranial mesenchyme (CM) above the eye is integral for calvarial growth and enclosure of the brain. The cellular behaviors and signals underlying the morphogenetic process of calvarial expansion are unknown. During apical expansion, we found that mouse calvarial primordia have consistent cellular proliferation, density, and survival with complex tissue scale deformations, raising the possibility that morphogenetic movements underlie expansion. Time lapse light sheet imaging of mouse embryos revealed that calvarial progenitors intercalate in 3D to converge supraorbital arch mesenchyme mediolaterally and extend it apically. In contrast, progenitors located further apically exhibited protrusive and crawling activity. CM cells express non-canonical Wnt/Planar Cell Polarity (PCP) core components and calvarial osteoblasts are bidirectionally polarized. We found non-canonical ligand, Wnt5a-/- mutants have less dynamic cell rearrangements, protrusive activity, and a flattened head shape. Loss of cranial mesenchyme-restricted Wntless (CM-Wls), a gene required for secretion of all Wnt ligands, led to diminished apical expansion of OSX+ calvarial osteoblasts in the frontal bone primordia in a non-cell autonomous manner without perturbing proliferation or survival. Calvarial osteoblast polarization, progressive cell elongation and enrichment for actin cytoskeleton protein along the baso-apical axis were dependent on CM-Wnts. Thus, CM-Wnts regulate cellular behaviors during calvarial morphogenesis and provide tissue level cues for efficient apical expansion of calvarial osteoblasts. These findings also offer potential insights into the etiologies of calvarial dysplasias.

developmental biology↗

bioRxiv2023

Fluctuations and entropy enable neural crest cell ingression

The second law of thermodynamics explains the dissipative nature of embryonic development as an exchange of energy-dependent order for proportionately greater output of heat and waste. Recent work on granular matter provides a path by which to define the roles of passive, stochastic mechanisms in nonequilibrium systems. Here, we apply such a framework to examine the role of thermodynamic parameters to cell ingression, the movement of cells from one tissue layer to another that has been attributed, in part, to directional cues. Using the murine neural crest as a model system, we provide evidence that a stochastic mechanism, rather than a proposed stiffness gradient, underlies cell ingression. Cortical fluctuations representing effective temperature and cell packing configurations generate an entropic trap that promotes cell ingression. The results imply dissipative mechanisms that transiently disorder tissue underlie some morphogenetic events.

developmental biology↗

bioRxiv2023

Apical expansion of calvarial osteoblasts and suture patency is dependent on graded fibronectin cues

The skull roof, or calvaria, is comprised of interlocking plates of bone. Premature suture fusion (craniosynostosis, CS) or persistent fontanelles are common defects in calvarial development. Although some of the genetic causes of these disorders are known, we lack an understanding of the instructions directing the growth and migration of progenitors of these bones, which may affect the suture patency. Here, we identify graded expression of Fibronectin (FN1) protein in the mouse embryonic cranial mesenchyme (CM) that precedes the apical expansion of calvarial osteoblasts. Syndromic forms of CS exhibit dysregulated FN1 expression, and we find FN1 expression is altered in a mouse CS model as well. Conditional deletion of Fn1 in CM causes diminished frontal bone expansion by altering cell polarity and shape. To address how osteoprogenitors interact with the observed FN1 prepattern, we conditionally ablate Wasl/N-Wasp to disrupt F-actin junctions in migrating cells, impacting lamellipodia and cell-matrix interaction. Neural crest-targeted deletion of Wasl results in a diminished actin network and reduced expansion of frontal bone primordia similar to conditional Fn1 mutants. Interestingly, defective calvaria formation in both the Fn1 and Wasl mutants occurs without a significant change in proliferation, survival, or osteogenesis. Finally, we find that CM-restricted Fn1 deletion leads to premature fusion of coronal sutures. These data support a model of FN1 as a directional substrate for calvarial osteoblast migration that may be a common mechanism underlying many cranial disorders of disparate genetic etiologies.

developmental biology↗

bioRxiv2023

Durotaxis bridges phase transition as a function of tissue stiffness in vivo

Physical processes ultimately drive morphogenetic cell movements. Two proposals are that 1) cells migrate toward stiffer tissue (durotaxis) and that 2) the extent of cell rearrangements reflects liquid-solid tissue phase. 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 by durotaxis or intercalate as a function of their location along a stiffness gradient. A unifying stiffness-phase transition model that is based on a Landau equation accurately predicts cell diffusivity upon loss or gain of fibronectin. Fibronectin is regulated by a WNT5A-YAP positive feedback pathway that controls cell movements, tissue shape and skeletal pattern. The results identify a key determinant of phase transition and show how durotaxis emerges in a mixed phase environment in vivo.

developmental biology↗

bioRxiv2022

Shape driven confluent rigidity transition in curved biological tissues

Collective cell motions underlie structure formation during embryonic development. Tissues exhibit emergent multicellular characteristics such as jamming, rigidity transitions, and glassy dynamics, but there remain questions about how those tissue scale dynamics derive from local cell level properties. Specifically, there has been little consideration of the interplay between local tissue geometry and cellular properties influencing larger scale tissue behaviours. Here we consider a simple two dimensional computational vertex model for confluent tissue monolayers, which exhibits a rigidity phase transition controlled by the shape index (ratio of perimeter to square root area) of cells, on surfaces of constant curvature. We show that the critical point for the rigidity transition is a function of curvature such that positively curved systems are likely to be in a less rigid, more fluid, phase. Likewise, negatively curved systems (saddles) are likely to be in a more rigid, less fluid, phase. A phase diagram we generate for the curvature and shape index constitutes a testable prediction from the model. The curvature dependence is interesting because it suggests a natural explanation for more dynamic tissue remodelling and facile growth in regions of higher surface curvature, without invoking the need for biochemical or other physical differences. This has potential ramifications for our understanding of morphogenesis of budding and branching structures.

biophysics↗

bioRxiv2021

Live imaging YAP signaling in vivo with fusion reporter mice

YAP protein is a critical regulator of mammalian embryonic development. By generating a near-infrared fusion YAP reporter mouse line, we have achieved high-resolution live imaging of YAP localization during mouse embryonic development. We have validated the reporter by demonstrating its predicted responses to blocking LATs kinase activity or blocking cell polarity. By time lapse imaging preimplantation embryos, we revealed a mitotic reset behavior of YAP nuclear localization. We also demonstrated deep tissue live imaging in post-implantation embryos and revealed an intriguing nuclear YAP pattern in migrating cells. The YAP fusion reporter mice and imaging methods will open new opportunities for understanding dynamic YAP signaling in vivo in many different situations.

developmental biology↗

bioRxiv2020

Patterning the embryonic pulmonary mesenchyme

Smooth muscle guides 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 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 distinct progenitors. Transcriptional variability correlates with 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. Cytoskeletal and Wnt signaling pathways are activated early in reconstructed differentiation trajectories. Consistently, Wnt activation stimulates the earliest stages of smooth muscle differentiation and induces local accumulation of mesenchymal F-actin, which influences epithelial morphology. Our single-cell approach uncovers the principles of pulmonary mesenchymal patterning during branching morphogenesis and identifies a morphogenetically active mesenchymal layer that sculpts the airway epithelium.

developmental biology↗

bioRxiv2018

Three-dimensional tissue stiffness mapping in the mouse embryo supports durotaxis during early limb bud morphogenesis

Numerous biophysical hypotheses invoke tissue stiffness as a key parameter for shaping tissue during development and for influencing cell behaviours during disease progression. However, currently available methods are insufficient to test hypotheses that concern the physical properties of bulk tissues. Here we introduce, validate and apply a new 3D magnetic device that generates a uniform magnetic field gradient within a space that is sufficient to accommodate a vertebrate, organ-stage embryo under live conditions. The device allows for rapid, nontoxic measurement of the spatial variation of absolute elastic modulus and viscosity deep within mesenchymal tissues and within epithelia. By applying the device to map the spatiotemporal variation of viscoelastic properties within the early mouse limb bud, we identified an anteriorly biased mesodermal stiffness gradient along which cells move collectively to shape the early bud. Tissue stiffness corresponds to the nascent expression domain of fibronectin that is Wnt5a-dependent. The findings challenge the notion that Wnt5a regulates cell movements by chemotaxis, and raises the possibility that Wnt5a modifies the tissue microenvironment to promote durotaxis in vivo. Importantly, the ability to precisely measure tissue stiffness in 3D has the potential to instigate and refine mechanisms of development and disease progression.

developmental biology↗

bioRxiv2018

Oscillatory cortical forces promote three dimensional cell intercalations that shape the mandibular arch

Multiple vertebrate embryonic structures such as organ primordia are composed of a volume of confluent cells. Although mechanisms that shape tissue sheets are increasingly understood, those which shape a volume of cells remain obscure. Here we show 3D mesenchymal cell intercalations, rather than cell divisions and biophysical tissue properties, are essential to shape the mandibular arch of the mouse embryo. Using a genetically encoded vinculin tension sensor, we show that cortical force oscillations promote these intercalations. Genetic loss and gain of function approaches show that Wnt5a functions as a spatial cue to coordinate cell polarity with cytoskeletal oscillation. YAP/TAZ and PIEZO1 serve as downstream effectors of Wnt5a-mediated actomyosin bias and cytosolic calcium transients, respectively, to ensure appropriate tissue form during growth. Our data support oriented 3D cell neighbour exchange as a conserved mechanism driving volumetric morphogenesis.

developmental biology↗