Guillaume Salbreux

Summary: Successful regeneration recovers lost tissues to their original form. Here, we investigate how proliferation of bone-forming osteoblasts is timely regulated to control tissue size during zebrafish scale regeneration. Regeneration of the scale osteoblast tissue proceeds in two phases: an initial osteoblast proliferative expansion followed by hypertrophic growth without cell division. First, we show that regeneration recovers scale size by amplifying the number of osteoblasts by a fixed factor, starting from a properly scaled tissue primordium. This fixed expansion is achieved by regulating proliferation such that osteoblast number increases roughly linearly, with the rate of increase scaling with initial osteoblast number, and halting cell proliferation at a given time. To understand the mechanisms underlying this proliferation control, we used live imaging of transgenic reporters and biosensors together with transcriptomics. We uncover that proliferation arrest is imparted by a moving front of osteoblast maturation travelling outward across the scale, with dynamics that scales with tissue size. The osteoblast maturation front coincides with onset of hypertrophy and bone deposition, and it is accompanied by reduction in expression of the fibroblast growth factor receptor 3 ( fgfr3 ) and activity of the kinases Erk (Extracellular signal-Regulated Kinase) and p38. Inhibition of Fgf/Erk signaling and osteoblast proliferation does not alter the progression of the maturation front. We show that the scaling maturation front can quantitatively explain the osteoblast proliferation dynamics and its scaling across different tissue sizes. Overall, we identify a moving front of cell maturation as a mechanism for controlling cell proliferation across a regenerating tissue.

Bilateral symmetry is a hallmark of many paired organs and often essential for optimal functionality. The vertebrate eyes are a prominent example of this, as the matched development of the two retinas is required for accurate visual processing. While macroscopic aspects of symmetry emergence across systems have been investigated, how bilateral matching is maintained once cells start to differentiate remains less understood. Here we address this question using the zebrafish retina as a model to follow neurogenic programs in vivo at single-cell resolution. We perform quantitative 3D live imaging of both retinas simultaneously and directly compare neurogenesis onset and propagation within and across embryos. We find that neurogenic waves initiate at the retinal poles and progress towards the mid-retina in a conserved spatiotemporal pattern. Within embryos, the two eyes exhibit highly similar neurogenesis dynamics when it comes to timing of neurogenesis onset, cell number increase, and spatial wave progression. Across embryos, however, variability is larger. While these observations hint at active inter-retinal coordination, a stochastic model predicts that a shared onset of neurogenesis can be sufficient to explain bilateral matching. Targeted genetic perturbation experiments support this prediction. We find that altering wave propagation affects patterning but not bilateral similarity. Disrupting neurogenesis onset timing, however, reduces bilateral symmetry between eyes. Thus, the combination of experiment and theory identifies synchronized neurogenesis onset as a key determinant of bilateral symmetry, revealing a minimal principle for how reproducible development of paired organs can emerge from stochastic processes.

Epithelia are tissues which envelop organs, exchange with and protect from their environment. The tight packing of cells within an epithelium guarantees its cohesiveness and impermeability, essential to its functions. Yet, how cells spatially arrange in three dimensions within an epithelium, and how the three-dimensional cell packing responds to geometrical constraints, is not fully understood. In a proliferating epithelium, cellular volumes vary from cell to cell, notably due to cell growth. At the same time, epithelia tend to display a smooth, continuous apical surface, indicating that cell shape determinants are spatially coupled. It is unclear how these and other factors modulate cell shape variability within the epithelium. Here, we segmented three-dimensional cell shapes of MDCK epithelia, grown in hollow spheres of alginate. With this assay, we could modulate the substrate adhesive properties, tissue curvature, and cell area density. We observed that as the tissue proliferates and cell density increases, average cell volume decreases. In contrast, cell height is relatively conserved over time, with an average value sensitive to the rigidity, curvature and adhesive strength of the substrate. We identified large and spatially correlated fluctuations in cell skewing, defined as the relative difference between apical and basal area that do not arise from curvature. Skewing is associated to spatial patterns of tilted cells whose apico-basal axis deviates from orthogonality to the substrate. Surprisingly, cell skewing and cell height are correlated, indicating internal rules for three-dimensional cell shapes. Altogether, our study identifies unexpected patterns of three-dimensional cell shape variation within a proliferative epithelium.

Curved geometries are a defining feature of epithelial tissues, yet how cells integrate curvature cues across spatial scales remains unclear. Here, we combined wavy hydrogels with inducible self-rolling substrates to readily and independently impose local and large-scale curvatures on epithelial monolayers, recapitulating key geometric features of bronchiolar epithelia. By controlling the orientation of local curvature relative to the large-scale curvature set by the tube axis, we show that multiscale curvature induces scale-dependent and anisotropic remodeling of cell shape, nuclear organization, and tissue thickness. In contrast, nuclei maintain a robust and conserved three-dimensional geometry, with curvature primarily regulating nuclear orientation rather than shape. This hierarchical induction of specific changes to the tissue architecture shows that multiscale curvature sensing is a fundamental physical principle governing epithelial architecture.

Shape changes are ubiquitous in biology, from cytokinesis at the single cell scale to tissue-scale morphogenesis involving coordinated changes in hundreds of cells. In all cases, morphogenesis is powered by gradients in mechanical tension that arise downstream of signalling. Many pathways converge on RhoGTPases that modulate the cytoskeleton and cell contractility to control cell mechanics and, subsequently, shape. Despite their physiological importance, we lack a quantitative understanding of how changes in signalling alter cortical mechanics to drive cell shape change. Here, we use optogenetics to quantitatively characterise the relationship between the amount of RhoGEF localised to the membrane, the downstream myosin recruitment, and the subsequent mechanical changes. We then show that cortical myosin amount and cortical tension increase linearly with the amount of membranous RhoGEF signalling. Based on these data, we develop a predictive model of the temporal evolution of RhoGEF membrane localisation, cortical myosin enrichment, and cortical tension in response to a pulse of light. Using this model together with an active surface model of the cell cortex, we show that the cellular shape changes induced by localised optogenetic recruitment of RhoGEF signalling can be predicted, directly linking gradients in signalling to shape change. Significance statement: Shape changes are ubiquitous in biology, during division in single cells and in tissue during embryogenesis. In all cases, shape change is powered by gradients in mechanical tension that arise downstream of changes in biochemical signals. Despite their importance, we lack a quantitative understanding of how changes in signals alter cell mechanics to drive cell shape change. Here, we control the location and amount of biochemical signal using light to quantitatively characterise the relationship between signals and their resulting biological and mechanical changes. We show that mechanical change scales linearly with the amount of biochemical signal. Based on this, we develop a mathematical model that predicts cell mechanical and shape changes from the location and amount of biochemical signals.

Blood vessels expand and contract actively as they continuously experience dynamic external stresses from blood flow. The mechanical response of the vessel wall is that of a composite material: its mechanical properties depend on its cellular components, which change dynamically as the cells respond to external stress. Mapping the relationship between these underlying cellular processes and emergent tissue mechanics is an ongoing challenge, particularly in endothelial cells. Here we assess the mechanics and cellular dynamics of an endothelial tube using a microstretcher that mimics the native environment of blood vessels. The characterization of the instantaneous monolayer elasticity reveals a strain-stiffening, actin-dependent and substrate-responsive behaviour. After a physiological pressure increase, the tissue displays a fluid-like expansion, with the reorientation of cell shape and actin fibres. We introduce a mechanical model that considers the actin fibres as a network in the nematic phase and couples their dynamics with active and elastic fibre tension. The model accurately describes the response to the pressure of endothelial tubes.

Morphogens are produced by a subset of cells to trigger a signalling gradient that provides positional information to surrounding tissues. At increasing distances from the source, the dwindling number of morphogen molecules is expected to constrain the useful range of morphogen gradients. We have identified a genetic circuit that counteracts this limitation in developing wings of Drosophila by boosting BMP signalling at the distal end of the gradient without amplifying the signal near the source. This circuit involves Brinker, a transcription factor that represses BMP target genes while itself being repressed by BMP signalling. We suggest that temporal averaging inherent to the production of the inverse Brk gradient contributes to the enhancement of the positional information far from the Dpp source. Despite being a core component of BMP signalling in flies, Brinker is exclusively found in insects, likely in all insect species. Genomic analysis across a wide range of insects and gene expression analysis in limb primordia of the apterygote Thermobia domestica suggests that Brinker is an insect-specific innovation that was subsequently wired into the BMP signalling network in pterygotes, perhaps to enable wing development.

How complex molecular mechanisms translate into diverse multicellular shapes remains unclear. By leveraging the bi-layered architecture of six cnidarian species that diverged 500 million years ago, we show that modularity in supracellular mechanics governs larval shape diversity. Using active surface theory, quantitative imaging, and an inducible genetic system, we identify species-specific variations in three biomechanical modules. Basally aligned stress fibers drive axial elongation, while oral geometry and aboral rigidity define shape polarity. Remarkably, manipulating these modules transforms one species' shape into another, demonstrating the causal relationship between module variation and shape diversity. Our analysis also uncovers instances of mechanical redundancies, where distinct module combinations generate similar shapes. These findings provide a general framework for how molecular complexity funnels into mesoscale mechanical determinants shaping morphological diversity.