Prof Alessandro De Simone

Regeneration restores a damaged body part to its original size, shape and structure. Research over the last decades identified signaling pathways, cell types and cellular processes that are key for regeneration. Moreover, mechanical cues and electric potentials are increasingly implicated in modulating regenerative processes. An intriguing open question regards how these chemical, mechanical and electric signals are dynamically organized to coordinate cell behaviors across large regenerating tissues and long regenerative timescales for proper morphogenesis. In addition, it is less explored how regeneration is stopped once tissues reach their proper final form. These questions and related models cross-talk with physical notions like information, pattern formation, self-organization, and control. An interdisciplinary approach combining methods and concepts of developmental biology and physics is offering new quantitative insights on these questions. In this approach, researchers characterize the spatial organization and temporal dynamics of chemical, mechanical and electric signal inputs and relate them to cell and tissue behaviors. Initial observations inform theory; in turn, theory guides experiments and data analysis, while state-of-the-art perturbations allow testing these models. After illustrating this approach, we provide examples of its application to animal regeneration in vivo. These works are extending the notion of "morphogen", contributing to establishing the emerging field of quantitative regeneration and uncovering principles of multicellular organization.

Regional and tissue-wide regulation of signaling pathways orchestrates cellular proliferation and differentiation during organ development. In this study, we established an imaging platform for longitudinal analysis of liver development in live developing zebrafish. We generated hepatocyte-specific transgenic lines for kinase translocation reporters of extracellular signal-regulated kinase (Erk) and c-Jun N-terminal kinase (Jnk) signaling, and with these we captured signaling dynamics that govern rapid expansion of hepatocytes toward creation of the functioning liver at single-cell resolution. Our findings reveal Erk signaling fluctuations as the liver develops and introduce methodology for investigating cell-type specific signaling dynamics during organ morphogenesis.

Identification of signaling events that contribute to innate spinal cord regeneration in zebrafish can inform new targets for modulating injury responses of the mammalian central nervous system. Using a chemical screen, we identify JNK signaling as a necessary regulator of glial cell cycling and tissue bridging during spinal cord regeneration in larval zebrafish. With a kinase translocation reporter, we visualize and quantify JNK signaling dynamics at single-cell resolution in glial cell populations in developing larvae and during injury-induced regeneration. Glial JNK signaling is patterned in time and space during development and regeneration, decreasing globally as the tissue matures and increasing in the rostral cord stump upon transection injury. Thus, dynamic and regional regulation of JNK signaling help to direct glial cell behaviors during innate spinal cord regeneration.

Regeneration requires the collective effort of multiple organ systems. A recent study of planarian whole-body regeneration finds that Erk kinase activity propagates rapidly across the entire animal through longitudinal muscle cells to coordinate animal-wide wound responses and that this signal propagation is required for regeneration.

In regeneration, a damaged body part grows back to its original form. Understanding the mechanisms and physical principles underlying this process has been limited by the difficulties of visualizing cell signals and behaviors in regeneration. Zebrafish scales are emerging as a model system to investigate morphogenesis during vertebrate regeneration using quantitative live imaging. Scales are millimeter-sized dermal bone disks forming a skeletal armor on the body of the fish. The scale bone is deposited by an adjacent monolayer of osteoblasts that, after scale loss, regenerates in about 2 weeks. This intriguing regenerative process is accessible to live confocal microscopy, quantifications, and mathematical modeling. Here, I describe methods to image scale regeneration live, tissue-wide and at sub-cellular resolution. Furthermore, I describe methods to process the resulting images and quantify cell, tissue, and signal dynamics.

The vertebrate body displays a segmental organization which is most conspicuous in the periodic organization of the vertebral column and peripheral nerves. This metameric organization is first implemented when somites, which contain the precursors of skeletal muscles and vertebrae, are rhythmically generated from the presomitic mesoderm (PSM). Somites then become subdivided into anterior and posterior compartments essential for vertebral formation and segmental patterning of the peripheral nervous system. How this key somitic subdivision is established remains poorly understood. Here we introduce novel tridimensional culture systems of human pluripotent stem cells (PSCs), called Somitoids and Segmentoids, which recapitulate the formation of somite-like structures with antero-posterior (AP) identity. We identify a key function of the segmentation clock in converting temporal rhythmicity into the spatial regularity of anterior and posterior somitic compartments. We show that an initial salt-and-pepper expression of the segmentation gene MESP2 in the newly formed segment is transformed into compartments of anterior and posterior identity via an active cell sorting mechanism. Our work demonstrates that the major patterning modules involved in somitogenesis including the clock and wavefront, AP polarity patterning and somite epithelialization can be dissociated and operate independently in our in vitro systems. Together we define a novel framework for the symmetry breaking process initiating somite polarity patterning. Our work provides a valuable platform to decode general principles of somitogenesis and advance knowledge of human development.

Erk signaling regulates cellular decisions in many biological contexts. Recently, we have reported a series of Erk activity traveling waves that coordinate regeneration of osteoblast tissue in zebrafish scales. These waves originate from a central source region, propagate as expanding rings, and impart cell growth, thus controlling tissue morphogenesis. Here, we present a minimal reaction-diffusion model for Erk activity waves. The model considers three components: Erk, a diffusible Erk activator, and an Erk inhibitor. Erk stimulates both its activator and inhibitor, forming a positive and negative feedback loop, respectively. Our model shows that this system can be excitable and propagate Erk activity waves. Waves originate from a pulsatile source that is modeled by adding a localized basal production of the activator, which turns the source region from an excitable to an oscillatory state. As Erk activity periodically rises in the source, it can trigger an excitable wave that travels across the entire tissue. Analysis of the model finds that positive feedback controls the properties of the traveling wavefront and that negative feedback controls the duration of Erk activity peak and the period of Erk activity waves. The geometrical properties of the waves facilitate constraints on the effective diffusivity of the activator, indicating that waves are an efficient mechanism to transfer growth factor signaling rapidly across a large tissue.

Regeneration is a complex chain of events that restores a tissue to its original size and shape. The tissue-wide coordination of cellular dynamics that is needed for proper morphogenesis is challenged by the large dimensions of regenerating body parts. Feedback mechanisms in biochemical pathways can provide effective communication across great distances1-5, but how they might regulate growth during tissue regeneration is unresolved6,7. Here we report that rhythmic travelling waves of Erk activity control the growth of bone in time and space in regenerating zebrafish scales, millimetre-sized discs of protective body armour. We find that waves of Erk activity travel across the osteoblast population as expanding concentric rings that are broadcast from a central source, inducing ring-like patterns of tissue growth. Using a combination of theoretical and experimental analyses, we show that Erk activity propagates as excitable trigger waves that are able to traverse the entire scale in approximately two days and that the frequency of wave generation controls the rate of scale regeneration. Furthermore, the periodic induction of synchronous, tissue-wide activation of Erk in place of travelling waves impairs tissue growth, which indicates that wave-distributed Erk activation is key to regeneration. Our findings reveal trigger waves as a regulatory strategy to coordinate cell behaviour and instruct tissue form during regeneration.

The synchronous cleavage divisions of early embryogenesis require coordination of the cell-cycle oscillator, the dynamics of the cytoskeleton, and the cytoplasm. Yet, it remains unclear how spatially restricted biochemical signals are integrated with physical properties of the embryo to generate collective dynamics. Here, we show that synchronization of the cell cycle in Drosophila embryos requires accurate nuclear positioning, which is regulated by the cell-cycle oscillator through cortical contractility and cytoplasmic flows. We demonstrate that biochemical oscillations are initiated by local Cdk1 inactivation and spread through the activity of phosphatase PP1 to generate cortical myosin II gradients. These gradients cause cortical and cytoplasmic flows that control proper nuclear positioning. Perturbations of PP1 activity and optogenetic manipulations of cortical actomyosin disrupt nuclear spreading, resulting in loss of cell-cycle synchrony. We conclude that mitotic synchrony is established by a self-organized mechanism that integrates the cell-cycle oscillator and embryo mechanics.

Osteoblasts are matrix-depositing cells that can divide and heal bone injuries. Their deep-tissue location and the slow progression of bone regeneration challenge attempts to capture osteoblast behaviors in live tissue at high spatiotemporal resolution. Here, we have developed an imaging platform to monitor and quantify individual and collective behaviors of osteoblasts in adult zebrafish scales, skeletal body armor discs that regenerate rapidly after loss. Using a panel of transgenic lines that visualize and manipulate osteoblasts, we find that a founder pool of osteoblasts emerges through de novo differentiation within one day of scale plucking. These osteoblasts undergo division events that are largely uniform in frequency and orientation to establish a primordium. Osteoblast proliferation dynamics diversify across the primordium by two days after injury, with cell divisions focused near, and with orientations parallel to, the scale periphery, occurring coincident with dynamic localization of fgf20a gene expression. In posterior scale regions, cell elongation events initiate in areas soon occupied by mineralized grooves called radii, beginning approximately 2 days post injury, with patterned osteoblast death events accompanying maturation of these radii. By imaging at single-cell resolution, we detail acquisition of spatiotemporally distinct cell division, motility, and death dynamics within a founder osteoblast pool as bone regenerates.