Notochord segmentation in zebrafish controlled by iterative mechanical signaling.

In bony fishes, patterning of the vertebral column, or spine, is guided by a metameric blueprint established in the notochord sheath. Notochord segmentation begins days after somitogenesis concludes and can occur in its absence. However, somite patterning defects lead to imprecise notochord segmentation, suggesting that these processes are linked. Here, we identify that interactions between the notochord and the axial musculature ensure precise spatiotemporal segmentation of the zebrafish spine. We demonstrate that myoseptum-notochord linkages drive notochord segment initiation by locally deforming the notochord extracellular matrix and recruiting focal adhesion machinery at these contact points. Irregular somite patterning alters this mechanical signaling, causing non-sequential and dysmorphic notochord segmentation, leading to altered spine development. Using a model that captures myoseptum-notochord interactions, we find that a fixed spatial interval is critical for driving sequential segment initiation. Thus, mechanical coupling of axial tissues facilitates spatiotemporal spine patterning.

Dynamic BMP signaling mediates notochord segmentation in zebrafish.

The vertebrate spine is a metameric structure composed of alternating vertebral bodies (centra) and intervertebral discs.1 Recent studies in zebrafish have shown that the epithelial sheath surrounding the notochord differentiates into alternating cartilage-like (col2a1/col9a2+) and mineralizing (entpd5a+) segments which serve as a blueprint for centra formation.2,3,4,5 This process also defines the trajectories of migrating sclerotomal cells that form the mature vertebral bodies.4 Previous work demonstrated that notochord segmentation is typically sequential and involves the segmented activation of Notch signaling.2 However, it is unclear how Notch is activated in an alternating and sequential fashion. Furthermore, the molecular components that define segment size, regulate segment growth, and produce sharp segment boundaries have not been identified. In this study, we uncover that a BMP signaling wave acts upstream of Notch during zebrafish notochord segmentation. Using genetically encoded reporters of BMP activity and signaling pathway components, we show that BMP signaling is dynamic as axial patterning progresses, leading to the sequential formation of mineralizing domains in the notochord sheath. Genetic manipulations reveal that type I BMP receptor activation is sufficient to ectopically trigger Notch signaling. Moreover, loss of Bmpr1ba and Bmpr1aa or Bmp3 function disrupts ordered segment formation and growth, which is recapitulated by notochord-specific overexpression of the BMP antagonist, Noggin3. Our data suggest that BMP signaling in the notochord sheath precedes Notch activation and instructs segment growth, facilitating proper spine morphogenesis.

Axial segmentation by iterative mechanical signaling.

In bony fishes, formation of the vertebral column, or spine, is guided by a metameric blueprint established in the epithelial sheath of the notochord. Generation of the notochord template begins days after somitogenesis and even occurs in the absence of somite segmentation. However, patterning defects in the somites lead to imprecise notochord segmentation, suggesting these processes are linked. Here, we reveal that spatial coordination between the notochord and the axial musculature is necessary to ensure segmentation of the zebrafish spine both in time and space. We find that the connective tissues that anchor the axial skeletal musculature, known as the myosepta in zebrafish, transmit spatial patterning cues necessary to initiate notochord segment formation, a critical pre-patterning step in spine morphogenesis. When an irregular pattern of muscle segments and myosepta interact with the notochord sheath, segments form non-sequentially, initiate at atypical locations, and eventually display altered morphology later in development. We determine that locations of myoseptum-notochord connections are hubs for mechanical signal transmission, which are characterized by localized sites of deformation of the extracellular matrix (ECM) layer encasing the notochord. The notochord sheath responds to the external mechanical changes by locally augmenting focal adhesion machinery to define the initiation site for segmentation. Using a coarse-grained mathematical model that captures the spatial patterns of myoseptum-notochord interactions, we find that a fixed-length scale of external cues is critical for driving sequential segment patterning in the notochord. Together, this work identifies a robust segmentation mechanism that hinges upon mechanical coupling of adjacent tissues to control patterning dynamics.

A mechanical model of early somite segmentation.

Somitogenesis is often described using the clock-and-wavefront (CW) model, which does not explain how molecular signaling rearranges the pre-somitic mesoderm (PSM) cells into somites. Our scanning electron microscopy analysis of chicken embryos reveals a caudally-progressing epithelialization front in the dorsal PSM that precedes somite formation. Signs of apical constriction and tissue segmentation appear in this layer 3-4 somite lengths caudal to the last-formed somite. We propose a mechanical instability model in which a steady increase of apical contractility leads to periodic failure of adhesion junctions within the dorsal PSM and positions the future inter-somite boundaries. This model produces spatially periodic segments whose size depends on the speed of the activation front of contraction (F), and the buildup rate of contractility (Λ). The Λ/F ratio determines whether this mechanism produces spatially and temporally regular or irregular segments, and whether segment size increases with the front speed.

A computational model of liver tissue damage and repair.

Drug induced liver injury (DILI) and cell death can result from oxidative stress in hepatocytes. An initial pattern of centrilobular damage in the APAP model of DILI is amplified by communication from stressed cells and immune system activation. While hepatocyte proliferation counters cell loss, high doses are still lethal to the tissue. To understand the progression of disease from the initial damage to tissue recovery or death, we computationally model the competing biological processes of hepatocyte proliferation, necrosis and injury propagation. We parametrize timescales of proliferation (α), conversion of healthy to stressed cells (ß) and further sensitization of stressed cells towards necrotic pathways (γ) and model them on a Cellular Automaton (CA) based grid of lattice sites. 1D simulations show that a small α/ß (fast proliferation), combined with a large γ/ß (slow death) have the lowest probabilities of tissue survival. At large α/ß, tissue fate can be described by a critical γ/ß* ratio alone; this value is dependent on the initial amount of damage and proportional to the tissue size N. Additionally, the 1D model predicts a minimum healthy population size below which damage is irreversible. Finally, we compare 1D and 2D phase spaces and discuss outcomes of bistability where either survival or death is possible, and of coexistence where simulated tissue never completely recovers or dies but persists as a mixture of healthy, stressed and necrotic cells. In conclusion, our model sheds light on the evolution of tissue damage or recovery and predicts potential for divergent fates given different rates of proliferation, necrosis, and injury propagation.