We then measured the angle of the neural epithelium at the level of the predicted or present DLHP at the position of the 5th somite pair ( STAR Methods), which constitutes the central region of the apoptotic domain. We treated the embryos with the broad-spectrum caspase inhibitor QVD-Oph (summarized in Figure 1F, see STAR Methods), which drastically reduces the number of apoptotic cells in the neural tube ( Figures S1F and S1G). In order to test this hypothesis, we inhibited apoptosis prior to the dorsal bending of the neural epithelium. This led us to hypothesize that apoptosis might influence neural tube morphogenesis by taking part in the bending of the neural epithelium at the DLHP. We found that the increase of apoptosis slightly precedes tissue bending at the DLHP, highlighting a tight spatial and temporal correlation between apoptosis and the neural plate bending at the DLHP region in the avian neural tube ( Figure 1E). We further plotted the number of apoptotic cells together with the angle of the apical surface in the DLHP region over time. As a consequence, we chose to focus on dorsal folds morphogenesis. At later stages (from 6 ss to 8 ss), the level of apoptosis increased and a preferential accumulation of apoptotic cells was visible both at the midline, which had already bent ( Figures 1C and 1D), and in the DLHP region and more generally in the whole dorsal folds region, which interestingly changed its shape from convex to concave at around 6 ss ( Figure 1C). From 3 ss to 5 ss, apoptosis was low and randomly distributed along the DV axis of the neural tube, with a frequency slightly higher in the most dorsal part (commonly called dorsal folds), particularly in the region of the future DLHP ( Figures 1C and 1D ). To precisely map the apoptotic pattern, we counted only cells that were in the process of dying ( STAR Methods) rather than the post-apoptotic fragments of cells, which tend to relocate within the epithelium. We next quantified the distribution of apoptotic cells along the dorsoventral (DV) axis of the neural ectoderm. Interestingly, the zone with high apoptotic density extends progressively to the posterior region following somite formation (see rectangles in Figures S1C and S1D). We found that apoptotic cells tend to appear in the trunk neural plate at the 3-somite stage and persist until the complete closure of the tube. Results Apoptosis is highly increased at DLHP and required for neural tube bendingĪs a first step to define how apoptosis participates in neural tube morphogenesis, we mapped the spatiotemporal occurrences of apoptotic cells during neural plate folding ( Figure S1A, orange and blue arrowheads Video S1) and neural tube closure ( Figure S1A, red arrowhead) of chicken embryos, focusing particularly on trunk neural tissue ( Figure S1B) where apoptosis is highly abundant ( Figures 1A and S1C and S1E). This transient force would lead to a progressive deformation of the epithelium and thus participate in epithelium bending. These deformations were partly maintained, leading us to propose that apoptotic neighbors keep some memory of the apoptotic force. The contraction of this cable coincides with a fast apico-basal displacement of the apoptotic nucleus dependent on myosin II contractility and with apical and basal deformations. We show that an actomyosin cable spreads along the apico-basal axis of the dying cell and generates an apico-basal force before cell extrusion. We found that apoptosis occurs at a high frequency at the DLHP of the neural tube shortly before or at the time of bending and revealed that apoptosis plays a role in DLHP morphogenesis. Here, we use neural tube bending in avian embryos as a model to explore the dynamics of apoptotic cells and decipher how they impact the surrounding tissue. Together with the morphological defects observed when apoptosis is prevented, these data strongly suggest that the neuroepithelium keeps track of the mechanical impact of apoptotic cells and that the apoptotic forces, cumulatively, contribute actively to neural tube bending. This force, which relies on a contractile actomyosin cable that extends along the apico-basal axis of the cell, drives nuclear fragmentation and influences the neighboring tissue. Using avian embryos, we found that apoptotic cells generate an apico-basal force before being extruded from the neuro-epithelium. Neural tube closure defects have been reported when apoptosis is impaired in vertebrates, although the cellular mechanisms involved are unknown. Here, we use the formation of the neural tube to determine how apoptosis contributes to morphogenesis in vertebrates. However, how this applies to vertebrate morphogenesis remains unknown. Apoptosis plays an important role in morphogenesis, and the notion that apoptotic cells can impact their surroundings came to light recently.
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