Morphogenesis is the process by which an organism, organ or tissue develops its shape/morphology. Realised through cellular activities, which are governed by gene regulatory circuits, morphogenesis can be reduced, at a larger spatial scale, to the spatiotemporal changes of three effective physical quantities within tissues: volumetric growth, tissue material properties and active forces. Estimated from experimental data, these three quantities can be implemented in a computational model to assess if they jointly recapitulate the dynamics of morphological changes observed in the real embryos.
Quantifying 3D growth within large tissues is relatively straightforward thanks to advances in tissue clearing and imaging. On the other hand, quantifying in 3D the anisotropic and spatially-inhomogeneous material properties of tissues, as well as the active forces they experience, remains highly challenging. However, high-resolution imaging of the 3D collagen network architecture of tissues might provide a good proxy for estimating their anisotropic material properties. Similarly, high-resolution imaging of the 3D arrangement of the supracellular actin network might greatly help estimating the spatial distribution of active forces inside the developing tissue. In the current study, we aim at producing such high-resolution imaging data (i.e., spatial distribution of proliferating cells, of collagen fibres and of supracellular actin bundles) required for estimating all three physical quantities. These data will later be implemented in a numerical model to attempt recapitulating the morphogenetic processes that lead to the development of overlapping skin scales in snakes.
In order to produce high resolution imaging of the collagen network architecture in very large samples (such as developing snake scales), we developed a simple and robust method of whole-mount collagen staining with the ‘Fast Green’ dye. We then use confocal and light-sheet microscopy, on samples stained with this new method, to study snake skin scale development in 3D, from the formation of a hexagonal pattern of placodes in the initially flat skin, to the point when scale anlagen acquire the overlapping morphology observed in adult skin scales. We demonstrate that the placode pattern appears row-by-row and transforms into a pattern of bump-shaped scale anlagen where each bump is bearing one placode. Meanwhile, we show that the so-called ‘dermoepidermal elevations’ reported in the literature as scale anlagen are artefacts of dehydration. We then study later stages of snake skin scale development and show that changes in the scale anlage geometry are associated with reorganisation of cell proliferation patterns as well as the development of dermal structures characterised here for the first time: supracellular bundles of dermal actin filaments, a collagenous ‘scale plate’ and a ‘bridge’. Once fully developed, the latter is composed of two parallel collagenous sheets, in the plane of the scale, that connect the two lateral sides of the scale. As we identify the presence of two nerves between the two collagenous sheets of the bridge, we suggest that this structure serves a mechano-sensory function. The complexity of the 3D architectures of the collagen and actin networks highlights the significance of tissue mechanics in the morphogenesis of overlapping scales.
