Abstract
During development, coordinated cell behaviors orchestrate tissue and organ morphogenesis. Detailed descriptions of cell lineages and behaviors provide a powerful framework to elucidate the mechanisms of morphogenesis. To study the cellular basis of limb development, we imaged transgenic fluorescently-labeled embryos from the crustacean Parhyale hawaiensis with multi-view light-sheet microscopy at high spatiotemporal resolution over several days of embryogenesis. The cell lineage of outgrowing thoracic limbs was reconstructed at single-cell resolution with new software called Massive Multi-view Tracker (MaMuT). In silico clonal analyses suggested that the early limb primordium becomes subdivided into anterior-posterior and dorsal-ventral compartments whose boundaries intersect at the distal tip of the growing limb. Limb-bud formation is associated with spatial modulation of cell proliferation, while limb elongation is also driven by preferential orientation of cell divisions along the proximal-distal growth axis. Cellular reconstructions were predictive of the expression patterns of limb development genes including the BMP morphogen Decapentaplegic.
Highlights
Morphogenesis, or the origin of biological form, is one of the oldest and most enduring problems in biology
We describe here the Massive Multi-view Tracker (MaMuT) software that allows visualization, annotation, and accurate lineage reconstruction of large multi-dimensional microscopy data
Three-day old transgenic embryos with fluorescently labeled nuclei were mounted for Light-sheet Fluorescence Microscopy (LSFM) in low melting agarose with scattered fluorescent beads
Summary
Morphogenesis, or the origin of biological form, is one of the oldest and most enduring problems in biology. As limbs develop in Parhyale, the MaMuT software tracks how cells behave, and how they are organized This analysis revealed that for cells to produce a limb bud, they need to split at an early stage into separate groups. Other mechanisms like differential cell proliferation and cell rearrangement could play a role in the formation of limb buds and their elongation along the proximal-distal (PD) axis These processes have not been possible to live image and quantify in direct developing arthropod limbs. We validated our cellular models of morphogenesis by studying the expression of developmental regulatory genes implicated in limb patterning and growth
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