Abstract
Quantitative analysis of the dynamic cellular mechanisms shaping the Drosophila wing during its larval growth phase has been limited, impeding our ability to understand how morphogen patterns regulate tissue shape. Such analysis requires explants to be imaged under conditions that maintain both growth and patterning, as well as methods to quantify how much cellular behaviors change tissue shape. Here, we demonstrate a key requirement for the steroid hormone 20-hydroxyecdysone (20E) in the maintenance of numerous patterning systems in vivo and in explant culture. We find that low concentrations of 20E support prolonged proliferation in explanted wing discs in the absence of insulin, incidentally providing novel insight into the hormonal regulation of imaginal growth. We use 20E-containing media to observe growth directly and to apply recently developed methods for quantitatively decomposing tissue shape changes into cellular contributions. We discover that whereas cell divisions drive tissue expansion along one axis, their contribution to expansion along the orthogonal axis is cancelled by cell rearrangements and cell shape changes. This finding raises the possibility that anisotropic mechanical constraints contribute to growth orientation in the wing disc.
Highlights
The Drosophila larval wing imaginal disc is a powerful model system in which to study the integration of diverse types of regulatory cues for tissue growth
Low levels of 20E stimulate cell division in cultured wing discs We directly tested the ability of low levels of 20E, with or without insulin, to maintain proliferation in explants from mid-third instar larvae [96 h after egg laying (AEL)]
We monitored the numbers of phospho-histone H3-positive proliferating cells in freshly explanted discs, and compared them with discs cultured in the absence of hormones, or in the presence of 20 nM 20E, 1 μM insulin, or both hormones (Fig. 1A,B)
Summary
The Drosophila larval wing imaginal disc is a powerful model system in which to study the integration of diverse types of regulatory cues for tissue growth. The wing disc is a relatively flat epithelial sac that grows during larval stages of development. As it grows, the rapidly proliferating cells on one side (the wing pouch) become pseudostratified, whereas cells on the other side become squamous. The size and shape of the wing are largely, but not entirely, determined during the larval growth phase. At this stage, signaling centers located at the anterior-posterior (AP) and dorsal-ventral (DV)
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