Volume conservation principle involved in cell lengthening and nucleus movement during tissue morphogenesis Michael A. Gelbarta,b, Bing Hec, Adam C. Martinc,d, Stephan Y. Thibergea, Eric F. Wieschausc, and Matthias Kaschubea,e,1 aLewis-Sigler Institute for Integrative Genomics, Carl Icahn Laboratory, Princeton University, Princeton, NJ 08544; bGraduate Program in Biophysics, Harvard University, Boston, MA 02115; cDepartment of Molecular Biology, The Howard Hughes Medical Institute, Moffett Laboratory 435, Princeton University, Princeton, NJ 08544; dDepartment of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139; and eFrankfurt Institute for Advanced Studies, Faculty of Computer Science and Mathematics, Goethe University, D-60438 Frankfurt am Main, Germany Edited by Thomas D. Pollard, Yale University, New Haven, CT, and approved October 8, 2012 (received for review March 30, 2012) Tissue morphogenesis is the process in which coordinated move- the basal end, resulting in cells of stereotypical columnar shape ments and shape changes of large numbers of cells form tissues, (Fig. 1B, Left). When gastrulation starts, individual cells constrict organs, and the internal body structure. Understanding morphoge- at their apical end and undergo elongation. Cells then shorten and netic movements requires precise measurements of whole-cell shape widen their basal ends, transforming shapes from columnar to changes over time. Tissue folding and invagination are thought to be wedge-like (6, 10–12) (Fig. 1B, Right). Cell nuclei are initially close facilitated by apical constriction, but the mechanism by which to the apical surface but move basally during the early phase of changes near the apical cell surface affect changes along the entire furrow formation (6, 13). These changes concerted across a large apical–basal axis of the cell remains elusive. Here, we developed number of cells are thought to underlie ventral furrow formation Embryo Development Geometry Explorer, an approach for quantify- in Drosophila (1). However, by what mechanism they are con- ing rapid whole-cell shape changes over time, and we combined it trolled and temporally coordinated with one another remained with deep-tissue time-lapse imaging based on fast two-photon mi- unclear from these studies, which were mostly based on fixed data. croscopy to study Drosophila ventral furrow formation. We found Characterizing the detailed dynamics of cell shape changes that both the cell lengthening along the apical–basal axis and the helps to elucidate the mechanism governing these changes. In movement of the nucleus to the basal side proceeded stepwise and a recent study (14), we observed that apical constriction in were correlated with apical constriction. Moreover, cell volume lost ventral furrow cells in Drosophila is highly dynamic. By com- apically due to constriction largely balanced the volume gained ba- bining live imaging, image analysis, and genetic methods, we sally by cell lengthening. The volume above the nucleus was con- identified a pulsatile actin–myosin network, which reduces apical served during its basal movement. Both apical volume loss and cell area in steps, suggestive of a ratchet-like mechanism. lengthening were absent in mutants showing deficits in the contrac- It is conceivable that initial changes in cell length and nuclear tile cytoskeleton underlying apical constriction. We conclude that position might be secondary consequences of apical constriction a single mechanical mechanism involving volume conservation and and that the associated contractile machinery might, therefore, apical constriction-induced basal movement of cytoplasm accounts constitute the major driving force of ventral furrow formation quantitatively for the cell shape changes and the nucleus movement (3, 15) (Fig. 1C, Left). However, alternative scenarios seem in Drosophila ventral furrow formation. Our study provides a com- plausible, in which separate, albeit sufficiently concerted, active prehensive quantitative analysis of the fast dynamics of whole-cell cellular processes drive the sequence of apical and basal cell shape changes during tissue folding and points to a simplified model shape changes (16–19) and the relocation of the nucleus (20, 21) for Drosophila gastrulation. (Fig. 1C, Right). For instance, fibroblast elongation is believed to involve ends of growing microtubules promoting actin poly- two-photon imaging | 4D reconstruction | segmentation merization (19). To shed light on the mechanisms underlying cell elongation uring development, sheets of cells undergo dramatic rear- and nuclear movement, a precise characterization of the fast Drangements to generate the complex 3D structures of the spatiotemporal dynamics of whole-cell shape changes in a large body and internal organs (1–3). This process, called tissue mor- number of cells is necessary. Deep-tissue live-imaging techniques phogenesis, results from coordinated cell shape changes and provide the means for visualizing outlines of entire cells with movements that collectively deform tissues (4–7). Although much a temporal resolution of a few seconds (8). However, most is known about the role of cell patterning and signaling in tissue previously proposed methods for studying cell shape changes are morphogenesis, our understanding of how tissues acquire their restricted to 2D cross-sections of cells (22), whereas those shapes is relatively immature. Shedding light on this central aspect methods enabling whole-cell reconstructions are limited to rel- atively simple and static shapes (23, 24) or rely heavily on manual of tissue morphogenesis requires detailed characterizations of the editing (25). Here, we therefore developed a cell shape analysis dynamics of shape changes on subcellular, cellular, and tissue levels. However, available methods for performing the necessary morphological analyses are still relatively rudimentary (8, 9). Author contributions: M.A.G., B.H., A.C.M., E.F.W., and M.K. designed research; M.A.G., An important example of tissue morphogenesis is tissue folding B.H., A.C.M., S.Y.T., E.F.W., and M.K. performed research; S.Y.T. contributed new and invagination. Gastrulation, the process in which the basic body reagents/analytic tools; M.A.G. and M.K. analyzed data; and M.A.G., B.H., A.C.M., E.F.W., plan is laid out, begins in Drosophila with the formation of the and M.K. wrote the paper. ventral furrow and subsequent invagination of the mesoderm The authors declare no conflict of interest. primordium (Fig. 1A), a section of tissue that eventually gives rise This article is a PNAS Direct Submission. to muscles and fat bodies (1). Before gastrulation, the Drosophila Freely available online through the PNAS open access option. embryo undergoes cellularization, during which time the syncytial 1To whom correspondence should be addressed. E-mail: kaschube@fias.uni-frankfurt.de. blastoderm is partitioned into individual cells. During cellulari- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. zation, cell membranes are formed progressing from the apical to 1073/pnas.1205258109/-/DCSupplemental. 19298–19303 | PNAS | November 20, 2012 | vol. 109 | no. 47 www.pnas.org/cgi/doi/10.1073/pnas.1205258109 Downloaded by guest on October 2, 2021 is suitable for deep-tissue imaging (Materials and Methods and A Anterior Fig. 1D). Cell membranes were visualized using a GFP-tagged transmembrane protein (E-Cadherin-GFP). Image stacks cov- Ventral μ – furrow ered a section of tissue of about 90 m in the ventral dorsal Posterior directions and 45 μm in the anterior–posterior directions ap- proximately centered on the ventral midline. The temporal res- olution (12–18 s) was chosen to be sufficiently high to capture the dynamics of apical constriction observed in ventral furrow cells Yolk (14) (Materials and Methods). We used EDGE to measure cell shapes from the two-photon Cells image stacks (Materials and Methods and Fig. 1 E and F)(a Apical constriction initiates Mesoderm invagination detailed description of EDGE is provided in SI Text and Figs. S1–S4, and the software is open source and freely available at B Cellularization Gastrulation C Apical Active cellular http://www.code.google.com/p/embryo-development-geometry- constriction forces explorer/). EDGE identifies cell outlines in individual 2D images Apical Nucleus based on a sequence of image-processing steps, including band- pass filtering, thresholding, and morphological thinning. Vertices Microtubule Basal and centroids of individual 2D cell outlines are extracted, and Nucleus cell outlines are then reduced to the polygons defined by the vertices. EDGE then tracks these polygons across z-stacks and D 2-Photon live imaging E-cad GFP Motors time to form 4D (i.e., three space dimensions and one time di- y mension) representations of cells. The starting point of tracking x is a reference image at some chosen depth and point in time (Fig. E 1E). Tracking was checked by visual inspection, and sporadic Acquire Filter, Extract tracking failures were hand-curated using EDGE’s built-in in- image threshold, vertices, terface for automated and manual error correction (SI Text and stacks thinning polygons Fig. S1). The accuracy of segmentation and tracking is assessed in Fig. S5. To use the capabilities of EDGE in understanding whole-cell shape changes, we first automatically estimated the apical and Make quantitative Stack polygons Track polygons basal limits of each cell at each point in time based on the la- measurements of to