Determining the role of patterned cell proliferation in the shape and size of the Drosophila wing Jaime Resino, Patricia Salama-Cohen, and Antonio Garcı´a-Bellido* Centro de Biologı´aMolecular Severo Ochoa, Universidad Auto´noma de Madrid, Cantoblanco 28049 Madrid, Spain Contributed by Antonio Garcı´a-Bellido,April 5, 2002 The present work is a detailed analysis of the numerical and growing disk corresponds with that of the clones seen in the adult positional parameters of cell proliferation in all of the derivatives wing, indicating that there are no major changes in the relative of the wing disk. We have made use of twin clones resulting from position of neighboring cells during the eversion of the disk at mitotic recombination events at three different ages of develop- metamorphosis (9). During the larval and pupal periods cell ment. The interfaces between twin clones indicate the relative death in the disk affects a very low number of cells in the hinge. position in the anlage of the mother cells. Interface types vary with There are therefore no major morphogenetic changes associated age of clone initiation and with wing regions. They are indicative with cell death in late larval or pupal stages (10). of the main allocation of postmitotic cells of the growing clones. But how do these morphogenetic parameters relate to the final Growth is exponential and intercalar, i.e., the progeny of ancestor constant wing shape and size? It has been proposed that cells becomes more and more separated. Clones are compact, compartment boundaries work as ‘‘organizers’’ of compartment indicating that daughter cells tend to remain side by side. The growth and patterning. Along the growing A͞P boundary, the shape of the clones is wing region characteristic. Subpopulations of selector gene engrailed (en), acting in the P compartment, elicits cells grow preferentially along veins and wing margins and show the expression of genes encoding for diffusible ligands. In the P characteristic shapes in different pleural regions. The shape and compartment en directs the expression of hedgehog (hh), and size of the adult wing regions largely depend on the shape of through it promotes the expression of decapentaplegic (dpp)ina clones and hence of the allocation of successive rounds of daughter few cells in the A compartment. These morphogens are proposed cells. The role of mitogenic morphogens in wing size and shape is to promote cell proliferation and later vein patterning (see refs. discussed. 11–14 for reviews). In a similar way, in the D͞V boundary the selector gene apterous (ap) expressed in the dorsal compartment n multicellular organisms morphogenesis highly depends on affects the regulation of the downstream effector wingless (wg), another diffusible ligand, which has been suggested to act as a Icell proliferation. Morphogenesis relates to the genetic mech- ͞ anisms that determine specific sizes and shapes. Morphogenetic morphogen in growth and patterning from the D V boundary analyses need a detailed description of growth in terms of cell (see refs. 11–13 for reviews). However, whereas the role of these lineages. Cell lineage studies reveal spatial and numerical pa- morphogens in the wing pattern formation is well established, rameters of ordered cell proliferation, an indication of genetic their role in the control of cell proliferation leading to size and control of cell behavior. The wing disk of Drosophila melano- shape of the wing remains elusive (13, 15, 16). gaster possibly is, in this sense, the best-studied growing anlage. In this work we analyze in detail the cell proliferation param- The imaginal wing disk is a monolayer of cells that give rise to eters of the wing anlage by using twin clones, the labeled the adult epidermis of the dorsal mesothorax, including notum offspring of the daughter cells of a cell in which a mitotic recombination event has taken place. In this way the topological and wing. Cell lineage analyses of the disk have been carried out position of the mother cell of a clone and its subsequent growth with mitotic recombination clones labeled with mutant but can be estimated and the geometrical parameters of cell prolif- gratuitous cell markers (1, 2). These clonal analyses have re- eration can be evaluated. vealed clonal restrictions that separate so-called ‘‘compart- ments,’’ subdividing the early anlage in four major compart- Materials and Methods ments, anterior͞posterior (A͞P) and dorsal͞ventral (D͞V). A Clonal Analysis. Clones in the adult. Mitotic recombination was subsequent subdivision separates notum and pleura from the generated by the FLP͞FRT technique (17), by heat shock wing proper (3, 4). New clonal restrictions, less stringent than in treatment in a water bath at 37°C for 10 min. f36a hs-FLP; mwh compartment boundaries, later symmetrically subdivide the dor- ϩ P{f } FRT ͞FRT larvae were treated at 38–62, 48–72, or sal and ventral wing compartments into sectors delimited by the 77a 80B 80B 60–84 h after egg laying (AEL). A total of 712 twin clones were veins (5, 6). Cell proliferation within these compartments and studied (230 at 38–62 h, 204 at 48–72 h, and 278 at 60–84 h). wing sectors is more undetermined with clone borders overlap- Clones in larval discs. Mitotic recombination was generated by ping in the same regions of different wings. The shape of these the FLP͞FRT technique, by heat shock treatment in a water bath clones is, however, region characteristic, symmetrical in both ϫ ͞ at 37°C for 30 min. hs-FLP; P{2 GFP} FRT40A FRT40A larvae dorsal and ventral surfaces and near symmetrical in both anterior were treated at 24–48, 48–72 or 70–96 h AEL. Larvae were and posterior compartments (1, 2); see ref. 7 for review. dissected during the third larval stage. Twin clones were visu- In the wing disk and the presumptive wing blade in particular, alized in a BioRad Radiance 2000 confocal microscope. Seventy cell proliferation increases the number of cells in an exponential twin ventral clones were studied. The multiple wing hair (mwh) mode, with an average cycle time of 8.5 h (8). The wing disk and forked36a (f36a) mutations and the 2ϫGFP and wild-type primordium in the embryo contains about 20 cells and the forked transgene (fϩ) are gratuitous genetic variants in prolif- proliferation period ends with about 50,000, the equivalent to erating cells. We have found no systemic differences in the 10–11 rounds of cell division (8). Direct observation of growing number of cells (size) of the twin clones. Thus, in the adult wing imaginal discs has shown that clusters of neighboring cells, not the f͞mwh size correlation coefficient is 1.0294 Ϯ 0.4. clonally related, enter both the S phase of the cell cycle and mitosis in synchrony (9). Anaphases in a cluster are randomly oriented in the planar axis, but subsequently the two daughter Abbreviations: A͞P, anterior͞posterior; D͞V, dorsal͞ventral; AEL, after egg laying; PBT, cells allocate along either the A͞Paxis(y axis) or the proximo- PBS͞0.1% Tween 20; GFP, green fluorescent protein. distal axis (x axis) (9). Moreover, the shape of the clones in the *To whom reprint requests should be addressed. E-mail: [email protected]. 7502–7507 ͉ PNAS ͉ May 28, 2002 ͉ vol. 99 ͉ no. 11 www.pnas.org͞cgi͞doi͞10.1073͞pnas.072208199 Downloaded by guest on September 26, 2021 location of the first mother cell of the twin. That must apply to parallel clones as well, and thus the topological position of the mother cell of the twin must be in the middle of the interface of parallel clones and by extension in the center of clones. Because twin clones are of similar size, proliferation is, in addition to intercalar, exponential in all of the wing cells of the wing blade. This finding applies equally to both dorsal and ventral wing surfaces. The ratio of longitudinal to transverse width (measured in number of cells) of clones varies with the age of clone initiation. This ratio is 9.78 in clones initiated at 50 h AEL, 5.51 for those at 60 h, and 2.83 for those at 72 h. Previous work had shown that whereas mitotic spindles appear in the planar axis at random in clusters of dividing cells, the daughter cells allocate either longitudinally or transversally in the wing anlage (9). Those ratios reflect the alternating orientation of the first cell division of the mother cell of twin clones. Thus, those figures indicate that the wing blade anlage grows preferentially distalward at the beginning and more isodiametrically at the end of development. The wing blade: Shape and size of clones. A plot of twin interfaces in the wing reveals some precision to these general trends. In Fig. 1C we observe mainly longitudinal interfaces in wing sector A and D whereas transversal interfaces are abundant in other regions. Longitudinal interfaces and clones bend in the distal wing margin to run parallel to it. Longitudinal clones tend also to be associated with certain pattern elements such as veins. This histotypic restriction can extend for hundreds of cells (Figs. 2A and 3 A and B). In fact, the veins are associated with late restriction borders (5). The same restriction applies to long narrow clones running along the wing margin in both most anterior and most posterior margins (Fig. 2A). There are some indications that the posterior wing margin may be considered a hidden vein, because of the expression along it of rhomboid (rho) Fig. 1. Clones in the adult wing. (A) Some examples of representative twin (a vein-specific marker) (20) and the absence of blistered (bs) (f and mwh) clones; A–D, wing sectors.