Determining the Role of Patterned Cell Proliferation in the Shape and Size of the Drosophila Wing

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 ‘‘compartments,’’ 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. They were initiated at 48–72 h AEL. (specific of intervein regions) (21). Thus, an early determination Arrows indicate main axes of the twin clones starting in the topological of cells to become vein histotype may be associated with a position of the mother cell (circle). (B) Plot of twins in both wing surfaces. (C) particular mode of cell proliferation. Interfaces of the twin clones. Those clonal restrictions may cause asymmetries in size of twin clone size, because one of the members of the twin cannot cross them. This situation applies mainly to clones between the basal Staining and Antibodies. Dissected larvae were fixed for 20 min in ͞ trunks of veins, where clones are smaller than in more distal a 4% paraformaldehyde solution in PBT (PBS 0.1% Tween 20) regions (80% of clones in proximal regions show these asym- and inmunostained with either anti-Engrailed antibody (1:4) or ͞ metries). These singularities also reflect postmitotic cell alloca- anti-Armadillo antibody (1:20) in PBT-BSA (PBT 0.3% BSA). tion and therefore are indicative of differential cell behavior An Alexa Fluor 546-conjugated secondary anti-mouse antibody during cell proliferation associated with imaginal histotypes. (Molecular Probes) was used to detect primary antibodies Interestingly, at 50 h AEL, when the disk contains about 200 localization. Anti-Engrailed (18) and anti-Armadillo (19) anti- cells, 58.3% of the twins that abut the D͞V compartment bodies were provided by the Developmental Studies Hybridoma boundary, the interface of the twin runs along the wing margin Bank, University of Iowa, Iowa City. To determine nuclei shape, boundary. Twin clones initiated later, after the D͞V clonal density, and localization, discs were stained for 20 min ina1mM restriction, are in either the dorsal or the ventral surface. To-Pro-3 iodide (Molecular Probes) solution in PBT and washed Clones are very compact. ‘‘Split’’ clones, members of a clone three times in PBT for 20 min. separated from the main body by more than one cell diameter are rare: 5.2% in early (50 h) clones (n ϭ 136; clone size of about 400 Results cells). These clones consist of groups of fewer than 10 cells. In Clones in the Adult Cuticle. The wing blade: Position of clone mother one case, a clone was split in two groups of 300 and 160 cells each. cells. The use of twin clones allows us to reveal the topological Thus split clones are preferentially late events. Reciprocally, few position of mother cells. That topological position corresponds (a total of 2–30) nonmarked cells within clone territories are also

to some point in the interface between the twin clones. Twin rare: 1.2% of early initiated (50 h) clones. They appear in the BIOLOGY clones may appear in tandem along the proximo-distal (x)axis borders of the clone. Related to these split clones is indentation DEVELOPMENTAL of the wing, run parallel along the A͞P(y) axis, or a combination in clone borders. This finding is very frequent in the most of both (Fig. 1A). In the first type, the interfaces are of few cells, posterior wing sector of the wing, perhaps reflecting a hidden in the second of many cells, indicating a preferential location of vein plexate, the ‘‘anal plexus.’’ daughter cells along the proximo-distal axis of the wing blade. A Notum and pleura. The same clonal analysis in regions of the plot of tandem clones on the wing shows that the position of the mesothorax other than the wing blade reveals new territorial mother cells can be anywhere in the anlage (Fig. 1B). Because specificities in cell proliferation. In the notum the cell marker f longitudinal twin clones extend at both sides of these positions is not very reliable for trichomes and the data correspond to mwh (Fig. 1B) it follows that cell proliferation is intercalar, with clones. In the notum, large (early initiated) clones are more successive mother cells being more and more separated from the isodiametric than clones in the wing blade, although they have a

Resino et al. PNAS ͉ May 28, 2002 ͉ vol. 99 ͉ no. 11 ͉ 7503 Downloaded by guest on September 26, 2021 Fig. 2. Different shapes of clones in different adult regions (A and B) and interfaces of the wing disk (C–F). (A) Outlines of clones running along the veins and the wing margin. (B) mwh clones in a notum and pleura lateral view (and coxa of ﬁrst and second legs). Blue background in second leg distinguishes imaginal discs derivatives. All thoracic regions, except mesothorax, are in green. Bases of wing (in mesothorax) and haltere (in metathotax) are in shadow. Black lines mark thorax sutures. sc, scutum; sct, scutellum; Abd., abdomen; L I, L II, and L III, ﬁrst, second, and third legs. (C) Twin (red and yellow) clones in the dorsal side of the disk. Arrows point to clones growing perpendicular to the A͞P compartment boundary. Green line separates dorsal from ventral wing surfaces. (D) Twin clones in the most ventral side of the wing disk. (E) Main directions of the clones. (F) Interfaces of twin clones. A, anterior; P, posterior compartments. (C–F) Blue lines mark A͞P boundary. (D–F) Dash lines separate squamous cells from cylindric cells. Dark gray marks posterior compartments.

main A͞P component (Fig. 2B) (1, 2). Clones running along the clones in the (dorsal) notum are, again like in the adult, more border between both heminota are narrow and long (see below). isodiametric and can grow along the A͞P compartment bound- Clones crossing the notal sutures do not show differences in ary or perpendicular to it but always with shapes in position shape, an indication that these infolded cuticles (apodemes) do characteristic ways. not represent clonal restrictions. In fact, direct counting of cells A more complex, albeit constant, clonal pattern appears in in the adult sutures shows very few cells (data not shown). the ventral side of the wing disk, which corresponds to the In the pleura, clones are also rather isodiametric although they adult pleural regions (Fig. 2 E and F). The pleural region in the run preferentially perpendicular to the notum͞pleura sutures disk contains about 600 squamous cells (the peripodial mem- (Fig. 2B). They run parallel to the junction between wing pleura brane; Fig. 3 D and E) as well as cylindrical cells like in the rest and the derivatives of the mesothoracic leg disk (Figs. 2B and of the epithelium. The borders of this cell shape discontinuity 3C). Thus, the regions connecting the contralateral discs and the are not sharp, but can be defined easily (dash lines in Fig. 2 E ipsolateral leg disk show a preferential elongated shape along the and F and 3D). In addition, there is a stripe of very elongated adult borders. This situation may extend to the junction between fusiform cells at both sides of the A͞P boundary, anterior to the wing and the haltere discs of the same side but we do not have the peripodial membrane (Fig. 3E). We have used gene enough large clones to confirm it. A survey of twin clones in the expression as a way of labeling the different pleural regions legs also indicates a preferential growth along the legs in narrow (Fig. 3D). Ultrabithorax (Ubx) is expressed mainly in squamous clones (data not shown) (1). cells of the peripodial membrane (22). The puckered-LacZ (puc-z) marker appears as a double horn (23), in the anterior Clones in the Imaginal Wing Disc. In the imaginal disk twin clones horn the cells are fusiform running to the stalk of the imaginal are marked by the expression of four doses of green fluorescent disk (Fig. 3D). Given the squamous shape of the cells of the protein (GFP) (4ϫGFP) vs. 0ϫGFP in a background of 2ϫGFP peripodial membrane, clones appear large in surface but (see Fig. 3 F–H). The twin clones visualized in the imaginal disk contain few cells. Clones including the fusiform cells run of late third instar larvae show the same features observed in the parallel to the A͞P boundary with a width of only one cell adult cuticle. This applies to the presumptive wing blade and (Figs. 2D and 3 G and H). This clone and cell elongation starts notum (Fig. 2C). Clones are compact with clone interfaces in early in development because it is noticed in early third larval tandem, in parallel, or composite. They are mainly elongated in instar discs (Fig. 3H). More distal cells of the same clone or its the proximo-distal axis in the wing blade, where, depending on twin expand to the rest of the ventral surface (Fig. 2D). Large age of clone initiation, they may cross the D͞V boundary or be clones seem to be distally restricted to either side of the separated by it (Fig. 2C). Clones in the wing base can extend to histologic border of the peripodial membrane (Fig. 2D). The more central regions of the hinge (arrows in Fig. 2C), although main trends in the shape and extension of clones in the ventral

7504 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.072208199 Resino et al. Downloaded by guest on September 26, 2021 Fig. 3. Representative clones in the adult and imaginal wing disk. (A and B) Cases of twin clones in the adult wing veins (A) and wing margin (B). Note in B that the f (blue) clone does not reach the wing margin. mwh has a red outline. (C) Clones running parallel to the border of the wing disk and second leg derivatives. Thick orange line separates prothorax from mesothorax. Thin orange line separates mesopleura from sternopleura. Dotted line marks wing A͞P boundary. TS, thorax spiracle; st, sternopleural chaetae. (D) Scheme of gene expression pattern of several histotypic markers in the ventral surface: puckered-Lacz (puc-z), Engrailed (En), Ultrabithorax (Ubx), Iroquois (Iro). (E) Expression of Armadillo (red) marking apical cell membranes and To-Pro-3 iodide (blue) marking cell nuclei, show the morphology of fusiform cells (white arrows) between cylindrical (pleura) and squamous cells (peripodial membrane). (F) Twin 2ϫGFP (red outline)͞0ϫGFP (white outline) clones in the peripodial and the posterior pleura. (G) Twin clone in the ventral horn of fusiform cells. Late third instar disk. (H) Similar twin clones in early third instar disk, showing the elongated clone shape with cell body shorter than later, as observed in G.(E–H) Wing discs are at the same magniﬁcation; (Insets) higher magniﬁcation.

surface of the wing disk are visualized in a plot in Fig. 2 E and growing wing disk. The interface between the twin clones reveals F. Clones expand in distal regions and become elongated and the topological position of the mother cell of both clones. It BIOLOGY narrow in proximal (stalk) regions, where they are defined by follows that for daughter single clones the mother cell is topo- DEVELOPMENTAL parallel bundles of cells of different lineages. During disk logically located in its center. Interfaces appear everywhere in eversion in metamorphosis, wing blade, notum, and pleura the adult wing but with shape, extent, and positions that are wing pass through this stalk to merge with the derivatives of sector and initiation age specific. Frequencies and position ipsolateral and contralateral discs. specificities correspond to the developmentally changing location of clusters of cells in the G2 stage of the cell cycle, when the Discussion cells are sensitive to induced mitotic recombination (5, 9). The use of twin clones has allowed us to get a detailed insight in Mitotic spindle orientations in the planar axis of the wing how ordered cell proliferation generates the shape and size of the epithelium are random, but postmitotic cells allocate preferen-

Resino et al. PNAS ͉ May 28, 2002 ͉ vol. 99 ͉ no. 11 ͉ 7505 Downloaded by guest on September 26, 2021 tially along the x or y axis of the wing blade (24). The shape of expression of transcription factors distalless (dll) (33) and vesti- interfaces reflects the main orientations of successive clonal cell gial (vg) needed for distal wing specification (34, 35). The same divisions. The fact that twin clones are of similar sizes and genes are operative later in the differentiation of pattern ele- mother cells appear anywhere in the wing indicate that growth ments, wg in the wing margin and dpp in veins. The role of these is exponential and intercalar. Clones are in the wing blade genes in growth or cell proliferation is less clear (see below). The preferentially longitudinal, along the proximo-distal axis, with absence of dpp or its receptors, such as thickveins (tkv), prevents ratios of length͞width always larger than one (9.8 in early clones cell proliferation in clones. The clonal overexpression of acti- and 2.8 in late ones). Thus, cell allocation and growth in the wing vated receptor tkvQ253D leads to extra growth in pleural regions occurs in a proximo-distal direction at the beginning, perpen- but less so in the wing blade, where mutant cells do not dicular to the base of the primordium, and more isodiametricaly intermingle with surrounding cells in mosaics (36). wg overex- or anterior-posteriorly later. This finding is in agreement with pression clones initiated during larval development are normal the expansion of distances between precursor cells in gynan- in the wing blade (37). These observations are compatible with dromorph maps when compared with the derived adult land- a permissive role rather than an instructive one of these two marks (25). classical morphogens in an ordered cell proliferation. There are regional differences in these general cell allocation We have not found in the present work any relationship trends. The preferential longitudinal orientation of growth in between the known gradient of morphogens emanating from intervein sectors changes to parallel to the wing margin in compartment borders in cell proliferation, either in mitotic rates anterior and posterior wing borders and perpendicular to the or in clone shapes or main orientations. We have seen that margin in the tip of the wing. In contrast, clones in the notum are growth seems to be related rather to regional specifications. We more isodiametric with the exception of a ribbon of cells along will see its relation to positional heterogeneities of the prolif- the thorax middle line between contralateral discs. Departures erating cells. Patterned cell proliferation varies in a number of from the regularity of the geometry of clones in wing sectors morphogenetic mutants. For example, en mutant wings show a appear in the wing veins and wing margin. Narrow clones may mirror image transformation of a posterior compartment into an run for hundreds of cells of vein histotype (5), revealing a anterior one. This substitution is correlated with a pattern of cell connection of cell allocation to hystotipical cell specification. proliferation like in the anterior compartment (38). The gene The rule of similar clone sizes in twins that applies to the wing nubbin (nub) encoding a transcription factor (39) causes in blade and notum is broken in several regions of the wing disk. mutant condition smaller wings reduced along the proximo- Clones abutting the base of the veins are smaller than those in distal axis (x axis). In nub mutant wings cells show preferential the distal wing sectors. The same applies to the D͞V wing margin alocation perpendicular to that axis (40). Mutations in genes (but not the A͞P boundary) and to cells that differentiate the related to ligands and receptors of several signaling pathways squamous territory of the peripodial membrane, its most distal reveal cell behaviors that point to the existence of positional border and the ‘‘horns’’ of fusiform cells anterior to the peri- values in wing sectors. Thus, in genetic mosaics, mutant clones podial membrane and connecting the pleura with the disk stalk. may show failures in the preferential allocation of daughter cells. These restrictions in growth may reflect, like in the veins, the Clones of Notch (N) mutant cells impair cell proliferation and appearance of differently specified (or differentiated) cell pop- tend to growth toward veins (41). Similar behavior applies to ulations that may act as clonal boundaries (see gene expression clones of mutations in extramacrochaetae (emc), encoding a patterns in Fig. 3D). These constraints to growth and shape of negative cofactor of basic helix–loop–helix transcription factors the clones indicate that the anlage is formed as a mosaic of (42). veinlet (rhove) mutations, which encodes for a transmem- differently specified cell territories with local limits related to cell brane protein of the Drosophila epidermal growth factor recep- proliferation constraints. tor signaling pathway, directs cells to grow away of veins, where Clones and members of twin clones appear very compact, with the wild-type gene is maximally expressed (43). Contrarily bs, all of the clone cells remaining side by side, with the exception encoding the Drosophila serum response factor (44) and ex- of rare ‘‘islands’’ and ‘‘lakes’’ that originate late in development. pressed in interveins, in the mutant condition in mosaics causes Why members of the clone remain together is difficult to explain cells to grow toward and differentiate as veins (21). In genetic whether daughter cells move freely in the epithelium. This mosaics, clones of nub mutant cells grow toward the base of the behavior may reflect an inertia or delay in the separation of wing (40). These mutant cell behaviors reveal properties of cells daughter cells, perhaps in the formation of new membranes (26) that relate to the generation of space and the acquisition of or the connection of cells by filopodia that retain cell recognition positional values (see below). Cell allocation possibly reflects or adhesion features. As a whole, it is the allocation of prolif- preferential cell adhesion and͞or affinity properties that relate erating cells in the disk that relates to the final shape of the wing. to position, as shown in cell dissociation and reaggregation Clone shape in imaginal discs (Fig. 2C) can directly be trans- experiments (45). Positional values seem to be intimately related formed into the shape of clones in the adult, indicating the to growth. vein (vn), a ligand Drosophila epidermal growth absence of morphogenetic movements or separation of neighbor factor receptor, causes in mutant combination with rhove,in cells in the eversion of the disk (9). However, the eversion of the mosaics, lower cell proliferation and reduction of the intervein wing imaginal disk through the stalk connecting it to the larval regions (43), as it occurs in emc territories that abut neighboring epidermis at metamorphosis may be an exception. The eversion veins (42). of the disk poses morphogenetic problems (27, 28). How is the A generative model of wing morphogenesis has been pre- stalk, which is few cells in circumference, enlarged to make a sented [the Entelechia model (6, 46)], in which dividing cells border of hundreds of cells with ipsolateral and contralateral acquire positional values intermediate between those of neigh- discs? boring cells and are allocated according to these values in We now turn to the question of the role of morphogens (such intermediate positions along the x and y axes by cell–cell as dpp or wg) in the growth and morphogenesis of the imaginal recognition to the best matching positions in this positional disk. The role of diffusible morphogenes in patterning of the values landscape. These values relate to the activity of so-called anlage is well documented. dpp concentration thresholds deter- ‘‘martial’’ genes, which determine highest values at restriction mines the expression of region-specific transcription factor genes borders and lowest away from the border. The highest values are like spalt (sal), spalt-related (sal-r) (29–31), and optomotor blind in compartment boundaries at the beginning and are substituted (omb) (32) in the wing blade in the early disk. On the other hand, by vein restriction boundaries later. With cell proliferation in the wg is operational in the definition of the wing margin and in the anlage, boundary values increase up to a species-specific maxi-

7506 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.072208199 Resino et al. Downloaded by guest on September 26, 2021 mum. Intercalar cell proliferation continues until intermediate sectors, because of mutant cells having similar (low or high) values reach a specific minimum where the value differences positional values. between neighboring cell become indistinguishable i.e., the Thus, the morphogenetic role of dpp (and wg) morphogens anlage reaches the Entelechia condition. could affect the wing vein patterning first, and the vein restric- This model accounts for and explains several observations. tion borders, or gene expression discontinuities, will act later as ͞ The formation of a new A P boundary in mosaics of en in the positional references to intercalary cell proliferation. In this later ͞ posterior compartment and of a new D V boundary in ap stage these morphogens will then be permissive rather than mosaics in the dorsal compartments is associated with nonau- instructive in cell proliferation. It seems from this work that wing tonomous extraproliferation. This observation was the basis for size and shape result from highly determined cell lineages with the proposal of a mitogenic role for the dpp and wg ligands a controlled postmitotic cell allocation in internal wing regions arising in those boundaries (11–13). The observations that the rather than from instructions of diffusible morphogens emanat- size of these overgrowths depends on the position of the clone ing from clonal restriction borders. Size and shape of the wing rather than on the extent of the border, and that this growth ends seem rather to be locally determined by cell interactions of the in pattern duplications, suggests a different interpretation. It proliferating cell population. may result from positional ‘‘accommodation’’ reminiscent of intercalar growth in regeneration experiments (43). This positive We thank S. Carroll, E. Bier, L. A. Baena, and other members of the lab accommodation can be negative in nub mosaics that nonauto- for reading and discussing this work. We also thank the Developmental nomously cause reduced growth distally to the mutant territory Studies Hybridoma Bank for providing antibodies. This work was (40). The activities of these mutations (emc, N, Drosophila supported by grants from the Direccioń General de Investigacioń epidermal growth factor receptor, and nub) are related to the Cientı´fica y Tećnica and an institutional grant from the F. Ramoń Areces acquisition of positional values. Low values (caused by Drosoph- to the Centro de Biologıá Molecular Severo Ochoa. J.R. is a fellow of the ila epidermal growth factor receptor) and high values (by emc) Comunidad Autońoma de Madrid, and P.S.-C. is a fellow of the may fail to elicit lack of intercalary cell proliferation in wing Universidad Autońoma de Madrid.

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