Cell divisions and EGFR activity in the male A7.

<p>(A–A″) Snapshots from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002874#pgen.1002874.s006" target="_blank">Video S1</a> (<i>esg</i>-Gal4 UAS-<i>nls-myc</i>-GFP pupa), from ∼15 to 29 h APF showing the expansion of the dorsal histoblast nests in the male A6 and A7 segments. In this and subsequent figures (except in adult cuticle preparations) the posterior part of the pupa is at the top. Note that the size of the A7 is smaller than that of the A6 (horizontal bars in A″). Approximate hours of development APF are also indicated. (B) <i>y</i>; <i>MD761</i>-Gal4 UAS-<i>y⁺</i>/+ female. Note the dark pigmentation in the A7 (arrow). (C) <i>MD761</i>-Gal4/<i>Abd-B^M1</i> male. See that the A7 (absent in the wildtype) is almost completely transformed into the A6. (D–D″) Posterior part of a <i>spi</i>-lacZ <i>esg</i>-Gal4 UASGFP pupa of about 26 h APF showing that <i>spi</i> levels (in red) are reduced in the A7 histoblasts (the A7/A6 ratio of signal intensity is 0,82±0,12; n = 10). Histoblasts are marked by <i>esg</i> in green. (E–E″) If we express a UAS-<i>Abd-BRNAi</i> (UAS-<i>Abd-Bi</i>) construct under the control of the <i>pannier</i> (<i>pnr</i>)-Gal4 driver (domain of expression in green), the levels of <i>spi</i>-lacZ are elevated in this domain (arrow). The arrowhead marks the lower <i>spi</i>-lacZ levels in A7 histoblasts where <i>Abd-B</i> expression is still high (note also the bigger cells). (F) UAS-Spi.m-GFP; <i>MD761</i>-Gal4/+ adult male (cross made at 17°C), with a small A7 segment (compare with a male expressing a UAS-<i>y+</i> construct (G), which, like the wildtype, has no A7); g, genitalia. (H) UAS-<i>Abd-BRNAi</i>/+; <i>MD761</i>-Gal4 UAS-<i>y⁺</i>/+adult male, showing the transformation of the A7 into the A4. (I) In UAS-<i>Abd-BRNAi</i>/+; <i>MD761</i>-Gal4/UAS-Raf^DN the size of the A7 segment is reduced as compared to that shown in H. (J–L′). Posterior abdomens of ∼22–24 h APF male pupae of the following genotypes: <i>His2A-RFP</i>/+; <i>MD761</i>-Gal4 UAS-GFP/+ (J, J′), UAS-Spi.m-GFP; <i>His2A-RFP</i>/+; <i>MD761</i>-Gal4 UAS-GFP/+ (K, K′), and <i>His2A-RFP</i>/UAS-Egfr^DN; <i>MD761</i>-Gal4 UAS-GFP/+ (L, L′), showing a slight reduction (L, L′) and an increase (K, K′) in histoblast number in the A7 with respect to the <i>His2A-RFP</i>/+; <i>MD761</i>-Gal4 UAS-GFP/+ pupae (J, J′): at about this time, in the wildtype, the histoblast number A7/A6 ratio is 0’47±0’07 (n = 4), it is 0’95±0’19 (n = 4) when we express <i>mspi</i> in the A7, and 0’31±0’04 (n = 4) when the Raf^DN product is present in the same segment. (M, M′) In <i>MD761</i>-Gal4 UAS-GFP/<i>Abd-B^M1</i> male pupa the number of histoblasts in the A7a dorsal nests approaches that of the A6a, and this number is reduced in <i>MD761</i>-Gal4 UAS-GFP/UAS-Raf^DN<i>Abd-B^M1</i> pupae (N, N′). At later stages, the A7 of the <i>MD761</i>-Gal4 UAS-GFP/<i>Abd-B^M1</i> pupae (O; marked in green) is bigger than the wildtype and it is strongly reduced when a Raf^DN protein is concomitantly expressed in this genetic background (P). Nuclei are marked in red and the A7 delimited by GFP expression (in green). In the lower panels (J′, K′, L′, M′, N′) the A7 dorsal nests are delineated in white and the A6 ones in green.</p

Inhibition of cell death does not prevent delamination of histoblasts.

<p>(A–A″″) Snapshots from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002874#pgen.1002874.s014" target="_blank">video S9</a>, from about 38 to 44 h APF, showing how the histoblasts from the male A7 left and right anterior dorsal nests of a (<i>p</i>)<i>esg</i>-Gal4 UAS-GFP male pupa delaminate as the nests meet at the central midline. The arrows indicate the delamination of some histoblasts. (B–B″″) Stills from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002874#pgen.1002874.s015" target="_blank">video S10</a>, made in a (<i>p</i>)<i>esg</i>-Gal4 UAS-GFP male pupa of about 42–50 h APF, showing delamination of A7 histoblasts (arrows). See how the “width” of the A7 segment (brackets at 42 h and 50 h) is reduced as delamination proceeds; g, genitalia. Numbers indicate approximate hours APF. (C, D) Two optical sections of a movie sequence in which the Apoliner construct, which reveals cell death <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002874#pgen.1002874-Bardet1" target="_blank">[59]</a>, is expressed under the control of the <i>MD761</i>-Gal4 driver in A7 histoblasts. The panel C is from about 40 h APF and panel D from about 42 h APF. The red arrows indicate three cells where the GFP reporter has nuclear localization (indicating apoptosis) and the white arrows point to what could be apoptotic bodies. (E–E′″) Snapshots from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002874#pgen.1002874.s016" target="_blank">video S11</a> showing the invagination of the male A7 in (<i>p</i>)<i>esg</i>-Gal4 UAS-GFP UAS-<i>Diap1</i> (<i>esg</i>-Gal4 <i>act</i>><i>y⁺</i>>Gal4/UAS-<i>Diap1</i>; UAS-<i>flp</i>/UAS-<i>Diap1</i>) pupae. The invagination takes place as in the wildtype although it may be delayed. Numbers indicate approximate hours APF. (F–I) Optical sections of (<i>p</i>)<i>esg</i>-Gal4 UAS-GFP (F, G) and (<i>p</i>)<i>esg</i>-Gal4 UAS-<i>Diap1</i> UAS-GFP (H, I) male pupae. Cross-sections (white lines) to the right and below in each figure show that in (<i>p</i>)<i>esg</i>-Gal4 UAS-GFP pupae there are some histoblasts under the epithelium at about 38 h APF (F), but they are not longer there by 48 h APF (G) (arrowheads). By contrast, in (<i>p</i>)<i>esg</i>-Gal4 UAS-<i>Diap1</i> UAS-GFP male pupae, the number of A7 histoblasts that remain under the epithelium is higher at about 38 h APF (H) and have not been completely eliminated by 48 h APF (I) (arrows). (J, K) The inhibition of cell death in UAS-<i>P35</i>/+; UAS-<i>P35</i>/<i>MD761</i>-Gal4 (J) and UAS-<i>Diap1</i>/+; UAS-<i>Diap1</i>/<i>MD761</i>-Gal4 (K) males does not prevent A7 elimination.</p

Genetic interactions between <i>emc</i> and the EGRF pathway.

<p>(A–B′) Stills from two videos (S14 and another one, not shown) done in pupae of the following genotypes: UAS-<i>Egfr</i>/+; <i>emc</i>-GFP <i>MD761</i>-Gal4/+ (A–A″) and <i>emc</i>-GFP <i>MD761</i>-Gal4/UAS-Spi.m-HRP (B, B′). The size of the A7 is increased in A–A″ and B, B′ with respect to the wildtype and <i>emc</i>-GFP expression seems slightly reduced in the A7 (compare with <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002874#pgen-1002874-g004" target="_blank">Figure 4E</a>). (C–C″″) Snapshots from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002874#pgen.1002874.s020" target="_blank">video S15</a> (∼35–48 h APF) showing a male pupa of the genotype UAS-Spi.m-GFP; <i>MD761</i>-Gal4 UAS-GFP/+. There is an excess of histoblasts in this segment (marked in green). The extrusion of LECs takes longer and that of histoblasts seems to be largely prevented. Numbers indicate approximate hours APF.</p

<i>emc</i> regulates extrusion of the male A7 histoblasts.

<p>(A, B) <i>emc^P5C</i> homozygous males show a small A7 segment (A), absent in <i>emc⁻</i> heterozygous conditions (B). (C) Males heterozygous for an <i>Abd-B</i> mutation present a small A7 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002874#pgen.1002874-SnchezHerrero1" target="_blank">[3]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002874#pgen.1002874-Tiong1" target="_blank">[4]</a>, and the <i>trans</i>-heterozygous combination <i>Abd-B^M1</i>/<i>emc¹</i> enhances this phenotype (D). Arrows indicate the A7 segment. (E) Distribution of <i>emc</i>-GFP in the A7 and A6 segments of a ∼38 h APF male pupa. There is <i>emc</i> expression in LECs and histoblasts, with higher levels in A7 than in A6 histoblasts. We have measured the difference in signal intensity between nuclei of the two segments and found that the A7/A6 signal ratio is 1, 32±0,15 (n = 4). See also higher levels at the periphery of histoblast nests. (F–F″) UAS-DsRed/+; <i>emc</i>-GFP <i>pnr</i>-Gal4/UAS-<i>Abd-BRNAi</i> male pupa of about 36 h APF showing that in the central region of <i>pnr</i> expression (red in F′, F″), where <i>Abd-B</i> levels are reduced, <i>emc</i>-GFP levels are also reduced (arrow in F). Levels remain high where <i>Abd-B</i> has not been eliminated (arrowhead in F) but also in midline cells (also with high expression in anterior segments; yellow arrow in inset). (G) <i>Abd-B^Fab7-1</i> homozygous adult male. The A6 disappears as it is transformed into the A7 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002874#pgen.1002874-Gyurkovics1" target="_blank">[20]</a>. (H) <i>Abd-B^Fab7-1 emc^P5C</i> homozygous male: there are small A6 and A7 segments (arrows), showing the <i>emc^P5C</i> mutation is epistatic over the <i>Abd-B^Fab7-1</i> mutation. (I–I″) Snapshots from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002874#pgen.1002874.s017" target="_blank">Video S12</a> in a ∼36–48 h APF <i>en</i>-Gal4 UAS-GFP/His2A-RFP male, showing the progressive disappearance of the A7 segment. Posterior compartments show <i>en</i> expression (in green), whereas nuclei are labeled in red. The arrow marks the A6p band of expression and the arrowhead the A8p cells. (J–J″) Snapshots from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002874#pgen.1002874.s018" target="_blank">Video S13</a>, taken at similar stages and with the same markers but in an <i>emc</i> mutant background (<i>en</i>-Gal4 UAS-GFP/His2A-RFP; <i>emc^P5</i>/<i>emc^P5</i>male). Note that, contrary to the previous panels (I–I″), the A6p en band does not move posteriorly, indicating that the A7 segment is not being extruded. Numbers indicate approximate hours APF.</p

Relationship between sex determination and Hox information in the development of an A7.

<p>(A) Wildtype female adult, showing the small A7 segment (arrow). (B–B″) Stills taken from a movie in which the histoblasts of a ∼15–27 h APF (<i>p</i>)<i>esg</i>-Gal4 UAS-GFP female pupa are marked in green. Note that at the end of this period the A7 is slightly smaller than the A6 (segments separated by white lines). vn and sn indicate ventral nest and spiracular nests, respectively. Numbers indicate approximate hours of development APF. (C, C′). Posterior abdomen of an <i>esg</i>-Gal4 UAS-GFP/<i>spi</i>-lacZ female pupa from ∼25 h APF showing that the expression of <i>spi</i>-lacZ in the A7 (C, C′ in red) is reduced compared with that of the A6. Histoblasts are marked by <i>esg</i> expression in green (C′). (D) In UAS-Spi.m-GFP/+; <i>MD761</i>-Gal4/+ females the A7 is slightly bigger than in the wildtype (arrow; compare with A). (E) Posterior abdomen of a <i>dsx</i>-Gal4/UAS-Raf^DN female, in which the A7 segment is reduced. The larvae were grown at 25°C and transferred to 29°C at the third larval stage. (F, G) The expression of <i>emc</i>-GFP (F) and <i>sqh</i>-GFP (G) in the A7 of female pupae of about 36–38 h APF is not higher than in the A6 (compare with the male expression in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002874#pgen-1002874-g004" target="_blank">Figs. 4E</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002874#pgen-1002874-g002" target="_blank">2F</a>, respectively). (H) Posterior abdomen of a female pupa marked with (<i>p</i>)<i>esg</i>-Gal4 UAS-GFP at about 36 h APF. The optical section below (the white line indicates the plane of section) shows a slight accumulation of histoblasts in the central region of the segment under the epithelium (arrows in H′). This central dorsal region is partially absent in the adult female (see A). (I) Posterior abdomen of a UAS-<i>emcRNAi</i>/+; <i>MD761</i>-Gal4/+ female showing a small size increase in the dorsal region of the A7 (arrow: compare with <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002874#pgen-1002874-g006" target="_blank">Figure 6A</a>). (J) Females expressing the MbsN300 protein in their A7 also show an enlarged dorsal domain (arrow). (K) In UAS-<i>emc</i>/+; <i>MD761</i>-Gal4/+ female pupae the expression of <i>sqh</i>-GFP is increased in the A7 (compare with G) and this segment disappears in the adult female (L). (M) X B^SY; <i>dsx¹</i>/<i>emc</i>-GFP <i>dsx¹</i> intersex pupa of ∼36 h APF, in which <i>emc</i> expression in the A7 is not up-regulated as in males. (N–Q) The expression of DsxM in the female A7 increases <i>emc</i>-GFP signal (N) and prevents the formation of the segment (O), whereas the expression of DsxF in the male A7 down-regulates <i>emc</i>-GFP expression (P) and develops an A7 (Q). (R, S) In female (R) or male (S) <i>dsx</i>-Gal4 UAS-GFP late pupae, the levels of <i>dsx</i> are higher in the A7 than in the A6. In the males, measurements show that the A7/A6 ratio in GFP signal intensity is 3,29±0,99 (n = 5). (T) In UAS-GFP/+; <i>dsx</i>-Gal4 <i>Abd-B^M1</i>/<i>Abd-B^iab-7MX2</i> male pupae, in which the A7 and A6 are transformed into A5, the levels of <i>dsx</i> in the A7 and A6 are similar. The round cells expressing <i>dsx</i>-Gal4 in R-T are probably fat cells <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002874#pgen.1002874-Aguila1" target="_blank">[63]</a>. (U) If the DsxM protein is expressed in an <i>Abd-B</i> mutant background (UAS-DsxM/+; <i>MD761</i>-Gal4/<i>Abd-B^M1</i>), it almost completely rescues the transformation induced by the loss of <i>Abd-B</i> (compare with <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002874#pgen-1002874-g001" target="_blank">Figure 1C</a>). 5 males of this genotype show this phenotype whereas 4 other males present a small A7 segment, sometimes in only one side. Cross made at 17°C. (V) In a male pupa of the UAS-DsxM/+; <i>emc</i>-GFP <i>MD761</i>-Gal4/<i>Abd-B^M1</i> genotype the levels of <i>emc</i>-GFP expression in the A7 are high.</p

Schemes of genetic regulation in male and female A7.

<p>Summary of the results obtained together with those of Ref. 15. The different regulatory inputs take place in some cases at different times in development. See text for details.</p

Dorsal histoblasts of the male A7 extrude through the epithelium.

<p>(A–B″′) Stills from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002874#pgen.1002874.s007" target="_blank">Videos S2</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002874#pgen.1002874.s008" target="_blank">S3</a> showing the progressive elimination, first of A8 LECs and then of A7 LECs and histoblasts, in His2A-RFP/<i>en</i>-Gal4 UAS-GFP male pupae from about 25 h till about 50 h APF. En is expressed in posterior compartments, (marked in green by GFP), and nuclei are marked in red. In this and other movies (and snapshots from them) marked with His2A-RFP, histoblasts are difficult to see under the moving cells (macrophages or hemocytes), which present a strong red signal. A histoblast nest is indicated by an arrow in A″. A8 cells disappear first and A7 cells follow, so that A6p histoblasts end up contacting the genitalia (G), which rotates during this period. Numbers indicate approximate hours APF. (C–C″′) Snapshots from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002874#pgen.1002874.s010" target="_blank">Video S5</a> (approx. 35–50 h APF) showing the apparent disappearance of A7 histoblasts in an <i>nrg</i>-GFP male pupa. The yellow arrows indicate the position of a bristle precursor and the white arrowheads shows the LECs separating the A6p and A7a histoblast nests. Note how both marks move posteriorly as the A7 histoblasts are eliminated. Numbers indicate approximate hours APF. (D) Snapshot from a movie showing a (<i>p</i>)<i>esg</i>-Gal4 UAS-GFP male pupa of about 40 h APF; cross sections, to the right and below (the plane of section indicated by white lines) show the accumulation of histoblasts as bulges under the epidermis (arrowheads). (E) Still taken from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002874#pgen.1002874.s011" target="_blank">video S6</a>, showing an <i>Abd-B^Fab7-1</i> homozygous male pupa in which Hh-RFP marks posterior compartments (in red) and the membrane marker <i>zcl22</i>-GFP <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002874#pgen.1002874-Morin1" target="_blank">[30]</a> is in green. E′, E″ are details of the squares in E, showing the constriction of cells in this optical section in two regions of the histoblast nests before histoblast invagination. (F) <i>sqh</i>-GFP expression is higher in the A7 histoblast nests of a ∼36 h APF male pupa than in the A6.</p

Hedgehog signaling and <i>Ultrabithorax</i> provide specific cell affinities to the cells.

<p>In <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057159#pone-0057159-g001" target="_blank">Figures 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057159#pone-0057159-g002" target="_blank">2</a> anterior compartments (A) of the imaginal discs are to the left and posterior ones (P) to the right. (A) <i>Ubx</i> mutant clones, marked by the absence of <i>arm</i>-lacZ expression (in green), are round and tend to segregate from the surrounding tissue. (B) An <i>Ubx</i>-expressing clone (arrow), marked with <i>yellow</i> and induced in the second thoracic segment also segregates from the rest of the notum. (C, C′) A <i>smo</i> clone in the anterior compartment of the wing pouch, marked by the absence of GFP signal (in green), penetrates into the posterior compartment, which is marked by <i>en</i>-lacZ expression (in red). (D) <i>hh</i>-lacZ expression in the haltere disc. (E–E′′) A <i>smo</i> clone in the anterior compartment of the haltere pouch, marked as in C also penetrates into the posterior compartment, marked with <i>hh</i>-lacZ (in red, E′). Merged image in E′′. Scale bars are 30 µm in A, D, E′′ and 60 µm in C′.</p

Differences in the amount of different Hox genes cause accumulation of <i>sqh</i>-GFP in imaginal discs.

<p>(A–A′′) Z-stack of an <i>Abd-B</i> mutant clone induced in the genital disc and marked by the absence of lacZ expression (in blue in A′) showing increased <i>sqh</i>-GFP expression around it (in green, A, A′). (B, B′) Z-stack of a control <i>Abd-B</i> clone similarly marked but induced in the wing disc, showing there is no increase in <i>sqh</i> levels. (C) In <i>ap</i>-Gal4 UAS-GFP/<i>ap^UGO35</i>; UAS-<i>Abd-B</i>/<i>tub-</i>Gal80^ts wing discs, the D/V boundary is smooth (compare with Fig. 3C). (D) A similar result is obtained in <i>ap</i>-Gal4 UAS-GFP/<i>ap^UGO35</i>; UAS-O<i>Ubx</i><b>/</b><i>tub-</i>Gal80^ts.</p

<i>Ultrabithorax</i> can maintain the A/P boundary in the absence of Hedgehog signaling.

<p> In all the panels of this Figure, the clones are marked by the lack of GFP in green, and the posterior compartment (to the right) is marked by either <i>hh</i>-lacZ or <i>en</i>-lacZ reporters in red. (A–B′′) Wing (A–A′′) and haltere (B–B′′) discs showing anterior clones double mutant for <i>smo</i> and <i>Ubx</i> that invade the posterior compartment. (C–C′′) <i>smo</i> clone induced in the anterior compartment of a <i>bx³/TM2, Ubx¹³⁰</i> haltere disc. See that it does not cross the compartment boundary. Note that a few cells in the A compartment weakly express <i>hh</i>-lacZ. (D–D′′) An anterior <i>smo</i> clone induced in the third leg disc cross the A/P compartment boundary. (E–E′′) A similar clone induced in the second leg disc does not cross the boundary. Scale bars are 30 µm except in A′′ (60 µm).</p