Model for the Coordinated Control of Growth and Patterning in the Drosophila Wing Disc

<p>A schematic representation of a growing (left) and a mature (right) wing disc is shown at the top. Corresponding cross-sections through the wing blade region are depicted below. The wing disc originates from the infolding of the embryonic ectoderm and consists of pseudostratified epithelial cells containing a basal–lateral side (yellow) and an apical side (red). The apical surface faces the disc lumen that is formed by the epithelium and the overlaying peripodial membrane (black), consisting of squamous epithelial cells. The morphogen and growth factor Dpp (yellow) is secreted basal–laterally by the Dpp-producing cells located anterior to the anterior–posterior compartment boundary (line through centre of wing disc). The Dpp concentration gradient from the anterior–posterior boundary to the periphery provides the anterior–posterior patterning cues. In addition, Dpp is also secreted apically into the disc lumen where is can diffuse freely. The model proposes that luminal Dpp acts as a growth-promoting factor stimulating disc growth in young discs. As the disc grows, a hypothetical growth inhibitor (blue dots) is also secreted apically and antagonizes the growth promoting activity of Dpp. Once the concentration of the inhibitor has reached a certain threshold, proliferation of wing imaginal disc cells ceases.</p

Changing the Patterning Mechansims during Wing Development Affects Growth

<p>Compared with a wild-type wing (A), loss of Dpp function results in reduced growth and loss of pattern elements (B). Ectopic expression of Dpp in a clone of cells results in pattern duplications associated with massive extra growth (C). The region of Dpp expression in (A) and (C) is indicated by the green color. (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0000086#pbio.0000086-Zecca1" target="_blank">Zecca et al. 1995</a>; pictures courtesy of B. Müller and K. Basler.)</p

The Insulin Signaling Activity Controls Organ Size in a Compartment-Specific Manner

<div><p>Mosaic Drosophila wings with compartment-specific manipulations of dAkt function display striking size defects but normal patterning.</p> <p>(A) Selective reduction of dAkt function in the posterior compartment by means of FLP-mediated mitotic recombination in posterior cells (using <i>engrailed–Gal4</i> to drive the expression of <i>UAS–Flp</i>) results in a small P compartment largely consisting of <i>dAkt³</i> mutant cells. The smaller compartment size is due to fewer and smaller cells.</p> <p>(B) Wild-type wing for comparison.</p> <p>(C) Expression of dAkt in posterior cells (<i>engrailed–Gal4 UAS–dAkt</i>) of wings with reduced dAkt function (<i>dAkt³</i>) restores the size of the P compartment, whereas the A compartment remains small. The red lines mark the anterior–posterior compartment boundary. Note that similar results in the wing disc have been obtained by <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0000086#pbio.0000086-Teleman1" target="_blank">Teleman and Cohen (2000)</a>.</p></div

Lig regulates organ size during development.

<p>(A–C) Scanning electron micrographs of the control (A), <i>lig</i> mutant overgrown eyes (B), and <i>lig</i> mutant eyes rescued by one copy of <i>Glig</i> (C). The mutant eyes were generated by eyFLP/FRT-mediated mitotic recombination. Scale bar represents 100 µm. (D) Quantification of ommatidia number from two independent experiments. Statistical analyses were done with a Student's t test (two-tailed, unpaired). Error bars indicate the standard deviations, (n) number of organs analyzed. Mean ± s.d. and p-values: Control (808±27 and 780±20), <i>lig</i> mutant eyes (856±23; p = 0.0027 and 806±28; p = 0.031) and <i>lig</i> mutant eyes with one copy of a genomic rescue transgene for <i>lig</i> (ND (not determined) and 768±9; p = 0.0011). (E–F) <i>lig¹</i> in combination with <i>lig^PP1</i>, a <i>lig</i> null mutant allele, causes long, slender pupae (F) in comparison to the control (E). Scale bar represents 500 µm. (G) Tangential eye sections of adult <i>lig¹</i> mosaic eyes reveal normal differentiation and cell size in <i>lig¹</i> mutant clones. The <i>lig¹</i> mutant cells are marked by the absence of pigmentation. (H–M) Scanning electron micrographs of adult control and <i>lig¹</i> mutant eyes generated by eyFLP/FRT-mediated mitotic recombination from flies grown on 25% (H–I), 100% (J–K) or 400% (L–M') yeast-containing food. Scale bar represents 100 µm. (N) Statistical analyses as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003598#pgen-1003598-g001" target="_blank">Figure 1D</a>: control (690±39 and 726±21) and <i>lig¹</i> mutant (729±27; p = 0.022 and 789±20; p = 2.35E-05) eyes at 25% yeast-containing food, control (763±23 and 749±23) and <i>lig¹</i> mutant (747±47; p = 0.33 and 761±43; p = 0.46) eyes at 100% yeast-containing food, and control (708±38 and 719±43) and <i>lig¹</i> mutant (688±53; p = 0.35 and 700±75; p = 0.48) eyes at 400% yeast-containing food. (O–R) Scanning electron micrographs of adult control (O), <i>lig¹</i> mutant (P), <i>DIAP1</i> overexpressing (Q) and <i>lig¹</i> mutant <i>DIAP1</i> overexpressing eye (R) generated by eyFLP, Actin-Flp out-Gal4/FRT-mediated mitotic recombination. Scale bar represents 100 µm. (S) Statistical analyses as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003598#pgen-1003598-g001" target="_blank">Figure 1D</a>: control (752±7 and 751±35), <i>lig¹</i> mutant (750±29; p = 0.85 and 770±40; p = 0.36), <i>DIAP1</i> overexpressing (768±12; p = 0.016 and 766±14; p = 0.34) and <i>lig¹</i> mutant <i>DIAP1</i> overexpressing (855±64; p = 0.0053 and 840±42; p = 0.0011) eyes. (T–U) Scanning electron micrographs of adult <i>p35</i> overexpressing (T) and <i>lig¹</i> mutant <i>p35</i> overexpressing eyes (U) generated by eyFLP, Actin-Flp out-Gal4/FRT-mediated mitotic recombination. Scale bar represents 100 µm. (V–X) Wings overexpressing the indicated UAS transgenes under the control of <i>nub-Gal4</i>. Scale bar represents 100 µm. (Y) Statistical analyses of wing area (as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003598#pgen-1003598-g001" target="_blank">Figure 1D</a>) of wings overexpressing a control <i>UAS-RNAi</i> transgene (371±17.7 and 363±12.9), <i>UAS-lig^{RNAi I}</i> (393±9.9; p = 1.28E-03 and 366.8±12.4; p = 0.41) and <i>UAS-lig^{RNAi II}</i> (418.5±7.6; p = 9.47E-06 and 399.3±16.6; p = 2.14E-04). (Z) The <i>lig</i> locus (drawn to scale) spans 11.5 kbp and consists of 14 protein-coding exons. <i>lig¹</i> and <i>lig²</i> are small deletions in the third exon resulting in a frameshift and premature stop codon. <i>lig³</i> contains a premature stop codon in the second exon. The RNAi lines I and II are specific for exon 3 and for exons 11 and 12, respectively. The genomic rescue construct <i>Glig</i> includes 12 kbp. <i>Glig^FS</i> lacks a nucleotide in exon 10 leading to a frameshift and a premature stop. Genotypes: (A) <i>y w eyFLP</i>/<i>y w</i>; <i>FRT42 cl w⁺</i>/<i>FRT42</i> (B) <i>y w eyFLP</i>/<i>y w</i>; <i>FRT42 cl w⁺</i>/<i>FRT42 lig¹</i> (C) <i>y w eyFLP</i>/<i>y w</i>; <i>FRT42 cl w⁺</i>/<i>FRT42 lig¹</i>; <i>Glig</i> [61B3]/+ (E) <i>y w</i>; <i>lig^PP1</i>/<i>FRT42</i> (F) <i>y w</i>; <i>lig^PP1</i>/<i>FRT42 lig¹</i> (G) <i>y w hsFlp</i>/<i>y w</i>; <i>FRT42 w⁺</i>/<i>FRT42 lig¹</i> (H, J and L) <i>y w eyFLP</i>/<i>y w</i>; <i>FRT42 P{SUPor-P}VhaAC45^KG02272</i> (cl)/<i>FRT42</i> (I, K, M and M') <i>y w eyFLP</i>/<i>y w</i>; <i>FRT42 P{SUPor-P}VhaAC45^KG02272</i> (cl)/<i>FRT42 lig¹</i> (O) <i>y w eyFLP</i>, <i>Act>CD2>Gal4</i>/<i>y w</i>; <i>FRT42 P{SUPor-P}VhaAC45^KG02272</i> (cl)/<i>FRT42</i> (P) <i>y w eyFLP</i>, <i>Act>CD2>Gal4</i>/<i>y w</i>; <i>FRT42 P{SUPor-P}VhaAC45^KG02272</i> (cl)/<i>FRT42 lig¹</i> (Q) <i>y w eyFLP</i>, <i>Act>CD2>Gal4</i>/<i>y w</i>; <i>FRT42 P{SUPor-P}VhaAC45^KG02272</i> (cl)/+; <i>EP-DIAP1</i>/+ (R) <i>y w eyFLP</i>, <i>Act>CD2>Gal4</i>/<i>y w</i>; <i>FRT42 P{SUPor-P}VhaAC45^KG02272</i> (cl)/<i>FRT42 lig¹</i>; <i>EP-DIAP1</i>/+ (T) <i>y w eyFLP</i>, <i>Act>CD2>Gal4</i>/<i>y w</i>; <i>FRT42 P{SUPor-P}VhaAC45^KG02272</i> (cl)/+; UAS-p35/+ (U) <i>y w eyFLP</i>, <i>Act>CD2>Gal4</i>/<i>y w</i>; <i>FRT42 P{SUPor-P}VhaAC45^KG02272</i> (cl)/<i>FRT42 lig¹</i>; <i>UAS-p35</i>/+ (V) <i>y w</i>; <i>nub-Gal4</i>/+; <i>UAS-CG1315^RNAi</i> (control)/+ (W) <i>y w</i>; <i>nub-Gal4</i>/+; <i>UAS-lig^{RNAi I}</i> [86Fb]/+ (X) <i>y w</i>; <i>nub-Gal4</i>/+; <i>UAS-lig^{RNAi II}</i> [86Fb]/+.</p

Capr cooperates with FMR1 and Rin to suppress growth.

<p>(A–B) Scanning electron micrographs of adult control (A) and <i>Capr²</i> (B) eyes generated by eyFLP/FRT Minute mediated mitotic recombination from flies reared on 25% yeast food (A–B). Scale bar represents 100 µm. (C) Statistical analyses as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003598#pgen-1003598-g001" target="_blank">Figure 1D</a>: control (772±22 and 765±15) and <i>Capr²</i> (754±18; p = 0.11 and 738±26; p = 0.04) mutant eyes from flies raised on 25% yeast-containing food. (D–K) Scanning electron micrographs of adult control (D), <i>Capr^RNAi</i> (E), <i>FMR1^D113M</i> (F), <i>Capr^RNAi FMR1^D113M</i> (G), <i>rin²</i> (H), <i>Capr^RNAi rin²</i> (I), <i>FMR1^D113M rin²</i> (J) and <i>Capr^RNAi FMR1^D113M rin²</i> eyes (K) generated by eyFLP, Actin-Flp out-Gal4/FRT-mediated mitotic recombination from flies grown on 25% yeast-containing food. Scale bar represents 100 µm. (L) Statistical analyses as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003598#pgen-1003598-g001" target="_blank">Figure 1D</a>: control (753±28 and 724±31), <i>Capr^RNAi</i> expressing (733±34 and 706±28), <i>FMR1^D113M</i> mutant (720±18 and 736±19), <i>Capr^RNAi</i> expressing <i>FMR1^D113M</i> mutant (789±20; p = 1.82E-05 and 787±14; p = 0.00015), <i>rin²</i> mutant (782±18 and 779±13) and <i>Capr^RNAi</i> expressing <i>rin²</i> mutant (837±52; p = 0.033 and 843±53; p = 0.018) eyes from flies raised on 25% yeast-containing food. Genotypes: (A) <i>y w eyFLP</i>/<i>y w</i>; <i>M(3)RpS17⁴ FRT80</i>/<i>FRT80</i> (B) <i>y w eyFLP</i>/<i>w</i>; <i>M(3)RpS17⁴ FRT80</i>/<i>Capr² FRT80</i> (D) <i>y w eyFLP</i>, <i>Act>CD2>Gal4</i>/<i>y w</i>; <i>FRT82 cl w⁺</i>/<i>FRT82</i> (E) <i>y w eyFLP</i>, <i>Act>CD2>Gal4</i>/<i>y w</i>; <i>UAS-Capr^RNAi</i>/+; <i>FRT82 cl w⁺</i>/+ (F) <i>y w eyFLP</i>, <i>Act>CD2>Gal4</i>/<i>y w</i>; <i>FRT82 cl w⁺</i>/<i>FRT82 FMR1^D113M</i> (G) <i>y w eyFLP</i>, <i>Act>CD2>Gal4</i>/<i>y w</i>; <i>UAS-Capr^RNAi</i>/+; <i>FRT82 cl w⁺</i>/<i>FRT82 FMR1^D113M</i> (H) <i>y w eyFLP</i>, <i>Act>CD2>Gal4</i>/<i>y w</i>; <i>FRT82 cl w⁺</i>/<i>FRT82 rin²</i> (I) <i>y w eyFLP</i>, <i>Act>CD2>Gal4</i>/<i>y w</i>; <i>UAS-Capr^RNAi</i>/+; <i>FRT82 cl w⁺</i>/<i>FRT82 rin²</i> (J) <i>y w eyFLP</i>, <i>Act>CD2>Gal4</i>/<i>y w</i>; <i>FRT82 cl w⁺</i>/<i>FRT82 FMR1^D113M rin²</i> (K) <i>y w eyFLP</i>, <i>Act>CD2>Gal4</i>/<i>y w</i>; <i>UAS-Capr^RNAi</i>/+; <i>FRT82 cl w⁺</i>/<i>FRT82 FMR1^D113M rin²</i>.</p

FoxO restricts growth and differentiation of cells with elevated TORC1 activity under nutrient restriction

<div><p>TORC1, a central regulator of cell survival, growth, and metabolism, is activated in a variety of cancers. Loss of the tumor suppressors PTEN and Tsc1/2 results in hyperactivation of TORC1. Tumors caused by the loss of PTEN, but not Tsc1/2, are often malignant and have been shown to be insensitive to nutrient restriction (NR). In <i>Drosophila</i>, loss of PTEN or Tsc1 results in hypertrophic overgrowth of epithelial tissues under normal nutritional conditions, and an enhanced TORC1-dependent hyperplastic overgrowth of <i>PTEN</i> mutant tissue under NR. Here we demonstrate that epithelial cells lacking Tsc1 or Tsc2 also acquire a growth advantage under NR. The overgrowth correlates with high TORC1 activity, and activating TORC1 downstream of Tsc1 by overexpression of <i>Rheb</i> is sufficient to enhance tissue growth. In contrast to cells lacking PTEN, <i>Tsc1</i> mutant cells show decreased PKB activity, and the extent of <i>Tsc1</i> mutant overgrowth is dependent on the loss of PKB-mediated inhibition of the transcription factor FoxO. Removal of FoxO function from <i>Tsc1</i> mutant tissue induces massive hyperplasia, precocious differentiation, and morphological defects specifically under NR, demonstrating that FoxO activation is responsible for restricting overgrowth of <i>Tsc1</i> mutant tissue. The activation status of FoxO may thus explain why tumors caused by the loss of Tsc1–in contrast to PTEN–rarely become malignant.</p></div

FoxO restricts growth and differentiation of cells with elevated TORC1 activity under nutrient restriction

<div><p>TORC1, a central regulator of cell survival, growth, and metabolism, is activated in a variety of cancers. Loss of the tumor suppressors PTEN and Tsc1/2 results in hyperactivation of TORC1. Tumors caused by the loss of PTEN, but not Tsc1/2, are often malignant and have been shown to be insensitive to nutrient restriction (NR). In <i>Drosophila</i>, loss of PTEN or Tsc1 results in hypertrophic overgrowth of epithelial tissues under normal nutritional conditions, and an enhanced TORC1-dependent hyperplastic overgrowth of <i>PTEN</i> mutant tissue under NR. Here we demonstrate that epithelial cells lacking Tsc1 or Tsc2 also acquire a growth advantage under NR. The overgrowth correlates with high TORC1 activity, and activating TORC1 downstream of Tsc1 by overexpression of <i>Rheb</i> is sufficient to enhance tissue growth. In contrast to cells lacking PTEN, <i>Tsc1</i> mutant cells show decreased PKB activity, and the extent of <i>Tsc1</i> mutant overgrowth is dependent on the loss of PKB-mediated inhibition of the transcription factor FoxO. Removal of FoxO function from <i>Tsc1</i> mutant tissue induces massive hyperplasia, precocious differentiation, and morphological defects specifically under NR, demonstrating that FoxO activation is responsible for restricting overgrowth of <i>Tsc1</i> mutant tissue. The activation status of FoxO may thus explain why tumors caused by the loss of Tsc1–in contrast to PTEN–rarely become malignant.</p></div

<i>Tsc1 FoxO</i> double mutant discs exhibit misfoldings due to massive overproliferation under NR.

<p><b>(A)</b> aPKC (in green), phalloidin (in red) and DAPI (in blue) stainings in orthogonal sections of eye discs with control, <i>FoxO</i>, <i>Tsc1</i> and <i>Tsc1 FoxO</i> mutant tissue dissected from larvae reared on normal food and NR <b>(A’)</b>. Scale bars are 25 μm except for <i>Tsc1</i>^<i>Q87X</i> <i>FoxO</i>^<i>25</i> on NR, where they are 75 μm. A lower magnification was used to give a better overview of the misfolded tissue. <b>(B)</b> aPKC (in green) and Dlg (in red) stainings in orthogonal sections of eye discs with hsFlp control, <i>FoxO</i>, <i>Tsc1</i>, and <i>Tsc1 FoxO</i> mutant clones (marked by the absence of GFP) dissected from larvae reared on normal food and NR.</p

The RNA-binding Proteins FMR1, Rasputin and Caprin Act Together with the UBA Protein Lingerer to Restrict Tissue Growth in <i>Drosophila melanogaster</i>

<div><p>Appropriate expression of growth-regulatory genes is essential to ensure normal animal development and to prevent diseases like cancer. Gene regulation at the levels of transcription and translational initiation mediated by the Hippo and Insulin signaling pathways and by the TORC1 complex, respectively, has been well documented. Whether translational control mediated by RNA-binding proteins contributes to the regulation of cellular growth is less clear. Here, we identify Lingerer (Lig), an UBA domain-containing protein, as growth suppressor that associates with the RNA-binding proteins Fragile X mental retardation protein 1 (FMR1) and Caprin (Capr) and directly interacts with and regulates the RNA-binding protein Rasputin (Rin) in <i>Drosophila melanogaster</i>. <i>lig</i> mutant organs overgrow due to increased proliferation, and a reporter for the JAK/STAT signaling pathway is upregulated in a <i>lig</i> mutant situation. <i>rin</i>, <i>Capr</i> or <i>FMR1</i> in combination as double mutants, but not the respective single mutants, display <i>lig</i> like phenotypes, implicating a redundant function of Rin, Capr and FMR1 in growth control in epithelial tissues. Thus, Lig regulates cell proliferation during development in concert with Rin, Capr and FMR1.</p></div

JAK/STAT signaling is activated in <i>lig</i> mutant cells.

<p>(A–C) <i>lig¹</i> mutant clones (induced with the FLP/FRT system, 72 h old, marked by the lack of lacZ staining) in eye (A and A''), antenna (B and B'') and wing (C and C'') imaginal discs of early third instar larvae. The JAK/STAT signaling reporter 10xSTAT92E-GFP is upregulated in <i>lig1</i> mutant clones in the posterior side of the eye imaginal disc (A' and A''), antenna imaginal disc (B' and B'') and in the hinge region of the wing disc (C' and C''). Note that the reporter signal is autonomously increased in the mutant clones. Scale bars represent 50 µm. (D) Schematic representation of the interactions shown in this study (left) and a working model of a Lig/Rin/FMR1/Caprin complex (right). Genotypes: (A–C) <i>y w hsFLP</i>/<i>y w</i>; <i>FRT42 arm-lacZ</i>/<i>FRT42 lig¹</i>; <i>10xSTAT92E-GFP</i>/+.</p