Scribble Acts in the Drosophila Fat-Hippo Pathway to Regulate Warts Activity

Epithelial cells are the major cell-type for all organs in multicellular organisms. In order to achieve correct organ size, epithelial tissues need mechanisms that limit their proliferation, and protect tissues from damage caused by defective epithelial cells. Recently, the Hippo signaling pathway has emerged as a major mechanism that orchestrates epithelial development. Hippo signaling is required for cells to stop proliferation as in the absence of Hippo signaling tissues continue to proliferate and produce overgrown organs or tumors. Studies in Drosophila have led the way in providing a framework for how Hippo alters the pattern of gene transcription in target cells, leading to changes in cell proliferation, survival, and other behaviors. Scribble (Scrib) belongs to a class of neoplastic tumor suppressor genes that are required to establish apical-basal cell polarity. The disruption of apical-basal polarity leads to uncontrolled cell proliferation of epithelial cells. The interaction of apical basal polarity genes with the Hippo pathway has been an area of intense investigation. Loss of Scrib has been known to affect Hippo pathway targets, however, its functions in the Hippo pathway still remain largely unknown. We investigated the interactions of Scrib with the Hippo pathway. We present data suggesting that Drosophila Scrib acts downstream of the Fat (Ft) receptor, and requires Hippo signaling for its growth regulatory functions. We show that Ft requires Scrib to interact with Expanded (Ex) and Dachs (D), and for regulating Warts (Wts) levels and stability, thus placing Scrib in the Hippo pathway network

<i>scrib</i> acts upstream of <i>wts</i>.

<p>(a–d) Panels show comparison of MARCM clones (GFP-positive) from wild-type (a), <i>scrib</i> mutant (b), <i>scrib wts</i> double mutant (c), and <i>wts</i> mutant (d) eye imaginal discs. DIAP1 expression is shown in red in a–d and in greyscale in a’–d’. Anterior is to the right, and magnification is same in all images. Genotypes: (a) <i>ey Flp; Act>y+>GAL4 UAS-GFP; FRT82B tub-GAL80/FRT82B</i> (b) <i>ey Flp; Act>y+>GAL4 UAS-GFP; FRT82B tub-GAL80/FRT82B scrib^j7b3</i> (c) <i>ey Flp; Act>y+>GAL4 UAS-GFP; FRT82B tub-GAL80/FRT82B scrib² wts^X1</i> (d) <i>ey Flp; Act>y+>GAL4 UAS-GFP; FRT82B tub-GAL80/FRT82B wts^X1.</i></p

<i>scrib</i> interacts upstream of <i>d</i> in the Fat-Hippo pathway.

<p>Panels show wing imaginal discs from <i>nub-GAL4 UAS-ft^RNAi</i> (a), and <i>nub-GAL4 UAS-ft^RNAi UAS-scrib^RNAi</i> (c), <i>nub-GAL4 UAS-D</i> (e) and <i>nub-GAL4 UAS-D UAS-scrib^RNAi</i> (g) larvae stained for DIAP1. Adult wings of the corresponding genotypes are shown in (b) <i>nub-GAL4 UAS-ft^RNAi</i>, (f) <i>nub-GAL4 UAS-D,</i> and (h) <i>nub-GAL4 UAS-D UAS-scrib^RNAi</i>. (d) Phenotype of <i>nub-GAL4 UAS-ft^RNAi UAS-scrib^RNAi</i> adults shows loss of wings (white arrows). (i) Semi-quantitative Western blot for Wts levels in wild-type[Lane 1], <i>scrib</i> mutants (<i>scrib²/scrib³</i>) [Lane 2], <i>ft</i> mutants (<i>ft^fd/ft⁴²²)</i>[Lane 3], and <i>ft; scrib</i> double mutant (<i>ft^fd/ft⁴²²</i>; <i>scrib²/scrib⁷</i>)[Lane 4] and (<i>ft^fd/ft⁴²²</i>; <i>scrib²/scrib³</i>)[Lane 5] is shown. Anti-α-Tubulin is the loading control. (j): Model of <i>scrib</i> in Hippo Pathway- <i>scrib</i> acts downstream of <i>ft</i> and mediates it effects on growth. Magnification of images in panels a-h is identical.</p

Effect of Ft-Scrib interaction on Ex localization.

<p>Ex expression in <b>(</b>a–c) <i>ft</i> mutant clones (GFP negative) and (d–f) in <i>ft</i> mutant clones (GFP negative) induced in <i>scrib</i> heterozygous condition is shown. (g,h) Panels show Ex levels in <i>scrib</i> mutant clones (g, GFP positive) induced using the MARCM system, and in (i) <i>scrib</i> homozygous mutant discs. Ex levels are shown in red (a,d,g,i) and in grey scale (c,f,h). All images are at identical magnification. Genotypes: (a–c) <i>ubx-Flp; ft^fd FRT40A/ubi-GFP FRT40A</i> (d–f) <i>ubx-Flp; ft^fd FRT40A/ubi-GFP FRT40A; scrib⁷red e/+</i> (g,h) <i>ey-Flp; Act>y+>GAL4 UAS-GFP; FRT82B scrib²/FRT82B TubGal80</i> (i) <i>scrib²/scrib³.</i></p

<i>scrib</i> interacts with <i>yki</i> to regulate growth.

<p>(<b>a</b>) <i>nub-GAL4 UAS-scrib^RNAi UAS-GFP</i> wing imaginal discs showing down-regulation of Scrib expression in the <i>nub-Gal4</i> domain (shown by GFP expression in the inset). (b–d) Panels show comparison of clone sizes of GFP negative clones from larvae of the following genotype: <i>ubx-Flp; FRT82B M(3)95A ubi-GFP/FRT82B</i> (b), <i>ubx-Flp; FRT82B M(3)95A ubi-GFP/FRT82B scrib²</i> (c), and <i>ubx-Flp; yki^B5/+; FRT82B M(3)95A ubi-GFP/FRT82B scrib²</i> (d). Diap1 expression in third instar wing imaginal disc from wild-type (e), <i>nub-GAL4 UAS-scrib^RNAi</i> (g), <i>nub-GAL4 UAS-Yki</i> (i), <i>nub-GAL4 UAS-Yki UAS-scrib^RNAi</i> (k) <i>nub-GAL4 UAS-sd^RNAi</i> (m), <i>nub-GAL4 UAS-scrib^RNAi UAS-sd^RNAi</i> (o) larvae. The corresponding adult phenotypes for all genotypes are shown in panels to the right of imaginal discs. Adult wings of wild-type (f), <i>nub-GAL4 UAS-sd^RNAi</i> (n), and <i>nub-GAL4 UAS-scrib^RNAi UAS-sd^RNAi</i> (p) are shown. Images of adult flies are shown for <i>nub-GAL4 UAS-scrib^RNAi</i> (h), <i>nub-GAL4 UAS-Yki</i> (j), <i>nub-GAL4 UAS-Yki UAS-scrib^RNAi</i> (l).</p

<i>ft</i> requires <i>scrib</i> to regulate growth.

<p>Wing imaginal discs from (a) wild-type, and homozygous mutant larvae of (b) <i>ex^BQ FRT40A^/ex^BQ FRT40A,</i> (c) <i>ft^fd FRT40A</i>/<i>ft^fd FRT40A</i>, and (d) <i>FRT82B scrib²</i>/<i>scrib⁷</i> genotypes are shown. (e,f) Panels show the wing imaginal from double mutant larvae of (e) <i>ex^BQ FRT40A</i>/<i>ex^BQ FRT40A</i>; <i>FRT82B scrib²</i>/<i>scrib⁷</i> and (f) <i>ft^fd FRT40A</i>/<i>ft^fd FRT40A</i>; <i>FRT82B scrib²</i>/<i>scrib⁷</i> genotypes. Note that the overgrowth induced by loss of <i>ft</i> (c) is suppressed by concomitant loss of <i>scrib</i> (f). The magnification and orientation of images is identical.</p

Hippo target genes are affected in <i>scrib</i> mutant cells.

<p>(a, e, g) Panels show <i>diap1-lacZ</i> (a), <i>ex-lacZ</i> (e), <i>fj-lacZ</i> (g) expression in wild-type wing imaginal discs. (b, f, h) <i>scrib²/scrib³</i> mutant homozygous discs showing <i>diap1-lacZ</i> (b), <i>ex-lacZ</i> (f), <i>fj-lacZ</i> (h) expression. (c–d’’’) <i>diap-lacZ</i> levels in scrib mutant clones in wing imaginal discs from <i>yw hs-Flp FRT82B scrib^2/FRT82B ubi-GFP</i> larvae (c). Note that a majority of the clones get eliminated 48h after induction in the wing pouch. (d–d’’’) Magnified view of the clone in the notum indicated by a yellow box. The clone (GFP negative) is smaller than its wild-type (2XGFP) twin-spot, and shows down-regulation of <i>diap-lacZ</i> (red in d’’ and greyscale in d’’’). The clone boundary is marked by yellow line (d,d”). The magnification and orientation of images in a–c, e–h is identical.</p