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. 2010 Jul;137(14):2397-408.
doi: 10.1242/dev.050013.

Influence of fat-hippo and notch signaling on the proliferation and differentiation of Drosophila optic neuroepithelia

B V V G Reddy  1 , Cordelia Rauskolb, Kenneth D Irvine
Affiliations

Influence of fat-hippo and notch signaling on the proliferation and differentiation of Drosophila optic neuroepithelia

B V V G Reddy et al. Development. 2010 Jul.

Abstract

The Drosophila optic lobe develops from neuroepithelial cells, which function as symmetrically dividing neural progenitors. We describe here a role for the Fat-Hippo pathway in controlling the growth and differentiation of Drosophila optic neuroepithelia. Mutation of tumor suppressor genes within the pathway, or expression of activated Yorkie, promotes overgrowth of neuroepithelial cells and delays or blocks their differentiation; mutation of yorkie inhibits growth and accelerates differentiation. Neuroblasts and other neural cells, by contrast, appear unaffected by Yorkie activation. Neuroepithelial cells undergo a cell cycle arrest before converting to neuroblasts; this cell cycle arrest is regulated by Fat-Hippo signaling. Combinations of cell cycle regulators, including E2f1 and CyclinD, delay neuroepithelial differentiation, and Fat-Hippo signaling delays differentiation in part through E2f1. We also characterize roles for Jak-Stat and Notch signaling. Our studies establish that the progression of neuroepithelial cells to neuroblasts is regulated by Notch signaling, and suggest a model in which Fat-Hippo and Jak-Stat signaling influence differentiation by their acceleration of cell cycle progression and consequent impairment of Delta accumulation, thereby modulating Notch signaling. This characterization of Fat-Hippo signaling in neuroepithelial growth and differentiation also provides insights into the potential roles of Yes-associated protein in vertebrate neural development and medullablastoma.

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Figures

Fig. 1.
Fig. 1.
Organization of the optic lobe. (A) Larval eye disc and brain stained for Chaoptin to reveal retinal axons. (B) Brain hemisphere stained for Dpn (green), E-cad (red) and Dac (yellow). (C) Schematic of the surface of the optic lobe, illustrating NE (red) with symmetrically dividing cells. NE cells of the OOA are recruited (curved arrows) to lamina neurons (yellow) along the lateral side, and medulla NBs (green) along the medial side. Medulla NB recruitment progresses (thick arrow) from medial to lateral. (D) Anterior VNC and brain hemispheres, stained for E-cad (red) and actin (cyan). In cross-section, both the IOA and OOA are visible. (E) Simplified schematic of transcriptional branch of Fat-Hippo signaling, depicting key components and regulatory connections.
Fig. 2.
Fig. 2.
Expression of Fat-Hippo pathway components in the brain. Brain hemispheres from third instar larvae. (A-H) Surface views; (I) cross section. Brains are photographed from different angles; for reference, NE cells are marked and insets depict schematically (green, NBs; red, NE; yellow, lamina) whether the brain is viewed from the posterior or lateral side (except A′,B′, where insets show higher magnification of the boxed region). Panels marked by prime show a single fluorescent channel. (A-C) Stained for Arm (green), Actin (phalloidin, blue) and (A) Fat, (B) Ex, or (C) Mer (red). (D) Ds (magenta) and Arm (green). (E) fj-lacZ (cyan), E-cad (red) and Dpn (green). (F) Higher magnification of NE, stained for Dachs:V5 (green), ds-lacZ (magenta) and E-cad (red). Arrows indicate Dachs:V5 on the membrane, which occurs on the low side of the ds gradient. (G-I) Yki (red) in NE cells is complementary to Dpn in NBs (green, G), and Dac in lamina (yellow, H). (I) Yki in NE is visible in both the IOA and OOA.
Fig. 3.
Fig. 3.
Regulation of Yki localization in optic neuroepithelia. Brains stained for Yki (red) and DNA (Hoechst, blue) with clones mutant for genes in the Fat-Hippo pathway: (A) wtsX1, (B) Mer4 exe1, (C) exe1, (D) fat8, (E) fat8 dachsGC13 and (F) wild type (control). In A,C-F, mutant clones are positively marked by GFP (green); in B, mutant clones lack GFP. Panels marked by prime show Yki only, and are higher magnifications of the boxed regions, with clones outlined. +, wild type; −, mutant.
Fig. 4.
Fig. 4.
Influence of activated Yki on growth and differentiation. Larval brains, panels marked by prime show stains without the clone marker, arrows indicate clone edges. (A) Control clones, expressing GFP (blue), stained for Dpn (green) and E-cad (red). (B-E) Clones expressing Yki:V5S168A under act-Gal4 control, stained for (B) Dpn and F-actin (red), (C) Dpn and Pros (magenta), (D) Arm (red) and Elav (cyan), and (E) E-cad and Dac (yellow). Clones highlighted by white arrows maintain NE morphology and fail to express markers of more differentiated cells. In B, yellow arrows highlight normal-looking clones in the central brain. (F-I) Brains expressing wild-type or activated Yki under C855a-Gal4 control. (F,G) Brain and VNC, stained for F-actin (blue) and Yki:V5 (red). (H,I) Brain stained for E-cad (green) and Yki:V5 (red). (F,H) Expression of wild-type Yki:V5, which has no effect. (G,I) Expression of Yki:V5S168A, which results in overgrowth. (J,K) Brains from animals mutant for (J) fat8 or (K) exe1; the NE (marked by Yki and E-cad) are enlarged (compare with Fig. 2). (L) Clones expressing Yki:V5S168A under act-Gal4 control, stained for Diap1 (green) and E-cad (red). (M) Brain with clones (GFP, blue) mutant for wtsX1 and expressing wild-type Yki:V5 under act-Gal4 control, stained for Dpn (green) and Yki (red).
Fig. 5.
Fig. 5.
Influence of Fat-Hippo pathway genes on NE differentiation. Close-ups of larval brains, centered on the edge of the OOA NE. Panels marked by prime show stains without the clone marker, selected clones are outlined. Arrows indicate examples of differentiation delay. In A-D, mutant clones are positively marked by GFP; in E, mutant clones are marked by absence of GFP. (A) wtsX1 clone, stained for Dpn (green) and F-actin (red). (B) wtsX1 clone, stained for Pros (magenta) and E-cad (red). (C) wtsX1 mutant clone, stained for Arm (blue) and L(1)sc (red). (D) fat8 clones, stained for Dpn (green) and F-actin (red). (E) ykib5 clones, stained for Dpn (green) and L(1)sc (red). The Minute technique was employed to enable clone recovery.
Fig. 6.
Fig. 6.
Relationship of Fat-Hippo to Jak-Stat and Notch signaling. Regions of larval brains, centered on the NE to NB transition. Panels marked by prime show stains without the clone marker, selected clones are identified by dashed outlines or arrows. In A-D,G-O, clones are marked by GFP (blue/green); brains are stained for Dpn (green) in A-D,G-H,J,N,O,Q; Yki (red) in A,B,I; E-cad (red/blue) in C-D,K-O,Q; Dl (red/green) in E,F,K,L; L(1)sc (red) in F,H,J. (A) Clones expressing hop under act-Gal4 control. (B) Clones co-expressing hop and yki RNAi, the effectiveness of RNAi is indicated by reduced Yki. (C) Stat92E85C9 mutant clones accelerate differentiation. (D) Stat92E85C9 wtsX1 double mutant clones delay differentiation. (E,F) Dl expression at the edge of the NE in wild type. (G-I) Clones expressing activated Notch block differentiation (G), block L(1)sc expression (H), but do not influence Yki localization (I). (J,K) Clones expressing activated Yki block L(1)sc expression (J) and downregulate Dl expression (K). (L) wts mutant clones delay Dl expression. (M) Clones expressing activated Notch upregulate Dl. (N-P) Clones mutant for Dlrev10. (N) Apical section in which both autonomous delay and non-autonomous acceleration of differentiation are visible. (O) Basal section of the same sample, autonomous acceleration of differentiation is visible; these premature NBs often drop basally. (P) Schematic interpretation of Delta expression and the consequences of a Dl mutant clone. In the interior of a clone, Notch activity is lost (1). We propose that these cells become NBs and drop basally (asterisk in O). Around the edges of a clone (2), autonomous inhibition of Notch activity is lost but Notch can be activated by Delta from neighboring cells. Notch activity therefore increases, delaying NB formation (yellow arrow in N). Outside the edges of a clone (3), Notch activity is reduced owing to decreased Dl signaling from neighboring cells, accelerating NB formation (white arrow in N). (Q) Accelerated NB formation in a Notch mutant clone. (R,S) Expression of Dl (R) and L(1)sc (S) at the edge of the NE is detected in wild-type cells that do not label with EdU (red). (T,U) Clones expressing CycD+Cdk4+E2f1+Dp do not directly affect Yki (T) or Fat (U) expression, but expression of Yki and Fat is maintained as long as cells remain NE. (V) Projection through apical and basal regions of a clone over-expressing Dl. Some cells within Dl-expressing clones become NBs and drop basally (arrow).
Fig. 7.
Fig. 7.
Cell cycle regulation in the optic lobe. Larval brains with selected clones, marked by GFP expression, outlined. (A) Wild type, with S phase cells labeled by EdU (blue/white). Arrows indicate the cell cycle arrest along the medial side of the NE. (B,C) Surface (B) and cross-sectional (C) images of PCNA-EGFP; arrows indicate reduced expression along the medial edge of the NE. (D) Yki:V5S168A-expressing clones (green), with S-phase cells labeled by EdU (blue/white). (E) wtsX1 mutant clones (green), with S-phase cells labeled by EdU (blue/white). (F-I) Higher magnifications of brains stained for E-cad (red) and Dpn (green), with clones marked by presence of GFP (blue), centered on the edge of the OOA NE. (F) CycD+Cdk4-expressing clones. (G) E2f1+Dp-expressing clones. (H) CycD+Cdk4+E2f1+Dp-expressing clones. (I) wtsX1 e2f1729 double mutant clones. (J) Brain with CycD+Cdk4+E2f1+Dp-expressing clones, stained for Dl (red) and E-cad (blue).
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