Figure 1.
Treh is essential for lamina and medulla development.
(A) Schematic diagram of the larval CNS. OL: optic lobe; CB: central brain; OPC: outer proliferation center; IPC: inner proliferation center; LF: lamina furrow; me: medulla; NE: neuroepithelial cell; NB: neuroblast in the optic lobe and central brain; VNC: ventral nerve cord. (B) Magnified view of boxed region in (A). NEs in the medial region of the OPC differentiate into medulla NBs; the NBs divide asymmetrically to generate a neuroblast daughter and a smaller ganglion mother cell (GMC) that generates medulla neurons. (C) Lateral view of the optic lobe showing the visual processing neuropils, the medulla (me), lamina (la) and lobula complex (lo). The optic lobe is connected with the eye imaginal disc (ED) through the optic stalk (OS). (D-F) Brains dissected from late-third instar larvae were stained with Dac and Elav to visualize the lamina and medulla, respectively. (D) Wild-type brains have a crescent-shaped lamina and a dome-shaped medulla. (E) c768-Gal4/UAS-TrehRNAi brains do not have a lamina. (F) c855a-Gal4/UAS-TrehRNAi brains do not have a lamina, but have an underdeveloped medulla with regions that contained no differentiated neurons (indicated by arrow). Scale bar: 20 µm.
Figure 2.
Treh regulates neuroepithelial cell maintenance and differentiation in the optic lobe.
(A-I) Time courses of neuroepithelial growth and expansion. (A-C) Wild-type brains at late-second (A), mid-third (B) and late-third instar (C). (D-F) c768-Gal4/UAS-TrehRNAi brains at late-second (D), mid-third (E) and late-third instar (F). The OPC neuroepithelium was normal at late-second instar (D), but became gradually disintegrated from mid-third (E) to late-third instar stages (F). (G-I) c855a-Gal4/UAS-TrehRNAi brains at late-second (G), mid-third (H) and late-third instar (I). The OPC neuroepithelium began to disintegrate around mid-third instar. (J, K) Treh RNAi brains had some enlarged, rounded cells that were Dpn+ and localized in the medulla cortex (K, K' indicated by yellow arrows), whereas wild-type brains have medulla neuroblasts localized on the medial surface of the optic lobe (J, J', indicated by white arrowhead). White arrow indicates IPC neuroblasts, which were not analyzed in this study. Scale bar: 20 µm.
Figure 3.
Treh suppresses the differentiation of neuroepithelial cells.
Late-third instar larval brains were stained with the antibodies indicated and flip-out clones expressing Treh RNAi were marked by GFP and dashed lines. (A) Cells in Treh RNAi clones in the medulla cortex were large and rounded (indicated by white arrowhead). (B) Multiple cells in each Treh RNAi clone expressed Dpn. (C) Treh RNAi mutant cells had asymmetric Mira localization in the cell cortex. (D) Treh RNAi clones generated only a limited number of neurons as revealed by Elav staining. (E) A wild-type control clone had a large lineage with some neuroblasts localized on the medial surface of the OPC. (F) Treh RNAi mutant cells underwent proliferation as revealed by PH3 staining. (G) No apoptotic cell death of Treh RNAi mutant cells was detected by activated caspase-3 staining. (H) Ectopic neuroblasts in Treh RNAi clones had asymmetric aPKC and Numb localization at opposite poles. The apical and basal poles (H') were reversed as compared with wild-type medulla neuroblasts (H”). (I) Tubulin staining of Treh RNAi mutant cells revealed that the spindle was aligned along the apicobasal axis. In (H) and (I), white and yellow arrows indicate Treh RNAi mutant neuroblast and normal medulla neuroblast, respectively; purple and green arrows indicates apical and basal pole, respectively. (J) Schematic showing Treh RNAi mutant neuroblasts with a reversal of apical and basal poles as compared with normal medulla neuroblasts. Scale bar: 20 µm.
Figure 4.
Treh loss-of-function mutations cause neuroepithelial disintegration and premature neuroblast formation.
(A, B) Treh18 and Treh41 homozygous late-third-instar larval brains had partly disintegrated OPC neuroepithelia, with some NEs transformed to rounded cells that expressed Dpn (indicated by arrow). (C) Treh18/Treh41 late-third-instar larval brains also had disintegrated NEs and premature formation of NBs (indicated by arrow). (D) Quantification of Treh mRNA levels in wild type and Treh mutants by real-time PCR analysis. Scale bar: 20 µm.
Figure 5.
Treh overexpression does not affect optic lobe development.
(A-E) Late-third instar larval brains expressing five different UAS-Treh lines under the control of c768-Gal4. Overexpression of Treh did not cause defects in the brain; and the proliferation and differentiation of NEs was normal. (F) Quantification of Treh mRNA levels in wild-type and c768-Gal4/UAS-Treh larval CNS by real-time PCR analysis. Scale bar: 20 µm.