Rap/Fzr promotes neuron formation. Third instar larval brains were stained with the early neuronal marker Dachshund (green) and the mitotic marker anti-phospho-histone H3 (red). Compared to wild-type brains (A), rap/fzr loss-of-function mutants w,rap3 (B) show a significant decrease in neuron density. (C) Ectopic overexpression of Rap/Fzr in brains results in an increase in neuron density. (A–C) Single optical sections from a confocal microscope. (A′–C′) Magnified areas of the brain (outlined squares in A–C) show densely packed neurons within third instar larval brains. (A″–C″) Quantification of surface neurons within the brain by Image J software. Projections with numerous colors represent individual cells that were counted. (D) Histogram of neuron density measured by the Volocity software (w,rap3, 1.08 × 10⁶ voxels ± 1.40 × 10⁵; wild type, 1.62 × 10⁶ voxels ± 1.41 × 10⁵, n = 17, *P < 0.017; UAS-rap/fzr; repo-GAL4, 3.07 × 10⁶ voxels ± 2.38 × 10⁵, n = 15, **P < 0.0003). (E) Histogram shows quantification of surface neurons (wild type, 398 neurons ± 33.3; w,rap3, 300 neurons ± 30.1; UAS-rap/fzr;repo-GAL4, 488 neurons ± 38.44, n = 12, n = 16, *P < 0.05 and *P < 0.05). Bar, 50 μm.

Rap/Fzr regulates development of surface glia. Single optical sections from a confocal microscope show MARCM clones that are GFP positive (green). (A–C) Examples of rap/fzr overexpression clones (arrows) showing that the majority of the clones located on the surface do not contain glial cells (red, anti-Repo); only 26% are Repo positive. (D–F) rap/fzr overexpression clones located within the neuropile region of the brain show that 81% are Repo positive. Bar, 50 μm.

Biochemical evidence shows that Loco interacts with Rap/Fzr and that Loco is ubiquitinated in vivo. (A) Protein lysates from the UAS-loco-GFP; repo-GAL4 third instar larval brain–eye complex were used to immunoprecipitate Loco-GFP protein. Western blot analysis using anti-GFP reveals a 100-kDa band corresponding to Loco protein in larvae. Negative control shows the absence of Loco protein (left). Rap/Fzr co-immunoprecipitates with Loco in the third instar larval brain–eye complex. A 53-kDa band is detected using Rap/Fzr antibody in larval lysates. The negative control shows the absence of Rap/Fzr when IgG beads were incubated without GFP antibody (right). (B) Protein extracts from UAS-loco-GFP; repo-GAL4 third instar larval brain–eye complex were used to immunoprecipitate Rap/Fzr protein. Western blot analysis using anti-Rap/Fzr reveals a 53-kDa band corresponding to Rap/Fzr protein in larval brain extracts (left). The negative control shows the absence of Rap/Fzr protein. Data in the right panel show that Loco physically interacts with Rap/Fzr in the third instar larval brain–eye complex. A 100-kDa band is detected using anti-GFP antibody in larvae, representative of Loco protein. The negative control shows the absence of Loco protein when IgG beads were incubated without Rap/Fzr antibody. (C) Loco is ubiquitinated in vivo. Protein lysates from UAS-loco-GFP; repo-GAL4 third instar larval brains were again used to immunoprecipiate Loco-GFP protein. Western blot analysis using anti-GFP reveals a 100-kDa band corresponding to Loco protein in larval extracts (left). The negative control shows the absence of Loco protein. Western blot analysis with GFP-Loco immunoprecipitate reveals the presence of ubiquitin in 80- and 100-kDa bands (right). The inset (bottom right) represents lighter exposure to visualize bands clearly.

Rap/Fzr and Loco are localized within neuroblasts located on the brain surface. Third instar larval brains showing immunolocalization of Loco (blue), Rap/Fzr (red), and Miranda (green) within neuroblasts in the superficial areas (A–C). A merge of the images A–C shows colocalization of Loco and Rap/Fzr in Miranda-positive cells (D); the square in D indicates the magnified area displayed in E. Bar, 50 μm. Images shown are single optical sections from a confocal microscope.

Overexpression of rap/fzr generates extra neuroblasts in third instar larvae brains. Confocal images of third instar larval brains were labeled with the neuroblast marker Miranda (red) and the glial marker Repo (green) antibody. Compared to wild type (A), rap/fzr gain-of-function UAS-rap/fzr; repo-GAL4 (C) brains show a significant increase in Miranda-positive cells (5416 neuroblasts compared to 3067 neuroblasts in normal larval brains, n = 15, *P < 0.002). Student t-test reveals no significant difference in neuroblast numbers between the loss-of-function mutant w,rap3 (B) and wild type (4356 neuroblast in w,rap3 and 3067 neuroblasts normal larvae, n = 15; P < 0.100). Arrows show some Repo-positive cells that are also Miranda positive in wild type, w,rap3, and UAS-rap/fzr;repo-GAL4 larvae. (D) Bar graph represents quantification of neuroblasts using Volocity software. Images are maximum projections taken from a Zeiss LSM 510 confocal microscope. Bar, 50 μm.

A proposed model for the interaction between Rap/Fzr and Loco. (A) In wild-type third instar larvae, neuroblasts located on the exterior surface of the brain divide asymmetrically, creating a larger daughter cell, a neuroblast (NB, yellow), and a smaller daughter cell, the GMC (blue). The GMCs will divide once to give rise to either two neurons (red) or two glia (green). Rap/Fzr and Loco are localized within the neuroblasts and function to control neuroblast and glia number. Rap/Fzr targets Loco for ubiquitination by APC/C and regulates its levels. (B) rap/fzr loss-of-function leads to elevated levels of Loco. Consequently, Loco accumulation within the GMCs leads to an increase in glia number. When Rap/Fzr is overexpressed (C) during the third instar larval stage, Loco is targeted for degradation, leading to an increase in neuroblast number. Furthermore, destruction of Loco within the GMCs leads to the formation of neurons.

Rap/Fzr regulates glial differentiation. (A–C) Confocal images of third instar larval brains stained with the glia-specific marker Repo (green) and the mitotic marker anti-phospho-histone H3 (red). (B) rap/fzr loss-of-function mutants, w,rap3, show a significant increase in glia number compared to (A) wild type (glia number for w,rap3 is 3431 cells ± 375; for wild type, 2390 cells ± 121, n = 17; P < 0.028). (C) Ectopic expression of rap/fzr shows a decrease in glia number (glia number for UAS-rap/fzr; repo-GAL4 is 724 cells ± 128, n = 17; **P < 0.00000014). (D) Histograms of glia number within the entire brain of third instar larvae. (E) Quantification of surface glia cells in wild type, w,rap3, and UAS-rap/fzr; repo-GAL4 larval brains (wild type, 380 ± 25; w,rap3, 682 ± 59; UAS-rap/fzr; repo-GAL4, 148 cells ± 35; n = 22, n = 21, P > 0.25, and P > 0.12; n = 13, n = 14, *P < 0.002, and **P < 0.0002). (F) Histogram of mitotic index quantified on the basis of phospho-H3 labeling of larval brains from w,rap3 and UAS-rap/fzr; repo-GAL4. No significant difference in the mitotic index was observed between wild type (1086 ± 117); w,rap3 (1621 ± 322); UAS-rap/fzr; repo-GAL4 (1554 cells ± 256; n = 22, n = 21, P > 0.25 and P > 0.12). Images are maximum projections taken from a LSM 510 confocal microscope. Bar, 50 μm.

Rap/Fzr functions cell autonomously to regulate glia number. Confocal microscope images of third instar larval brains stained with anti-β-galactosidase (red) and anti-Repo (green). (A) An example of a rap/fzr− clone located within the larval brain marked by the lack of anti-β-galactosidase (red) staining and a white outline. The corresponding wild-type tissue is outlined in yellow. (B) Magnified image of the outlined area from the rap/fzr− clone in A. (C) Magnified image of wild-type tissue shown as yellow inset in A. (D) Histogram shows that the number of glial cells in rap/fzr-- clones are increased compared to wild-type tissue (rap/fzr− clones, 19 glia cells ± 2.7; wild type, 8 glia cells ± 1; n = 10; *P < 0.004). rap/fzr mosaic clones were induced by the FLP/FRT technique (see materials and methods for details). Bar, 10 μm.

rap/fzr and loco genetically interact to regulate glial differentiation. Confocal images of third instar larval brains stained with the glia-specific marker Repo. Compared to wild-type brains (B), loco^P452, a loss-of-function mutant (A), shows a significant decrease in glia number (1628 ± 161 Repo-positive cells compared to 2390 ± 126 for wild type, n = 15, *P < 0.002). Conversely, the hypormorphic allele rap^E6 (C) shows a marked increase in glia number compared to wild-type brains (3627 ± 388 Repo-positive cells, *P < 0.02, n = 15). The rap^E6 glia phenotype is suppressed with (D) one copy of loco^P452 (brains analyzed for rap^E6; loco^P452/+ show 2464 ± 175 Repo-positive cells; n = 12, *P < 0.03). Images are maximum projections from a confocal microscope. Bar, 50 μm. (E) Histogram of glia number in third instar larval brains.

Loco and Rap/Fzr colocalize within a subset of glial cells in third instar larval brains. Single optical sections from a confocal image show Loco (blue), Rap/Fzr (red), and Repo (green) distribution within the exterior surface of the larval brain (A–C). A merged image shows triple-labeled brain with Rap/Fzr (red), Loco (blue), and Repo (green) localized within a subset of glia cells (D). Loco and Rap are localized within the cytoplasm of glia indicated by the arrow in E. (A′–C′) Deeper regions of the brain. Loco (blue) expression is diffuse in the brain's interior and is restricted to the ventral region of the brain. Unlike Loco, Rap/Fzr (red) expression is apparent in two regions, the optic lobe and the ventral edge of the brain. Images in D and D′ are a merge of images in A–C and A′–C′, respectively. Squares in D and D′ outline magnified regions displayed in E and E′, respectively. Bar, 50 μm.