Figure 1.
An RNAi screen for neurogenic genes that are regulated by mTOR signalling identifies unkempt.
(A) A wild-type antennal-eye imaginal disc stained with phalloidin (green) to mark F-actin at the apical surface and Prospero (red) to visualise R7 and cone cells. The left part of the disc forms the antenna, the right part the eye. Photoreceptors differentiate posterior to the morphogenetic furrow (MF), which forms an indentation in the disc that moves from posterior (P) to anterior (A). (B, B′) Precocious differentiation of R1/6 (shown by the expression of Bar, red) in Tsc1Q600X mutant clones (arrows). (C) Schematic of the unk genomic region showing unk and adjacent genes (top) and the domain structure of the Unk protein (bottom). Exons are shown as black rectangles and non-coding regions as white rectangles. The regions deleted in each of the mutants are represented by dotted lines. Transposon insertions are represented by triangles. Conserved domains in the protein are shown as circles. (D-F′) Precocious differentiation of R1/6 (marked by Bar expression, red in (D, D′)) and R7/cone cells (marked by Prospero expression, red in (E, E′) and D-Pax2, red in (F, F′)) in unkex24 mutant clones (arrows). Note also the increased expression of D-Pax2 in unk mutant clones. (G, G′) Loss of unk does not affect the differentiation of R3/4 (marked by the expression of Spalt (Sal, red)). (H, H′) Loss of unk does not affect the differentiation of R2/5 (marked by the expression of Rough (Ro, red)). (I) unkex24 clones in the adult eye cause photoreceptor rotation and morphogenesis defects. Mutant cells are marked by the lack of dark pigment surrounding each ommatidium. Dotted line indicates the equator. Black arrows indicate mis-rotated ommatidia; red arrow indicates an ommatidum with missing photoreceptors; black arrowheads indicate elliptical rhabdomeres; red arrowheads indicate split rhabdomeres. (J-K′) The delay in differentiation of R1/6 (marked by Bar expression, red), caused by loss of Rheb (J, J′), is suppressed in unkex24, Rheb2D1 mutant clones (K, K′). Mutant clones are marked by loss of GFP expression (green) in (B), (D–H), (J) and (K) and the differentiation front is marked by a white dotted line. Anterior is to the left in all images.
Figure 2.
Unk expression is regulated by Tsc1 in differentiating photoreceptors.
(A) A confocal projection of a wild-type eye disc stained for Unk protein expression. Unk is expressed throughout the disc, but its expression is stronger posterior to the morphogenetic furrow (MF, arrow). Scale bar: 50 µm. (B) High magnification single confocal section of Unk expression (red) in differentiating photoreceptors. Unk has a cytoplasmic, partially punctate distribution. Prospero expression marking R7 and cone cells is shown in green. Arrowheads mark examples of puncta. N: nucleus. Scale bar: 10 µm. (C–D) Unk expression (white in (C′),(D′) and red in (D)) is decreased in Tsc1Q600X mutant clones posterior to the MF (arrows) both in photoreceptors (C, showing apical level) and photoreceptor precursor cells (D, showing basal level), but not in clones anterior to the MF (arrowheads in (D)). Prospero expression (red in (C)) marks R7/cone cells. Mutant clones are marked by loss of GFP expression (green) in (C) and (D). Anterior is to the left.
Figure 3.
unk does not regulate cell growth.
(A–C) Phosphohistone H3 expression (PH3, green) posterior to the MF (arrowheads) is similar in eye discs from control, homozygous unkex24, or transheterozygous unkex24/unkDf larvae. (D–F) Examples of control, unkex24 or Tsc1Q600X clones in the eye disc. Homozygous mutant cells are marked by the loss of GFP (green) and the adjacent twin spot (ts) has stronger GFP expression than the surrounding heterozygous tissue. (G) Quantification of mutant clone versus twin spot size (n = 7 clones for each genotype). (H) Quantification of photoreceptor cell area (control n = 11, Tsc1Q600X n = 17, unkex24 n = 15, hdc43 n = 27, unk, hdc overexpression (o/e) n = 17). (I–K) Representative examples of individual cells within control, unkex24 or Tsc1Q600X MARCM clones in the eye disc expressing membrane-tagged GFP (green) and stained for Bar expression (red). Note the larger size of Tsc1 mutant cells in (K). Scale bar: 2 µm. (L–N) Phospho-AKT expression (red in (L),(M),(N) and white in (L′),(M′),(N′)) is not changed in control (L, L′) or unkex24 mutant clones (M, M′), but is decreased in Tsc1Q600X mutant clones (N, N′) in the eye disc. Clones marked by loss of GFP expression (green). Anterior is to the left. Data are represented as mean +/− SEM, ***p≤0.001.
Figure 4.
Hdc physically interacts with Unk and negatively regulates neurogenesis.
(A) Hdc physically interacts with Unk. Venus-Unk or FLAG-HdcS were expressed alone or together in S2 cells and immunoprecipitated with GFP or FLAG antibodies. (B, B′) hdc43 mutant clones showing precocious differentiation of R1/6 (arrows), marked by the expression of Bar (red). (C-C′′′) hdc43 mutant clones showing precocious differentiation (arrow) of R7 and cone cells (marked by the expression of Prospero (red)) and decreased expression of Unk (white in (C″) and (C′′′)). The differentiation front is marked by a dotted line. (D, D′) Precocious differentiation of cone cells (marked by the expression of D-Pax2, red) in a hdc43 mutant clone. Arrow indicates cone cells that have differentiated precociously. Note also the increased expression of D-Pax2 in hdc mutant clones. (E, E′) Loss of hdc does not affect the differentiation of R3/4 (marked by the expression of Spalt (Sal, red)). (F, F′) Loss of hdc does not affect the differentiation of R2/5 (marked by the expression of Rough (Ro, red)). (G) hdc43 mutant clones in the adult eye cause defects in ommatidial rotation and morphogenesis. Mutant cells are marked by the lack of dark pigment. Black arrows indicate mis-rotated ommatidia; red arrows indicate ommatidia with missing photoreceptors; black arrowheads indicate elliptical rhabdomeres; red arrowhead indicates split rhabdomeres. Mutant clones are marked by loss of GFP expression (green) in (B)–(F). Anterior is to the left.
Figure 5.
Hdc expression is controlled by InR/mTOR signalling and unk.
(A) A confocal projection of a wild-type eye disc stained for Hdc protein expression showing Hdc expression is enriched posterior to the morphogenetic furrow (MF). Scale bar: 50 µm. (B) High magnification single confocal section of Hdc expression (green) in differentiating photoreceptors showing cytoplasmic localisation. Bar staining marking R1/6 is shown in red. N: nucleus. Scale bar: 10 µm. (C, C′) Hdc expression (red in (C) and white in (C′)) is increased in unkex24 mutant clones. (D, D′) Close up of boxed region in (C) showing that Hdc expression (red) is increased in the unkex24 mutant clone behind the differentiation front for R1/6, marked by the expression of Bar (white). (E, E′) Hdc expression (red in (E) and white in (E′)) is increased in Tsc1Q600X mutant clones. (F, F′) Close up of boxed region in (E) showing that Hdc expression (red) is increased in the Tsc1Q600X mutant clone behind the differentiation front for R1/6, marked by the expression of Bar (white). White lines mark clone outlines. (G, G′) Hdc expression (red) is decreased in a Rheb2D1 mutant clone (arrow). Mutant clones are marked by loss of GFP expression (green). Anterior is to the left. (H–K) Quantification of expression levels in mutant clones versus adjacent wild-type tissue in posterior clones. Data are represented as mean +/− SEM, **p≤0.01,***p≤0.001.
Figure 6.
unk and hdc act together to control the timing of photoreceptor differentiation.
(A, A′) An unkex24, hdc43 double mutant clone causes a similar precocious differentiation of R1/6 phenotype (arrow) to either single mutant. (B) An unkex24, hdc43 double mutant clone in the adult eye causes similar ommatidial rotation and morphogenesis defects to unk and hdc mutant clones. Mutant cells are marked by the lack of pigment. Black arrow indicates a mis-rotated ommatidium; red arrows indicate ommatidia with missing photoreceptors; black arrowheads indicate elliptical rhabdomeres; red arrowheads indicate split rhabdomeres. (C, C′) Overexpression of hdcFL does not affect the differentiation of R1/6. (D, D′) Combined overexpression of unk and hdcFL cause a delay in the differentiation of R1/6 (arrow). (E−H) Combined overexpression of unk and hdcFL affects eye development. Eyes from GMR-Gal4 control (E), or GMR-Gal4 driving the expression of unk (F), hdcFL (G), or unk, hdcFL (H) in female flies. Note the glassy appearance in (H). (I, I′) Overexpression of unk in a Tsc1Q600X clone does not affect the precocious differentiation of R7 and cone cells. (J, J′) Overexpression of unk and hdc in a Tsc1Q600X clone completely suppresses the precocious differentiation of R7 and cone cells. MARCM was used to generate clones in (A), (C), (D), (I) and (J) and so clonal cells are marked by GFP expression (green). Bar (red) marks R1/6 in (A), (C) and (D), while Prospero expression (Pros, red) marks R7 and cone cells in (I) and (J). The differentiation front is marked by a dotted line. Anterior is to the left.
Figure 7.
Unkl is expressed in the developing mammalian eye and SVZ.
(A-A″) COS-7 cells overexpressing HA-tagged mouse Unkl stained for HA expression (red), Unkl expression (green) and DAPI (blue). (B) Hematoxylin and eosin (H&E) (blue) stained coronal section from a mouse E14.5 retina showing strong Unkl expression (brown) in the retina. (C, D) Serial H&E (blue) stained sagittal sections of the lateral SVZ from a P0 mouse showing Unkl expression (brown in (C)) and P-4E-BP expression (brown in (D)). (E-G″) Sagittal sections of the lateral SVZ from a P0 mouse showing Unkl expression (red in (E′, E″), (G′, G″), (F′, F″)) and NSCs (stained for GFAP, green in (E, E″)), TAPs (stained for Mash1, green in (F, F″)), or neuroblasts (stained for Dcx, green in (G, G″)). DAPI staining is shown in blue in (E″), (F″), (G″).
Figure 8.
A model for the regulation of the timing of neuronal differentiation by the Unk/Hdc complex acting downstream of InR/mTOR signalling.
Unk and Hdc form a complex that is negatively regulated by mTOR signalling. The Unk/Hdc complex then negatively regulates the expression of D-Pax2 and potentially other neurogenic factors to control the timing of photoreceptor differentiation. See the Discussion for details.