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First published online 13 March 2008
doi: 10.1242/dev.019117

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Drosophila optic lobe neuroblasts triggered by a wave of proneural gene expression that is negatively regulated by JAK/STAT

Tetsuo Yasugi^1,2,*, Daiki Umetsu^1,2,*,, Satoshi Murakami^1,2, Makoto Sato^1,2, and Tetsuya Tabata^1,2,

¹ Laboratory of Pattern Formation, Institute of Molecular and Cellular Biosciences, the University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-0032, Japan.
² Graduate program in Biophysics and Biochemistry, Graduate School of Science, the University of Tokyo, Hongo7-3-1, Bunkyo-ku, Tokyo 113-0033, Japan.

View larger version (90K): [in this window] [in a new window]   Fig. 1. Medullar neuroblast development. All images are of L3 stage unless otherwise noted. (A) Schematic of central nervous system. ED, eye disc; OL, optic lobe; VNC, ventral nerve cord. (B) Schematic of the optic lobe (ventrolateral view). Lamina neurons (La, gray), NE cells (blue), medulla NBs (light pink) and central brain (CB) are shown. (C) Schematic horizontal section of the optic lobe (section of blue plane in B). Lamina neurons (gray), NE cells (blue), medulla NBs (magenta), ganglion mother cells (GMCs, yellow), medulla neurons (orange), lamina furrow and R axons (R1-6 terminate in the lamina, while R7 and R8 project their axons through lamina to medulla) are shown. Cells located lateral most in the NE differentiate into lamina neurons (red arrow). (D) Confocal microscopic image of the optic lobe immunostained for lamina neurons (Dachshund, Dac, gray), NE cells (tkv-lacZ, blue) and medulla NBs (Dpn, magenta). (E,F) Lateral view (E) and horizontal section (F) of the optic lobe. Lamina neurons (Dac, white), NBs (Dpn, magenta), lobula neurons (Elav, yellow in E) and medulla neurons (Elav, yellow in F) are depicted. (G-I) The swath of NBs (Dpn, magenta) widens and the expanse of NE cells (Tailless, Tll, blue) decreases as the optic lobe matures. Optic lobe of early- (G), mid- (H) and late- (I) stage larvae. Tll is expressed in NE cells and lamina neurons. (J) Schematic of NB differentiation in G-I.

View larger version (90K): [in this window] [in a new window]   Fig. 2. L(1)sc is expressed in medial edge of the NE and can induce medulla NB differentiation. (A-F') L(1)sc is expressed in the medial edge of NE cells. Early (A,B), mid (C,D) and late (E,F) stages are shown. (A,C,E) Lateral view. Expression of L(1)sc (green), Dpn (magenta) and Dac (white) are shown. Broken white line in A indicates the border between optic lobe and central brain. (B,D,F) Horizontal section. Expression of L(1)sc (green), Dpn (magenta) and tkv-lacZ (blue) are shown. White arrowheads show cells expressing L(1)sc. (B',D',F') L(1)sc expression in B,D,F. (G) Schematics of the horizontal section as in Fig. 1C. L(1)sc-expressing cells (green) and `proneural wave' (green arrow) are shown. (H-M) Uniform and premature NB differentiation associated with ectopic l(1)sc expression driven by NP6099 Gal4. Early (H,K), mid (I,L) and late (J,M) stages are shown. NBs are marked by Dpn (magenta) and neurons by Elav (blue). Broken white lines indicate the border between optic lobe and central brain.

View larger version (117K): [in this window] [in a new window]   Fig. 3. Proneural genes regulate medulla differentiation. (A) L(1)sc (green) and Ase (magenta) are expressed in NE cells and NBs, respectively. (B) Schematic of lateral view of the optic lobe. Lamina neurons (yellow), L(1)sc-expressing cells (green), NE cells (blue) and NBs (magenta) are shown. (C) Expression pattern of proneural genes in square of B. Among the proteins of the AS-C, Sc is expressed in both NE cells and NBs, L(1)sc in the medial edge of NE cells, and Ase in NBs, respectively. (D,D') Initiation of NB differentiation was delayed in da10 clones. Dpn expression (magenta) almost disappeared in the da10 clones, shown by the absence of GFP (blue in D) and of white in D'. (D') Higher magnification of square in D. (E,E') Expression of L(1)sc (green) was not affected in da10 clones, shown by the absence of GFP (blue), while onset of Dpn expression was delayed (magenta). (E') Higher magnification of square in E. Clones are indicated by the absence of white signal. L(1)sc was shown in green and Dpn in magenta. (F) Genomic locus of AS-C and deficiency lines used. Deleted genes are depicted by crosses. (G) Onset of Dpn expression (magenta) was delayed in Df(1)260-1 clones, indicated by the absence of white signal. (H) Onset of Dpn expression (magenta) was not affected in Df(1)sc10-1 clones, indicated by the absence of white signal. (I) Onset of Dpn expression (magenta) was slightly delayed in Df(1)sc19-1 clones shown by the absence of white signal. Expression of L(1)sc is shown by green. Broken yellow line shows the border between NE and NBs, and yellow arrowheads indicate cells not expressing Dpn in the clones. (J) Onset of Dpn expression (magenta) was not affected in Df(1)ase1 clones shown by the absence of white signal. (K) Summary of the phenotypes. Onset of Dpn expression was delayed by 4-6 rows of cells in the clones of da10 or Df(1)260-1 (left), and delayed by one or two rows in the clones of Df(1)sc19 (right). Clones are within the black lines. NBs (magenta), L(1)sc-expressing cells (green), NE cells (blue) and cells yet to express Dpn (gray) are shown. L(1)sc expression remained in da10 clones but not in Df(1)260-1 clones (half gray and half green circles).

View larger version (73K): [in this window] [in a new window]   Fig. 4. JAK/STAT signal is activated in the NE cells. (A-C') upd-GAl4 visualized with UAS-GFP.nls is expressed in the lateral side of NE cells in early (A) to mid (B) L3 stages. In the late L3, strong Gal4 expression is restricted to lamina neuron (C). NE cells are visualized by strong expression of Arm (blue) and NBs with Dpn (magenta). (A'-C') Only upd-GAl4 channel is shown. (D-F') 10xSTAT-GFP is expressed in the lateral side of NE cells during early (D) to mid (E) L3 stages. The signal is higher in the lateral side. In the late L3, GFP signal was restricted in lamina neurons (F). NE cells are visualized by strong expression of Arm (blue) and NBs with Dpn (magenta). (D'-F') Only 10xSTAT-GFP channel is shown. Broken white line in A and D indicates the border between optic lobe and central brain.

View larger version (75K): [in this window] [in a new window]   Fig. 5. JAK/STAT signal is required for the production of proper number of medulla neurons and lamina formation. (A,B) Mid L3 optic lobes of wild type (A) and hop2 (B). NE cells are marked by Arm (Blue) and NBs by Dpn (magenta). Both NE cells and NBs expressed high levels of Arm and Dpn in the hop2 optic lobe (B), while only cells at the transition from NE cells to NBs express both in the wild type (A). Broken white line in B indicates the border between optic lobe and central brain. (C-F) Late L3 optic lobes of wild type (C,E) and hop2 (D,F). (C,D) In hop2 mutant, NBs (Dpn, magenta) disappeared, neurons (Elav, blue) were fewer and lamina (Dac, green) was disrupted. (E,F) In hop2 mutant optic lobe, most neurons expressing Elav also express ap-lacZ, which is a marker for the medulla neurons, as in the wild type (F, compare with E). Broken white line in F indicates the border between optic lobe and central brain.

View larger version (73K): [in this window] [in a new window]   Fig. 6. JAK/STAT signal negatively regulates the progression of proneural wave. (A-A'') Loss of Stat92E function lead to earlier progression of proneural wave and NB development. Stat92E85C9 clones were shown by the absence of GFP (blue). (A') Enlarged view of square in A. Clones were circled by yellow lines. Broken white line indicates border between NE and NB. White arrows show earlier (more lateral) expression of Dpn (magenta) and L(1)sc (green). L(1)sc was ectopically expressed in the M(3)w124/+ cells next to the clones (white arrowheads). (A'') Projection of XX stacking confocal planes including A. Yellow arrows show earlier expression of Dpn (magenta) and L(1)sc (green). (B,B') Ase (magenta) was expressed in more lateral cells and ectopic neurons (Elav, blue) were observed associated with the Stat92E6346 clones. The clones are shown by the absence of GFP (green) in B and yellow line in B'. (C-C'') JAK/STAT signal regulates the number of lamina neurons and medulla NBs. Stat92E85C9 clones are shown by the absence of GFP (green; circled by a yellow line in C'). (C'') Projection of XX stacking confocal planes including C. In and around the Stat92E85C9 clone, putative lamina cells [marked by Dac (blue)] were replaced by medulla NBs [marked by Dpn (magenta)], so that the lamina region was reduced in contrast to the normal optic lobe (arrow in C'', compare with Fig. 1D or Fig. 5C). (D,D') L(1)sc (green) and Dpn (magenta) remained more medial; nearby cells expressing hopTum-l are marked by the expression of GFP (blue) or arrowhead in D'. (E,E') Expression of Arm (blue) remained in the hopTum-l-expressing clone. Clones were marked by the expression of GFP (green) and NBs by Dpn (magenta). (F,F') L(1)sc (green) and Dpn (magenta) remained more medial; nearby cells expressing upd are marked by the expression of GFP (blue) or arrowhead in F'. (G,G') Expression of Arm (blue) remained near the upd-expressing clone marked by GFP (green). NBs were marked by Dpn (magenta). (H) Summary of loss-of-function and gain-of-function mutation of the JAK/STAT signal. Proneural wave extends more medially with elevated JAK/STAT signaling and laterally with decreased signaling. Clones are within the black lines. NBs (magenta), L(1)sc-expressing cells (green) and NE cells (blue) are shown. (I) A model for the ordered formation of NBs in medulla development. JAK/STAT signaling negatively regulates the progression of the proneural wave, which induces NB formation and hence regulates the number of medulla NBs.

View larger version (18K): [in this window] [in a new window]   Fig. 7. A model for the progression of proneural wave. Schematic horizontal sections as in (Fig. 1C, Fig. 2G). JAK/STAT signaling is activated in the NE cells and its activation is high in the lateral and low in the medial side of NE. As the NE cells proliferate, Upd-expressing NE cells move laterally, and so does the activation of the JAK/STAT signaling. Medial NE cells freed from negative regulation of JAK/STAT signaling express L(1)sc and proneural wave moves laterally.

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