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First published online 14 March 2007
doi: 10.1242/dev.001529

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Focal adhesion kinase controls morphogenesis of the Drosophila optic stalk

¹ Laboratory of Morphogenesis, Institute of Molecular and Cellular Biosciences, the University of Tokyo, 1-1-1 Yayoi, Bunkyo-Ku, Tokyo 113-0032, Japan.
² Graduate Program in Biophysics and Biochemistry, Graduate School of Science, the University of Tokyo, 7-3-1 Hongo, Bunkyo-Ku, Tokyo 113-0033, Japan.

View larger version (66K): [in this window] [in a new window]   Fig. 1. SG cells form the optic stalk, the monolayered tubular structure that surrounds the R axon bundle. Unless otherwise noted, all specimens are late third instar viewed from a lateral perspective. Gal4 expression was detected with UAS-mCD8GFP (green) and glial cells with anti-Repo (Blue), except where noted. (A) Developing Drosophila visual system. Photoreceptor cells extend their axons (R axons; white) toward the target region, the lamina (green, anti-Dachshund), in the optic lobe. R axons form a bundle in the optic stalk. (B) R axons (green, visualized with GMRGal4) are surrounded with glial cells (white). (C) Processes of WG (green, visualized using ptc-Gal4) enwrap R axons (magenta, anti-HRP) in the optic stalk (arrow). Note that ptc-Gal4 also labels a subset of epithelial cells of the eye disc (arrowhead). (D) SG cells (green, visualized using NP4702-Gal4) form the outer sheath (arrows) that surrounds the R axons (magenta, anti-HRP). (E) An optic stalk carrying NP4702-Gal4 and UAS-GFP. SG cells (green) are arranged along the AP axis, and form a sheath by tightly contacting each other. (E') A single SG cell, which is clonally labeled by mCD8GFP (green), extends two long processes anteroposteriorly. (F) An optic stalk carrying NP4702-Gal4, viewed from a horizontal perspective. SG cells (green) form a single-cell layer. LamininA (magenta, anti-LamininA) is detected on the surface of the SG cell layer. (G,H) Quantitative analysis of the optic stalk morphology. (G) An optic stalk stained with anti-Perlecan (magenta). The green bar indicates the height of the optic stalk, while the white bar indicates the diameter. (H) Quantification of the average length of height (A/P) and the diameter (D/V) of wild-type optic stalks. The error bars indicate s.d. (I,J) Schematic drawings of the optic stalk. (I) WG cell processes (gray) loosely enwrap several R axons (magenta). SG cells (green) surround the entire R axon bundle, forming an outer sheath. (J, left) The optic stalk consists of SG cells (green), which form a monolayered tubular structure, and is surrounded by the BM (gray). (J, right) The optic stalk viewed from a horizontal perspective (top) or a lateral perspective (bottom). (A,B,C,D,E,G) Anterior is upwards and dorsal is to the right. (F) Lateral down, dorsal right.

View larger version (37K): [in this window] [in a new window]   Fig. 2. The optic stalk expands before R axon innervation. (A-D) Optic stalks at the early larval stages before R axon projection. (A,B) Optic stalks carrying ombP1 at the late second instar (A,A') and the early third instar (B,B'). lacZ expression is visualized with anti-ß-galactosidase (ß-gal) antibody (white). The BM is visualized with anti-Perlecan. (A) At the late second instar before R axon innervation, glial cells (white) form a tubular structure around Bolwig's nerves (magenta, anti-HRP). (B) At the early third instar, the optic stalk expands and glial cells increase in number. One ommatidial column is detected in this specimen, but R axon innervation is not observed (arrow). (C,D) A late second instar (C) or an early third instar (D) larval optic stalk carrying NP4702-Gal4 and UAS-mCD8GFP, viewed from horizontal perspective. (E,E') Cells in mitosis are visualized with anti-phospho-Histone H3 (magenta) in an early third instar optic stalk. Glial cells (blue, anti-Repo) in the optic stalk undergo mitosis (arrow). Repo-positive glial cells are shown alone in E'. (F,F') An early third instar larval optic stalk carrying NP4702-Gal4, UAS-mCD8GFP and ptc-lacZ (visualized with anti-ß-gal). The optic stalk consists solely of SG cells (green), while no WG cells (white) are seen in the optic stalk. (G-G'') Clonally labeled glial cells surrounding the optic lobe (green) never entered into the optic stalk (white, visualized with -Tubulin). (A-G'', except for C and D) Anterior is to the up, dorsal right. (C,D) Lateral is down, dorsal right. os, optic stalk.

View larger version (57K): [in this window] [in a new window]   Fig. 3. The optic stalk is formed independently of R axon projection. (A-D) Wild-type (A,C) and sine oculis1 (so1) (B,D) optic stalks at the early pupal stage, viewed from a lateral (A,B) or a horizontal (C,D) perspective. Glial cells are visualized with anti-Repo (blue). The BM is visualized with anti-Perlecan (green). In a so1 mutant, the optic stalk was observed, despite a complete lack of photoreceptor cells (magenta, anti-HRP). (E) Quantification of the optic stalk growth in wild-type (white column) and so1 mutant animals (gray column). A cross-sectional area surrounded by the BM (green in C,D) is measured in each specimen at larval or early pupal stages. Columns represent the mean cross-sectional area. The mean cross-sectional area of so1 optic stalk is not significantly different from that of wild type at the early pupal stage (P=0.57). The error bars indicate s.d.; n.s. indicates no significant difference. (F) A late third instar so1 mutant optic stalk carrying NP4702-Gal4 and UAS-mCD8GFP, viewed from a horizontal perspective. SG cells (green) form a monolayered tubular structure. Arrows in B,D and F indicate Bolwig's nerves. (A,B) Anterior is to the up, dorsal right. (C,D,F) Lateral is down, dorsal right.

View larger version (35K): [in this window] [in a new window]   Fig. 4. Disruption of optic stalk morphology in the Fak56D mutant. (A,B) R axons in a wild type (A) and a Fak56DCG1 homozygous mutant (B), visualized with mAb24B10 (white). In a Fak56D mutant, R axons failed to be bundled (arrow). (C) Optic stalks in a wild type (0, left) and in Fak56D mutants (1-4), visualized with anti-Perlecan (white). Fak56D mutants exhibited defects in optic stalk morphology. Defects were classified into five groups (0-4), according to the severity of the phenotype. A dashed line indicates the eye disc-optic lobe boundary. Arrows indicate the optic stalk/optic lobe boundary. (D) Quantification of optic stalk defects in wild type and Fak56D mutants. The percentage of the brain that shows optic stalk defect is plotted as a function of severity level of the phenotype (0-4 in C). (E) Quantification of optic stalk defects in wild-type animals or Fak56DCG1 homozygous animals at various developmental stages. Defects are not detected until the early third instar larval stage in Fak56D mutants, becoming progressively more severe during third instar larval stages. (F) Fak56D expression in the late third instar larvae. Brains and imaginal discs were stained with the Fak56D sense or the Fak56D antisense RNA probe. Fak56D is ubiquitously expressed. (A-C) Anterior up, dorsal right. AP, the height of the optic stalk; DV, the diameter of the optic stalk.

View larger version (55K): [in this window] [in a new window]   Fig. 5. Rescue of optic stalk defects in Fak56D mutants by SG-cell-specific expression of a Fak56D transgene. (A-C) Expression of Gal4 lines used for rescue experiments, visualized with UAS-mCD8GFP (green). (D-F) Fak56D optic stalks carrying NP2109-Gal4, UAS-Fak56D (D) or NP4702-Gal4, UAS-Fak56D (E) or GMR-Gal4, UAS-Fak56D (F). Arrows indicate optic stalks (white, visualized with anti-Perlecan). (G) Quantification of optic stalk defects in animals carrying constructs as described. Expression of exogenous Fak56D in SG cells significantly rescued optic stalk disruption, while photoreceptor cell expression of Fak56D did not rescue the phenotype. Note that rescued optic stalks with NP4702-Gal4 exhibit the thinner morphology than those of wild type (asterisk). (H) Fak56D overexpression in wild type. Columns represent mean length of optic stalks carrying NP4702-Gal4 (white column) or NP4702-Gal4, UAS-Fak56D (gray column). Fak56D overexpression resulted in thinner optic stalks (***P>0.001), while length of the optic stalks did not change (P=0.73). The error bars indicate s.d. (A-F) Anterior up, dorsal right. Genotypes for rescue: Fak56DCG1/Fak56DCG1; NP2109/+, Fak56DCG1/Fak56DCG1; NP2109/UAS-Fak56D, Fak56DCG1/Fak56DCG1; NP4702/+, Fak56DCG1/Fak56DCG1; NP4702/UAS-Fak56D, Fak56DCG1/Fak56DCG1; GMR-Gal4/+, Fak56DCG1/Fak56DCG1; GMR-Gal4/UAS-Fak56D. Genotypes for Fak56D overexpression: NP4702/+, NP4702/UAS-Fak56D.

View larger version (49K): [in this window] [in a new window]   Fig. 6. SG cells fail to be organized into a tubular structure in Fak56D mutants. (A,B) A wild-type (A) and a Fak56D mutant (B) optic stalk at late third instar larval stage were stained with anti--Tubulin (white). In a wild-type optic stalk, SG cells express a high level of -Tubulin (white, anti--Tubulin) (arrow in A). In a Fak56D mutant, SG cells failed to be organized into a tubular structure and instead spread over the surface of the optic lobe (arrow in B). (C-D') A wild-type (C) or a Fak56D mutant (D) optic stalk of an early third instar larva carrying ombP1 (white, visualized with anti-ß-gal). The BM is visualized with anti-Perlecan (green). Anti-HRP (magenta) stained only Bolwig's nerves. In a Fak56D mutant at the early third instar, SG cells spread over the surface of the optic lobe (arrow in D). (C',D') Glial cells are shown alone in these panels. The outlines of the optic stalks are indicated with dashed yellow lines. (E-G) Subsets of SG cells were clonally labeled with UAS-mCD8GFP (green) in a wild type (E) or Fak56D mutants (F,G). SG cells in the optic stalk were visualized with anti--Tubulin (white). SG cells distributed along the AP axis and made contacts with each other via their processes (arrowhead in E). In Fak56D mutants, SG cells exhibited normal elongated morphology (arrowheads in F) but occasionally failed to distribute properly (arrows in G). (H) Quantification of SG cell distribution along the AP axis in wild type (yw) and Fak56D mutants. Positions of SG cells in clones containing a few cells were classified into four groups.

View larger version (36K): [in this window] [in a new window]   Fig. 7. Identification of CdGAPr as a gene involved in optic stalk morphogenesis. (A) Expression of NP3053-Gal4, which is inserted in the first intron of the CdGAPr, is visualized with UAS-mCD8GFP (green). NP3053-Gal4 exhibits restricted expression in SG cells (arrow) and WG cells (arrowhead). R axons are visualized with anti-HRP (magenta). (B) CdGAPr gene organization, showing the positions of P-element insertions in the CdGAPNP3053 and CdGAPr EY13451 lines. (C,D) Optic stalk morphology in a wild type (C) and a CdGAPrNP3053 homozygote (D), visualized with anti-Perlecan (magenta). A CdGAPrNP3053 homozygote exhibited optic stalk disruption (D). Arrows indicate optic stalks. (E) Quantification of optic stalk defects in various allelic combinations of CdGAPr alleles. (F) Quantification of optic stalk defects in animals carrying NP3053-Gal4, UAS-CdGAPr-dsRNA. (G) Quantification of the optic stalk defects in trans-heterozygous combinations of Fak56D and CdGAPr alleles, or trans-heterozygous mutants for Fak56D and myospheroid (mys). (A,C,D) Anterior up, dorsal right.

View larger version (24K): [in this window] [in a new window]   Fig. 8. A model of Fak56D and CdGAPr control of optic stalk morphogenesis. SG cells distribute along the AP axis. This might be important for anteroposteriorly-elongated morphology of the optic stalk. The mutations of either Fak56D or CdGAPr may cause random distribution of SG cells, which results in a broadened and shortened optic stalk. Cells in the same linage are visualized with the same color. Red arrows indicate directions towards which proliferated cells migrate. Cells in the optic lobe are shown as orange circles. Anterior up, dorsal right.

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