To investigate Gogo's role in axonal pathfinding, its expression during visual system development was assessed by gogo in situ hybridization performed by Satoko Suzuki and antibody staining performed together with Takashi Suzuki. gogo mRNA expression was detected both in the eye disc and the brain of third instar larvae. In eye discs, the region posterior to the morphogenetic furrow, where differentiating R neurons reside, is stained in a dotted manner (Figure 3-5A). The dotted expression in the eye disc was investigated in more detail by double staining of gogo mRNA and Elav antibody which labels all R cell types. gogo mRNA is detected predominantly in the center of each ommatidium, where R8 is located (Figure 3-5B-Bi’). The single gogo expressing cell in the ommatidial center was identified as R8 by double staining using the R8 specific marker Senseless (Sens). The R8 nuclei stained by Senseless are nicely enclosed by gogo mRNA, localizing to the cytoplasm of the cell (Figure 3-5D-Di’). In addition to photoreceptor expression, presumed medulla neurons, whose cell bodies lie outside the crescent shape formed by innervating R7/8 neurons (Figure 3-5C), showed high levels of gogo mRNA expression.
Figure 3-5 gogo in situ staining in eye discs and optic lobes
(A-D) in situ hybridization of gogo in third instar larvae. In the eye-disc (A,B,D), gogo mRNA is
expressed in developing photoreceptors posterior to the morphogenetic furrow (arrowheads) (A). Staining of gogo mRNA (magenta) and Elav protein (green) shows that gogo mRNA is expressed in one
cell per ommatidium (B). In the optic lobe, gogo mRNA is expressed in a crescent shape surrounding the optic lobe center (C) A 3D image of simultaneous staining gogo mRNA (magenta) and Senseless
protein (green) shows that gogo mRNA is localized around the Senseless positive nuclei of R8s (D). Magnifications: merge (Di); magenta (Di’). a; anterior, p; posterior. Scale bars 10μm
A Gogo antibody was generated against the extracellular domain by Takashi Suzuki. This Gogo antibody labels R axons, shown by colocalization with labeled R axons. R cells were specifically labeled with mKOrange (monomeric Kusabira Orange) (Karasawa et al., 2004) under the control of the GMR promoter. In third instar larvae, Gogo seems to be absent in the lamina, which is innervated by R1-R6 (Figure 3-6B- B’), but highly expressed in R axons in the medulla (R7 and/or R8). As the majority of R axons innervating the medulla are R8 axons at this stage of development, this staining is consistent with the in situ which shows strong gogo expression in R8s in the eye disc. In particular, antibody staining at the tip of axons is clearly visible (Figure 3-6A-Ai’ arrows), suggesting a role for Gogo in navigating growth cones. The most outer axons (arrowhead in Figure 3-6A), which represent the youngest in- growing axons show the strongest Gogo expression along the axon. Strong staining is also observed below the lamina plexus (Figure 3-6A, B, bracket), in the lobula and in the lobula plate. Since the in situ also shows robust expression in the region outside the medulla crescent where medulla neurons arise, it is very likely that the antibody staining in the lobula/lobula plate is mainly caused by the localization of Gogo to the processes of unidentified medulla neurons.
Figure 3-6 Gogo expression during larval development
(A-B) Gogo antibody staining of WT third instar optic lobes (A). Gogo (green) localizes predominantly
along the axon (arrowhead in A) and at the growth cones of R7/8 (arrow in A). The staining in the medulla colocalizes with the growth cones marked by GMR-mCD8mKOrange, stained with anti-KO (magenta); arrows in Ai, Ai). 3D projection image (using the software AMIRA) shows that Gogo
expression is not observed on R1-R6 axons, which terminate in the lamina (arrowhead). R axons are stained using GMR-mCD8mKOrange (red) and Gogo antibody (green) (B-B’). Scale bars 10μm
Results
During early pupal stages, Gogo expression is unambiguously detected in all photoreceptor types stained with mKOrange. At 24APF (24hr after puparium formation), Gogo is observed in R8 axons and the axonal termini of R7 and R1-6 (Figure 3-7A-Aii’). Thus, Gogo is expressed in all the photoreceptors at this stage (Figure 3-7H, H’). Interestingly, at 40APF, when R1-6s are still involved in lamina cartridge formation, Gogo is expressed on all R1-6 axons (Figure 3-7C, C’, D, D’). From the mid-pupal stage onward, Gogo expression becomes reduced in the photoreceptor axons. At 40APF faint staining can be observed around the M3 layer (Figure 3-7E-Ei’). Since the Gogo staining and R axons do not overlap perfectly, it is likely that the staining derives from lamina neurons or higher order neurons.
Similarly, in late stages of pupal development Gogo seems to be present in R neurons at a very low level only. At 72APF, Gogo can be rarely seen on the axons of R7/8, together with a faint staining of supposedly lamina or medulla neurons (Figure 3-7F-Fi’). In contrast to the observed protein reduction in photoreceptor axons, Gogo protein levels seem not to be altered in the photoreceptor cell bodies. In the retina Gogo protein was detected in all photoreceptor types throughout pupal development (Figure 3-7G-L’). This observation rather suggests the presence of a mechanism regulating Gogo protein level within the axon or growth cone rather than transcriptional regulation.
The specificity of the generated Gogo antibody was confirmed in gogo- eyFLP larval
mosaics and by labeling small heterozygous WT clones in an otherwise homozygous gogo mutant retina during pupal development. Anti-Gogo staining is not detected in gogo mutant retinal cells at 24APF, 40APF and 48APF (Figure 3-7B-B’ and data not shown).
Figure 3-7 Gogo is dynamically presented on R axons during pupal stages
Anti-Gogo staining is shown in green (A-L) and R axons are visualized by GMR-mCD8mKOrange in magenta (A-F) or blue (G-L), respectively. (A-B’) The optic lobe (A-Aii’) and the retina (B-B’) of 24APF pupae. (A) Strong Gogo expression is observed on R axons in the lamina (i) and the medulla (ii). Magnifications of the lamina (Ai, Ai’) and the medulla (Aii, Aii’). In the lamina, Gogo localizes to the
termini of R1-6 (arrowheads in Ai, Ai’). In the medulla, Gogo strongly overlaps with the termini of R7 (arrowheads in Aii, Aii’) and R8 (arrows in Aii, Aii’). In gogoH1675eyFLP retina, WT R cells marked with
GMR-mCD8mKOrange (magenta) completely overlap with Gogo positive cells (green), whereas gogo- cells lack Gogo protein (B, B’). (C-Fi’) The optic lobe at 40APF (C-D’), 48APF (E-Ei’) and 72APF (F-Fi’):
At 40 APF termini of R7 and R8 show Gogo staining (R7, arrowheads; R8, arrows in Ci and Ci’). Gogo is strongly localized to the R1-6 axons during lamina cartridge formation (D, D’). At 48APF, overall expression of Gogo becomes reduced. Punctate Gogo is seen around the M3 layer to which R8 extends its filopodia at this stage (arrows in Ei, Ei’). Gogo expression can be vaguely detected on R axons at
72APF (arrow in Fi, Fi’). Medulla layers are indicated in Ci, Ei, and Fi. (G-L’) In contrast all photoreceptor cell bodies are Gogo protein positive during the pupal stages 10APF, 24APF, 42APF, 48 APF, 52 APF and 55 APF. Scale bars 10μm.
Results