Chen YY, Hehr CL, Atkinson-Leadbeater K, Hocking JC, McFarlane S.

	Figure 1. Xadam10 mRNA is expressed in the developing dorsal diencephalon and midbrain at the time RGC axons grow to the optic tectum. Expression of Xadam10 mRNA in a whole-mount brain and sections through the eye and brain at stages 32–37/38 as visualized by in situ hybridization with an antisense Xadam10 riboprobe is shown. A, Lateral view of a stage 33/34 whole-mount brain. Xadam10 mRNA is expressed in the dorsal diencephalon and optic tectum, but signal is much reduced in the ventral diencephalon. Similar expression is seen at stage 32 (data not shown). The inset shows a stage 40 brain with an HRP-labeled optic projection to compare the path taken by RGC axons with the expression of Xadam10 mRNA. B, Stage 32. Expression in the retina is widespread, including the RGC layer (brackets indicate the RGC layer, which is just forming) as indicated by immunolabeling for the Islet-1 transcription factor (inset), although little or no signal is observed in the developing outer nuclear layer (arrowhead). C, D, Stage 35/36. Xadam10 mRNA is still expressed in the dorsal diencephalon (C) and the optic tectum (D). The expected location of the distalmost part of the optic projection is indicated by asterisks, and insets show the location of HRP-labeled RGC axons (red-brown) in comparable brain sections. E, F, Stage 35/36. In a section through the more peripheral retina (E), Xadam10 mRNA is expressed in the innermost layers of the retina, including the RGC layer (indicated by brackets), as well the proliferative ciliary marginal zone. In sections through the central retina (F), Xadam10 mRNA expression is patchy (compare labeling in the RGC layer in both panels). G, Sense control at stage 33/34. H, By stage 37/38, Xadam10 mRNA is mainly localized to the inner nuclear layer and absent or greatly reduced in the outer nuclear and RGC layers. T, Optic tectum; Pi, pineal gland; Di, diencephalon; Ve, ventricle; ONL, outer nuclear layer; INL, inner nuclear layer; PE, pigment epithelium; cmz, ciliary marginal zone; L, lens; D, dorsal; V, ventral; Tel, telencephalon. Scale bar: (in H) A, 100 μm; C, D, 75 μm; B, E–H, 40 μm.
	Figure 2. A–I, Pharmacological inhibition of ADAM10 causes defects in target recognition. HRP-labeled optic projections in stage 40 brains exposed at stages 31 or 33/34 to either a control solution (A, G) or a solution containing the ADAM10 inhibitor, GI254023X (B–F, H, I) are shown. Dotted lines show the approximate anterior border of the optic tectum. A, Control DMSO solution. Axons grow through the diencephalon, make a turn in the mid-diencephalon (asterisk), and innervate the optic tectum. The inset shows the behavior of axons at the border of the optic tectum. B, GI254023X (0.1 μM). The white arrow indicates the normal trajectory of RGC axons after the mid-diencephalic turn (asterisk) and has been included in D–F for comparison purposes. C, GI254023X (0.5 μM). Many RGC axons fail to recognize their target and turn and grow along the anterior border of the optic tectum (black arrowheads). The inset shows a high-power view of the optic projection at the border of the optic tectum. D, E, GI254023X (1 μM). Some axons fail to recognize the optic tectum (black arrowheads) or make an earlier guidance error at the mid-diencephalic turn (black arrows). F, GI254023X (5 μM). Most axons fail to turn in the mid-diencephalon (asterisk; compare trajectory of axons to that of the white arrow) and never reach the anterior border of the optic tectum. G–I, HRP-labeled RGC axons in stage 39 brains where only the optic chiasm was exposed at stage 31 to either a control solution (G) or a solution containing 5 μM GI254023X (H, I). In the G1254023X-treated brain (H), as in control (G), there is no obvious ipsilateral projection, and the optic nerve crosses over at the optic chiasm to the contralateral side of the brain and grows normally within the diencephalon that is not exposed to the inhibitor (I). Tec, Tectum; Pi, pineal gland; ot, optic tract; Di, diencephalon; Hb, hindbrain; on, optic nerve; Ipsi, ipsilateral; Contra, contralateral; D, dorsal; A, anterior; Tel, telencephalon. Scale bar: (in B) A, C, G–I, 50 μm; B, D–F, 30 μm.
	Figure 3. The ADAM10 pharmacological inhibitor has a dose-dependent effect on RGC axon guidance. A, Graph showing the percentages of embryos at different doses of GI254023X that have a normal RGC axon projection to the optic tectum. B, Graph showing the percentages of embryos at different doses of GI254023X that exhibit either defects in target recognition, guidance at the mid-diencephalic turn (Miss turn), or axon extension (Short). Note that some embryos show more than one defect and that the number of embryos showing target recognition defects decreases at 5 μM GI254023X because axons fail to make the turn in the mid-diencephalon and never reach their target. The numbers in parentheses are the numbers of embryos.
	Figure 4. The pharmacological inhibitor GI254023X does not cause gross defects in the patterning of the diencephalic neuroepithelium. Neuroepithelial marker immunostaining of 12 μm cross sections of the diencephalon/midbrain regions of stage 40 embryos exposed at stage 33/34 to either 0.05% DMSO control medium (A, C, E, G) or 5 μM GI254023X (B, D, F, H). In all panels, the exposed side of the brain is on the left, and the unexposed side is on the right. Arrowheads point to the dorsal and ventral midline. Representative images are shown for the ventrally expressed transcription factor, Islet-1 (A, B), general neuronal markers NCAM (C, D) and Zn-12 (E, F), and the neurotransmitter GABA, which is expressed in three dorsal-to-ventral arrayed populations of neurons in the diencephalon (G, H, asterisk). No gross alterations in the pattern of expression of the antigens were observed between control and inhibitor-treated brains. Ex, Exposed side of brain; UnEx, unexposed side of brain; Ve, ventricle; Di, diencephalon; E, eye; np, neuropil; D, dorsal; V, ventral. Scale bar, 100 μm.
	Figure 5. Misexpression of DN-ADAM10 in the dorsal neuroepithelium causes target recognition defects. A, Schematic diagram of the DNADAM10 constructs used in the electroporation and transfection experiments. S, Signal sequence; Pro, prodomain; MP, metalloproteinase domain; Dis, disintegrin domain; Cys, cysteine-rich region; TM, transmembrane domain; IC, intracellular domain; N, N terminal; C, C terminal. B–J, Transgene-expressing cells and HRP-labeled optic projections in lateral views of stage 40 brains. mRNA for GFP (B–D), GFP+DNXadam10 (E–I), and DNXadam10-mt (J) were injected into the anterior brain vesicles and electroporated into the dorsal neuroepithelium. At stage 40, the optic projections were anterogradely labeled with HRP, and the brains were processed for whole-mount immunochemistry with anti-HRP and anti-GFP or anti-myc. D and G are high-power views of the images shown in B and E, respectively. Note that many more axons enter the optic tectum in the control (D) than in the DNADAM10-expressing (G) brain. H shows an HRP-labeled optic projection in a brain electroporated with GFP+DNXadam10, and I illustrates the same projection along with expression of the coelectroporated GFP transgene. J shows immunolabeling with an antibody against the myc tag to visualize DNXADAM10-MT expression (green) and an antibody against HRP to visualize RGC axons (red). The arrows and arrowheads in H and J indicate target recognition and turning defects, respectively. The dotted lines outline the embryonic brain (C, F) and indicate the approximate anterior border of the optic tectum (B, D, E, G, H, J). Di, Diencephalon; Pi, pineal gland; Tec, tectum; D, dorsal; A, anterior. Scale bar: B, C, E, F, 50 μm; D, G–J, 25 μm.
	Figure 6. Misexpression of DN-ADAM10 in developing RGCs has no effect on RGC axon extension or guidance. Eye primordia were transfected in vivo with either CS2GFP or CS2DNXadam10-mt DNA at stage 18, and embryos were left to develop until stage 40. Dissected brains and transverse cryostat sections were processed with antibodies against GFP or the myc protein to visualize transgene-expressing axons. In C and D, transfected RGC axons are immunopositive for GFP; in A, B, E, and F, transfected RGC axons are immunopositive for the myc tag of the DNADAM10 protein. A, DNADAM10-MT-expressing axon extends along the vitreal surface of the retina (arrowheads) and in the optic nerve head in a transverse section through the retina. B, DNADAM10-MT-expressing axons in the optic nerve and crossing the midline at the optic chiasm in a transverse section through the retina and ventral diencephalon. A few DNADAM10-MT expressing cells in the mesenchyme and retina are myc immunopositive. C–F, GFP-expressing (C, D) and DNADAM10-MT-expressing (E, F) RGC axons in lateral views of whole-mount brains. D and F are higher-magnification views of boxed areas in C and E, respectively. Dotted lines indicate the approximate anterior border of the optic tectum. Both GFP- and DNADAM10-MT-expressing axons show normal guidance to and innervation of the optic tectum. Tec, tectum; on, optic nerve; onh, optic nerve head; oc, optic chiasm; ot, optic tract; PE, pigment epithelium; Di, diencephalon; E, eye; L, lens. Scale bar: C, E, 50 μm; A, B, D, F, 25 μm.

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