Webber CA, Hocking JC, Yong VW, Stange CL, McFarlane S.

	Fig. 1. RGC axons make improper guidance decisions in the presence of the metalloprotease inhibitor GM6001.A–G, Representative examples of stage 40 whole-mount brain preparations showing the HRP-labeled optic projection in a control brain (A, B) and when GM6001 was applied to the exposed brain (C–G). B andD are higher-power views of A andC, respectively. C, D, A 1 μM concentration of GM6001. E, A 10 μM concentration of GM6001. F, A 20 μM concentration of GM6001. G, Axons cross over the dorsal midline into the contralateral brain of a 1 μM GM6001-treated brain. Tec, Tectum;Pi, pineal gland; Di, diencephalon;Tel, telencephalon; ot, optic tract;Hb, hindbrain; D, dorsal;A, anterior. Arrowheads mark the midbrain–hindbrain isthmus; the asterisk marks the caudal turn of the optic projection in the mid-diencephalon, andarrows in F show axons growing aberrantly in the telencephalon. White dots show the approximate anterior border of the optic tectum. Scale bars: C, 50 μm; A, C, 65 μm for E andF; and G, 50 μm for B,D, and G.
	Fig. 2. Defects in RGC axon pathfinding and target recognition caused by the application of GM6001. A qualitative description of the different defects in axon behavior observed in optic projections treated with different doses of GM6001 is given.Numbers in parentheses represent the number of embryos. % Abnormal is the percentage of embryos with some defect in the optic projection; % Failure to Turn refers to optic projections in which the majority of axons show a guidance defect in the mid-diencephalon, where RGC axons normally make a caudal turn toward the optic tectum; % Extension Defect refers to a shortened optic projection;% Normal Target Innervation means that the majority of RGC axons enter the optic tectum; % Midline Crossingrefers to axons that extend aberrantly across the dorsal midline into the contralateral brain.
	Fig. 3. Quantitation of the effects of GM6001 on axon guidance and extension. Control and GM6001-treated brains were exposed at stage 33/34 and fixed at stage 40. Camera lucida representations of the brains and optic projections were normalized; two features of each optic projection were measured: length and the angle of the turn made in the mid-diencephalon. A, To measure the turning angle, a line (L2) was drawn at a 60° angle (θ1) to a standard reference line (L1) between the optic chiasm and the midbrain–hindbrain isthmus, bisecting the optic tract at the level of the turn made in the mid-diencephalon. The turning angle (θ2) was measured between L2 and a third line (L3) drawn through the middle of the distalmost portion of the optic projection. Measurements are shown for a control exposed brain (A) and a metalloprotease inhibitor exposed brain (B). C, Mean turning angle in control embryos and embryos treated with different doses of GM6001.D, Effects on optic projection length (in micrometers) of increasing doses of the GM6001. Tract length was measured in normalized b.r.u.s and then converted to micrometers (1 b.r.u. ≅ 620 μm in an unfixed brain) (Chien et al., 1993). At lower concentrations, at which defects in pathfinding are observed, the extension of RGC axons is only moderately affected by GM6001. At the highest concentrations of GM6001, the optic projection is significantly shorter than in control exposed brains. *p < 0.05; **p < 0.01; ***p < 0.001; ANOVA, Student–Newmann–Keuls post hoc test. The numbers of embryos are shown in parentheses, and error bars are SEM.
	Fig. 4. A compound related to GM6001 that does not inhibit metalloproteases fails to influence formation of the optic projection. An L-tryptophan methylamide compound, GM6001 negative control, was applied to the developing optic projection using the exposed brain preparation. A, B, HRP-labeled projections in stage 40 brains exposed at stage 33/34 to control (A) and 5 μM GM6001 negative control solutions (B). Tec, Tectum; D, dorsal; A, anterior. C, D, Graphs of the mean turning angle (C) and mean normalized optic projection length (D) in brains exposed to 0.2% DMSO (Cont), 5 μM GM6001 negative control [Gm(−)], or 5 μM GM6001 (Gm) for 3 or 20 hr. The numbers of embryos are shown in parentheses, and error bars are SEM. ***p < 0.001; ANOVA, Student–Newmann–Keuls post hoc test). Scale bar: (inB) A, B, 50 μm.
	Fig. 5. A second metalloprotease inhibitor, BB-94, has effects similar to those of GM6001 on RGC axon guidance and extension.A–D, HRP-labeled optic projections in stage 40 whole-mount brains exposed at stage 33/34 to 0.1 μM(A) and 1.0 μM(B–D) BB-94. At the low dose of BB-94 (A) the optic projection forms normally. At 1.0 μM, BB-94 RGC axons do not turn in the mid-diencephalon (B, C) and fail to innervate the optic tectum (D). D is a higher-power view ofC. The dotted white line shows the approximate anterior border of the optic tectum. Tec, Tectum; Pi, pineal gland; D, dorsal;V, ventral. Scale bars: C (forA–C), D, 50 μm.
	Fig. 6. Defects in RGC axon pathfinding and target recognition caused by the application of BB-94. A, Qualitative description of the different defects in axon behavior observed in optic projections treated with BB-94.Numbers in parentheses represent the number of embryos. The description of the defects is the same as presented in the legend to Figure 2. B, C, Control and BB-94-treated brains were exposed at stage 33/34 and fixed at stage 40. Measurements were made as described in the legend to Figure 3.B, Effect on mean turning angle of increasing doses of BB-94. C, Dose–response of the effects of BB-94 on optic tract length (in micrometers). At lower concentrations, at which defects in pathfinding are observed, the extension of RGC axons is unaffected by BB-94. At the highest concentrations of BB-94, the optic projection is significantly shorter than in control exposed brains. *p < 0.05; **p < 0.01; ***p < 0.001; ANOVA, Student–Newmann–Keuls post hoc test. The numbers of embryos are shown in parentheses, and error bars are SEM.
	Fig. 7. Xenopus Kuzbanian is expressed in the developing diencephalic brain neuroepithelium.A, Western blot of adult rat cerebellar (left) and stage 42 Xenopus head (right) tissue using a rabbit antibody developed against a human ADAM10 peptide. Molecular masses are indicated based on standards run at the same time as the sample tissue. A prominent protein band with an approximate molecular mass of 82 kDa was observed in rats, and a similarly sized band of ∼75 kDa was observed inXenopus. B, C, Immunolabeling of stage 35/36 Xenopus transverse sections with the ADAM10 antibody shows labeling of cells in the diencephalon (B) and in the RGC layer (C). Note that there is no immunoreactivity in the neuropil or the optic nerve. Np, Neuropil;Di, diencephalon; PE, pigment epithelium;RGCL, RGC layer; L, lens;D, dorsal; V, ventral. Scale bars, 50 μm.
	Fig. 8. Metalloprotease inhibitor treatment does not grossly affect growth cone morphology or the patterning or morphology of the neuroepithelium. A, B, Control (A) or 5 μM GM6001-treated (B) growth cones in culture. C–H, Cross sections through the diencephalon/midbrain regions of stage 40 embryos exposed at stage 33/34 to control (0.4% DMSO) or 10 μM GM6001 bathing solutions. In allpanels, the exposed side of the brain is on theleft, and the unexposed side is on theright. Note that the left eye is removed during the exposure. Sections were immunolabeled with markers of the neuroepithelium. C, D, Zn-12 immunolabeling of control brains (C) and brains exposed to GM6001 (D). E, β-tubulin immunoreactivity of a GM6001-treated brain. F, Islet-1 immunolabeling of ventrally located neurons (arrows) in a GM6001-treated brain showing that dorsoventral polarity is maintained. G, H, Immunolabeling of control (G) and GM6001-treated (H) brains with a rabbit polyclonal GABA antibody. GABAergic neurons found in the mid-diencephalon are present in both control and experimental brains. R, Retina;Di, diencephalon; Ve, ventricle;Mb, midbrain; D, dorsal;V, ventral. Scale bar: (in H)A, B, 10 μm; C–H, 50 μm.
	Fig. 9. Defects in RGC axon target recognition caused by the late application of GM6001. Brains were exposed at stage 37/38 to either a control solution (A) or 5 μM GM6001 (B–E), and optic projections were HRP-labeled at stage 40. Target-recognition defects are evident in the GM6001-treated brains. Axons grow dorsally along the anterior border of the optic tectum and then cross over the dorsal midline into the contralateral brain (D).E is a higher-power view of the boxed area in C. Scale bars: C (forA–C), D, 50 μm; E, 25 μm.

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