Nedivi E , Javaherian A , Cantallops I , Cline HT .

R29 EY011894-06 NEI NIH HHS , R29 EY011894 NEI NIH HHS

	Fig. 1. Sequence alignment of CPG15. A: Sequence alignment of rat and Xenopus CPG15. Identical amino acids are boxed and similar amino acids are shaded gray. Conserved cysteine residues are marked with asterisks. The N-terminal and C-terminal signal sequences for secretion and GPI linkage, respectively, are underlined. Sequence analysis shows an overall 67% identity between the two species and 73% identity in the mature protein. B: Northern blot analysis from whole tadpole Poly A1 RNA results in the detection of a single band of approximately 1.7 kb, similar to the size of mammalian cpg15.
	Fig. 2. Whole-mount in situ hybridizations on albino Xenopus embryos. A: cpg15 can be detected in dorsal spinal cord at stage 24. B: cpg15 expression increases in the spinal cord and can be detected in the forebrain (FB), midbrain (MB), and hindbrain (HB) at stage 33. C: Higher magnification of spinal cord of stage 33 embryo. cpg15 expression is detectable in both dorsal (arrow) and ventral cells (arrowhead) within the spinal cord. D: Sense probe. Rostral is to the left in all images. Scale bar 5 500 mm in A,B,D; 250 mm in C.
	Fig. 3. In situ hybridization of cpg15 in early stages of tadpole development. Darkfield images show mRNA (A,C,E,G) and brightfield images show toludine blue counterstain (B,D,F,H). A,B: Horizontal sections through stage 33 tadpoles. Horizontal sections through brains from stage 38 (C,D), stage 42 (E,F), and stage 45 (G,H). cpg15 is expressed in the optic tectum (OT) , olfactory pits (OP), olfactory bulb (OB), telencephalon (TL), midbrain (MB), thalamus (Th), and auditory ganglion cells (AGC), as well as vestibulo-ocular nuclei (VON) and hindbrain (HB). Rostral is up in all sections. Scale bar 5 100 mm.
	Fig. 4. In situ hybridization of cpg15 in brains of midstage Xenopus tadpoles. Darkfield images show mRNA (A,C,E,G) and brightfield images show toludine blue counterstain (B,D,F,H). A,B: Horizontal brain sections through dorsal brain of stage 47 tadpoles show cpg15 expression in the olfactory bulb (OB), dorsal thalamus (Th), optic tectum (OT), and hindbrain (HB). C,D: Sections through ventral brain show labeling in thalamus (Th), tegmentum (Te), and hindbrain. E,F: Stage 53 tadpoles continue to express cpg15 in the olfactory bulb, thalamus, and optic tectum, as well as the pallium (Pa). G,H: High magnification of stage 47 optic tectum. Arrows show rostrolateral and caudomedial cells indicating the higher level of mRNA in rostrolateral tectum. Arrowheads indicate cells within the neuropil that have no detectable levels of cpg15. Dorsal lines in H show rostral and caudal regions of tectum used for quantification. Rostral is up in all sections. I: Quantification of cpg15 expression as mean pixel density in rostrolateral and caudomedial tectum on a 0–255 scale where 255 is white (mean 6 standard error). *, P , 0.001. Scale bar 5 100 mm.
	Fig. 5. In situ hybridization of cpg15 in the retina. Darkfield images (A,C,E,G) show mRNA detection and brightfield images (B,D,F,H) show toludine blue counterstain for the same sections. cpg15 is expressed in retinal ganglion cells (RGC) but not in the proliferative zone (PZ) of retinae from stage 38 tadpoles and at all later stages examined. In G and H, sense probe did not label any cells. Scale bar 5 100 mm.
	Fig. 6. Western blot analysis of Xenopus eye and brain. CPG15 antiserum detects a band of approximately 12 kD in eye and brain homogenates from stages 47, 55, and 60 and postmetamorphic (PM) Xenopus. Proteins were loaded in equal amounts (25 mg) and overloaded (75mg) for stage 47 eye homogenate (first lane only, OL). Higher molecular weight band (;50 kD) is not detected consistently and is also present in blots incubated in preimmune serum (Nedivi et al., 1998).
	Fig. 7. CPG15 is targeted to retinal ganglion cell axons. A: CPG15 immunoreactivity in stage 42 is detected in the optic nerve (ON) and optic chiasm (OX). B: Retinal axons within the optic nerve in stage 47. C: High-magnification image of the optic nerve head of retina from stage 47. Note the CPG15 IR around cells in the RGC layer and the concentrated immunoreactivity in the optic nerve. D: Preimmune serum does not label RGC somata or axons. E: In retinal explants, CPG15 IR can be observed all along the length of axons, including the growth cone. F: Double staining with rhodamine-phalloidin highlights actin-based axonal structure. Scale bar 5 100 mm for A,B,D; 50 mm for C.
	Fig. 8. CPG15 expression in tadpole brain. A–E: Horizontal cryostat sections from a stage 45 tadpole shown in sequence from dorsal through ventral. CPG15 immunohistochemistry shows cell bodies labeled in a honeycomb pattern throughout the brain. Cells are labeled in the olfactory bulb (OB), optic tectum (OT), telencephalon (TL), and tegmentum (Te). Intense labeling occurs in axon tracts, notably the optic chiasm (OX), retinotectal axons (RT) in the optic tectal neuropil, tectotegmental axons (TT), and tectospinal (TS) axons coursing toward hindbrain (HB) and spinal cord. Scale bar 5 100 mm.
	Fig. 9. CPG15 expression in spinal cord. A: Immunohistochemistry for CPG15 labels commissural axons in the ventral spinal cord of stage 34 tadpole (arrows). B: Large somata (arrowhead) as well as axons (arrows) in spinal cord label intensely for CPG15 at stage 47. Scale bar 5 30 mm in A; 50 mm in B.
	nrn1 (neuritin 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 33, lateral view, anterior left, dorsal up.

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