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	Fig. 1 Experimental design of behavioral analysis and swimming trajectory. a Schematic diagram of the experimental apparatus: 1, monitor; 2, computer system; 3, camera; 4, glass aquarium; 5, biological sample (Xenopus larvae); 6, illuminator. b Quantification of swimming behavior. The maximum and the minimum values of the swimming trajectory (red line) in both the X- and Y-axis (XMAX, XMIM, YMAX, YMIM) were obtained from the recorded trajectory. Swimming area (light gray) is derived from max-min value of swimming trajectory
	Fig. 2 slit2- or robo2-MO-injected tadpoles indicates an abnormality in swimming behavior. a–c, g–i Red lines show the swimming trajectory of tadpoles: a, g Un-injected control, b Slit2-control-MO, c Slit2-MO, h Robo2-control-MO, i Robo2-MO. d–f, j–l Quantification of the swimming area (d, j), swimming distance (e, k) and swimming speed (f, l). In the slit2- or robo2 MO-injected larvae, values of all items measured are significantly decreased. d The average swimming areas of control, Slit2-control-MO and Slit2-MO are 126.59 cm2, 108.92 cm2 and 16.40 cm2 respectively. e The average swimming distances of control, Slit2-miss-control and Slit2-MO are 67.44 cm, 64.24 cm and 23.83 cm respectively. f The average swimming speeds of control, Slit2-control-MO, and Slit2-MO are 2.28 cm/s, 2.06 cm/s and 0.82 cm/s, respectively. j The average swimming areas of control, Robo2-control-MO, and Robo2-MO are 126.59 cm2, 110.84 cm2 and 19.42 cm2, respectively. k The average swimming distances of control, Robo2-control-MO, and Robo2-MO are 67.44 cm, 59.24 cm and 21.99 cm, respectively. l The average swimming speeds of control, Robo2-control-MO, and Robo2-MO are 2.28 cm/s, 1.94 cm/s and 0.83 cm/s, respectively. Error bars are shown as standard deviation (SD). Data denoted by the same letter are not significantly different (P > 0.05) by Scheffé test after one-way analysis of variance
	Fig. 3 External morphology of Xenopus larvae. Blue staining show nucleus labeled by DAPI. Myotomes (a–c) and nerves (d–s) are visualized by immunohistochemistry (shown in green). a–c Lateral view of the trunk in un-injected control (a), slit2-MO-injected (b) and robo2-MO-injected (c) specimens. Development of myotomes is normal in all conditions. d–g Lateral view of the trunk in slit2-control-MO-injected (d), slit2-MOinjected (e), robo2-control-MO-injected (f), robo2-MO-injected (g) specimens. Arrowheads indicate segmentally organized spinal nerves. h–k Dorsal view of the anterior trunk in slit2-control-MO-injected (h), slit2-MO-injected (i), robo2-control-MO-injected (j), robo2-MO-injected (k) specimens. Arrowheads indicate segmentally organized spinal nerves. l-o Dorsal view of the head in slit2-control-MO-injected (l), slit2-MO-injected (m), robo2-control-MO-injected (n), robo2-MO-injected (o) specimens. p-s Dorsal view of the optic and cranial nerves in slit2-control-MO-injected (p), slit2-MO-injected (q), robo2-control- MO-injected (r), robo2-MO-injected (s) specimens. Arrowheads indicate the optic nerves. The peripheral nerves visualized by immunohistochemistry represent a normal innervation pattern. Scale bars: A–K, P-S, 200 μm; L-O, 500 μm
	Fig. 4 Expression patterns of Xlslit2 and Xlrobo2 and morphology of axonal tracts in Xenopus larvae. a–c Transcripts of Xlslit2 are detected through the dorsal midline in the diencephalon at stage 32 (a), stage 40 (b) and stage 44 (c). Xlslit2-weak gap is found between diencephalon and mesencephalon (arrows in A and C, A’ is a dorsal view). In the metencephalon (met), expression domains of Xlslit2 are observed along the antero-posterior axis (arrowheads). d–f Expression pattern of Xlrobo2 at stage 32 (d), stage 40 (e) and stage 44 (f). (d) At early tail-bud stage, Xlrobo2 is expressed in the telencephalon (te), diencephalon (di), mesencephalon (mes), dorsal metencephalon (met) and notochord (nc). e, f At middle and late tail-bud stage, XlRobo2 expression is detected at high levels in the dorsal CNS. g, h Dorsal (g) and lateral (h) view of the developing Xenopus larva at stage 46. Axons in the PNS and CNS are visualized by anti-acetylated tubulin antibody. Habenular and posterior commissures are located the diencephalon (HC and PC). Scale bars: A-F, 200 μm; G and H, 500 μm
	Fig. 5 Morphology of axonal tracts and Xlslit2 expression domain in the neural tube. Dorsal (a-c) and lateral (d-f) view of the developing Xenopus larva at stage 42. (A’-F’) High-magnification images of white squares in A-F. a, c Transcripts of Xlslit2. Xlslit2 mRNA is discontinuously expressed in the dorsal diencephalon. The anterior limit of Xlslit2 attach to the superficial region of the brain (arrow in D). b, e Axons in the CNS are visualized by immunohistochemistry (shown in green). Habenular and posterior commissures are visible. c, f Merged image of Xlslit2 (purple) and nerves (green). The anterior end of the PC corresponds to the anterior limit of Xlslit2 expression domain (arrow in F). Scale bars: 100 μm
	Fig. 6 Morphology of axonal tracts and Xlrobo2 expression domain in the diencephalon. a, b Coronal sections of the tadpole brain at stage 45, in which NeuroVue chips were inserted on the dorsal diencephalon and labeled neurons are shown in green. Blue staining shows nucleus labeled by DAPI. a Labeled axons (green), which correspond to the HC, are observed in the dorsal region of the diencephalon. b Labeled PC axons are observed in the dorsal diencephalon and labeled cell bodies are located ventral to the PC. This region is the presumptive nucleus of the tract of the PC (nTPC). (B’) High-magnification image of the red box in B. c, d Expression pattern of Xlrobo2 on coronal sections of the tadpole brain at stage 40. c and d are the slice of anterior and posterior diencephalon, respectively. d Xlrobo2 expression domain is observed in the nTPC (asterisk). Scale bars: 100 μm
	Fig. 7 slit2/robo2-MO injected larvae change the width of the posterior commissure. a, b, d, e dorsal views of the tadpole brain at stage 44. a In slit2-control-MO injected larva, three commissures (habenular commissure: HC, posterior commissure: PC and cerebellar commissures: CC) are observed. PC indicated by an arrowhead. b In Slit2-MO injected larva, morphology of the habenular and cerebellar commissure appear to be normal, whereas nerve bundle of the posterior commissure become wider than the control larva (open arrowhead). c Quantification of the width of nerve bundle in Slit2- control- (blue)/Slit2-MO (red). slit2-MO-injected larva is significantly changed the width of the posterior commissure compared to that of slit2-control-MO (**P < 0.01). d, e In robo2-control- (D)/robo2-MO (e) injected larvae, the former represents three commissures but the latter shows unclear PC bundle (open arrowhead). f Quantification of the width of three commissures. The posterior commissure of robo2- MO-injected larva is significantly changed the width of the PC. Error bars are shown as standard deviation (SD). *P value was obtained by ANOVA (P < 0.05 is significant). (**P < 0.01). Scale bars: 200 μm
	Fig. 8 slit2/robo2-MO injected larvae represent the abnormal morphology of the posterior commissure. Neurons are visualized by immunohistochemistry (shown in green). a Lateral view of the brain in Xenopus larva. Dashed line indicates the outline of the brain regions. b–e Coronal sections at the level of the posterior commissure: b Slit2- control-MO, c Slit2-MO, d Robo2-control-MO and e Robo2-MO. In slit2/ robo2-MO-injected larva, the bundle of the posterior commissure is thinner than that of control-MO. Blue staining show nucleus labeled by DAPI. Scale bars: 100 um
	Fig. 9 Median sections of the tadpole brain. Neurons are visualized by immunohistochemistry (shown in green). a Dorsal view of the Xenopus larva that performed the fluorescent immunostaining. Dashed line indicates the cutting plane in (b–e). b’–e’ High magnification images of the box in (b–e): b, b’ Slit2-control-MO, c, c’ Slit2-MO, d, d’ Robo2-control-MO, e, e’ Robo2-MO. In the Slit2/Robo2-control-MO, the posterior commissures make a tight bundle, whereas in the Slit2/Robo2-MO injected larvae, the width of PC is elongated anteroposteriorly. Blue staining shows nucleus labeled by DAPI. Scale bars: 100 um
	Fig. 10 Schematic view of the result of the experiment. In the control, Robo2-expressing PC axons extend through the Slit2-weak region in the posterior diencephalon and result in a tight bundle. However, in the slit2-/robo2-MO-injected larvae, the PC makes elongates anteriorly with a defasciculated tract, due to the absence of repulsive interaction between Slit2 and Robo2. Disappearance of gene expression indicated by the dashed line
	Fig. 11 Evolutionary process of Slit/Robo mediated axonal wiring. In amphibians and amniotes, Slit2-weak region is present in the superficial part of the diencephalon, in which posterior commissure is formed. Robo2 is expressed in axons of the posterior commissure originates from nTPC in the posterior diencephalon. In the zebrafish (24 h post fertilization), slit2 expression domain in the dorsal diencephalon is similar to those of tetrapods, whereas robo2 transcripts are appear to be absent in the homologous region of nTPC. In mammals, Slit/Robo is also used for the development of the newly acquired commissure, such as corpus callous (indicated by the blue dashed ring). cc, corpus callous; Di, diencephalon; Mes, mesencephalon; Met, metencephalon; nTPC, nucleus of the tract of the posterior commissure; PC, posterior commissure; Te, telencephalon; vcc, ventrocaldal cluster

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