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Fig. 6. Robo2 and Robo3 have roles in RGC axon extension and guidance. (Aã¢â â B) Brains dissected out of stage 35/36 embryos following wholemount in situ hybridization with antisense riboprobes to slit1 (A) or slit2 (B). (C) For comparison, a stage 35/36 horseradish peroxidase-labelled optic tract (brown) is shown enroute to the tectum. RGC axons cross the midline at the optic chiasm, travel dorsally through the diencephalon, make a caudal turn (*) in the mid-diencephalon, and enter the tectum in the midbrain (approximate tectal border is shown with dotted line). Slit1 and slit2 are expressed near the optic chiasm and the mid-diencephalic turn, and are present dorsal to where the axons enter the tectum. (Dã¢â â I) Retinas of stage 27 Xenopus embryos were electroporated with GFP alone or together with dnRobo2-MT or dnRobo3-MT. Embryos were fixed at stage 40, and brains were removed and processed for anti-GFP or anti-myc immunochemistry. The black cells are melanophores. (Dã¢â â I) Lateral views of axons travelling in the contralateral optic tract of a control brain with GFP-expressing RGC axons (D), or of brains with axons expressing dnRobo2 (Eã¢â â F) or dnRobo3 (Gã¢â â I). In the control, axons come across the optic chiasm, make a caudal turn in the diencephalon (*) and innervate the tectum. Axons expressing dnRobo2 (Eã¢â â F) or dnRobo3 (G) often end in the ventral or mid-diencephalon (arrowheads show the locations of the growth cones). Axon guidance errors were sometimes seen: In (F) one axon (arrows) makes an aberrant turn just anterior to the rostral border of the optic tectum (high power view of the area boxed on the wholemount brain shown in inset), while in (Hã¢â â I), one of the dnRobo3-expressing axons missed the mid-diencephalic turn and travelled dorsally towards the pineal gland. I is a high power view of the boxed area in H, and numbers identify the three axon tips. Scale bar in D is 50ã â ã â¼m for Dã¢â â E and Gã¢â â H, 25ã â ã â¼m for F and 10ã â ã â¼m for I. A, anterior; D, dorsal; di, diencephalon; hb, hindbrain; oc, optic chiasm; pi, pineal gland; P, posterior; tec, tectum; tel, telencephalon; V, ventral. (J) Graph showing defects in axon extension upon inhibition of Robo signalling. The location of the growth cone for each GFP, dnRobo2 or dnRobo3-expressing axon was scored as being in the ventral diencephalon (di), mid-diencephalon, dorsal diencephalon, or at the tectum. The numbers in brackets represent the total numbers of axons analyzed for each condition. Data were pooled from four independent experiments. dnRobo2 and dnRobo3 each caused a significant change in the extension of RGC axons (pã â <ã â 0.001, Chi-square test). |
