N-SHH specifically suppress the growth of RGC axons. E5 chick neural retina (NR) (A-D,I), dorsal neural tube explants (NT) (E,F) and E12 dorsal root ganglia (DRG) (G,H) were cultured in collagen gel matrix 100 μm from control (A,E,G), N-SHH-soaked beads (B,F,H) or floor plate explants (I). In presence of N-SHH-soaked beads, both the length and the number of neurites from retinal explants (B) were decreased when compared with the growth in the presence of control beads (A). Similarly, addition of soluble N-SHH directly to the culture medium reduced RGC axon growth (D) when compared with controls (C). Similar results were obtained when retinal explants were co-cultured in the presence of floor plate (FP) explants (I). Neurite outgrowth from dorsal neural tube explants (E,F) and dorsal root ganglia (G,H) was unaffected by the presence of N-SHH. (J) Quantification of neurite outgrowth (both neurite lengths and numbers) from (n) explants. Neurite outgrowth is expressed as the number of pixels occupied by neurites normalised for the explant size. (K) Quantification of cell proliferation in the explants, as assessed by staining with an antiserum the mitotic marker phospho-histone H3. (L) Quantification of cell differentiation in the explants, as determined by counting the number of Islet1-positive cells in the explants. Scale bars: 50 μm in A-H; 55 μm in I.

Ptc1 expression and SHH activity at the growth cone. (A) In situ hybridisation of E5 chick retina with antisense probe shows low levels of Ptc1 mRNA in the retinal neuroepithelium and moderate in the retinal ganglion cell layer (RGC). PE, pigment epithelium. (B) Hybridisation with the sense probe is shown for comparison. (C-E) E5 chick retina explants were cultured on laminin substrate for 12 hours and the behaviour of neurites was observed by means of digital images, captured at fixed intervals during the next 2 hours. Colour-coded asterisks identify individual neurites. (C) Control explant treated with vehicle where neurites continued to extend at a steady pace. (D) Explant treated with purified N-SHH (2.5 μg/ml) already shows neurite retraction 15 minutes after N-SHH addition. Neurite retraction persisted for the following hour, thereafter growth cone slowly reassumed their extension. (E) Statistical analysis of neurite growth rate in cultured explants treated with vehicle (blue) or with N-SHH (red) shows a clear reduction in the total neurite length after N-SHH treatment, neurite extension is slowly reassumed 2 hours after the treatment. Scale bar: 24 μm in A,B; 100 μm in C,D.

N-SHH induces the retraction on newly initiated neurites and lowers the cAMP levels in the growth cone. E5 chick retina (A-D) or E12 DRG (E-G) explants were cultured on laminin substrate for 20 hours. (A) The behaviour of neurites was observed with time lapse video recording of individual growth cones. Addition of N-SHH to the culture medium induced a slowing down of the growth cone movement which was followed by a partial retraction of the axon shaft. Growth cone collapse is reversible and re-inducible as observed after N-SHH washout (45 minutes later), and N-SHH re-addition (2 hours later). (B-D) Retinal and DRG (E-G) growth cones immunostained with anti-cAMP antibody. Note the intense staining of RGC growth cone after incubation with forskolin (B) and medium intensity in control conditions (C). Note how 20 minutes exposure to N-SHH greatly reduces the levels of cAMP in retinal (D) but not DRG (G) growth cone, which presents an intensity similar to those observed in control (F) or after forskolin addition (E). Scale bar: 12 μm in A; 6 μm in B-G.

RCAS-mediated ectopic expression of Shh alters RGC axon pathway in vivo. HH13-14 chick embryos were infected in the ventral forebrain and analysed at E5-E6 when many RGC axons have already passed through the chiasm. DiI crystals were placed into the right eye of each embryo to visualise RGC axon pathway. In control or RCAS-AP infected embryos, Shh expression is normal (A) and DiI-labelled axons grew through the optic disc into the optic nerve (ON) and crossed the midline (broken white line) to invade the contralateral optic tract (arrowhead in B). RCAS-Shh infected embryos presented several alterations as compared to this normal pathway. (C) In most cases (78%, n=32) Shh expression was ectopically extended into the hypothalamic region (red arrows) and into the optic nerve (ON). (D) In these animals, DiI labelling shows that axons advanced very little along the nerve (ON) and they barely reached the chiasm region. (E,G) In 15% of treated embryos, viral infection and therefore Shh ectopic expression was confined to the midline hypothalamic region. (F,H) In these animals, few axons grew across the midline (broken white line) into the contralateral optic tract (arrowheads in F,H) and a consistent small proportion of axons was misrouted into the ipsilateral optic tract (arrows in H). Scale bar: 79 μm in A,C,E,G; 37 μm in B,D,F,H.

Patterning and neural differentiation are not affected in the RCAS-Shh infected eyes. Images illustrate the expression of PAX2 (A,D), Shh (B,E) and Tuj (C,F) on consecutive sections of control (A-C) and infected (D-F) embryos. (A,D) PAX2 expression is normal in the optic nerve without extending into the Shh infected neural retina (E). Tuj1 immunostaining highlighted that the RGG axons avoided the portion of the optic disk that ectopically expressed Shh (compare C with F). NR, neural retina; PE, pigment epithelium. Scale bar: 67 μm.