Franco PG , Paganelli AR , López SL , Carrasco AE .

	Fig. 1. RA exposure during gastrulation increased the expression of activators and repressed the expression of inhibitors of primary neurogenesis, while the retinoid antagonist Ro produced the opposite results. Embryos were untreated (Control column), treated with RA (RA column) or treated with Ro 41-5253 (Ro column) and the effect on the expression of different components of the primary neurogenesis cascade was evaluated by in situ hybridization at neurula stage. All panels are dorsal views (anterior up). (A-C) Ntubulin (N-tub) domains were strongly expanded by RA (B; 54%, n=24 for 1 mMRA; 100%, n=44 for 10 mM RA). The stripes of sensory neurons and interneurons were merged (compare arrowheads between A and B) and shifted anteriorly. Ro certainly reduced Ntubulin expression (C, 83%, n=12 for 1.5 mM Ro) and stripes appeared more distant than in control embryos. m, i and s, primary motor neurons, interneurons and sensory neurons, respectively. (D-F) X-MyT1 domains were expanded and shifted anteriorly after RA treatment (E, 100%, n=8 for 1 mM RA; 100%, n=6 for 10 mM RA). Ro clearly reduced X-MyT1 expression in the neural plate (F, 100%, n=8 for 1.5 mM Ro). (G-I) X-ngnr-1 expression was increased, stripes were merged (compare black arrowheads in G and H) and shifted anteriorly, while trigeminal expression (white arrowhead in G) was lost in RA-treated embryos (H, 61%, n=33 for 1 mM RA; 72%, n=29 for 10 mM RA). Ro reduced X-ngnr-1 expression in the neural plate but enlarged the trigeminal domain (white arrowhead; I, 21%, n=14 for 1.5 mM Ro; 20%, n=10 for 4 mM Ro). (J-L) X-Delta-1 domains were increased, merged and shifted anteriorly in response to RA treatment, while the most-anterior domain (arrowhead in J) was lost probably due to posteriorization (K, 50%, n=16 for 10 mM RA, 38% n=16 for 1 mM RA). Ro changes the expression pattern of XDelta- 1 resolving the longitudinal stripes into a bilateral one towards the dorsal midline, while the anterior domains (arrowhead) converge (L, 30%, n=10 for 1.5 mM Ro). (M-O) RA treatment resulted in a widespread expansion of Gli3 over the mediolateral axis in the posterior neural plate, and the anterior domain was markedly reduced (N, 100%, n=10 for 1 mM RA; 100%, n=10 for 10 mM RA). Ro treatment only reduced the posterior expression (O, 67%, n=9 for 4 mM Ro; 63%, n=8 for 7.5 mM Ro). The low levels of Gli3 expression in the posterior domain in control embryos makes the comparison with Ro-treated embryos difficult. (P-R) RA produced a dosedependent abolishment of Zic2 expression (Q, complete lost, 100%, n=10 for 10 mM RA; posterior reduction and anterior lost, 100%, n=10 for 1 mM RA, see inset). Ro treatment dispersed Zic2 expression over the mediolateral axis in the posterior neural plate (R, 30%, n=10 for 4 mM Ro; 40%, n=10 for 7.5 mM Ro). (S-U) X-shh expression was strongly reduced in the posterior level and was completely abolished in the anterior notochord and floor plate after RA treatment (T, 100%, n=23 for 10 mM RA). Ro-treated embryos showed a clear increase of X-shh along the dorsal midline (U).
	Fig. 2. X-shh and X-bhh overexpression increased the expression of inhibitors and repressed the expression of activators of primary neurogenesis, and reduced neural crest markers without impairing neural development. Embryos were unilaterally injected with 1 or 2 ng of nuc-b galactosidase mRNA as a negative control (b-gal column), full-length X-shh mRNA (X-shh column) or full-length Xbhh mRNA (X-bhh column) plus 100 pg of nuc-bgal mRNA as tracer. They were analyzed at neurula stage by whole-mount in situ hybridization with different neural markers. All are dorsal views (anterior up). The injected side is demarcated by the pale blue staining and is oriented to the left. IS, injected side. NIS, noninjected side. (A-C) Suppression of primary neuron formation as revealed by the differentiation marker N-tubulin (N-tub) in X-shhinjected embryos (B, 90%, n=21 for 2 ng; 79%, n=14 for 1 ng; 57%, n=21 for 0.125 ng) and X-bhh-injected embryos (C, 100%, n=24 for 2 ng; 93%, n=14 for 1 ng; 28%, n=22 for 0.125 ng). Notice the absence of N-tubulin expression from the trigeminal ganglion in the injected side. m, i and s, primary motor neurons, interneurons and sensory neurons, respectively; arrowhead, trigeminal ganglion. (D-F) Expansion of the neural plate as revealed by the general neural marker nrp-1 in X-shh-injected embryos (E, 100%, n=10 for 2 ng) and X-bhh-injected embryos (F, 100%, n=10 for 2 ng). (G-I) Abolishment of Gli3 expression in the posterior neural plate in Xshh- injected embryos (H, 67%, n=21 for 2 ng; 15%, n=13 for 1 ng) and X-bhh-injected embryos (I, 50%, n=26 for 2 ng). Notice that nuc-b-gal dark-blue staining may interfere with the appreciation of Gli3 decrease on the injected side. (J-L) Widespread expansion of Zic2 domain in X-shh-injected embryos (K, 71%, n=34 for 2 ng; 44%, n=18 for 0.25 ng) and X-bhh-injected embryos (L, 80%, n=35 for 2 ng; 32%, n=63 for 0.25 ng). Notice the absence of Zic2 expression from the medial cranial neural crest domain (arrow) in the injected side. (M-O) Reduction and ventral displacement of the Slug domain in X-shh-injected embryos (N, 82%, n=22 for 2 ng; 64%, n=11 for 1 ng; 35%, n=17 for 0.25 ng) and X-bhh-injected embryos (O, 50%, n=6 for 1 ng; 32%, n=25 for 0.25 ng). Arrows, neural crests. (P-R) Downregulation of Krox-20 in r5 and caudal displacement of r3 domain in X-shh-injected embryos (Q, 60%, n=10 for 2 ng) and X-bhh-injected embryos (R, 60%, n=10 for 2 ng). r3, third rhombomere; r5, fifth rhombomere.
	Fig. 3. Cross sections of X-shh- and X-bhh-injected embryos at neurula stage (A-E) and tadpole stages (F,G). Dorsal side is up. IS, injected side; NIS, non-injected side; n, notochord; ne, neural ectoderm; s, somites. (A) N-tubulin distribution shows the ventral displacement of the primary sensory neurons stripe (arrowhead) in the IS. (B) Hoechst nuclear labeling revealing the increased cell number in the IS including the neural ectoderm (compare bars). (C,D) Zic2 expression. Note the expansion in the neural ectoderm and mesoderm in the IS in C. The inset shown at higher magnification in D shows the very active transcription of Zic2 in nuclei (arrowheads) of the IS. (E) The same section as in D revealed for Hoescht staining, confirms the nuclear location (arrowheads) of Zic2 transcripts. (F,G) Xsal-1 expression is downregulated in the VIIth cranial ganglion (arrow in F) and reveals an expansion of ventral secondary neurons within the neural tube (arrowheads in F and G) in the IS.
	Fig. 4. RA acts upstream of lateral inhibition, Zic2 and X-shh in the primary neurogenesis cascade. Embryos were coinjected unilaterally with 100 pg nuc-b-gal mRNA as tracer plus NotchICD, Zic2 or X-shh mRNAs and left untreated (control column) or treated with RA during gastrulation (RA column). N-tubulin distribution was revealed by in situ hybridization at neurula stage. All are dorsal views (anterior up). Dotted line, dorsal midline; IS, injected side (blue staining); NIS, non-injected side. Sensory neuron stripes are marked with arrows. Percentages below indicate the reduction of N-tubulin expression in the IS. (A,B) RA treatment cannot overcome the inhibitory effect of NotchICD on N-tubulin expression. NotchICDinjected embryos, untreated (A, 93%, n=11 for 1 ng NotchICD; 75%, n=14 for 0.5 ng NotchICD). NotchICD-injected embryos, treated with RA (B, 100%, n=10 for 1 ng NotchICD + 10 mM RA; 70%, n=10 for 1 ng NotchICD + 1 mM RA; 73%, n=11 for 0.5 ng NotchICD + 10 mM RA; 100%, n=10 for 0.5 ng NotchICD + 1 mM RA). (C,D) RA treatment cannot rescue the inhibitory effect of Zic2 on N-tubulin expression. Zic2-injected embryos, untreated (C, 20%, n=22 for 2 ng Zic2; 15%, n=32 for 1 ng Zic2). Zic2-injected embryos, treated with RA (D, 22%, n=23 for 1 ng Zic2 + 10 mM RA). (E,F) RA treatment cannot rescue the inhibitory effect of X-shh on N-tubulin expression. X-shh-injected embryos, untreated (E, 17%, n=6 for 0.25 ng X-shh). X-shh-injected embryos, treated with RA (F, 30%, n=10 for 0.25 ng X-shh + 10 mM RA). All RA-treated embryos showed the previously described enhancement of N-tubulin expression in the uninjected side.
	Fig. 5. Proposed model for the molecular interactions involving RA and hedgehog signaling leading to terminal primary neuronal differentiation.