Figure 4: Loss of uORF-mediated translational regulation of Ptch1 disrupts Shh signalling and neurogenesis.

(a) The position guide RNA (gRNA) targets for the generation of Ptch1^{ΔuORF/ΔuORF} homozygous mutant mESC line with CRISPR/Cas9 is shown on the schematic accompanied by the sequence flanking the deletion. gRNA target sequence is indicated in blue. Schematic of monolayer differentiation of mESCs into neural progenitor cells (NPCs) is depicted. RA (Retinoic Acid) treatment is initiated on day 3.0. Shh pathway activation to generate ventral neurons is achieved by stimulation with either recombinant SHH protein or SAG beginning on day 3.5 of differentiation—assays in c–f are performed at day 3.5+0, 4, 12 h. Differentiation is terminated at day 5 (36 h) (g–k). (b) Schematic illustrates graded Shh signal across the ventral-to-dorsal axis of the developing neural tube. Highest concentration of SHH specifies FOXA2⁺ FP, progressively lower quantities of SHH specify NKX2.2⁺ p3 and OLIG2⁺ pMN neurons. PAX6⁺, IRX3⁺ and IRX5⁺ neurons form in the dorsal portion of the neural tube and are not SHH dependent. (c–d) Western blot analysis reveals increased PTCH1 protein at 0 and 4 h after SAG treatment. Analysis of (e) Gli1 and (f) Ptch1 mRNA expression at 0, 4 and 12 h after addition of SHH protein (solid lines) or SAG (dashed lines) on day 3.5 of NPC differentiation. (g) Analysis of mRNA expression of ventral neuron marker genes (Foxa2, Nkx2.2 and Olig2) and (h) dorsal neuron maker genes (Pax6, Irx3 and Irx5) by RT-qPCR in wildtype and Ptch1^{ΔuORF/ΔuORF} cells at day 5 of neuron differentiation. (i–k) Concomitant immunofluorescence staining and quantitation confirms decreased numbers of FOXA2⁺ and NKX2.2⁺ neurons at day 5 of differentiation. (c–k) Error bars represent s.d. (t-test, **P<0.01; *P<0.05; NS, not significant, n≥3).