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Figure S1. Hairy2 depletion using morpholinos targeting both Hairy2a and b pseudolleles efficiently inhibits NC formation. (A) Hairy2 MOs efficiently and specifically inhibits the translation of both Hairy2 pseudoalleles. Design of the Hairy2a and Hairy2b MOs (a). GFP fluorescence of embryos injected with 500 pg of Hairy2a-GFP or Hairy2b-GFP mRNA alone (b,c) or in combination with 5 ng of the corresponding MO (d,e). No effect was observed with Hairy2b MO 5mismatch (f). Hairy2a+b MOs have no effect on GFP translation (g).
(B) Hairy2 MOs (10 ng MO) effciently inhibits Snail2 expression (76%, n = 47) in neurula embryos (st14-15) (a). The phenotype of Hairy2 knockdown is rescued (67% normal, n = 29) by coinjection of a low dose (50 pg) of Hairy2-GR mRNA lacking the MOs binding site (b). Hairy2 MOs (10ng) blocks the expression of Slug, Sox10, Sox9 and Trp2 in tailbud embryos (respective inhibition: 53%, n = 26 ; 57%, n = 17 ; 75%, n = 24 and 69% n = 16 respectively)(c-f).
(C) Hairy2 inhibitory phenotype on NC is not due to an increase in primary neurons nor to cell fate changes. Note that in Hairy2 MOs (10ng total) injected embryos, the expression of neuronal (a, MyT1 n = 24; b, N-tubulin n = 14; c, NeuroD n = 27), neural plate (d, Sox3 n = 22; e, Nrp1 n = 22), mesodermal (f, MyoD n = 14), placodal (g, Six1 n = 18; h, Six3 n = 31; I, Pax8 n = 19), antero-posterior patterning markers (j, Otx2 n = 21; k, HoxB9 n = 20; l, XAG1 n = 22) or epidermal (m, Epi. k n = 34) is unaffected.
Dorso-anterior views with injected side to the right (Ba,b,C) or lateral views (Bc,f) of embryos are shown. β-galactosidase was used as a lineage tracer.
