Figure 4

In vivo knockdown of n4bp3 in Xenopus laevis results in abnormal cranial nerve development. (A) Spatiotemporal expression profile of n4bp3 during early X. laevis embryogenesis. At stages 28, 32 and 34, n4bp3 transcripts are detected in different cranial ganglia as indicated. (B) Coinjection of n4bp3 morpholino oligonucleotide green fluorescent protein (n4bp3 MO-GFP) RNA together with a control MO results in GFP expression, whereas coinjection with n4bp3 MO results in a block of GFP translation. (C) Western blot shows that n4bp3 protein level is strongly decreased upon n4bp3 MO injection compared to n4bp3 protein level in wild-type (WT) embryos. β-tubulin served as a loading control. (D) Unilateral injection of 20 to 25 ng of n4bp3 MO results in disturbed cranial nerve formation (yellow arrows), whereas control embryos (WT or control MO-injected) show normal cranial nerve development. (E) Quantitative presentation of the results shown in (D). Statistical evaluation of all visible points of arborization of all cranial nerves (F) or the trigeminal nerve (G). (E) through (G) Black bars, WT; dark gray bars, control MO; light gray bars, n4bp3 MO. For statistical evaluation in (F) and (G), WT was compared with n4bp3 MO using Student’s t-test. **P < 0.01. egVII, facial epibranchial ganglion; egIX, glossopharyngeal epibranchial ganglion; egXI, first vagal epibranchial ganglion; gVPL, cells contributing to the vagal and posterior lateral line ganglion; n, independent experiments; N, number of investigated embryos; Nf, facial nerve; Nh, hypoglossal nerve; Nm, mandibular nerve; No, optical nerve; Noc, oculomotor nerve; st, stage.