Pnx is involved in primary neurogenesis. (A-D) Specific inhibition of the translation of pnx RNA by antisense morpholino oligonucleotide (MO). Two nanograms of the MO recognizing the 5′ untranslated region of pnx (pnx MO; A,B) or the control MO (4-mis; C), which contains four mispaired bases, were coinjected with RNA for Pnx-GFP (B,C) or Myc-tagged GFP (MTGFP; A) into one-cell-stage embryos. (D) Pnx-GFP-expressing control embryo. The expression of Pnx-GFP and MTGFP was detected by fluorescence microscopy. pnx MO (F,H,J,L,N,P,R,T,V) and 4-mis MO (E,G,I,K,M,O,Q,S,U)-injected embryos were stained with markers for neural precursors and primary and secondary neurons. (F,H) ngn1- and elavl3-expressing cells were diminished in the posterior neuroectoderm of pnx MO-injected embryos at the one- to three-somite stage. (G,H) elavl3 expression in trigeminal ganglions was not significantly affected in the pnx MO-injected embryos (marked by arrowheads in insets). (I,J) At the five-somite stage, pnx MO-injected embryos displayed a strong reduction in islet1-expressing primary motoneurons and a less efficient reduction in islet1-positive Rohon-Beard neurons. (K,L) At 24 hpf, the pnx MO-injected embryos (L) displayed a reduction in islet2-expressing primary motoneurons and defects in axon outgrowth from motoneurons (stained with the znp1 monoclonal antibody, R). (M-P) olig2 expression was not affected in the pnx MO-injected embryos at the one-somite stage (N) and at 24 hpf (P). (S,T) zn5-immunoreactive secondary motoneurons (48 hpf) and (U,V) zn 12-immunoreactive Rohon-Beard neurons (25 hpf) appeared to be normal or only marginally affected in the pnx MO-injected embryos (T,V). (E-J,M,N) Dorsal views. (K,L,O-R) Lateral views. (S,T) Horizontal sections in the spinal cord region. (U,V) Dorsal views in the spinal cord region.
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