Calarco et al., 2009 - Regulation of vertebrate nervous system alternative splicing and development by an SR-related protein. Cell   138(5):898-910 Full text @ Cell

Fig. 6 nSR100 Regulates Vertebrate Nervous System and Sensory Organ Development

(A–C) RNA in situ hybridization assays monitoring ZnSR100 expression in the developing zebrafish embryo.

(A and A′) expression at 24 hpf, lateral and frontal views, respectively.

(B and B′) nSR100 expression at 36 hpf, lateral and dorsal views, respectively.

(C and C′) nSR100 expression at 48 hpf, lateral and dorsal views, respectively.

(D) p53 morpholino antisense oligonucleotide (MO) control-injected embryo at 50 hpf.

(E) p53 + ZnSR100 MOspl-injected embryo at 50 hpf.

(F) p53 + ZnSR100 MOspl-injected embryo at 50 hpf, rescued through coinjection of ZnSR100 mRNA.

(G–I) RNA in situ hybridization assays monitoring islet-1 expression in 50 hpf embryos. p53-MO-injected control (G), nSR100spl morphant embryo (H), and nSR100spl morphant embryo rescued through coinjection of 20 pg ZnSR100 mRNA (I) are shown.

(J–M) Confocal projections of trigeminal ganglion in Tg(sensory:GFP) transgenic embryos. Control embryo at 30 hpf (J), representative nSR100spl morphant embryo at 30 hpf (K), control embryo at 36 hpf (L), and representative nSR100spl morphant embryo at 36 hpf (M) are shown.

(N) nSR100-regulated alternative exons conserved between zebrafish and mouse display altered splicing patterns in nSR100spl morphant embryos, relative to control embryos. Total RNA was isolated from the heads of wild-type and nSR100spl morphant embryos at 50 hpf, and semiquantitative RT-PCR was performed to analyze AS patterns. Percent exon inclusion values are shown below each lane.

Fig. S12 ZnSR100 pre-mRNA splicing is blocked in embryos treated with splice blocking morpholino oligonucleotide (MOspl). ZnSR100 transcript splicing patterns in the vicinity of exon 5 and intron 5 were analyzed in WT and nSR100 MOspl morphant embryos at 50hpf. A near complete disappearance of fully spliced transcripts along with the accumulation of transcripts with retained intron 5 could be observed in morphant embryos relative to WT embryos.

Fig. S13 Phenotype of nSR100 MOspl morphant embryos. A-B. Lateral views of control p53MO-injected (A) and nSR100 MOspl-injected (B) embryos at 30hpf. nSR100 morphant embryos exhibit only a slight delay in embryonic development, as exhibited by morphogenesis of the midbrain-hindbrain boundary (asterisks). C-D. Lateral (C, D) and ventral (C′, D′) views of control p53MO-injected (C, C′) and nSR100 MOspl-injected (D, D′) embryos at 48hpf. Note the expanded ventricle (arrowhead) and convoluted body axis of nSR100 morphant embryos. However, general embryonic patterning and morphogenesis of nSR100 morphants, including heart formation (arrows), appears relatively normal.

Fig. S14 Morphology and islet-1 expression in control and nSR100 MOspl embryos at 24hpf. A. p53MO-injected embryo at 24hpf. B. p53 + ZnSR100 MOspl injected embryo at 24hpf. C-D. RNA in situ hybridization assays monitoring islet-1 expression in 24hpf embryos. C. p53-MO injected control. D. ZnSR100 MOspl morphant embryo.

Fig. S15 Injection of ZnSR100 mRNA rescues neurite outgrowth defects in ZnSR100 MOspl morphant embryos. A-C. Confocal projections of trigeminal ganglion in Tg(sensory:GFP) transgenic embryos at 30hpf. A. p53 + ZnSR100 MOspl injected embryo. B-C. p53 + ZnSR100 MOspl embryo, co-injected with ZnSR100 mRNA.

Unillustrated author statements

EXPRESSION / LABELING:
Gene:
Fish:
Stage Range: 1-cell to 26+ somites
Acknowledgments:
ZFIN wishes to thank the journal Cell for permission to reproduce figures from this article. Please note that this material may be protected by copyright.

Reprinted from Cell, 138(5), Calarco, J.A., Superina, S., O'Hanlon, D., Gabut, M., Raj, B., Pan, Q., Skalska, U., Clarke, L., Gelinas, D., van der Kooy, D., Zhen, M., Ciruna, B., and Blencowe, B.J., Regulation of vertebrate nervous system alternative splicing and development by an SR-related protein, 898-910, Copyright (2009) with permission from Elsevier. Full text @ Cell