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Figure 1. Expression pattern of select RNA binding proteins during development. A,B,E,F,H,I,K,L,N,O,Q,R,T,U,W,X: Stage 20 embryos from dorsoanterior and dorsal views. C,D,G: Stage 25 tail bud embryos. J,M,P,S,V,Y: Stage 32 tadpoles. A-G were cleared in BB/BA. A-D: Rbmx/hnRnpG. A,B: Stage 20 embryos show hnRnpG expression all along the anterior-posterior extent of the neural tube with strong expression in the anterior neural domains, and placodal regions. C,D: Stage 25 tail bud embryos; Rbmx/HnRnpG is expressed in the fore-, mid-, and hindbrain, neural crest tissue of the branchial arches, and in the somites. E,F: Stage 20 embryos; Rbm4 is expressed in the neural tube, and at the tail bud stage. G: Rbm4 is expressed in the brain and eye. H-J: Sfrs3 is expressed ubiquitously, but expression is enriched in the neural tube and somites at the neural tube stage, and this pattern is maintained into the tadpole stage. K-M: SRrp35 is expressed in the neural tube and weakly in the somites along the entire anterior-posterior axis. N-P: Sam68 is expressed along the entire anterior-posterior axis of the neural tube and in the neural crest adjacent to the hindbrain. Q-R: hnRnpL expression is present in the neural tube at stage 20 but is restricted to more anterior neural regions in the tadpole (S). T-V: hnRnpC is expressed at low levels throughout the embryo early and is slightly enriched in the neural tube and somites at stage 20 (T,U), but is restricted primarily to the neural tube and somites by the tadpole stage (V). Expression of hnRnpA1 is enriched in the neural tube and somites at stage 20, and this expression is maintained into the tadpole stage (W-Y).
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Figure 2. Effect of overexpression of RNA binding proteins on midneurula development. Whole-mount in situ hybridization for gene expression during neurula stages on uninjected control embryos or embryos injected with 50 pg of mRNA. A-E: Uninjected control embryos showing expression of Sox2 marking all neural tissue (A); N-tubulin in differentiating neurons (B); Pax2 in the forebrain, MHB, and differentiating neurons of the spinal cord (C); Slug marking neural crest (D); and MyoD marking mesoderm (E). F-J: RBMX mRNA injected embryos have normal neural plate morphology (F), but inhibited neural differentiation (G), and perturbed expression of Pax2 (H) and Slug (I), whereas mesoderm is largely unaffected (J). K-N: SFRS3 mRNA injection does not affect Sox2 expression (K), but neurogenesis is inhibited (L), although Pax2 expression in the brain (M) and Slug expression in the neural crest is intact (N). O: MyoD expression is reduced as response to SFRS3. P-T: Sam68 injection does not alter Sox2 (P), Pax2 (R), or MyoD expression (T), but inhibit neurogenesis (Q) and Slug expression in neural crest (S). Embryos were injected with 50 pg of mRNA in one cell at the two-cell stage. Coinjected beta-galactosidase mRNA developed with red-gal was used as a lineage tracer. Embryo orientation is dorsal up, anterior facing.
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Figure 3. Effect of overexpression of RNA binding proteins on muscle structure. Embryos were injected with 50 pg of mRNA and beta-gal lineage tracer into one cell at the two-cell stage and cultured to stage 37 followed by immunostaining with 12/101 to mark skeletal muscle. A-C: Uninjected controls in whole view (A), magnified view of paraxial muscle (B), and schematic outline of the segmentation pattern (C). D-L: Embryos injected with mRNA for RNPA1a (D-F), RNPC1/C2 (G-I), or Rbm4 (J-L) have normal overall amount of muscle (D,G,J), but show irregular segmentation (E,F,H,I,K,L). M-O: Injection of Sam68 mRNA stains weaker for 12/101 (M) and show almost completely absent segmentation (N,O). P-R: Misexpression of SRrp35 caused severe lateral kinking of the embryo (P) and highly irregular segmentation (Q,R). S-U: Injection of RNPL mRNA as a control did not affect overall muscle amount (S) or muscle segmentation (T,U). Embryos were injected with 50 pg of mRNA in one cell at the two-cell stage. Coinjected beta-galactosidase mRNA developed with red-gal was used as a lineage tracer.
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Figure 4. Rbmx is necessary for normal neural development independent of neural induction and mesoderm formation. Embryos are either uninjected controls, injected with Rbmx MO in one cell at the one-cell stage (B,D,F,H,J,L) or one side at the two-cell stage Injected (N,M,P; injected side to the right). Embryos were analyzed at stage 14 (A-L) or 17 (M-R). A,B: N-tubulin-positive primary neurons do not form in Rbmx MO-injected embryos. C,D: Sox2 expression shows normal morphology of the neural plate. E-H: Early mesoderm formation is normal as judged by MyoD (E,F) and Xbra (G,H) expression. I,J: Otx5 expression in the anterior neural plate is not excluded from the presumptive forebrain. K,L: Noggin expression is absent in the anterior neural ridge (red arrow) indicating perturbed anterior neural development. M,N: Pax2-expressing interneurons in the spinal neural plate fail to form on the injected side. O,P: Reduced Slug expression on the injected side indicate defect in neural crest formation. Q,R: Mediolateral patterning of the spinal neural plate proper is normal judged by Pax6 expression.
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Figure 5. Rbmx is required for normal eye development and neuronal differentiation. A-H: Embryos injected unilaterally with Rbmx MO and cultured to stage 31 (A-D), stage 28 (E-F), or stage 35 (H). A,B: In situ hybridization for Xrx1. C,D: In situ hybridization for Pax6; red lines indicate Pax6 expression domain in normal forebrain (C), which is absent in the Rbmx MO injected side (D). E-H: Dorsal view of head and anterior trunk of Rbmx morphants. Injected side is to the right. E-H: In situ hybridization using antisense probe for En1 (E), Lbx1 (F), Lim1 (G), and Slug (H). Red arrowhead in F indicates Lbx1 expression in hypaxial muscle on the uninjected side. Yellow line in H indicates the midline and red arrow indicates melanocytes on the uninjected side. Notably Slug expression is absent in the neural tube and trunk on the injected side.
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Figure 6. Rbmx is required for normal muscle formation and segmentation. Embryos shown are injected with Rbmx MO in one cell at the two-cell stage and cultured to stage 38. A,B: In situ hybridization for MyoD. C,D: 12-101 antibody staining for skeletal muscle. E,F: Outline of muscle segmentation pattern shown in (C,D). Hypaxial muscle migrated from the muscle block in (E, colored solid) is absent on the injected side (F).
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lbx1 (ladybird homeobox 1) gene expression in Xenopus laevis embryo via in situ hybridization, NF stage 28, dorsal view, anterior up.
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hnrnpa1 (heterogeneous nuclear ribonucleoprotein A1 ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 20, anterior view, dorsal up.
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hnrnpa1 (heterogeneous nuclear ribonucleoprotein A1 ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 25, dorsal view, anterior left.
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hnrnpa1 (heterogeneous nuclear ribonucleoprotein A1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 32, lateral view, anterior left, dorsal up.
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khdrbs1 (KH domain containing, RNA binding, signal transduction associated 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 20, anterio view, dorsal up.
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khdrbs1 (KH domain containing, RNA binding, signal transduction associated 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 25, dorsal view, anterior left.
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khdrbs1 (KH domain containing, RNA binding, signal transduction associated 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 32, lateral view, anterior left, dorsal up.
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srsf12 (serine/arginine-rich splicing factor 12) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 20, anterior view, dorsal up.
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srsf12 (serine/arginine-rich splicing factor 12) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 25, dorsal view, anterior left.
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srsf12 (serine/arginine-rich splicing factor 12) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 32, lateral view, anterior left, dorsal up.
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