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The collaspin response mediator proteins (CRMPs) are a family of cytosolic phosphoproteins which play a critical role in the establishment of neuronal polarity and growth cone guidance. Here, we describe the temporal and spatial expression of CRMP-4 during early Xenopus embryogenesis. CRMP-4 transcripts were first detected by whole mount in situ hybridization at the end of gastrulation in the prospective neuroectoderm. During open neural plate stages, CRMP-4 was expressed broadly throughout the anterior neural plate and in the three bilateral stripes of the posterior neural plate where primary neurons arise. The expression in the territories of primary neurogenesis prefigures that of the post-mitotic neuronal marker N-tubulin. At tadpole stages, expression was maintained throughout the central nervous system and in the retina of the eye. Consistent with the observed expression, CRMP-4 transcripts are positively regulated by X-ngnr-1 and negatively by Notch signaling. The observed expression and regulation of CRMP-4 differ from that of the CRMP-2, which is induced by the events of neural induction.
Fig. 2. Expression of Xenopus CRMP-4. (A) Temporal expression of CRMP-4 determined by RT-PCR at various stages of Xenopus development. The ubiquitous marker Histone H4 served as a loading control. (B-O) Xenopus embryos at the indicated stages were analyzed by whole mount in situ hybridization using a digoxigenin- labelled CRMP-4 antisense probe. (B) Stage 11.5 d., (C) stage 11.5, veg., (D) stage 12.5 d., (E) stage 14 d., (F) stage 15 d., (G) stage 15 ant., (H) stage 16 d-ant., (I) stage 19 ant., (J) stage 24, (K) stage 28, (L) transversal section of (K) through the spinal chord, (M) transversal section of (K) showing through the punctuate anterior-
lateral cells, (N) stage 37, lat., (O) transversal section of (N) through the midbrain region.(p shown separately)
anp, anterior neural plate; ant, anterior; cmz, cilliary marginal zone; d, dorsal; i, intermediate; l, lateral; mz, marginal zone; m, medial; r, retina; svz, subventricular zone; tg, trigeminal placode; veg, vegetal; vz, ventricular zone; IX, glossopharngeal ganglion; X, vagus ganglion. Red arrowheads indicate scattered cells.
Fig. 2. (P) N-tubulin antisense probe, stage 27. Red arrowheads indicate scattered cells.
tubb2b (tubulin, beta 2B class IIb ) gene expression in Xenopus tropicalis embryo, via in situ hybridization, NF stage 27, lateral view, dorsal up, anteriorleft.
Fig. 3. Regulation of CRMP-4. (A) Xenopus embryos were injected bilaterally with Noggin (50 pg) or X-ngnr-1 (25 pg) and animal caps excised at the blastula stage. When sibling embryos reached stage 14, the caps were collected, total RNA isolated and RT-PCR analysis performed. Histone H4 served as a loading control. (B) Whole mount in situ hybridization of stage 14 embryos injected with sense RNA encoding X-ngnr-1 (25 pg) or Notch-ICD (50 pg) in one blastomere at the two-cell stage.
dpysl3-b (dihydropyrimidinase-like 3) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 15, anterior view, dorsal up.
