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Early embryogenesis in Xenopus development depends on correct anterior-posterior (A-P) neural patterning during gastrulation. It is known that high levels of retinoic acid (RA), a major intracellular signaling molecule, determine posterior cell fate, also reflecting an involvement in A-P neural patterning. Here we show that the known RA effector, xCOUP-TF, plays important roles in head development of Xenopus embryo. xCOUP-TF-B injection into the dorsal region of embryos induced formation of an abnormal head with small eyes. Analysis of brain marker gene expression revealed that xCOUP-TF-B injection induced slight anteriorization in embryos and attenuated the effects of RA treatment. This anteriorization effect was enhanced when xCOUP-TF-B was co-injected with xCyp26A or xCyp26C, which are known RA metabolizing factors. Furthermore, xCOUP-TF-B injection enhanced xCyp26A/C transcription. Together, these results suggest that xCOUP-TF and xCyp26 are both regulated by Wnt signaling, and cooperatively function in RA signaling to affect A-P neural patterning.
Fig.1 Phenotype caused by xCOUP-TF-B mRNA injection. XCOUP-TF-B mRNA was injected into the dorso-animal (B, F, G) or ventro-animal (C) regions of 4-cell embryos, and the superficial phenotype (A,B,C) of 3-day tadpoles was observed. Thin lines in A or B indicate the position of thin sections. (D-G) Transverse section of embryo injected with EGFP (D,E) or xCOUP-TF-B (F, G). (H-K) Whole-mount in situ hybridization of embryos injected with 100 pg of xCOUP-TF-B mRNA. En2 and Krox20 (H), Xslug (I), Xotx2 (J), and xSox2 (K) expression was observed at the mid-late neurula stage.
Fig. 2. COUP-TF-B could attenuate the retinoic acid (RA) effects. (A-F) Superficial phenotype of xCOUP-TF-B-injected embryos treated with 10-7 M (B,E) or 10-6 M (C,F) of RA. (G) RT-PCR analysis showing gene expressions of xBF1 (column 1) and Xotx2 (column 2). (H,I) Whole-mount in situ hybridization with xCOUP-TF-B-injected embryos treated with 10-7M RA. Injected side was marked with Red-gal staining by co-injection with lacZ mRNA. Transcription of En2 and Krox20 (H) or xSlug (I) were shown.
Fig. 3. Brain marker expression in xCOUP-TF-B-inject- ed embryo. (A) RT-PCR analysis with cDNA derived from the anterior half of stage-13 embryo injected with 1000 pg (lane1) or 500 pg (lane 2) of bcatenin mRNA, and 10 ng (lane 4) or 20 ng (lane 5) of bcat MO. (B) Whole-mount in situ hybridization with b-catenin-injected embryo. For tracing the injected area, lacZ mRNA was co-injected and injected embryos were stained with Red-Gal.
Fig. 4. Comparison of xCOUP-TF-B and xCyp26C expression in brain region of tadpole. (A-D) Whole mount in situ hybridization was carried out with COUP-TF-B (A,B) and xCyp26C (C,D) probe. We observed Stage 28 (A,C) or Stage 34 (B,D) embryo. MHB: midbrain-hindbrain boundary. AD: anterodorsal lateral line pracode. AV: anteroventral lateral line pracode. Ev: eye vesicle. Ov: otic vesicle. IX: Epibranchial IX placode. PP: pharyngeal pouch. R2/3: rhombomere 2/3. R6: rhombomere 6.
Fig. 5. Co-injection of xCOUP-TF-B enhanced the head defect pheno- type induced by ectopic expression of xCyp26A or xCyp26C. (A-F) 500 pg of xCyp26A (C, D) or 500 pg of xCyp26C (E, F) was co-injected with xCOUP-TF-B (D, F), and injected embryos were observed at stage 40. (G) The open graph bar indicates head defect index (left column; Michiue et al., 2004) and gray bar indicates average eye size of injected embryo (right column). 26A and 26C indicates xCyp26A and xCyp26C, respectively. Error bar indicates S.E.
Fig. 6. xCOUP-TF-B enhanced transcription of both xCyp26A and xCyp26C. (A) RT-PCR was performed with stage-13 embryos injected with 50 pg (lane 2) or 100 pg (lane 3) of xCOUP-TF-B. We observed gene expression of Cyp26A (column 1), Cyp26C (column 2), XBF-1 telencephalon marker: column 3), Xotx2 (forebrain marker: column 4), xSlug (neural crest marker: column 5), and ODC (quantitative control: column 6). (B,C) Whole mount in situ hybridization of embryos injected with 75 pg of xCOUP-TF-B with xCyp26A (B,B or xCyp26C (C,C.The injection side was marked with Red-Gal. (D) Schematic model of xCOUP-TF function.
