XB-ART-45256Int J Dev Biol January 1, 2012; 56 (4): 239-44.
xCOUP-TF-B regulates xCyp26 transcription and modulates retinoic acid signaling for anterior neural patterning in Xenopus.
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.
PubMed ID: 22562199
Article link: Int J Dev Biol
Species referenced: Xenopus laevis
Genes referenced: ctnnb1 cyp26a1 cyp26c1 egr2 en2 foxg1 gal.2 nr2f2 nr2f5 odc1 otx2 snai2 sox2 tbx2 tf
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|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.|