XB-ART-39551Genes Cells May 1, 2009; 14 (5): 583-95.
Expression cloning of Xenopus zygote arrest 2 (Xzar2) as a novel epidermalization-promoting factor in early embryos of Xenopus laevis.
In vertebrates, BMPs are known to induce epidermal fate at the expense of neural fate. To further explore the molecular mechanisms of epidermal differentiation, we have developed an expression cloning system for isolating cDNAs that encode intrinsic proteins with epidermal-inducing activity. Under our conditions, 92.5% of the dissociated animal cap cells treated with the conditioned medium from H(2)O-injected control oocytes differentiated into neural tissue, which developed neural fibers and expressed a neural marker (NCAM). In contrast, when dissociated animal cap cells were treated with the supernatant collected from the culture of BMP-4 mRNA-injected oocytes, the microcultures differentiated into epidermal tissue, which developed cilium. The cells expressed an epidermal marker (keratin), but not NCAM. Using the dissociated animal cap cells in a functional screening system, we cloned a cDNA encoding a novel polypeptide, Xenopus zygote arrest 2 (Xzar2). Over-expression of Xzar2 caused anterior defects and suppressed expressions of the neural markers. The epidermalization-promoting activity of Xzar2 was substantially not affected by over-expression of the BMP signaling antagonists Smad6 and 7, and a dominant negative receptor for BMP (tBR). Our results suggest that Xzar2 is involved in epidermal fate determination mainly through signaling pathways distinct from that of BMP-Smad during early embryogenesis.
PubMed ID: 19371384
Article link: Genes Cells
Genes referenced: bmp2 bmp4 bmp7.1 gdf6 krt12.4 ncam1 nog odc1 rax smad6 smad7 sox2 tbxt zar1 zar2
Antibodies: Epidermis Ab3 Map2 Ab2
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|Figure 1 Experimental design for isolating epidermal inducer cDNA from Xenopus laevis. A cDNA library was prepared from Xenopus oocytes at stage 8. For sib selection, plasmid DNA pools were prepared from the cDNA library after subdivision and transformation. RNAs were transcribed in vitro and microinjected into X. laevis oocytes. After 16 h, the incubation medium was collected and used for culture of dissociated animal cap cells. Epidermal inducing activities contained in the medium were assessed by morphology of the animal cap cells.|
|Figure 2 Differentiated cells under the microculture condition. (A) Differentiated cell morphologies. Dissociated animal cap cells cultured in different conditions were observed under phase-contrast. Representative morphologies are shown. a: Dissociated animal cap cells cultured in control condition. The cells developed nerve fibers (arrowheads). Bar = 200 μm. b, c: Dissociated animal cap cells incubated in CMFM with 40 ng/mL rhBMP-4. Bar = 100 μm. (b) An aggregate of ciliated cells. (c) A sheet of ciliated cells attached to the culture dish. (B) Immunostaining of the differentiated cells. Cells were fixed after incubation for 36 h and examined for the presence of AP-20 or E3 antigens. a, b: Cells with nerve fibers are stained with mAb AP-20, which is highly reactive with the microtubule-associated protein 2 (MAP2). This antibody stains throughout dendrites and somas. c, d: Ciliated cells stained with mAb E3, which selectively recognizes differentiated epidermal cells. Nuclear staining was carried out with PI (b, d). Bar = 100 μm. (C) RT-PCR analysis of the differentiated cells. Dissociated animal cap cells prepared from stage 8 were incubated in CMFM for 4 h. Then, the cells were cultured for 18 h in NAM/2 before assessing the cell differentiation by RT-PCR. NCAM is a general neural marker: keratin for epidermis: Xbra for mesoderm: and EF1-α for loading control. The animal cap cells that were treated with the culture medium derived from 1.5 ng BMP-4 mRNA- and H2O-injected oocytes expressed keratin (lane 1) and NCAM (lane 2), respectively. Intact animal caps expressed keratin (lane 3), while the whole embryos at stage 25, which is accounted for the processing time of 18 h from stage 8, exhibited all the marker transcripts examined thus far (lane 4). The ‘–RT’ contained all reagents except reverse transcriptase and was used as a negative control (lane 5).|
|Figure 3 Expression cloning of Xzar2 in cDNA pools. The cDNA library was divided into 20 pools (~5000 clones per pool), from which mRNA was synthesized in vitro. Each mRNA pool was assessed by epidermal inducing activity and presence of the known secreted epidermal inducers, such as BMPs and GDF6. mRNAs generated from each pool were injected into oocytes and the incubation medium of the oocytes was subjected to the microculture assay. The epidermal inducing activities of the various pools from the first sib selection are indicated (plus or minus). Plasmid DNAs of each pool were assayed by RT-PCR for the presence of the clones encoding BMP-2, -4, -7 and GDF6. First strand cDNA synthesized from the stage 26 embryos was used for positive control of RT-PCR. We used the culture medium of BMP-4 mRNA injected oocytes for positive control of the microculture assay.|
|Figure 4 Amino acid sequence alignment of Zar2 and Zar1 proteins from Xenopus, pufferfish, zebrafish, rat, mouse and human. Alignment was carried out using the Clustal W program by the Megalign software component of Lasergene version 7.2.1. Sets of four or more identical residues at one aligned position are shown in black boxes. Asterisks denote the conserved cysteins of the apical PHD motif. Gen- Bank accession numbers are as follows: Xenopus Zar2, AB190316; Xenopus Zar1, AY283176; Fugu rubripes Zar1, AY283177; Danio rerio Zar1, AY283178; Rattus norvegicus Zar1, AY283175; Mus musculus Zar1, AY191415; and Homo sapiens Zar1, AY191416.|
|Figure 5 Expression and localization of Xzar2 RNA. (A) RT-PCR analysis of Xzar2 expression during early development. The ‘–RT’ lane contained all reagents except for reverse transcriptase and was used as a negative control. Ornithine decarboxylase (ODC) was used as a loading control. (B) Whole-mount in situ hybridization using anti-sense Xzar2 probe. At blastula and gastrula stages, Xzar2 was expressed in the ectodermal and the mesodermal regions. (a) Lateral view of a blastula embryo (stage 8). (b) Lateral view of an early gastrula embryo (stage 10). (c) Vegetal view of a late gastrula embryo (stage 12), as even shown in the corresponding vertical section (c′). (C) Intracellular localization of Xzar2. HEK-293 cells were transfected with EGFPN1- Xzar2 construct. Images were obtained using a confocal microscope. (a) Phase contrast. (b) Distribution of Xzar2 in a nucleus. (c) Merge image of (a) and (b). Bar = 100 μm.|
|Figure 6 Role of Xzar2 in Xenopus embryo. (A–C) Xar2 inhibited neuralization and induced epidermis in animal cap calls. (A) RTPCRs were performed as in Fig. 2C. Dissociated animal caps were microcultured for 4 h in the conditioned oocyte medium collected from Xzar2 mRNA-injected (lane 1), BMP4 mRNA-injected (lane 2) or H2O-injected (lane 3) oocytes. Oocyte medium generated from Xzar2 and BMP4 converted the cell fate from neural to epidermal. Lane 4 is from the intact animal caps, which expressed the epidermal keratin and did not express the neural gene (NCAM). Lanes 5 and 6 are whole embryos with (lane 5) and without (lane 6) reverse transcriptase during the RT reaction. (B) In vitro-coupled transcription/translation reactions with plasmid encoding the Xzar2 ORF in the presence of either control MO (50 ng) or perfect-match Xzar2 MO (50 ng). Protein synthesis was assessed by [35S]methionine incorporation. (C) Control MO (9.6 ng, lane 5), Xzar2 MO (9.6 ng, lanes 1 and 3; 4.8 ng, lanes 2 and 4) and Xzar2 mRNA (lanes 3 and 4) were injected near the animal pole of two-cell stage embryos. Animal caps were isolated from embryos at the blastula stage and cultured to stage 26 before assessing the cell differentiation by RT-PCR. Whole embryos were referred as control for the RT reactions with (lane 6) and without (lane 7) reverse transcriptase. Xzar2 knock down expressed the neural marker induced by Xzar2 MO (lanes 1 and 2), while the expression was blocked by Xzar2 mRNA (lanes 3 and 4). (D–G) Suppressions of eye formation and early neural marker expression in embryos by injecting Xzar2 mRNA. (D) Experimental design for mRNA injection into a single animal-dorsal blastomere at the 8-cell stage (arrow); the blastomere developed into the left head was injected with 320 pg of Xzar2 or control preprolactin mRNA, each with 10 pg of GFP mRNA as a tracer. (E) Embryos injected with Xzar2 mRNA showed defects in head and eye development. (a) Control preprolactin mRNA-injected embryos. (b) Xzar2 mRNA injected embryos. All views are injected on the left side of stage 32 embryos. (F) Embryos of stage 32 were sectioned and stained in hematoxylin and eosin. Transverse sections through the head structure of preprolactin (a) or Xzar2 (b) mRNA-injected embryos are shown. (G) Whole-mount in situ hybridization was performed to stage 20 embryos; anterior views are shown. Expression of Xrx1A (a, b) and Sox2 (c, d) are suppressed in the Xzar2 mRNA-injected sides of embryos (b, d), but control mRNA-injected embryos (a, c). Red staining shows GFP expressed region. Blue staining represents the anterior neural (eye) marker gene (Xrx1A) expression or the neural plate marker (Sox2) expression.|
|Figure 7 Inhibitions of the BMP-Smad signaling pathway by I-Smads and tBR were not involved in the epidermal differentiation promoted by Xzar2. (A) mRNA encoding Smad6 (lane 1), Smad7 (lane 2), Smad6 and BMP4 (lane 3), Smad6 and Xzar2 (lane 4), Smad7 and BMP4 (lane 5), Smad7 and Xzar2 (lane 6), or control (lane 7) was injected near the animal pole of two-cell stage embryos. Animal caps were isolated from embryos at the blastula stage and cultured to stage 26 before assessing the cell differentiation by RT-PCR. Whole embryos were referred as control for the RT reactions with (lane 8) and without (lane 9) reverse transcriptase. Over-expression of Smad6 and Smad7 in animal cap cells did not inhibit the expression of the epidermal marker induced by Xzar2 (lanes 4 and 6), while epidermal induction of BMP4 was blocked by Smad6 (lane 3) and Smad7 (lane 5). (B) Dissociated animal cap cells were microcultured for 4 h in the conditioned oocyte medium collected from H2O-injected (lane 1), Xzar2 mRNA-injected (lanes 2 and 5) or BMP4 mRNA-injected (lanes 3 and 6) oocytes. tBR mRNA was injected into both blastmeres of 2-cell-etage embryos (lanes 4, 5 and 6). Then the animal cap cells of injected embryos were used for the microculture. Lanes 7 and 8 are whole embryos with (lane 7) and without (lane 8) reverse transcriptase during the RT reaction.|
|Supplemental Figure S1 Xzar1 inhibited neuralization and promoted epidermal differentiation in microcultured cells from animal caps. RT-PCRs were performed as Fig. 2C. Dissociated animal caps were microcultured for 4 h in the conditioned oocyte medium collected from Xzar1 mRNA-injected (lane 1), Xzar2 mRNA-injected (lane 2) or H2O-injected (lane 3) oocytes. Lane 4 is from the intact animal caps, which expressed the epidermal keratin and did not express the neural gene (NCAM). Lanes 5 and 6 are whole embryos with (lane 5) and without (lane 6) reverse transcriptase during the RT reaction.|
|Supplemental Figure S2 Noggin blocked epidermalization-promoting activity of Xzar2. RT-PCRs were performed as Fig. 2C. Animal cap cells were dissociated for 4 h in the conditioned oocyte medium collected from H2O-injected (lane 1), Xzar2 mRNA-injected (lane 2 and 4) or BMP4 mRNA-injected (lane 3 and 5) oocytes, containing 1mg/ml noggin protein (lanes 4 and 5). Lanes 6 and 7 are whole embryos with (lane 6) and without (lane 7) reverse transcriptase during the RT reaction.|
|Zar2 (ZAR1-like protein) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 12, lateral view of sagittal section, animal up, dorsal axis on right.|