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J Biomed Biotechnol
2010 Jan 01;2010:134764. doi: 10.1155/2010/134764.
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Identification, characterization, and effects of Xenopus laevis PNAS-4 gene on embryonic development.
Yan F, Ruan XZ, Yang HS, Yao SH, Zhao XY, Gou LT, Ma FX, Yuan Z, Deng HX, Wei YQ.
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Apoptosis plays an important role in embryonic development. PNAS-4 has been demonstrated to induce apoptosis in several cancer cells. In this study, we cloned Xenopus laevis PNAS-4 (xPNAS-4), which is homologous to the human PNAS-4 gene. Bioinformatics analysis for PNAS-4 indicated that xPNAS-4 shared 87.6% identity with human PNAS-4 and 85.5% with mouse PNAS-4. The phylogenetic tree of PNAS-4 protein was also summarized. An analysis of cellular localization using an EGFP-fused protein demonstrated that xPNAS-4 was localized in the perinuclear region of the cytoplasm. RT-PCR analysis revealed that xPNAS-4, as a maternally expressed gene, was present in all stages of early embryo development. Whole-mount in situ hybridization showed that xPNAS-4 was mainly expressed in ectoderm and mesoderm. Furthermore, microinjection of xPNAS-4 mRNA in vivo caused developmental defects manifesting as a small eye phenotype in the Xenopous embryos, and as a small eye or one-eye phenotype in developing zebrafish embryos. In addition, embryos microinjected with xPNAS-4 antisense morpholino oligonucleotides (MOs) exhibited a failure of head development and shortened axis.
Figure 1. Comparison of the PNAS-4 protein sequence from X. laevis (GenBank protein_id: AAH87412) with those of other species. (a) Alignment of xPNAS-4 and hPNAS-4 protein sequences (GenBank protein_id: NP_057160) showing that they are homologous proteins. There is a DUF862 domain (underlined) in the hPNAS-4 and xPNAS-4 proteins. Asterisks (∗) indicate amino acid residues that are conserved across species. Colons (:) indicate strong similarity between protein xPNAS-4 and hPNAS-4. Dots (·) indicate weak similarity. (b) Phylogenetic tree of the PNAS-4 nucleotide sequences.
Figure 2. Cellular localization, and temporal and spatial expression patterns of xPNAS-4. (a) HEK293 cells were transfected with pEGFP-N1 or pEGFP-N1-xPNAS-4 plasmids as explained in materials and methods. Left: pEGFP-N1 transfected cells (200×); Right: pEGFP-N1-xPNAS-4 transfected cells (200×). (b) Analysis of the temporal expression pattern of xPNAS-4 gene by semiquantitative RT-PCR. ODC: X. laevis ornithine decarboxylase as internal control gene. RT (–): control reverse transcription without template. (c) Whole mount in situ hybridizations showing the spatial expression pattern of xPNAS-4 gene. The upper-left image shows the expression pattern of the xPNAS-4 gene in a stage-2 embryo. The upper-right image shows the expression pattern of the xPNAS-4 gene in a stage-10 embryo (beginning of gastrulation). An ubiquitous expression pattern (purple staining) can be seen. The lower image shows the expression pattern of the xPNAS-4 gene at stage 35. xPNAS-4 transcripts were present in the head, dorsal neural tissue, and prospective heart.
Figure 3. Developmental defects in Xenopus embryos caused by microinjection of xPNAS-4. Embryos were injected once into the dorsal marginal zone at the one-cell stage with 450 pg or 900 pg of xPNAS-4 mRNA. (a) Lateral view of stage 35 tadpoles showing (bottom to top) an unaffected embryo (Wt, bottom); a mildly affected embryo (Lae, middle) and a strongly affected embryo (Sae, upper). Arrows indicate embryos' eyes. (b), (c), and (d) are magnified views from local regions in the pictures. Abbreviations: Wt, wild type; Lae, mildly affected embryo; Sae, strongly affected embryo; My, myotome; Pi, pigment. (e) Dorsal view of an uninjected stage 43 embryo. (f) Dorsal view of stage 43 embryo which was injected with 900 pg of xPNAS-4 mRNA. (g) Graphical representation of the percent of embryos injected with xPNAS-4 mRNA with the small eye phenotype at stage 35 (n = the number of embryos in each group).
Figure 4. Heterologous expression of xPNAS-4 causes eye defects in zebrafish embryos. Embryos were injected at the one-cell stage with 300 pg or 600 pg of xPNAS-4 mRNA. Figures 4(a)–4(f) show embryos at 24 hours post fertilization (hpf). (a) Ventral view of a wild type zebrafish embryo with anterior to the bottom right. (b) Ventral view of a wild type zebrafish embryo with anterior to the top. (c) Lateral view of the normal axis of a wild type embryo. (d) Ventral view of a reduced-eye embryo. Arrow indicates the reduced eye. (e) Ventral view of a one-eyed embryo with anterior to the top. Arrow indicates the lost eye. (f) Lateral view of bent axis defects (arrow) of xPNAS-4 mRNA injected embryo. Figures 4(g)–4(i) show embryos at 48 hpf with anterior to the top. (g) Dorsal view of a wild type zebrafish embryo. (h) Dorsal view of an embryo with a reduced size eye. (i) Dorsal view of a one-eyed embryo. (j) Population of wild type zebrafish embryos at 48 hpf. (k) Population of zebrafish embryos with eye defects at 48 hpf. (l) Quantitative data for eye defects in injected and uninjected zebrafish embryos at 48 hpf. N is the number of embryos examined for each treatment. (m) Whole-mount TUNEL staining of a wild type embryo at 24 hpf. (n) Whole-mount TUNEL staining of an embryo with an eye defect at 24 hpf. A high number of TUNEL-positive cells were detected on the side of injection (black arrow). Dotted circle indicates the developing eyes.
Figure 5. Effect of xPNAS-4 MOs microinjection. (a) Embryos microinjected with 20 ng of xPNAS-4 MO exhibited failure of head development and shortened axis defects (left), but embryos microinjected with 20 ng of control MO embryos were normal (right). (b) The embryos were partially rescued by co-injected with 450 pg of xPNAS-4 mRNA and 20 ng of xPNAS-4 MO (the second row) in comparison with embryos microinjected with 20 ng of xPNAS-4 MO alone (the first row). Better rescue was observed after co-injection of 20 ng of xPNAS-4 MO plus 900 pg of xPNAS-4 mRNA (the third row). The fourth row shows a normal embryo microinjected with 20 ng of control MO. (c) The rate of defective embryos seen in the experiment.
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