Identification, Characterization, and Effects of Xenopus laevis PNAS-4 Gene on Embryonic Development

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.


Introduction
Apoptosis, which protects organisms by removing potentially damaged cells and unnecessary cells after differentiation, is a widely occurring phenomenon in animal development [1]. Recently, reports about apoptosis during embryonic development in various model organisms have flourished. Apoptosis occurs in blastocyst cavitation and continues during gastrulation in mammal and chicken cells [2,3]. Increased caspase-like activity and DNA fragmentation occurs in Xenopus laevis (X. laevis) embryos at the gastrula stage [4,5]. Stress-induced apoptosis has also been characterized in zebrafish embryos [6]. Moreover, studies have revealed that apoptosis may contribute to development of a variety of organs such as brain, heart, lungs, and kidneys [7][8][9][10].
X. laevis is considered as one of the most important model organisms for studying vertebrate development and diseases. In contrast with mouse embryos, the large and robust X. laevis embryos are accessible in the stages during which most important decisions of development are taken so that a large variety of methodologies can be used to analyze the genetic regulation of many different developmental processes.
PNAS-4 was previously identified as a novel apoptosisrelated protein in human acute promyelocytic leukemia NB4 cells. Recently, a report showed that the human PNAS-4 gene was activated during the early response to DNA damage [11]. In our previous studies, we proved that the overexpression of PNAS-4 induced cancer cell apoptosis via the activation of a mitochondrial pathway [12]. The potential antitumor effects of human PNAS-4 were also demonstrated [13,14]. To understand the biological function of the PNAS-4 gene in development, we first cloned the homologous PNAS-4 gene from X. laevis based on PNAS-4 bioinformatics analysis. In the present work, we addressed the preliminary functional annotation of X. laevis PNAS-4 (xPNAS-4) including protein sequence characterization, subcellular localization, gene expression profiles in developing embryos, and the impacts of microinjection of xPNAS-4 mRNA or antisense morpholino oligonucleotides (MOs) on embryonic development. Our primary studies on xPNAS-4 showed that xPNAS-4 may play important roles in embryo development.

Cell Lines and Animal
Models. The HEK (Human embryonic kidney) 293 epithelial cell line was purchased from ATCC. Female Xenopus laevis were injected with 300-500 IU human chorionic gonadotropin (hCG) on the evening before egg collection. Nine to 12 hours after injection, eggs were collected and transferred into a fresh petri dish with culture medium. Staging of embryos was performed according to Nieuwkoop and Faber [15]. Zebrafish embryos were obtained and maintained as described in the zebrafish book [16].

Identification of xPNAS-4 cDNA and Plasmid Constructs.
The Xenopus homolog of PNAS-4 (xPNAS-4) was identified in silico by blasting the X. laevis database with the full-length sequence of the human PNAS-4 protein (GenBank protein id NP 057160). The cDNA of xPNAS-4 gene (GenBank accession no. BC087412) was amplified using the primer combination 5 -GGATCCATGGCCAA-CCAGCCCATCATC-3 and 5 -CTCGAGCTATAGTTTTG-TGTGGCGCCCAGG-3 . RT-PCR amplification was carried out in a MyCycler thermal cycler (Bio-Rad) using the following program: reverse transcription at 50 • C for 40 minutes, and denaturing at 94 • C for 2 minutes, followed by a standard touchdown PCR regime of 94 • C for 30 seconds, 60 • C for 30 seconds, and 72 • C for 40 seconds (15 cycles, −1 • C/cycle), and 94 • C for 30 seconds, 48 • C for 30 seconds, and 72 • C for 40 seconds (35 cycles). The PCR product was cloned directly into the pGEM-T easy vector (Promega) to obtain pGEM-T-xPNAS-4 plasmid. DNA sequencing was performed by BigDye Terminator Cycle sequencing and the sequences were obtained with a 3730 DNA Analyzer (Applied Biosystems). In order to construct the pEGFP-N1-xPNAS-4 plasmid, the following primers were used, the sense primer: 5 -CCGCTCGAGATGGCCAACCAGCCCATCA-3 and the antisense primer: 5 -CGGGATCCCGTAGTTTT-GTGTGGCGCCCAG-3 , containing the Xho I and BamH I restriction sites (underlined), respectively. The termination codon was removed to be in-frame with the enhanced green fluorescent protein (EGFP) sequence. The following amplification procedure was applied: initial PCR activation step at 94 • C for 4 minutes, 35 PCR amplification cycles at 94 • C for 30 seconds, 55 • C for 30 seconds, 72 • C for 40 seconds and a final extension cycle of 72 • C for 10 minutes.

Bioinformatics.
Nucleotide sequence and conserved domain analysis were carried out using BLAST programs (http://www.ncbi.nlm.nih.gov/blast/). Alignment of amino acid sequences was constructed using the Clustal W method (http://www.ebi.ac.uk/clustalw/) [17]. Homology analysis and molecular evolutionary analysis were generated using Lasergene Software and the MegAlign program (DNASTAR).

RT-PCR Analysis.
Semiquantitative RT-PCR analysis of xPNAS-4 expression was performed using the abovementioned primers. As an internal control for RNA quality, the housekeeping gene ornithine decarboxylase (ODC) was also amplified with sense primer 5 -GTCAATGAT-GGAGTGTATGGATC-3 and antisense primer 5 -TCC-ATTCCGCTCTCCTGAGCAC-3 according to published document [18]. Both reactions were amplified by using the same number of extension cycles (30 cycles). The samples were resolved on a 1.5% agarose gel with 1 μg/mL of ethidium bromide, and analyzed in a UV-Transilluminator mini darkroom using Quantity One Software (Bio-Rad).

In Situ RNA Hybridization.
Whole-mount in situ hybridizations were performed according to the method of Harland [19]. Digoxigenin-rUTP-labeled antisense probes for xPNAS-4 were synthesized from pGEM-T-xPNAS-4 plasmid via Sp6 RNA polymerase. Hybridized transcripts were visualized in situ with antidigoxigenin antibodies and photographed using a Nikon dissecting microscope with an attached camera.
2.6. Cellular Localization. 5 × 10 5 HEK293 cells which had been grown in DMEM culture medium containing 10% FBS, 100 units/mL penicillin, and 100 mg/mL streptomycin were seeded on 6-well plates in a 37 • C incubator with 5% CO 2 . Transfections were performed according to the manufacturer's recommendations by using 10 μL of lipofectamine 2000 (Invitrogen) and either 4 μg of pEGFP-N1 or pEGFP-N1-xPNAS-4 plasmid DNA. The culture medium was changed after 6 hours with growth medium and maintained for another 24 hours to allow expression of the fusion protein. The transfected cells were examined under a Zeiss Axiophot fluorescence microscope.

In Vitro Transcription and Microinjection.
Capped mRNAs used for microinjection in vitro were produced using an mMessage mMachine kit (Ambion). The reaction was set up in a total volume of 50 μL containing 2.5 μg Xho I-linearized pGEM-T-xPNAS-4 plasmid, 1×transcription buffer, 0.5 mM dNTPs, 2.5 mM RNA cap structure analogue, 10 mM DTT, 20 U RNAsin, and 40 U T7 RNA polymerase, and then incubated for 2 hours at 37 • C. The templates were digested with 10 U of RNase free DNase I by incubating the samples for 30 minutes at 37 • C. The volume was increased with RNase free water to 100 μL, and then the samples were applied on Quick spin columns (Ambion) to purify the products. Xenopus embryos at the one-cell stage were used for microinjection.

Whole-Mount TUNEL Staining.
Published procedures for the whole-mount TUNEL staining of embryos were followed [20]. In brief, embryos were fixed overnight at 4 • C in a 4% solution of formaldehyde in PBS. The samples were washed twice in 100 mM Tris-HCl buffer, pH 7.5, containing 150 mM sodium chloride and 0.1% Tween 20 (TBST), and fixed in methanol at −20 • C for an additional 24 hours. The samples were washed three times at room temperature for 15 minutes in TBST to rehydrate the embryos following methanol fixation. The samples were transferred to TUNEL buffer (25 mM Tris-HCl, pH 6.6, containing 200 mM sodium cacodylate, 5 mM cobalt chloride, and 0.25% bovine serum albumin), and washed for 30 minutes. Fluorescent labeling of fragmented DNA in the samples was carried out for 1 hour at 37 • C in TUNEL buffer containing 1 mM fluorescein-dUTP and 50 units/mL terminal deoxynucleotidyl transferase. The reaction was stopped by washing the sample in TBST buffer five times for 5 minutes at room temperature. The fluorescein-labeled TUNEL-positive cells were photographed using an Olympus microscope with excitation at 460-490 nm and emission at 510 nm.   : Q9D291), the identity was 88.1%, 87.6%, 87.6%, 87.0%, 86.0%, 85.5%, and 85.5%, respectively. This confirmed that these genes share a common ancestor. Based on the homology analysis, a phylogenetic tree of PNAS-4 proteins was constructed (Figure 1(b)).

Cellular Localization of xPNAS-4.
To examine xPNAS-4 cellular localization, we fused xPNAS-4 to EGFP. Expression of xPNAS-4-EGFP in HEK293 cells appeared in the cytoplasm and was closely located around the nuclei, but the control (EGFP) protein was evenly distributed throughout the whole cell without any compartmentalization (Figure 2(a)). Thus, xPNAS-4 was a cytoplasmic protein which was localized in the perinuclear region of the cytoplasm.

Temporal and Spatial Expression of xPNAS-4 in X. Laevis
Embryos. In order to determine the temporal expression pattern of xPNAS-4, the mRNA level at different developmental stages was detected. Semiquantitative RT-PCR analysis showed that xPNAS-4 mRNA could be detected at all stages of development, from the unfertilized egg to the tadpole stage (Figure 2 there is xPNAS-4 message of maternal origin, since zygotic transcription starts at the mid-blastula transition (MBT) [21]. Indeed, many other apoptosis-related genes showed similar transcription patterns [22,23]. Consequently, xPNAS-4 may play an important role in Xenopus embryo development.
For a better understanding of the expression of xPNAS-4 mRNA in embryonic tissues, whole mount in situ hybridization was performed. Figure 2(c) shows that xPNAS-4 was mainly present along ectoderm and mesoderm. The expression of xPNAS-4 could be detected at stage 2. This was consistent with the result of semiquantitative RT-PCR. Up to the gastrula stage (stage 10), the expression of xPNAS-4 was present ubiquitously in the embryo. By the tail bud stage (stage 35), the expression of xPNAS-4 mainly appeared in the head and dorsal neural tissue. Furthermore, a weak expression of xPNAS-4 could also be observed in the prospective heart of the tail bud embryo. Thus, xPNAS-4 is a maternally expressed gene. Its transcripts are present ubiquitously in gastrula stage embryos, while tissue specific expression can be detected in later embryogenesis.

Microinjections of xPNAS-4 mRNA Caused Developmental Defects.
To explore the role of xPNAS-4 during embryonic development, the synthesized xPNAS-4 mRNA was injected into Xenopus embryos at the one-cell stage. As shown in Figures 3(a), 3(e), and 3(f), the eyes were poorly developed and were smaller than normal. Some embryos also showed defective myotomes and reduced pigmentation (Figures 3(b)-3(d)). The abnormal eye phenotype was dose dependent. About 26% small eye embryos were produced by injection of xPNAS-4 mRNA at 450 pg/embryo; while about 38% small eye embryos were observed when xPNAS-4 mRNA was injected at 900 pg/embryo. The uninjected embryos only produced 2% eye abnormalities (Figure 3(g)). This result demonstrated that xPNAS-4 plays an important role in embryonic eye development.
Taking into consideration the high conservation of xPNAS-4 protein with that of zebrafish, we hypothesized that xPNAS-4 would act in a similar manner in zebrafish, another developmental system used to study eye development [24]. To test this postulation, we injected xPNAS-4 mRNA into zebrafish embryos at the one-cell stage. The resulting phenotype was quite similar to what occurred with xPNAS-4 mRNA injection in Xenopus. Following injection of 300 pg xPNAS-4 mRNA 38% of embryos had abnormal eyes, and after injection of 600 pg mRNA 86% of embryos exhibited abnormal eye development. Unlike Xenopus embryos, however, zebrafish embryos with eye defects could be divided into two groups: the less affected embryos had reduced eye size, while the more strongly affected embryos exhibited a one-eye phenotype (Figures 4(d), 4(e), and 4(h)). In addition to the eye defect, some strongly affected embryos had a bent axis (Figures 4(f) and 4(i)). Similarly, the defects produced by heterologous expression of xPNAS-4 were also dose dependent. Injection of 300 pg of xPNAS-4 mRNA resulted in 34% reduced eye embryos and 4% oneeye embryos, while injection of 600 pg of xPNAS-4 mRNA resulted in 24% reduced eye embryos and 62% one-eye embryos (Figures 4(j)-4(l)). All of the uninjected embryos were normal (Figures 4(a)-4(c), and 4(g)).

xPNAS-4 Induces Eye Developmental Defects Via Apoptosis.
To confirm that the developmental defects caused   by heterologous injection of xPNAS-4 resulted from apoptosis, whole-mount TUNEL (terminal deoxynucleotidyl transferase-mediated nicked-end labeling) staining, a highly sensitive indicator of DNA fragmentation in situ was employed to detect the presence of apoptotic cells. TUNELpositive cells were present at high frequency on the side of zebrafish embryos on which eye development was affected (Figure 4(n)) when compared with the control embryos (Figure 4(m)).
It is widely accepted that apoptosis is involved in many embryological processes and plays a significant role in animal development. If apoptosis is disturbed, tissues develop abnormally and various syndromes or cancers develop [25]. The tumor suppressor protein p53, a widely known trigger of apoptosis, has been implicated as an important protein in embryonic development. Inappropriate overexpression or underexpression of p53 can lead to embryonic lethality or increased risk of malformations [26]. Furthermore, an effect of apoptosis on eye development has also well been documented. Inactivation of p53 function by gene knock-out [27] or through lens specific promoter-driven expression of the viral gene E6, interrupts normal apoptosis in the lens leading to formation of a cataract [28,29]. The caspase family including caspase-1, -2, -3, -4, -6, and -9, is involved in eye differentiation in different species of vertebrates [30]. During development of the chicken lens, members of the Bcl-2 family, such as Bcl-2, Bax, Bad, and Bcl-Xs/l, are abundantly expressed between stages E12 and E16 [31]. In our experiment, we found a considerable number of apoptotic cells appeared in abnormal embryos by whole mount in situ hybridization, suggesting that abnormal eye development resulted from the overexpression or heterologous expression of xPNAS-4. This further confirmed that xPNAS-4 is involved in apoptosis during embryonic development. 8 Journal of Biomedicine and Biotechnology

Depletion of xPNAS-4 Affects the Normal Process of Embryos.
To further define the function of xPNAS-4 during embryogenesis, we performed translational inhibition of xPNAS-4 by microinjecting antisense morpholino oligonucleotides (MOs). Control and xPNAS-4 MOs (20 ng) were microinjected into the animal blastomere of stage-2 embryos. As development proceeded, 86.7% of embryos (n = 98) microinjected with xPNAS-4 MO had a failure of head development and a shortened axis (Figures 5(a) and 5(c)). All embryos which had a failure of head development exhibited a shortened axis. Embryos with failed head development died in the late tailbud stage, while control MO microinjected embryos (98.4%, n = 128) developed to the next stages normally.
In order to confirm that the phenotypes were due to translational inhibition of xPNAS-4, we co-injected xPNAS-4 MO and in vitro transcribed capped xPNAS-4 mRNA. As expected, the failure of head development and the shortened axis were rescued to some extent after co-injection of 20 ng xPNAS-4 MO plus 450 pg xPNAS-4 mRNA. About 56.5% of embryos (n = 92) exhibited a failure of head development. The defective embryos had longer axes than those microinjected with xPNAS-4 MO alone. A greater extent of rescue was observed after co-injection of 20 ng xPNAS-4 MO plus 900 pg xPNAS-4 mRNA. Only 32.1% embryos (n = 78) showed the failure of head development. The defective embryos had longer axes than those microinjected with xPNAS-4 MO plus 450 pg xPNAS-4 mRNA ( Figures  5(b) and 5(c)). These results revealed that the failure of head development and shortened axis caused by xPNAS-4 MO microinjection were due to depletion of xPNAS-4.
We also found similar defects using zebrafish as a model system in our previous studies. Loss of function of the PNAS-4 gene caused gastrulation defects with a shorter and broader axis, as well as a posteriorly mis-positioned prechordal plate, due to the defective convergence and extension movement [32]. In the present work, we found that loss of function of the PNAS-4 gene in X. laevis embryos resulted in the failure of head development and a shortened axis. The phenotypic differences were probably due to the different species or differences in the inhibition rate of morpholino oligonucleotides.

Conclusion
PNAS-4 protein is well conserved across species. Here we identified the PNAS-4 homolog from X. laevis based on the amino acid sequence of human PNAS-4 protein.
The xPNAS-4 protein is very similar to human PNAS-4. The high similarity suggested that the human and X. laevis proteins might have analogous functions. Cellular localization suggested that xPNAS-4 is in the cytoplasm and is located around the nucleus. We also proved that xPNAS-4 is a maternally expressed gene. Investigation of the developmental expression pattern showed that xPNAS-4 is present throughout embryogenesis and undergoes pronounced changes in its level of mRNA during embryo development. In addition, microinjections of xPNAS-4 mRNA into Xenopus laevis embryos resulted in embryonic developmental defects including a small eye phenotype in developing X. laevis embryos, and a reduced eye size or one-eye phenotype in the developing zebrafish embryos. Whole mount in situ hybridization further confirmed that the developmental defects resulted from apoptosis during embryonic development. Furthermore, embryos microinjected with xPNAS-4 MO had a failure of head development and shortened axis.
In conclusion, our results provide important clues about the molecular mechanisms by which xPNAS-4 regulates developmental processes.