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Genes to Cells (2009) 14, 583-595. doi:10.1111/j.1365-2443.2009.01291.x
© 2009 Blackwell Publishing or its licensors
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Expression cloning of Xenopus zygote arrest 2 (Xzar2) as a novel epidermalization-promoting factor in early embryos of Xenopus laevis

 Yuka Nakajimaa, Harumasa Okamotob and Tai Kubo*

Neuroscience Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), AIST Central 6, 1-1-1 Higashi, Tsukuba 305-8566, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
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 H2O-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.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
All tissues in animals are derived from the three primary germ layers: ectoderm, endoderm and mesoderm. The ectodermal cells can develop into either epidermal or nervous tissues. Much of the understanding of ectodermal differentiation has come from classical and molecular studies in amphibian embryos. In Xenopus, blastula ectodermal explants (animal caps) develop into epidermis when cultured in isolation. In contrast, neural tissue can be induced when animal cap cells are dissociated and maintained in dissociated condition for a few hours (Sato & Sargent 1989). Addition of BMP-4 to the dispersed Xenopus cells can restore epidermal differentiation (Wilson & Hemmati-Brivanlou 1995), leading to the generally accepted proposal that during cell dissociation, endogenous BMPs diffuse into the culture medium (De Robertis & Kuroda 2004). BMP inhibitors, such as noggin (Smith & Harland 1992), follistatin (Hemmati-Brivanlou et al. 1994), chordin (Sasai et al. 1994) and Cerberus (Bouwmeester et al. 1996), block the BMP-Smad signaling pathway and this affects ectodermal fate to direct differentiation from epidermis to neural tissue.

However, knock-out mice lacking the BMP antagonists, Cerberus, follistatin, noggin and/or chordin, still develop a nervous system (Vonica & Brivanlou 2006). BMP inhibition is only required as a late step during neural induction in the chicken (Linker & Stern 2004). In Xenopus, BMP antagonists cannot induce neural markers when FGF signaling is blocked (Launay et al. 1996; Sasai et al. 1996). Recently, BMPs and the ligands in the BMP pathways are shown to signal in dissociated cells at the same level as in intact animal cap cells (Kuroda et al. 2005).

To further explore the molecular mechanisms of epidermal differentiation, we have developed a functional screening system for isolating cDNAs that encode proteins with epidermal-inducing activity in the early vertebrate embryo. None of the known epidermal factors have been identified using expression cloning to isolate proteins with epidermalizing activity. Among the transforming growth factor-β (TGF-β) family, BMPs were originally identified as molecules that can induce ectopic bone and cartilage formation in rodents (Wozney et al. 1988). Wilson & Hemmati-Brivanlou (1995) showed that BMP-4 inhibits neuralization of dissociated animal caps, promoting the formation of epidermis. BMP-2 and BMP-7 were identified as a homolog of BMP-4 (Suzuki et al. 1997); DGF6 was identified by a differential screening using TGF-β probes (Chang & Hemmati-Brivanlou 1999). Many genes involved in epidermal induction through the BMP-Smad signaling pathway were characterized: BMPs and their downstreams including receptors and Smads (Muñoz-Sanjuán & Brivanlou 2002; De Robertis & Kuroda 2004).

In contrast to profound insight into the BMPs and the BMP-involved signaling pathways, little is known about endogenous factor(s) that have epidermal-inducing and/or neutralization-inhibiting activities in the development of embryos. Therefore, we first established culture conditions and an assay system to evaluate the epidermal-inducing activity. A cDNA library was prepared from Xenopus-developing embryos, transcribed in vitro and the RNAs were subjected to the functional assays. Thus, we identified Xenopus zygote arrest 2 (Zar2), which is homologous to Zar1, as an epidermalization-promoting factor during early embryogenesis. We demonstrated that Xzar2 can promote epidermal and inhibit neural differentiation in dissociated animal cap cells and embryos of Xenopus. Our results suggest that Xzar2 is involved in epidermal fate determination mainly through signaling pathways distinct from that of BMP-Smad during early embryogenesis.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
Establishment of microculture system for expression cloning of epidermal inducers

Animal cap cells dissected and dissociated from blastulae at stage 8: 50% of the cells develop into neural tissue extending nerve fibers (Godsave & Slack 1989). We modified the procedures to improve the neural fate ratio, so that we could use it for expression cloning of epidermal inducers. The experimental scheme is shown in Fig. 1. Briefly, cells were taken from the inner surface of the animal cap of stage 8 midblastulae and incubated in CMFM for 4 h. Then the cells were dispensed into about 20 cells per microculture well. Most cells were divided and aggregated to make spheroids. Then nerve fibers extended out in 92.6% of the microculture wells (Table 1 upper section, arrowheads in Fig. 2A,a). These results suggested that cells from animal caps of blastula embryos were directed to develop into neural tissue under our standard culture condition.


Figure 1
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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.

 

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  		Table 1  Differentiation of animal cap cells in a microculture system
 

Figure 2
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  		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-{alpha} 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).

 
Animal cap cells of the blastula stage of Xenopus laevis can be re-specified from a nerve cell fate to an epidermal fate by treatment with epidermal inducers, such as BMP-4 (Wilson & Hemmati-Brivanlou 1995). Cilia are present in the epidermal cells developed from single cells of blastulae animal cap cells (Godsave & Slack 1989). Thus to determine whether our dissociated animal cap assay can reconstitute epidermal induction, we treated dissociated animal cap cells with rhBMP-4 protein. Under the conditions, the cells were differentiated into tiny spheroids (Fig. 2A,b) or sheets of cells (Fig. 2A,c), which formed cilia. The response to the BMP-4 concentration was graded: the rate of ciliated cells increased and the rate of nerved cells decreased with rhBMP-4 concentration up to 40 ng/mL (Table 1 upper section). These results indicated that our culture conditions could reconstitute epidermal induction in vitro.

To examine whether cDNAs coding for epidermal inducers, if present, could change the neural fate of the cells to epidermis into our microculture system, we used the BMP-4 cDNA: mRNA was synthesized, injected into Xenopus oocytes, and the culture medium of the oocytes was subjected to the assay (Table 1 lower section). In the case of treatment with the supernatant of BMP-4 mRNA-injected oocytes, cells with cilia were present in 87.3% of the microculture wells. Following treatment of the cells with the supernatant of H2O-injected oocytes, however, 2.5% of the microcultures showed ciliate development and mostly extended nerve fibers. These results suggested the feasibility of the assay to identify novel epidermal inducers on the basis of morphological changes.

Neural cell and epidermis have been identified using antibodies AP-20 and E3, respectively; AP-20 is highly reactive with the microtubule-associated protein 2 (MAP2), which is localized in dendrites and somas, and E3 selectively recognizes differentiated epidermal cells (Mitani & Okamoto 1989). A neural antigen was detected in the cells with nerve fibers (Fig. 2B, a and b) and an epidermal antigen was detected on the surface of the cells with cilia (Fig. 2B, c and d). To further assess whether each marker gene was expressed, we performed RT-PCR after the microculture with oocyte medium, in which BMP-4 mRNA- or H2O-injected oocytes were maintained. RNA was extracted from cells cultured under our conditions and assayed for the expression of cell-type-specific molecular markers by RT-PCR. Intact animal caps expressed the epidermal keratin gene and did not express the general neural marker, neural cell adhesion molecule (NCAM, Fig. 2C, lane 3). In the absence of epidermal inducer, NCAM was turned on under the microculture condition (Fig. 2C, lane 2). The epidermal keratin gene was switched off, indicating that these cells had neuralized. Transcripts of Xenopus Brachyury (Xbra), which is a marker of both notochord and ventral/posterior mesoderm at this stage were detected in extracts of whole embryo (Fig. 2C, lane 4). When supernatant collected from BMP-4 mRNA-injected oocytes was included during dissociation in CMFM, the epidermal keratin was expressed and the neural gene was repressed (Fig. 2C, lane 1), indicating their differentiation into epidermis. Mesoderm was not induced under this condition. The recognition and expression of neural and epidermal markers indicate that proteins secreted by the oocytes had altered the developmental fate of the dissociated animal cap cells, from nerve tissue to epidermis under our culture conditions.

Expression cloning of Xzar2 assayed by its epidermaling effects

We used the assay in a screen for secreted proteins capable of causing epidermal induction in the dissociated animal cap cells of blastula embryos. cDNAs were prepared from the blastula embryo poly(A)+ RNA and the sized cDNA (0.7–5.0 kbp) was ligated into the plasmid vector, pSD64TRER. The cDNA library was divided into 20 pools (~5000 clones per pool), from which mRNAs were synthesized in vitro. Each mRNA pool was assessed by two criteria: epidermal-inducing activity and the presence of a known soluble epidermal inducer, 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 dissociated microculture assay. The presence of the clones encoding BMP-2, -4, -7, and GDF6 was analyzed by RT-PCR (Fig. 3). About 50 000 clones have been screened (50% of the library). Two pools, no. 3 and no. 8, met the criteria out of the seven active pools with epidermal-inducing activity (Fig. 3). Pool no. 8 was selected for subsequent sib selections until a single active clone. We isolated several active clones. One of the active clones showed high similarity to Xenopus zygote arrest 1 (zar1; Wu et al. 2003b); the similarity was 50.8% (Fig. 4). Therefore, we refer to it as Xenopus zygote arrest 2 (Xzar2). The full-length Xzar2 cDNA encodes a protein of 303 amino acids. Comparison of amino acid sequences of Xzar2 and zar1 from six species revealed conserved regions (Fig. 4). As is evident, the C-termini of the proteins were highly conserved. Similar to zar1 (Wu et al. 2003a,b), an atypical PHD motif was also identified in the C-terminal of Xzar2 protein (asterisks in Fig. 4).


Figure 3
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  		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
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  		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. GENBANK 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.

 
Distribution of Xzar2 in Xenopus embryos and eukaryotic cells

To explore the temporal pattern of Xzar2 expression, RT-PCR analysis was performed on embryos harvested during embryonic development (Fig. 5A). Xzar2 RNA was expressed maternally and maintained its level until stage 10. The expression decreased significantly around stage 12 (Fig. 5A). The spatial distribution of Xzar2 transcripts was investigated by whole-mount in situ hybridization (Fig. 5B). At the blastula and gastrula stages, Xzar2 was expressed uniformly in the ectodermal and the mesodermal regions. After neurula and tailbud stages, no localized Xzar2 transcripts were observed (data not shown). No signal was detected with a sense control probe at any stage (data not shown). Next, we transfected HEK-293 cells with pEGFP-N1-Xzar2 and examined its intracellular localization. Xzar2 was localized mainly in the nucleus (Fig. 5C).


Figure 5
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  		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 EGFP-N1-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.

 
Epidermalization-promoting and neural inhibition by Xzar2

Xzar2 cDNA was identified by its epidermal promoting activity after morphologic observation. To confirm the Xzar2 activity, dissociated cells were treated with the culture medium of synthetic Xzar2 mRNA-injected oocytes for 4 h. Cells were dispensed at the mid-gastrula stages and then cultured for 3 days in NAM/2. RNA was extracted and assayed for the expression of cell-type-specific molecular markers by RT-PCR (Fig. 6A). Cells from control embryos dissociated in this manner showed a strong expression of NCAM, and no expression of epidermal keratin (Fig. 6A, lane 3). Cells dissociated in the presence of BMP-4 expressed epidermal keratin (Fig. 6A, lane2). Like cells treated with BMP-4, cells treated with the culture medium of Xzar2 mRNA-injected oocytes did not express NCAM, but expressed epidermal keratin (Fig. 6A, lane 1). The mesodermal marker, Xbra, was not induced in these cells. These results suggested that Xzar2 has a role in early epidermal gene expression and an inhibition of neural gene expression.


Figure 6
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  		Figure 6  Role of Xzar2 in Xenopus embryo. (A–C) Xar2 inhibited neuralization and induced epidermis in animal cap calls. (A) RT-PCRs 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.

 
To investigate whether Xzar2 is indispensable for epidermal differentiation, we performed a loss-of-function analysis using the anti-sense morpholino oligonucleotide (MO), which prevents the translation of Xzar2 mRNA (Xzar2 MO). We also designed the control 5 mis MO (control MO), which has five altered nucleotides in the target sequence. As shown in Fig. 6(B), the Xzar2 MO strongly repressed in vitro translation of synthetic Xzar2 mRNA, whereas a control MO had much less effect. Xzar2 MO or control MO was microinjected into the animal pole of two-cell stage embryos (Fig. 6C). Injection of control MO had no effect of on the expression of neural marker (NCAM, Fig. 6C, lane5). In contrast, Xzar2 MO stimulated expression of NCAM in a dose-dependent manner (Fig. 6C, lanes 1 and 2). The stimulatory effect was abolished by co-injection of Xzar2 mRNA (Fig. 6C, lanes 3 and 4). These results suggest that endogenous Xzar2 is involved in suppression of neural induction in the cells of presumptive ectoderm.

Over-expression of Xzar2 in anterior brain region results in neural inhibition

Next, we performed an over-expression of Xzar2 to examine the effect of Xzar2 in neurogenesis. The Xzar2 mRNA was injected into a single animal-dorsal blastomere of an eight-cell stage embryo (Fig. 6D). Injection of Xzar2 mRNA caused a failure of head formation (Fig. 6E,b), especially normal eye formation in the injected embryos, while no defects were observed in embryos injected with control mRNA (Fig. 6E,a) at stage 32. Histological examination confirmed that lens vesicle was defective and eye vesicle was decreased in the Xzar2 mRNA-injected embryos (Fig. 6F,b). These defects were strongly observed in the injected side in particular (Fig. 6F,b). The other side of the injection was also had a minor defect: the size of the eyes were decreased (compare left sides of a with b in Fig. 6F). Furthermore, in situ hybridization revealed a reduction in the expression of neural markers; Xrx1A (Fig. 6G,b) and Sox2 (Fig. 6G,d) in the embryos derived from Xzar2 mRNA-injected blastomeres. The expressions were not affected in the control embryos (Fig. 6G,a and c). These results suggest that over-expression of Xzar2 may suppress normal development of anterior.

Xzar2 promotes epidermal differentiation through signaling pathway(s) different from the BMP pathway

BMPs transduce their signals via two types of serine/threonine kinase receptors, type I and type II, both of which are required for signal transduction. Inhibitory Smads (I-Smads), consisting of Smad6 and Smad7, can inhibit the BMP signaling pathway (von Bubnoff & Cho 2001). Smad6 has been reported to be associated with BMP type I receptors, thereby preventing receptor-mediated activation of receptor-regulated Smads (R-Smads: Smad1, 2, 3, 5 and 8 in vertebrates). Smad7 can likewise bind stably to type I receptors, thus precluding activation of R-Smads. Smad6 also competes with Smad4 for binding to receptor-activated Smad1.

To examine whether I-Smads could inhibit the epidermal differentiation concerned with Xzar2, we tested the effects of the corresponding transcripts, I-Smads and Xzar2, on the expression of epidermal markers in the animal cap assay. Initially, we injected mRNAs encoding Smad6 (1.2 ng), Smad7 (1.2 ng), Xzar2 (640 pg) and/or BMP4 (640 pg) into two-cell embryos. Animal caps were isolated at stage 8 and expressions of each marker genes were analyzed at stage 26 by RT-PCR. Over-expression of Smad6 and Smad7 induced expression of NCAM in animal caps and did not induce epidermal keratin (Fig. 7A: lanes 1 and 2 compared with 7). Smad6 and 7 inhibited the epidermal induction by BMP-4 (Fig. 7A: lanes 3 and 5). However, the epidermal differentiation by over-expression of Xzar2 was not inhibited by co-injection of Smad6 or 7 (Fig. 7A: lanes 4 and 6).


Figure 7
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  		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.

 
Next, we investigated whether a dominant negative BMP receptor (tBR) blocks the epidermal differentiation by Xzar2 in the dissociated animal cap cells. Dissociated animal cap cells from embryos injected with tBR mRNA at the two-cell stage were cultured with the culture medium of oocytes injected with either Xzar2 or BMP4 mRNA (Fig. 7B). Induction of keratin by the culture medium of BMP4 mRNA-injected oocytes was blocked in the tBR-expressed animal cap cells, and the expression of neural marker NCAM was restored (Fig. 7B, lane 6). On the other hand, the expression of keratin by the culture medium of Xzar2 mRNA-injected oocytes was only slightly suppressed in tBR mRNA injected animal cap cells (Fig. 7B, compare lanes 2 and 5). Taken together, our results indicate that Xzar2-induced epidermalizing factor(s) promotes epidermal differentiation mainly through the signaling pathways other than that regulated by type I BMP receptor and I-Smads.


   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
Expression cloning is a useful technique for screening novel proteins based on their functions (Seed 1995), and it has been applied in developmental biology. As a recent progress, a novel ectodermal factor was identified by expression cloning and its inhibitory effect on mesodermal differentiation was unveiled (Sasai et al. 2008). We have distinctively developed an expression cloning system for epidermal inducers using a functional expression in Xenopus oocytes combined with a dissociated animal cap assay (Fig. 1). mRNAs synthesized from Xenopus embryo cDNAs were injected into Xenopus oocytes, and the incubation medium of the oocytes were subjected to the developmental assay (Fig. 2). This expression cloning system has several advantages. First, neural cells are developed with nerve fibers in quite high proportion in the uninduced animal cap cells (Table 1). Second, the assay faithfully reconstitutes epidermal induction by epidermal inducer, such as BMP-4 (Table 1). Furthermore, the response is dose-dependent in a range of the inducers. Third, a fate of dissociated animal cap cells can be assessed simply by morphological observation, since induced explants are always clearly distinguishable from control (Fig. 2A).

Using the cloning system we have identified several cDNA, each of which is responsible for the epidermal differentiation of the dissociated animal cap cells. In this study, we have characterized one of the clones, Xzar2, which has sequence similarity to Xenopus zygote arrest 1 (Zar1: Wu et al. 2003b). Zar1 is an ovary-specific maternal factor that plays an essential role during the oocyte-to-embryo transition in mouse (Wu et al. 2003a), but the actual function of the protein is unknown. Our results show for the first time that Xzar2 in a Zar family can be involved in neural development at an early stage of embryogenesis. In Xenopus, Zar1 expression is detected in the mature oocyte through the tailbud-stage of embryogenesis, although the level of expression decreases dramatically during the neurula stage and eventually disappears altogether in tadpole-stage embryos (Wu et al. 2003b). Xzar2 expression is also detected during early zygotic stages (Fig. 5A). Moreover, the expression was detected in the ectodermal and the mesodermal regions (Fig. 5B). The Zar1 from the six species contains a highly conserved atypical PHD motif, which is a conserved C8 pattern (C-X2-C-X13-C-X2-C-X4-C-X1-C-X17-C-X2-C; Wu et al. 2003b). Similar to Zar1, an atypical PHD motif was also identified in the C-terminus of Xzar2 (Fig. 4 [asterisks]). The PHD domain is found in two major classes of proteins: (i) transcriptional activators, repressors, or cofactors (Aasland et al. 1995; Gibbons et al. 1997; Capili et al. 2001) and (ii) subunits of complexes that modulate chromatin (Aapola et al. 2002). Xzar2 is localized in the nucleus of HEK-293 cells (Fig. 5C). Thus, Xzar2 may play a role as a transcriptional regulator of secreted proteins concerned with epidermal differentiation.

The present model for neural induction (the ‘default model’) proposes neural fate as the default pathway that ectodermal cells would acquire in the absence of any signal (Chang & Hemmati-Brivanlou 1998). This fate is inhibited in the ectoderm by an active BMP signaling. The organizer secretes BMP antagonists, which block BMP signaling in adjacent cells, allowing them to follow their default neural pathway (Muñoz-Sánjuan & Brivanlou 2002). Dissociated animal cap cells that subsequently re-aggregate develop into neural tissues (Godsave & Slack 1989; Sato & Sargent 1989), and this induction can be blocked by the addition of BMP-4 protein (Wilson & Hemmati-Brivanlou 1995). Misexpression of BMP in the prospective neural plate ventralizes the embryo, as well as suppressing neural markers. In this study, we clearly showed that Xzar2 can promote the epidermal differentiation at the expense of the neural differentiation (Fig. 6A,C). Misexpression of Xzar2 induces anterior defects, such as dysgenesis of an eye (Fig. 6E,F), and suppression of neural markers (Sox2 and Xrx1A; Fig. 6G). These indicate that Xzar2 may play a critical role for epidermal differentiation in ectoderm by inducing the expression of secretory factor(s). However, we cannot rule out the possibility that Xzar2 affects anterior neural development directly or through the other pathway(s) at the moment.

An important question is whether the other family member Zar1 has analogous activity to Zar2. Dissociated animal cap assay shows that the secretory factor(s) activated by Xzar1 has a role in early epidermal gene expression and an inhibition of neural gene expression (Supporting Fig. S1). Because the other Zar family member may compensate for the loss of Xzar2, the injection of Xzar2 MO alone could not inhibit the expression of epidermal keratin (Fig. 6C). Recently, a novel protein, which is structurally related to Zar1, was found in human and cattle, and was named Zar1-like (Sangiorgio et al. 2008). On the basis of our result, it is interesting to suppose that the Zar related genes may be involved in differentiation of vertebrate.

Epidermal differentiation, using the supernatant derived from the Xzar2-mRNA-injected oocytes, suggests that Xzar2 promotes epidermal differentiation via soluble factor(s) that are activated through the epidermal signaling pathway. At present, BMP and the following signaling pathway are well studied for the epidermal induction event. Inhibitory Smads (I-Smads) consist of vertebrate Smad6 and Smad7, which are members of the TGF-β superfamily. I-Smads inhibit BMP signaling by binding with the intracellular domain of activated BMP type I receptors (Miyazono et al. 2005), and by competing with Smad4 for binding to receptor-activated Smad1, thereby preventing formation of an active Smad1-Smad4 complex, and can function as a transcriptional co-repressor to antagonize BMP signaling in the nucleus. Recently, in the chick, misexpression of BMP-4 or BMP-7 in the prospective neural plate did not block neural induction (Streit et al. 1998). In Xenopus, endogenous BMPs continue to signal in dissociated animal cap ectodermal cells, at the same levels as in undissociated cells (Kuroda et al. 2005). These results suggest that BMP inhibition may not be absolutely required for neural induction, and that the other molecules may be implicated in the process. In addition, we have shown that over-expression of Smad6 or Smad7 did not inhibit epidermal differentiation by Xzar2 (Fig. 7A). When tBR was over-expressed, Xzar2 could still induce epidermis in the dissociated animal cap assay (Fig. 7B). Our results indicate that the pathway(s) different from BMP pathway is involved in epidermal differentiation during early embryogenesis.

From our results, Xzar2 may play an important role in the early patterning of Xenopus embryo. Nevertheless, it remains unclear what kind of secretory factor(s) is activated by the injection of Xzar2 mRNA in oocytes. We show that noggin blocks promotion of epidermal differentiation by Xzar2 (Supporting Fig. S2). Furthermore, when the culture medium collected from Xzar2-mRNA injected oocytes was mixed with the noggin-conjugated beads, epidermal differentiation promoted by Xzar2 was abolished by incubation with the medium (data not shown). Our data show that noggin can bind to and inhibit secretory factor(s) that is activated by injection of Xzar2 mRNA in oocytes. Further studies will be required to reveal the target molecule(s) that Xzar2 interacts with and the signaling cascade thereafter through epidermalization.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Animal care

Methods for keeping frogs and for obtaining embryos followed those described by Mitani & Okamoto (1989). All experiments were performed in compliance with the guidelines of the National Institute of Advanced Industrial Science and Technology.

Cell culture

Midblastula embryos (stage 8 according to Nieuwkoop & Faber 1967) were dejellied in 2% cysteine hydrochloride (pH 7.8) and kept in a modified Barth solution (MBS) on ice until the subsequent operation. Operations were carried out in NAM/2 (Godsave & Slack 1989) on a 1% agarose cushion. Embryos were manually removed from the vitelline membrane and were cut out discs of tissue from the center of the animal pigmented hemisphere using hair loops. Then the animal caps were placed in a dissociating medium (PhoNaK: Godsave & Slack 1989). Unpigmented inner cells were separated by a gentle stream of medium. Dispersed cells were cultured for 4 h at 22 °C in calcium- and magnesium-free medium (CMFM: 5 mM HEPES substituted for Tris; Wilton & Melton 1994) with or without recombinant human BMP-4 (rhBMP-4, GT) or supernatants of culture medium from H2O- or mRNA-injected oocytes. The incubations were carried out in agarose-coated four-well dishes (Nunc); cells equivalent to two caps were induced in a single well containing 500 µL of solution. Then about 20–30 cells were transferred to a well of Terasaki plate (Nunc) containing 15 µL of NAM/2 and cultured for 68 h at 22 °C. The wells were pretreated with 2 µL of 100 µg/mL laminin (Invitrogen) and 100 µg/mL fibronectin (Invitrogen) in NAM/2 (Godsave et al. 1988). After removing the solution, the wells were allowed to dry and were filled with NAM/2 for culture.

Functional expression in Xenopus oocytes

This was done essentially as previously described (Kimura & Kubo 2003). For mRNA synthesis, plasmid DNAs pSDRER-XBMP4, pSDRER-Xzar2, pSDRER-Smad6, and pSDRER-Smad7, pSP64T-tBR, pSDRER-Xzar1, pSDRER-noggin were linearized with XbaI or BamHI. In the case of mRNA synthesis from the cDNA pools, we used the circular DNAs as templates. Capped synthetic RNAs were generated by in vitro transcription with SP6 RNA polymerase using a MEGAscript transcription kit (Ambion). Injected oocytes were incubated in CMFM for 1 day before collecting the supernatant.

cDNA library construction

Total RNA was isolated from X. laevis embryos at stage 8 by TRIZOL® (Invitrogen) and poly(A)+ RNA was selected by OligotexTM-dT30 (Takara). cDNA library construction was performed as previously described (Inagaki et al. 2004), with slight modifications. First-strand cDNAs were synthesized by ReverTra Ace (Toyobo) and Superscript II (Invitrogen) with NXho(dT)30 primer [5'-AAGCAGTGGTAACAACGCAGAGTCTCGAG (T)30 (GAC) (GTAC)-3']. The second strands were synthesized with DNA polymerase I, RNase H and Escherichia coli DNA ligase (Takara). After both ends of the double-stranded cDNAs were filled with a DNA blunting kit (Takara), EcoRI adapters (Clontech) were added to the cDNAs. The cDNAs were then digested with XhoI and fractionated by agarose gel electrophoresis. The cDNA fragments with length of 0.7–5.0 kbp were cloned into the EcoRI and XhoI sites of pSDRER vector (modified pSD64TR: Zhao et al. 1994) which contained 5' and 3' UTRs of Xenopus β-globin and SP6 promoter. Aliquots from the unamplified library were plated on LB-agar plates containing 50 µg/mL ampicillin. After an overnight incubation at room temperature and 37 °C, the transformants were collected from each plate and pooled (~5000 colonies per plate). The bacteria were stored as a 15% glycerol stock at –80 °C.

Staining of cultured cells

Indirect immunostaining was performed as described elsewhere (Mitani & Okamoto 1991) with the following modifications: cells were fixed with 0.5% paraformaldehyde in NAM/2 for 2 h on ice. After fixation, culture plates were washed by gentle dipping into potassium phosphate-buffered saline (PBS; pH 7.4), and PBS containing 50 mM glycine for 1 h at 4 °C. For indirect immunofluostaining, each culture well received 10 µL of a first layer monoclonal antibody (mAb) containing 1% (for E3, specific for epidermis; Mitani & Okamoto 1989) or 0.2% (for AP-20, specific for dendrites and somas) of NP-40 (Sigma). After incubation for 2 h at room temperature, the plates were washed by gentle dipping into PBS. The washing solution was changed four times each for 20 min. The plates were finally removed from PBS solution and the excess PBS in the plates was aspirated off. Each well then received 10 µL of Alexa Fluor® 488 goat anti-mouse IgG (H + L: Molecular Probes) containing 1% BSA. After a 1-h incubation at room temperature and washing as before, the cells were stained with 5 µg/mL propidium iodide (PI: Dojindo) after treatment with RNase (4 mg/mL). Finally, the cells were observed under a laser scanning confocal microscope (Lsm 410: Zeiss).

Microinjection and RT-PCR

Microinjection of mRNA was performed as previously described (Hongo et al. 1999). mRNAs were injected in 640 pg per blastomere at the two-cell stage, or 320 pg per blastomere at the eight-cell stage, of Xenopus embryos. Cells were collected from two culture plates or six animal cap explants for each experimental point and RNA was extracted by TRIZOL® reagent (Invitrogen) as described above. RT-PCR was carried out as previously described (Hongo et al. 1999). Primers used for RT-PCR were as follows: BMP-2, 5'-CAGAGGAAGGCAAACGCAAG-3' and 5'-AGCCGCCGC TGGCTTGACAA-3'; BMP-4, 5'-GCATGTAAGGATAAGTC GATC-3' and 5'-GATCTCAGACTCAACGGCAC-3'; BMP-7, 5'-ACAGTAAAGAGAAGATTGCC-3' and 5'-CTGCGGAGAT GGATATCTGA-3'; GDF6, 5'-GAATACGGATCTGCTTTGC AGG-3' and 5'-GGACTCAGTTTGAACAGGTGCC-3'; Xzar2, 5'-TACAGACTGTGCGCGCTTCTCCCGC-3' and 5'-TTCCT CTTCATTGGGCTCCTCAGC-3'; EF1{alpha}, 5'-CAGATTGGTGC TGGATATGC-3' and 5'-ACTGCCTTGATGACTCCTAG-3'; ODC, 5'-GTCAATGATGGAGTGTATGGATC-3' and 5'-TCC ATTCCGCTCTCCTGAGCAC-3'; Epidermal keratin, 5'-CAC CAGAACACAGAGTAC-3' and 5'-CAACCTTCCCATCAAC CA-3'; NCAM, 5'-CACAGTTCCACCAAATGC-3' and 5'-GGAATCAAGCGGTACAGA-3'; Xbra, 5'-GGATCGTTATC ACCTCTG-3' and 5'-GTGTAGTCTGTAGCAGCA-3'.

Whole-mount in situ hybridization

The technique was performed as previously described (Watanabe et al. 2005). Xzar2 probe was synthesized with SP6 RNA polymerase on KpnI linearized pSPT19-Xzar2 (591 bp 5' region, 1 to 591) template. The markers used in this experiment were Sox2 and Xrx1A.

Transfection

HEK 293 cells (human embryonic kidney cell line) were cultured in Dulbecco's modified Eagle's medium (DMEM, Sigma) supplemented with 10% fetal calf serum and 5 mM penicillin-streptomycin (Gibco). A coding region of Xzar2 plus Kozak sequence was cloned into pEGFP-N1 (Clontech). In preparation for transfection experiments, cells were cultured in 35-mm dishes for 24 h. The next day, the culture medium changed to DMEM without penicillin-streptomycin. Cells were transfected with pEGFP-N1 or pEGFP-N1-Xzar2 using LipofectamineTM 2000 (Invitrogen). After a 3-h incubation, the culture medium changed to DMEM supplemented with 10% fetal calf serum and 5 mM penicillin-streptomycin. Cells were analyzed with the confocal scanning system (Olympus, FluoView FV1000) after 48 h.

Histological examination

Embryos cultured until stage 32 were fixed in Bouin's solution for 1 h. They were dehydrated through ethanol series, embedded in paraffin (Wako) and sectioned at 6–10 µm. The sections were stained in hematoxylin (Lab Vision) and eosin (Chroma-Gesellschaft).

Morpholino oligonucleotide experiments

Antisense morpholinos (Xzar2 MO 5'-ATGGCGGGGTTTGTG TATTCTCCGT-3' and control MO 5'-ATGCCGGCGTTTC TGTATTGTCGGT-3', which contains nucleotide exchange, underline) were directed against sequences the 5' end of the Xzar2 transcript. MOs were dissolved in water and injected into blastomeres at varying concentrations of oligonucleotide. In vitro transcription/translation was performed using the TNT® Coupled Reticulocyte Lysate System (Promega) and [35S] methionine (20 mCi/reaction). In one set of experiments, control or Xzar2 MO was injected into both cells of the two-cell embryo. An Xzar2 cDNA that mutated (underline, 5'-ATGGCTGGATTCGTCTACGCC CCTTAC-3') was used to rescue MO treatment.


   Acknowledgements
 
We would like to thank Dr Masanori Yamagishi for teaching microinjection techniques, Dr Ikuko Hongo for technical advice on whole-mount in situ hybridization and Mrs Yoshiko Shimoyama for art works in Fig. 4. This study was partly supported by the Sasagawa Scientific Research Grant and Special Coordination Funds for Promoting Science and Technology from the Ministry of Education, Culture, Sports, Science and Technology of Japan.


   Footnotes
 
Communicated by: Noriko Osumi

aPresent address: Graduate School of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba 305-8572, Japan Back

bPresent address: Department of Life Science, Faculty of Science, Gakushuin University, 1-5-1 Mejiro Toshima-ku, Tokyo 171-8588, Japan Back

* Correspondence: tai.kubo{at}aist.go.jp


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Received: 5 November 2008
Accepted: 16 February 2009





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