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Keywords:

  • Reticulon;
  • RTN;
  • XRTN2-B;
  • XRTN2-C;
  • XRTN3;
  • endoplasmic reticulum;
  • Xenopus laevis

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. CONCLUSIONS
  6. EXPERIMENTAL PROCEDURES
  7. REFERENCES

The reticulon (RTN) family of proteins has been described as a new eukaryotic protein family. We have isolated Xenopus cDNA homologues of RTN2 and RTN3 and examined their expression patterns during Xenopus development. XRTN2 has two transcripts, XRTN2-B and XRTN2-C, which encode 321 and 191 amino acids, respectively. XRTN3 has only one transcript that encodes 214 amino acids. We detected the XRTN2-B transcript in the neural tissues and brain from the early neurula stage. XRTN2-C is strongly expressed in the myotome, future skeletal muscle. The XRTN3 mRNA is localized in the animal hemisphere of the egg and blastula stage embryos and then subsequently restricted, mainly in the neural tissues. At the subcellular level, The XRTN proteins are expressed in the endoplasmic reticulum network structure of the animal cap cells as well as COS-7 cells. Our results suggest the potential roles of XRTN2s and XRTN3 during Xenopus embryogenesis. Developmental Dynamics 233:240–247, 2005. © 2005 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. CONCLUSIONS
  6. EXPERIMENTAL PROCEDURES
  7. REFERENCES

The reticulon (RTN) family of proteins has been described during recent years as a new protein family member, although the functions of most members still remain to be elucidated. The first discovery was the neuroendocrine-specific protein (NSP/RTN1), recognized by lung cancer monoclonal antibodies, which are specific for small-cell lung carcinoma (SCLC) cell lines (Roebroek et al., 1993). Three transcripts were identified and named NSP-A, NSP-B, and NSP-C as alternative splicing variants. These proteins are known to be associated with the membranes of endoplasmic reticulum (ER) (van de Velde et al., 1994) and have been called reticulons (RTNs) (Roebroek et al., 1996). Since then, four members of RTN genes (RTN1, RTN2, RTN3, and RTN4/Nogo) have been isolated and characterized in mammals (reviewed in Oertle and Schwab, 2003). Each of the RTN protein members has one to three alternative splicing isoforms and is ubiquitously expressed in nearly all animal species (Oertle et al., 2003). The RTN family of proteins shares homology within the C-terminal region called the reticulon-homology domain (RHD), which contains two putative transmembrane domains and an ER retention motif. This RHD region is attributed to their association with the ER membranes.

Most RTN members are expressed in the nervous tissues. The RTN1 genes are expressed mainly in the neurons and neuroendocrine cells (Roebroek et al., 1993; van de Velde et al., 1994; Senden et al., 1996; Ninkina et al., 1997). In the case of the RTN2 genes, RTN2-B is localized in the neural tissues, but RTN2-C is highly expressed in the skeletal muscle (Geisler et al., 1998; Roebroek et al., 1998). RTN3 is widely expressed in many tissues, with the highest expression in the brain and neurons (Moreira et al., 1999; Hamada et al., 2002). RTN4-A/Nogo-A is expressed mainly in the oligodendrocytes and neurons as well. In the case of the other splicing forms of RTN4/Nogo genes, RTN4-B/Nogo-B has an ubiquitous expression, and RTN4-C/Nogo-C is enriched in the skeletal muscle, as is RTN2-C (Josephson et al., 2001; Li et al., 2001; Huber et al., 2002; Liu et al., 2002; Wang et al., 2002). Recently, the Xenopus homologues of RTN4/Nogo genes have been reported with their expression patterns (Klinger et al., 2004). The expressions of XRTN4/XNogo genes show almost the same pattern as those of mammalian RTN4/Nogo.

Among the members of RTN family proteins, the function of RTN4-A/Nogo-A has been well-characterized. Upon injury, the RTN4-A/Nogo-A protein inhibits the regeneration of the central nervous system (CNS) by blocking neurite outgrowth as well as myelin-associated glycoprotein (MAG) and myelin oligodendrocyte glycoprotein (OMgp) (Chen et al., 2000; GrandPré et al., 2000; Prinjha et al., 2000). The inhibitory function of RTN4-A/Nogo-A is mediated through the Nogo/Nogo-66 receptor (NgR), coreceptor p75NTR, and the downstream signaling molecules, such as Rho GDP dissociation inhibitor (Rho-GDI), RhoA, and Rho kinase (reviewed in McGee and Strittmatter, 2003; Oertle and Schwab, 2003; Yamashita and Tohyma, 2003). In a recent study, it has been shown that the RTN4-C/Nogo-C protein delays nerve regeneration in the same manner as RTN4-A/Nogo-A (Kim et al., 2003). On the other hand, RTN4-B/Nogo-B has been implicated in the apoptosis as a proapoptotic protein by interacting with both Bcl-xL and Bcl-2 and reducing the antiapoptotic activity of Bcl-xL and Bcl-2 (Tagami et al., 2000). This apoptotic activity is verified in the human cancer cell line (Li et al., 2001). Additionally, it has been shown that RTN4-B/Nogo-B interacts with RTN3 (Qi et al., 2003). This finding suggests that there is a possibility that RTN3 may also be involved in the regulation of RTN4-B/Nogo-B–mediated apoptosis. Some other possible functions of the RTN proteins can be formulated in connection with their localization within ER membranes. The RTN proteins could function as pores or transporter complexes, in the transport of constituents from the ER to other membrane compartments, and in the structural stabilization of the ER network (Oertle and Schwab, 2003). However, the functions of each RTN member still remain to be discovered. Especially, the functional information of RTN2 and RTN3 proteins is largely unknown.

Here, we report the isolation of Xenopus orthologues of RTN2 and RTN3 and the examination of their temporal and spatial expression patterns during embryogenesis. We also show the subcellular localization of RTN2 and RTN3 proteins in both animal cap cells and mammalian cells.

RESULTS AND DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. CONCLUSIONS
  6. EXPERIMENTAL PROCEDURES
  7. REFERENCES

Identification of the Xenopus Homologues of RTN2 and RTN3 Genes

To identify Xenopus homologues of RTN2 and RTN3, we performed an expressed sequence tag (EST) database search using the human and mouse RTN cDNA sequences as described in the Experimental Procedures section. We found three EST sequences that contain a full-length open reading frame (ORF) sequence for each RTN protein. The full-length ORF cDNA clones of XenopusRTN2 and RTN3 (XRTN2 and XRTN3) were isolated by polymerase chain reaction (PCR). We obtained two transcripts of the XRTN2 gene, XRTN2-B (GenBank accession no. AY495962) and XRTN2-C (GenBank accession no. AY495963), which encode 321 (Fig. 1A) and 192 (Fig. 1B) amino acids (aa), respectively. For XRTN3 (GenBank accession no. AY495964), we isolated only one transcript that encodes 214 aa (Fig. 1C). The XRTN2s and XRTN3 proteins contain a RHD, which is conserved among the RTN family of proteins and includes two transmembrane domains and an ER retention motif. Sequence analysis demonstrates that the XRTN2 proteins have identical C-terminal regions containing RHD but different N-terminal regions to those found in other species (Fig. 1D). The nucleotide sequences of the 3′ untranslated regions (UTRs) of XRTN2-B and XRTN2-C vary from each other significantly, indicating that these two transcripts may have originated from different ancestral genes rather than derived from alternative splicing. The XRTN2-B protein is shorter than those of human (472 aa; Roebroek et al., 1998) and mouse (471 aa; Geisler et al., 1998) because of missing nucleotides in the N-terminal region. However, the XRTN2 proteins share 50.8% amino acid sequence identity with human and 48.6% identity with mouse RTN2s in the RHD region. The longest transcript, RTN2-A, does not seem to exist in Xenopus laevis as it dose in mouse (Oertle et al., 2003). In the case of human RTN2-B, it was derived from the elimination of the fifth exon of RTN2-A isoform (Roebroek et al., 1998). Therefore, if the XRTN2-A gene exists in the Xenopus genome, two different sizes of DNA fragments would be detected when performing PCR with XRTN2-B primers—a large one for XRTN2-A and a small one for XRTN2-B. However, we observed a single DNA fragment for XRTN2-B and were not able to find any sequence information regarding the XRTN2-A EST data. Taken together, we suggest that the XRTN2-A gene does not exist in Xenopus genome as does in mouse.

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Figure 1. A–C: Multiple sequence alignment of the predicted amino acid sequences of XRTN2-B (A), XRTN2-C (B), and XRTN3 protein (C) with their homologues from human and mouse. Identical amino acid residues are dark-highlighted and similar residues light-highlighted. Dashes indicate gaps inserted for maximal alignment score. The boxes represent the reticulon-homology domain (RHD) region, and the double arrows indicate the endoplasmic reticulum retention motif. The dots show the antisense probe region for whole-mount in situ hybridization. These sequences are aligned using the ClustalW server, which uses the BLOSUM62 scoring matrix. D: Schematic representation of XRTN2 encoded proteins, XRTN2-B and XRTN2-C. These two proteins share the RHD region and contain specific regions within the N-terminal region, respectively. The hydrophobic domains in the C-terminal regions are shown as black boxes. E: Phylogenetic tree representing the relationships among the RTN gene family based on alignment of the RHD region. This tree diagram is obtained from the TreeTop phylogenetic tree prediction server.

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The XRTN3 protein is also shorter than mammalian RTN3 and shares 69.5% and 69.9% sequence identity with human and mouse RTN3, respectively, within the RHD region. Although the sequence identities of XRTN2 and XRTN3 proteins are lower than those of human and mouse RTN homologues, the phylogenetic tree of the RTN gene family clearly demonstrates that each XRTN gene family that we found encodes Xenopus homologues of each RTN member (Fig. 1E, XRTN1 is unpublished data).

Differential Expression of XRTN2 and XRTN3 Gene

To investigate the expression of XRTN2s and XRTN3 during Xenopus developmental stages, we first analyzed the temporal expression patterns of XRTN2s and XRTN3 transcripts by reverse transcriptase-PCR (RT-PCR) using the isoform-specific primers (Fig. 2). Both XRTN2-B and XRTN2-C are expressed zygotically from the early neurula stage (St. 13), and the expressions are maintained until the tadpole stage. In contrast, XRTN3 is expressed maternally, and its expression remains until the tadpole stage.

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Figure 2. The temporal expression patterns of XRTN2-B, XRTN2-C, and XRTN3 during Xenopus embryo development. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis shows that XRTN2-B and XRTN2-C transcripts are expressed zygotically from the neurula stage, and XRTN3 is both maternally and zygotically expressed. Ornithine decarboxylase (ODC) is used as a loading control. The −RT lane is the negative control of the RT-PCR on stage 45 whole embryo RNA in the absence of a reverse transcriptase.

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Next, we carried out whole-mount in situ hybridization to study the spatial expression patterns of XRTN2s and XRTN3. As for XRTN2-B, the complementary nucleotide sequence of the N-terminal region was used for the specific probe (Fig. 1A). The XRTN2-B transcript begins to appear in the anterior structure at the mid-neurula stage (St. 16) (Fig. 3A,a). At the late neurula stage (St. 19), the XRTN2-B expression is localized in the neural plate and eye anlagen with high intensity (Fig. 3A,b). The neural plate-specific expression is ascertained by embryo dissection (Fig. 3A,c,d). This XRTN2-B expression pattern persists through to the early tail bud stage (St. 21) (Fig. 3A,e). In the St. 25 embryo, the XRTN2-B mRNA is detectable in the head and neural tissues, including the eye and spinal cord (Fig. 3A,f,g). As Xenopus embryos develop to the tadpole stage, the localization of XRTN2-B is restricted in the specific regions of the developing brain, prosencephalon, mesencephalon, and rhombencephalon, as well as the eye and branchial arch (Fig. 3A,h,i). This pattern of expression is maintained until the beginning of the organogenesis stage (St. 40) (Fig. 3A,j).

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Figure 3. The spatial expression patterns of XRTN2s and XRTN3.A: XRTN2-B. a: Stage (St.) 16, anterior side view. b: St. 19, dorsal side view, anterior to the left. c: St. 20, dissection view. d: Magnified view of c. The XRTN2-B transcript is localized in neural tissues, specifically the eye anlagen and neural plate (arrows). e: St. 21, dorsal view. f,g: St. 25, lateral view (f), dorsal view (g). h: St. 30, lateral view. i: St. 32, lateral view. XRTN2-B expression is restricted in the specific regions of the brain such as the prosencephalon, mesencephalon, and rhombencephalon. j: St. 40, lateral view. B: XRTN2-C. a: St. 17, dorsal side view, anterior to the left. b: St. 18, dorsal view. Arrowheads indicate the paraxial mesoderm. c: St. 19, dissection view. d: Magnified view of c. e,f: St. 20, anterior view (e), dorsal view (f). As the neural tube closes, XRTN2-C expression spreads posterior–lateral side, which is the posterior paraxial mesoderm. g,h: St. 22, lateral view (g), dorsal view (h). i: St. 24, dissection view. j: Magnified view of i. The XRTN2-C expression is restricted in myotome of somite. k: St 30, lateral view. l: St. 35, lateral view. The XRTN2-C expression is specified in the specific regions of the brain, the prosencephalon, mesencephalon, and rhombencephalon, as well as the myotome. m: St. 40 lateral view. C: XRTN3. a: Four-cell stage, dorsolateral side view. b: St. 6, dorsolateral view. c: St. 8, lateral view. d,e: St. 10.5, animal view (d), ventrolateral view (e). The arrow in e indicates the blastopore lip. The maternal XRTN3 transcript is detected in the animal hemisphere of the blastula and gastrula stage embryos. f,g: St. 18, dorsal view (f), dissection view (g). h: Magnified view of g. At the neurula stage, the XRTN3 expression commences in neural tissues, including the neural plate (arrow) and anterior head structure. i: St. 21, dorsal view. j,k: St. 22, lateral view (j), dissection view (k). l: Magnified view of k. m: St. 23, dorsal view. Specific expression in the optic nerve and spinal cord can be observed. n: St. 25, lateral view. As the mid–tail bud stage is passed over, the XRTN3 transcript is expressed in the somite and branchial arch. o: St. 30, lateral view. In addition to the optic nerve, the otic vesicle has XRTN3 expression. p: St. 35, lateral view. The XRTN3 expression is roughly maintained in the neural tissue, including the head. D: Sense. No positive signals were detected with sense probes of XRTNs. a: XRTN2-B sense in situ. b: XRTN2-C sense in situ. c: XRTN3 sense in situ. ba, branchial arch; ea, eye anlagen; ey, eye; mc, mesencephalon; my, myotome; nc, notochord; np, neural plate; nt, neural tube; on, optic nerve; ov, otic vesicle; pc, prosencephalon; pm, paraxial mesoderm; rc, rhombencephalon; sc, spinal cord; sm, somite.

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In the case of the other transcript of XRTN2, XRTN2-C, the 3′ UTR was used for the antisense probe. The expression pattern of XRTN2-C is quite different from that of XRTN2-B. The XRTN2-C transcript is also detected first at the mid-neurula stage (St. 17) in the paraxial mesoderm region (Fig. 3B,a). As the neural fold closes (St. 18–20), the XRTN2-C expression spreads to the posterior–lateral side, which is the posterior paraxial mesoderm (Fig. 3B,b–d) and appears around the anterior head region (Fig. 3B,e,f). At the tail bud stage (St. 22), the XRTN2-C message is strongly expressed in the somitic mesoderm, which will later become the myotome (Fig. 3B,g,h). Truly, XRTN2-C is specifically localized in the myotome of somite (Fig. 3B,i,j). At the later stage of development, a similar expression pattern with XRTN2-B is seen in the brain (prosencephalon, mesencephalon, and rhombencephalon), while the strong expression in the somite is maintained through to the tadpole stage (Fig. 3B,k–m). It is worth noting that the expression patterns of XRTN2s are similar to those of human and mouse RTN2s. In human, RTN2-C has been shown to express greatly in the skeletal muscle, and XRTN2-B in the brain (Roebroek et al., 1998). In the case of mouse, RTN2-C is expressed at high levels in the skeletal muscle, at moderate levels in the heart and at lower levels in the smooth muscle, lung, and white adipose tissue, whereas RTN2-B has been detected in the brain and testes (Geisler et al., 1998).

For the expression pattern of XRTN3, the entire ORF sequence was used for the specific probe (Fig. 1C). As predicted from the RT-PCR, the maternal XRTN3 mRNA is present in the animal hemisphere at the four-cell stage (Fig. 3C,a). This animal pole localization of the maternal XRTN3 persists until the blastula stage (St. 8) (Fig. 3C,b,c). During gastrulation (St. 10.5), the XRTN3 expression becomes restricted in the prospective neuroectoderm (Fig. 3C,d,e). When the neural tube closes (St. 18), the XRTN3 transcript is detected in the neural plate along the anterior–posterior axis and the anterior-most region (Fig. 3C,f–h). At the early tail bud stage (St. 21), XRTN3 is expressed in the head structure, including the eye anlagen and optic nerve (Fig. 3C,i,j) with constant expression in the neural tube, which will become the spinal cord (Fig. 3C,k–m). As the embryos passed over the mid–tail bud stage (St. 25), XRTN3 is detectable in the somite and branchial arch at low levels (Fig. 3C,n). At the tadpole stage, the XRTN3 transcript is localized in the head and neural tissues, including the otic vesicle and optic nerve (Fig. 3C,o,p). The expression pattern of XRTN3 is also very similar to those of human and mouse RTN3. The RTN3 is widely expressed in human tissues with the highest expression in the brain (Moreira et al., 1999), and mouse RTN3 is observed dominantly in the brain and neurons (Hamada et al., 2002). Conversely, no positive signals of each XRTN were detected with sense probes.

Subcellular Localization of XRTN2 and XRTN3 Proteins

One of the common features of RTN proteins is their association with the ER membranes. This localization is attributed to the RHD region, which contains two transmembrane domains and an ER retention motif (van de Velde et al., 1994). This subcellular localization has been shown for RTN1 (Senden et al., 1994; van de Velde et al., 1994), RTN3 (Hamada et al., 2002; Qi et al., 2003), and RTN4/Nogo (Chen et al., 2000; Grand-Pré et al., 2000). To determine whether the expression of XRTN proteins corresponds with those of mammalian RTNs at the subcellular level, we microinjected green fluorescent protein (GFP) -tagged XRTN mRNAs into the two-cell stage Xenopus embryos, and analyzed their expressions in the animal cap cells of St. 9 embryos by observing the GFP images under a confocal fluorescence microscope. The expression of XRTN2-B protein in the animal cap cells is localized to the ER-like network structure (Fig. 4B). The XRTN2-C and XRTN3 proteins also show similar distribution in the cytoplasm (Fig. 4C,D). In the case of XRTN2-C, however, additional GFP-positive signals were detected in the areas of the plasma membrane and nuclear envelope. These subcellular localizations of the XRTN proteins in the animal cap cells resemble that of the ER network structure of the Xenopus oocyte (Marchant et al., 2002). The subcellular localizations of XRTN proteins in the ER network structure were further demonstrated in COS-7 cells transfected with GFP-tagged XRTN plasmids. The GFP-positive signals were localized in the ER network structure of GFP-XRTNs transfected cells (Fig. 4F–H). On the other hand, the control animal cap and COS-7 cells exhibited ubiquitous expression of GFP throughout the nucleus and cytoplasm as well as the plasma membrane (Fig. 4A,E).

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Figure 4. Subcellular localization of the XRTN2 and XRTN3 proteins. Both animal poles of two-cell stage embryos were injected with total 500 pg of each synthetic green fluorescent protein (GFP) -XRTN mRNAs. A–D: Then, animal caps were isolated from stage 9 embryos and fixed to observe the GFP confocal images. E–H: COS-7 cells were transfected with total 1 μg of individual GFP-XRTN plasmids. A,E: GFP control. B,F: XRTN2-B. C,G: XRTN2-C. D,H: XRTN3. The XRTN2 and XRTN3 proteins are localized in endoplasmic reticulum membranes. Original magnification, ×800. Scale bars = 50 μm in A (applies to A–D), in E (applies to E–H).

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CONCLUSIONS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. CONCLUSIONS
  6. EXPERIMENTAL PROCEDURES
  7. REFERENCES

We have identified the full-length ORF sequences of two transcripts of the XRTN2 gene and one transcript of the XRTN3 gene. These three XRTN proteins are composed of the C-terminal RHD region and N-terminal–specific region and share sequence homology within the C-terminal RHD region as the reticulon family of proteins. XRTN2-B and XRTN2-C proteins encode 321 and 192 aa, respectively, and hold the RHD region in common. The expressions of both XRTN2s commence from the early neurula stage as zygotically expressed genes. The XRTN2-B transcript is mainly expressed in the specific regions of the brain such as prosencephalon, mesencephalon, and rhombencephalon, as well as in the eye and branchial arch. The expression of the XRTN2-C is quite different from that of XRTN2-B. The XRTN2-C transcript is strongly expressed in the myotome, future skeletal muscle, which is consistent with that of human and mouse homologues. This specific and strong expression of XRTN2-C would make this gene a great somitic marker gene. XRTN2-C mRNA is also detectable in specific brain regions. In the case of the XRTN3 protein, it encodes 214 aa and expressed both maternally and zygotically. XRTN3 transcript is expressed mainly in the neural tissues such as the brain, spinal cord, eye, and optic nerve, as well as the somite and branchial arch. The expressions of these three XRTNs are quite similar to those of human and mouse. Compared with expression patterns of human and mouse RTNs, the expression of XRTN2-B and XRTN3 in the brain and neural tissues suggests that XRTN2-B and XRTN3 proteins may have certain relations in the specification within developing brain and neural tissues. In addition, XRTN2-C proteins may function within the skeletal muscle. Last, we have corroborated the association of the XRTN proteins with the ER membranes by microinjection of the GFP-tagged XRTN mRNAs into the animal cap and transfection of the GFP-tagged XRTN plasmids into COS-7 cells. In this report, we have first demonstrated the subcellular distribution of XRTN2 family proteins. These results indicate that the basic functions of the XRTN2 and XRTN3 proteins may have been conserved to a large degree among the vertebrate species. In addition, this study provides important information for future studies on the functions of the RTN family of proteins in vertebrate development, because the amphibian X. laevis is an excellent model organism for investigating the functions of such genes.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. CONCLUSIONS
  6. EXPERIMENTAL PROCEDURES
  7. REFERENCES

Embryo Manipulations

X. laevis was purchased from Xenopus I, Inc. (Ann Arbor, MI). Eggs were obtained from adult female X. laevis primed with 800 IU of human chorionic gonadotropin (Sigma). In vitro fertilization and culture were performed as described by Sive et al. (2000), and the embryos were staged according to Nieuwkoop and Faber (1994).

Identification of XRTN2 and XRTN3

XenopusRTN2s and RTN3 clones were isolated by BLAST searches of the GenBank (http://www.ncbi.nlm.nih.gov/BLAST/) and TIGR EST (http://www.tigr.org/) database using human and mouse RTN2 and RTN3 cDNA sequences. For XRTN2-B, the coding regions of human (Roebroek et al., 1998) and mouse (Geisler et al., 1998) RTN2-B genes were used as queries for the database search. As a result, the common corresponding clone (TC153270) for the XRTN2-B gene was obtained from the TIGR database. The other corresponding clone for the XRTN2-C gene (GenBank clone ID, CD252970) was acquired by the EST database search as well, using those of human (Roebroek et al., 1998) and mouse (Geisler et al., 1998) RTN2-C ORF sequences. In the same manner, the XRTN3 gene (TIGR clone ID: TC73017) was obtained from human (Moreira et al., 1999) and mouse (Hamada et al., 2002) RTN3. All three entire ESTs contained complete ORFs and little of the 5′ and 3′ UTRs. The ORF regions of each XenopusRTN gene were isolated by RT-PCR and sequenced. The amino acid sequences were encoded by these genes, aligned using the ClustalW (http://www.ch.embnet.org/software/ClustalW.html), and the phylogenetic tree was displayed with the TreeTop software (http://www.genebee.msu.su/services/phtree_reduced.html).

RT-PCR and Whole-Mount In Situ Hybridization

Xenopus total RNA was prepared from whole embryos with the TRI reagent (Molecular Research Center) and treated with the RNase-free DNase I (Roche) to remove genomic DNA. The extracted RNA was reverse transcribed by using M-MLV reverse transcriptase (Promega) with random hexameric primers (Promega), according to the manufacturer's instructions. PCR amplification was performed using Taq polymerase (Super Bio) with [α-32P]dCTP, and 8 μl of 25-μl reaction volume were analyzed on 6% polyacrylamide and 8 M urea gel electrophoresis by means of autoradiography. The reaction parameters were 94°C for 3 min, followed by 25 cycles of 94°C for 30 sec, 55°C for 30 sec, and 72°C for 1 min. A final hold of 72°C for 5 min was carried out. The PCR primers are as follows; XRTN2-B forward, 5′-CTGCCCTCTTGAAGGGGAAG-3′ and reverse, 5′-GTCTCCTCCCTCGGTGCTTC-3′; XRTN2-C forward, 5′-TGGTCCCAGAATCAAGGCAC-3′ and reverse, 5′-GCACCCGTAAGCGAACACAC-3′; XRTN3 forward, 5′-GCATTCAGCATTATTAGCGT-3′ and reverse, 5′-CTTCAGAGCATGGTTGACAT-3′. Ornithine decarboxylase (Bouwmeester et al., 1996) was used as a loading control.

Whole-mount in situ hybridization using the digoxigenin (DIG) -UTP–labeled antisense mRNA was performed as previously described (Harland, 1991). The N-terminal–specific region of XRTN2-B (Fig. 1A), 3′ UTR region of XRTN2-C, and the full-length ORF of XRTN3 gene (Fig. 1C) were used for each specific antisense probe site. The entire ORF sequences of each XRTN were used for the sense probes. The labeled probe was detected with Fab fragments from the anti-DIG antibody from sheep (Roche), conjugated with alkaline phosphatase, and visualized with the BM purple AP substrate (Roche).

mRNA Microinjection and Fluorescence Microscopy

The entire coding regions of XRTN2s and XRTN3 genes were amplified by PCR and cloned into pCS2+ EGFP-C1 plasmid, which we designed. The expression constructs pCS2+ GFP-XRTN2-B, pCS2+ GFP-XRTN2-C, and pCS2+ GFP-XRTN3 were linearized with Asp 718, and capped synthetic mRNAs were synthesized using the mMESSAGE mMACHINE SP6 kit (Ambion, Inc.). Microinjection of embryos was carried out in 0.33X MR containing 4% Ficoll-400 (Amersham).

The subcellular localization of proteins was monitored as previously described by Miller et al. (1999). The two-cell stage embryos were injected with 500 pg of mRNAs into the animal pole region, then GFP-expressing animal caps were excised at St. 9. Dissected explants were fixed in MEMFA for 1 hr, rinsed with phosphate buffered saline (PBS), and directly mounted. Image analysis was performed using a confocal laser scanning fluorescence microscope (Zeiss LSM 510).

Cell Culture and Transfection

COS-7 cells were cultured in Dulbecco's modified Eagle's medium supplemented with heat-inactivated 10% fetal bovine serum under humidified 5% CO2 and 95% air at 37°C. Subconfluent cells were plated onto poly-L-lysine–coated cover glasses in six-well plates. Transfection was carried out with a total of 1 μg of indicated plasmids using Lipofectamine Transfection Reagent (Invitrogen). Thirty hours after transfection, cells were fixed in 4% paraformaldehyde, rinsed with PBS, and directly mounted.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. CONCLUSIONS
  6. EXPERIMENTAL PROCEDURES
  7. REFERENCES
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