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Mechanisms of Hormone Action |
Department of Biology,3 York University, Toronto, Ontario, Canada M3J 1P3
Department of Obstetrics & Gynecology,4 University of British Columbia, Vancouver, British Columbia, Canada V6H 3V5
Department of Obstetrics and Gynecology,5 University of Ottawa, Ottawa Health Research Institute, Ottawa, Ontario, Canada K1Y 4E9
Samuel Lunenfeld Research Institute,6 Mount Sinai Hospital, Toronto, Ontario, Canada M5G 1X5
ABSTRACT
Members of the transforming growth factor (TGF) ß family play critical roles in regulating placental functions. Using polymerase chain reaction (PCR)-based strategies, we have cloned four transcripts encoding full-length activin receptor-like kinase 7 (ALK7) and three novel ALK7 isoforms from the human placenta. The full-length ALK7 has 493 amino acids and exhibits all characteristics of TGFß type I receptors, including an activin receptor-binding domain, a transmembrane domain, a GS domain, and a serine/threonine kinase domain. The three ALK7 isoforms identified include a truncated ALK7 (tALK7) and two soluble proteins designated as soluble ALK7a (sALK7a) and soluble ALK7b (sALK7b). The tALK7 lacks the first 50 amino acids of the full-length ALK7, resulting in a truncated receptor-binding domain. Both sALK7a and sALK7b lack transmembrane and GS domains. The ALK7 gene, located on chromosome 2q24.1, is composed of at least nine exons and eight introns. The isoforms of ALK7 are generated by alternative splicing. Transcripts encoding the sALK7 isoforms differ from the full-length transcript by lacking exon III or both exons III and IV in sALK7a and sALK7b, respectively. The transcript for tALK7 uses an alternative exon located within the first intron of the full-length transcript. These results indicate that four distinct proteins are encoded by the human ALK7 gene. Both reverse transcription-PCR and Western blot analysis showed that ALK7 and its isoforms are expressed in human placentae of different stages of pregnancy and that their expression is developmentally regulated. In addition, mRNA expression of Nodal, a ligand for ALK7, was also detected in placentae of different gestational age. The role of Nodal and ALK7 in human placenta is currently under investigation.
activin receptor-like kinase 7, alternative splicing, Nodal, placenta
INTRODUCTION
The transforming growth factor (TGF) ß family consists of a large group of growth and differentiation factors, including TGFßs, activins/inhibins, bone morphogenetic proteins (BMPs), growth and differentiation factors, as well as Nodal and its related proteins (for review, see [1–4]). These growth factors play critical roles in many developmental and physiological events [1–4]. In the human placenta, the TGFß family has been shown to regulate trophoblast proliferation and differentiation [4–9], invasion [7, 9], and production of several hormones, such as hCG [8, 10–12], gonadotropin-releasing hormone [11], as well as progesterone and estradiol [5, 11–15].
Members of the TGFß family exert their functions by interacting with two types of membrane proteins (type I and type II receptors), both of which are serine/threonine kinases [1–4, 16–18]. Type II receptors carry out the initial ligand recognition and binding and facilitate the presentation of ligands to type I receptors. Type I receptors, although unable to recognize free ligands, are critical in receptor signaling. When ligands are associated with the corresponding type II receptors, type I receptors are recruited into the ligand/receptor complex and become phosphorylated by type II receptors [1–4, 16–18]. On activation by the type II receptors, type I receptors phosphorylate receptor-regulated Smads (R-Smads), which in turn form complexes with common Smad (Smad4). Subsequently, the R-Smad/Smad4 complex is translocated into the nucleus to regulate target gene transcription [1, 2, 16–18].
Seven type I receptors, which are referred to as activin receptor-like kinase (ALK) 1 through 7, have been cloned in mammals. Both ALK1 and ALK5 are TGFß receptors, whereas ALK2, ALK3, and ALK6 mediate BMP signals [1, 2, 16–18]. Both ALK2 and ALK4 have been shown to bind to activins [1, 2]. In addition, ALK4 interacts with Nodal-related proteins [19, 20]. The ALK7 was initially cloned from the rat and was identified as an orphan receptor [21, 22]; however, recent studies have demonstrated that ALK7 can bind with mouse Nodal and Xenopus Nodal-related protein-1 (Xnr-1), and mediates Nodal signaling during embryonic development [19].
During our previous studies of activin receptors in human placentae [23, 24], a polymerase chain reaction (PCR) product with high homology to rat ALK7 was unexpectedly obtained. Using PCR and bioinformatic tools, we have now obtained full-length sequences of four ALK7 transcripts and have demonstrated that they are derived from alternative splicing of the ALK7 gene. Three of the transcripts encode novel isoforms of ALK7. Furthermore, we have provided the first evidence, to our knowledge, that ALK7 and its ligand, Nodal, are expressed in human placenta throughout pregnancy.
MATERIALS AND METHODS
Human Placenta and Trophoblast Cell Line
First-, second-, and third-trimester placental tissues were obtained from therapeutic abortion or at birth from Mount Sinai Hospital, British Columbia Women's Hospital, and the Ottawa Hospital. The collection and use of human placental tissues has been approved by the Office of Research Service of the University of Toronto, the Clinical Screening Committee for Research and Other Studies Involving Human Subjects of the University of British Columbia, and the Research Ethics Board of the Ottawa Hospital. Gestational ages were established by sure last menstrual period or else by first-trimester ultrasound. A choriocarcinoma cell line, JEG-3, was obtained and cultured as previously described [14, 15].
Total RNA Preparation and Reverse Transcription-PCR
Total RNA was extracted from JEG-3 cells and placental tissues using Trizol reagent (Invitrogen Canada, Inc., Burlington, ON). One to two micrograms of total RNA were reverse transcribed into cDNA in a total volume of 15 µl using First Strand cDNA Synthesis Kit (Amersham Pharmacia Biotech, Inc., Oakville, ON, Canada) using either oligo dT or random primers. Primers used in PCR are listed in Table 1, and the locations of ALK7 primers are indicated in Figure 2. The PCR was carried out in the presence of 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2.0 mM MgCl2, 50 µM dNTPs, 1 U of Platinum High Fidelity Taq DNA polymerase (Invitrogen) or Hotstart Taq (Qiagen, Inc., Mississauga, ON, Canada), and 5 pmol of primers for 40 cycles. Annealing temperature for PCR ranged from 55 to 65°C, depending on the primer sets used.
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Cloning and Sequence Analysis
The initial PCR product obtained from amplification of placental cDNA with ALK4 primers was cloned into a pDirect vector (BD Biosciences Clontech, Palo Alto, CA). The sequence was compared to the first draft of the human genome sequence. Genscan (http://bioweb.pasteur.fr/seqanal/interfaces/genscan-simple.html) was used to predict the coding region of human ALK7, and a sense primer (ALK7-10) based on the prediction was used with an antisense primer (ALK7-6) to obtain a larger fragment. The resulting PCR product was cloned into TOPO PCRII vector (Invitrogen). Subsequently, 5' and 3' rapid amplification of cDNA ends (RACE) were performed using RACE-ready human placenta cDNA (Ambion, Inc., Austin, TX). For 5' RACE, primer ALK7-11 was used with an anchor primer 5' outer (Ambion) in the first run of PCR, and the resulting PCR product was subjected to the second run using primer ALK7-12 and a nested anchor primer, 5' inner. For 3' RACE, primer ALK7-17 was used in the first run of PCR in conjunction with an anchor primer, 3' outer (Ambion). This was followed by another run of PCR with two nested primers, ALK7-18 and 3' inner. All RACE products were again cloned into the TOPO PCRII vector. Finally, end-to-end PCR was performed to confirm the presence and the sequence of the transcripts. Three transcripts were cloned using JEG-3 cDNA samples and primers ALK7-3 and ALK7-2. Another transcript was obtained by PCR using primers ALK7-20 and ALK7-2 and a full-length cDNA from 21 human tissues (Panomics, Inc., Redwood City, CA) as the template. All sequencing was performed using an ABI 373A Sequencer (Applied Biosystems, Foster City, CA) at York University's Core Molecular Biology Facility. Comparison of cDNA sequences with genomic sequence was done using the Blast program (http://www.genome.ucsc.edu). Prediction of opening reading frame was done using Genscan, and analysis of protein structural domain was performed using Single Modular Architecture Research Tool (SMART; http://Smart.embl-heidelbberg.de) [25]
Northern Blot Hybridization
The 688-base pair (bp) PCR product originally obtained from human placenta using primers ALK4-1 and ALK4-2 was labeled with [32P]dATP (Amersham) using a NEBlot kit (New England BioLabs, Inc., Beverly, MA). This was then used to hybridize with a human multiple-tissue Northern blot (BD Biosciences Clontech) using rapid hybridization buffer (BD Biociences Clontech) according to the manufacturer's instructions.
Western Blot Analysis
Placental tissues (50–100 mg) were cut into small pieces and were added to 1 ml of boiling lysis buffer (1% SDS, 1.0 mM sodium orth-vanadate, and 10 mM Tris [pH 7.4]). The samples were then sonicated and centrifuged (14 000 x g, 4°C, 20 min). The protein content of the supernatant was determined with the Bio-Rad DC protein assay kit (Bio-Rad Laboratories, Inc., Mississauga, ON, Canada) after the samples were diluted at least 10-fold to attain a final SDS concentration of 0.1%. An aliquot of each sample and an internal control (50 µg protein/lane) were resolved by SDS-PAGE and electrotransferred to nitrocellulose membranes. The membranes were blocked (1 h, room temperature) in blotto (Tris-buffered saline at pH 7.8 with 0.05% Tween [TBS-T] and 5% dehydrated nonfat milk). After rinsing with TBS-T, membranes were incubated with anti-rat ALK-7 monoclonal antibody (overnight, 4°C, 2 µg/ml; R&D Systems, Inc., Minneapolis, MN) and, subsequently, with human horseradish peroxidase-conjugated anti-mouse immunoglobulin G second antibody (1:4000; Bio-Rad) in blotto for another hour and then washed. Chemiluminescent signal was visualized with an enhanced chemiluminescence kit according to the manufacturer's instructions (Amersham). The intensity of specific protein band was densitometrically determined using Molecular Analyst software (Bio-Rad) and normalized with an internal control on the same membrane.
Statistical Analysis
To compare ALK7 protein contents in the first, second, and third trimesters, one-way analysis of variance was performed, followed by Newman-Keuls multiple comparison test. A level of P < 0.05 was considered to be significant.
RESULTS
Cloning of ALK7 cDNAs from Human Placenta
A pair of primers initially designed to detect ALK4 mRNA resulted in a PCR product with the expected size (688 bp). However, Southern blot hybridization of the PCR product with an oligonucleotide probe specific for ALK4 only showed weak signals (data not shown), suggesting that the PCR products may not be true ALK4. The DNA fragment was subsequently cloned. Sequencing analysis of several clones indicated that the DNA fragment had 75% nucleotide identity with ALK4. Interestingly, the sequence is highly similar to the rat ALK7, with 96% of amino acid residues being identical. We therefore designated this clone as the human ALK7. The open reading frame of rat ALK7 was then used to compare with the first draft of the human genome sequence, and based on this prediction, we designed another primer (ALK7-10) and used it, together with ALK7-6, to amplify JEG-3 cDNA. The PCR generated three DNA fragments. On sequencing, all fragments were found to have identical 5' and 3' sequences, but the two small bands lacked 240 and 471 bp of the sequence found in the largest band, respectively. To obtain full-length sequences, we then conducted 5' and 3' RACEs. Two DNA fragments were obtained from 5' RACE, which had identical 3' but different 5' sequences. However, 3' RACE consistently generated one single fragment. All these fragments were cloned and sequenced. The sequences were then assembled and confirmed by end-to-end PCR, followed by cloning and sequencing. Thus, we have obtained full-length sequences for four transcripts, and all sequencing data have been submitted to the GenBank database under the accession numbers AY127050, AF525679, AF525680, and AF525681.
Transcript 1 (GenBank accession no. AY127050) (Fig. 1A) encodes for the full-length receptor, which consists of 493 amino acids and has a signal peptide, an extracellular activin-receptor domain, a transmembrane domain, a GS domain that is conserved in all type I receptors of the TGFß family, and a serine/threonine kinase domain. The nucleotide sequence is very similar to an est clone recently deposited in the GenBank, designated as "similar to TGFß type I receptor" (GenBank accession no. BC022530). The protein sequence of this transcript has recently been annotated in the GenBank database as "similar to type I TGFß receptor (accession nos. NM145259 and XM_065712). Transcript 2 (GenBank accession no. AF525679) (Fig. 1B) has a different 5' sequence when compared to transcript 1. It lacks the first ATG used in transcript 1; therefore, translation likely commences at the second ATG. Prediction of the open reading frame using Genscan reveals that the transcript encodes for an N-terminal truncated protein: The first 50 amino acids of the full-length receptor are missing. Analysis of the deduced amino acid sequence by the SMART program suggests that the first 22 amino acids may function as a signal peptide. Thus, the truncated ALK7 (tALK7) differs from the full-length ALK7 by missing part of the activin receptor-binding domain. Transcript 3 (Fig. 1C) lacks 240 bp found in the coding region of ALK7; thus, the encoded protein has no transmembrane and GS domains and, therefore, is named soluble ALK7a (sALK7a; GenBank accession no. AF525680). Transcript 4 (Fig. 1D) has a 471-bp deletion of the coding region. Consequently, the transmembrane domain, GS domain, and part of the kinase domain are missing in the translated product, which we have designated as soluble ALK7b (sALK7b; GenBank accession no. AF525681).
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When compared with human ALK1 to ALK6, the kinase domain of ALK7 is the most conserved region. It shares approximately 82% identity with ALK4 and ALK5 and 61–63% identity with ALK1, ALK2, ALK3, and ALK6. The extracellular receptor-binding domain of ALK7 has less than 41% identity with other human ALKs (Table 2). The human ALK7 is very similar to the rat counterpart, with an overall 94% identity at the amino acid level.
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Genomic Organization of ALK7
Comparison of the full-length ALK7 cDNA (transcript 1) sequence to the human genome database using the Blast program reveals that the ALK7 gene is approximately 100 kilobases (kb) in size and is located at chromosome 2q24.1. The gene consists of at least nine exons and eight introns. Exon I encodes for the 5' untranslated region (UTR) and the signal peptide, and exon II encodes for the receptor-binding domain. The transmembrane and GS domain are contained in exon III. The intracellular region is encoded by exons IV–VIII and by part of exon IX, which also encodes for the 3' UTR (Fig. 2A). All other transcripts are derived from alternative splicing. Transcript 2 has a small exon (Ia) located within the first intron of transcript 1 (Fig. 2B). Transcript 3 has no exon III, whereas transcript 4 lacks both exon III and exon IV (Fig. 2A).
Expression of ALK7 and Nodal in Human Placenta
Using a human multiple-tissue Northern blot containing poly A+ RNA, the distribution of ALK7 mRNA in several human tissues was first examined. As shown in Figure 3, three transcripts, estimated to be 8, 6, and 4.5 kb, were detected. The 4.5-kb transcript was found in most of the tissues tested, such as the pancreas, kidney, brain, muscle, liver, and placenta, but the 8-kb transcript could only be seen in the brain, pancreas, and muscle. A weak 6-kb band was found in the placenta and pancreas.
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Using primers that span introns and are specific to different transcripts, we conducted RT-PCR to determine if all four ALK7 transcripts, as well as Nodal mRNA, are expressed in human placenta. As shown in Figure 4, three DNA fragments, corresponding to transcripts 1, 3, and 4, were observed using primers ALK7-7 and ALK7-6 (Fig. 4A), whereas a single band of the expected size for transcript 2 was detected when primers ALK7-20 and ALK7-6 were used (Fig. 4B). These DNA fragments have been sequenced and confirmed to represent the four transcripts we have identified. All transcripts were detected in JEG-3 cells and in some of the placental samples, but other placental samples expressed only two or three transcripts. To determine Nodal mRNA expression, once again, primers that span introns were used in PCR to amplify cDNA samples prepared from placentae. A single DNA fragment was observed in JEG-3 cells and placental samples from various gestational weeks (Fig. 4C).
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To confirm the presence of multiple ALK7 isoforms and to further determine the expression profile of ALK7 during placental development, Western blot analysis was conducted using placentas from first-, second-, and third-trimester pregnancies. An antibody directed against the extracellular region of rat ALK7 detected two major species of 58 and 52 kDa, respectively. In addition, two small bands of approximately 42 and 36 kDa were also observed in some placental samples (Fig. 5A). The 58- and 52-kDa bands are slightly larger than the calculated molecular mass of the full-length ALK7 and tALK7. However, a glycosylation site is found on the extracellular region of ALK7, and such glycosylation will result in an increase in molecular weight. The 42- and 36-kDa bands correspond to the sizes of sALK7a and sALK7b, respectively. Quantification of the 58- and 52-kDa bands in the first, second, and third trimester revealed that the full-length ALK7 content remained constant through gestation; however, tALK7 showed a significantly higher level in the third trimester compared to the earlier stages (Fig. 5, B and C). The 42- and 36-kDa bands were not always detected and, therefore, were not quantified.
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DISCUSSION
In the present study, we have cloned four transcripts of ALK7 from the human placenta and identified three novel ALK7 isoforms derived from alternative splicing. Furthermore, we have provided the first evidence, to our knowledge, that ALK7 and Nodal are expressed in the human placenta throughout pregnancy.
The human ALK7 is highly similar to the rat ALK7 and exhibits all characteristics of a type I receptor of the TGFß family. Although the nucleotide sequence has not been released, Bondestam et al. [26] recently reported the amino acid sequence of human ALK7 cloned from the brain, and the sequence is identical to the amino acid sequence deduced from our transcript 1. Similar to rat ALK7 [21, 22], the extracellular ligand-binding domain of human ALK7 also has very low similarity with other ALKs. This suggests that the ligand of ALK7 is different from those of other known ALKs. Indeed, the rat ALK7 is unable to bind to activin, TGFß, or BMP-7 [21, 22]. On the other hand, ALK7 has been recently shown to interact with mouse Nodal and Xnr1 and to mediate their activity in mesoderm formation [19], demonstrating that Nodal and its related proteins are physiological ligands of ALK7. The kinase domain of ALK7 is closely related to those of ALK4 and ALK5, suggesting that these receptors may have common substrates. Both ALK4 and ALK5 are known to phosphorylate Smad2 and Smad3, which in turn form complexes with Smad4 and are then translocated into the nucleus [1, 2]. The rat ALK7 has also been shown to regulate the activation and nuclear localization of Smad2 and Smad3 [22, 23] and to induce transcriptional activity of Smad2/3-dependent promoter constructs, such as PAI-1 [27].
The most novel and exciting finding of the present study is that the ALK7 gene generates, through alternative splicing, multiple transcripts that encode for four distinct proteins. The presence of multiple transcripts and isoforms of ALK7 in human placenta has been further confirmed by both RT-PCR and Western blot analysis. The full-length receptor encoded by transcript 1 is expected to be a functional receptor, as studies in the rat [27] and Xenopus [19] have demonstrated. Although the roles of ALK7 isoforms remain to be investigated, it is possible that they modulate the function of the full-length receptor. The open reading frame predicted for transcript 2 of the ALK7 gene is 50 amino acids shorter than the full-length receptor. This truncated receptor has a partial deletion of the ligand-binding domain. This may result in a decrease or absence of ligand-binding affinity. Alternative splicing that generates insertion or deletion of amino acids in the extracellular domain has been reported for other members of the TGFß-receptor family. The mouse activin-receptor IIB has four isoforms generated from alternative splicing of two regions, one in the cytoplasmic and the other in the extracellular domain [28]. Different binding affinities for activin-A have been observed for isoforms generated from alternative splicing of the extracellular region [28]. Similarly, the human TGFß type II receptor (TßRII) also has an isoform (TßRII-B) that contains an additional 25 amino acids in the receptor-binding domain [29, 30], derived from an insertion of a small exon within the first intron [30]. Unlike TßRII, which does not bind with TGFß2 in the absence of type III TGFß accessory receptors, the TßRII-B mediates type III TGFß receptor-independent signaling of TGFß2 [30]. Interestingly, another TGFß type II receptor isoform (TGFßRIIß), which is 70 amino acids shorter in the extracellular region, has also been reported [31]. However, to our knowledge, no functional studies on this isoform have been published.
Proteins encoded by transcripts 3 and 4 do not have the transmembrane domain but contain an intact ligand-binding domain. Therefore, they are predicted to be soluble proteins capable of binding with the ligands of ALK7. Ample examples of soluble receptors generated from alternative splicing have been published [32–36]. Some of these soluble receptors contain only the extracellular binding domain and lack the membrane spanning and cytoplasmic domains [33, 34], whereas others lack only the transmembrane domain [32]. In general, the soluble receptors function as either agonists or antagonists to enhance or to block, respectively, receptor signaling [32–34]. Two soluble receptors have previously been reported within the TGFß family. The first is a soluble form of TGFß type I receptor (sTßRI) found in the rat kidney that contains only the extracellular domain of the full-length receptor [36]. Interestingly, the sTßRI is a functional protein that binds to TGFß in the presence of TßRII and that potentiates TGFß signaling [36]. The other is a soluble form of inhibin B coreceptor in the rat [35]. However, to our knowledge, its function has not yet been characterized. It is possible that, similar to other soluble growth factor receptors [32–34], the soluble ALK7 isoforms may function as binding proteins to either inhibit or enhance signaling by the full-length ALK7.
Although four transcripts were obtained by RT-PCR, Northern blot analysis using a probe that is common in all four transcripts detected only two bands from the human placenta. This likely is because some of the transcripts (transcripts 3 and 4) differ from transcript 1 by less than 500 bp, and such a difference may not be detectable on the Northern blot. It is also possible that the placental sample on the Northern blot did not express all four transcripts, because we found in RT-PCR that not all placental samples expressed four transcripts. The sizes of transcripts estimated from the Northern blot analysis are bigger than the sequences we have obtained from the human placenta; therefore, it is possible that additional 5' and/or 3' untranslated sequences exist for these transcripts. Consequently, we cannot rule out the possibility of additional exons/introns for the ALK7 gene. In addition to the placenta, ALK7 transcripts were also detected in other tissues, such as the pancreas, kidney, brain, muscle, and liver. This suggests that ALK7 may also regulate the function of these organs. A recent study has shown that ALK7 affects proliferation and morphological differentiation in a neuronal cell line [37], suggesting that ALK7 plays a role in regulating neuronal functions. However, the role of ALK7 in pancreas, kidney, muscle, and liver is not known at present.
Although Nodal has been identified as a physiological ligand for ALK7, it is possible that other TGFß ligands can also bind to ALK7. Currently, more than 30 members of the TGFß family have been identified; however, only a limited number of receptors (five type II and seven type I) have been characterized [16–18]. Therefore, it is possible that one receptor can mediate the function of multiple ligands. Indeed, ALK4 has been shown to mediate both activin and Nodal functions [3]. Similarly, ALK2 can bind to activin as well as BMP, whereas activin receptors IIA and IIB also function in the BMP signaling pathway [16–18].
Members of the TGFß family have been shown to regulate human placental functions. The TGFß1 has an antiproliferative effect on cytotrophoblast cells [6, 15], whereas TGFß3 inhibits the differentiation of trophoblast cells into the invasive pathway [9]. On the other hand, activin-A stimulates the differentiation of invasive extravillous trophoblasts [5]. The TGFß1, activin-A, and BMP-7 also regulate production of placental hormones, such as hCG, human placental lactogen, progesterone, and estradiol [5, 8, 10–15, 38]. The present demonstration that Nodal and ALK7 are expressed in the human placenta suggests that additional members of the TGFß family, such as Nodal, are also involved in regulating placental development and function. Studies in rodents have shown that Nodal regulates differentiation of trophoblast cells. Homozygous mutations of the Nodal gene in mice resulted in excessive numbers of trophoblast giant cells [39]. On the other hand, overexpression of Nodal in a rat choriocarcinoma cell line, Rcho-1, decreased giant cell numbers [40]. These results suggest that Nodal inhibits the differentiation of stem trophoblast cells into giant cells. Interestingly, we found that whereas the full-length ALK7 and tALK7 appear to be expressed during all stages of gestation, the soluble forms of ALK7 are not always detected. Furthermore, the protein level of tALK7 significantly increased during the third trimester of pregnancy. These results suggest that expression of ALK7 spliced variants is developmentally regulated and that they may play important roles in ALK7 signaling. Because the tALK7 may have a reduced affinity for the ligand and, therefore, may function as an antagonist of the full-length ALK7, the increased level of tALK7 in third-trimester placentae suggests that ALK7 may be more functionally active during early pregnancy. It is possible that Nodal and ALK7 may regulate trophoblast proliferation and/or differentiation.
In summary, we have identified several novel isoforms of ALK7 generated from alternative splicing of the human ALK7 gene. We have also provided the first evidence, to our knowledge, that both Nodal and ALK7 are expressed in human placenta throughout pregnancy. The functions of ALK7 and its isoforms in human placenta are currently under investigation.
ACKNOWLEDGMENTS
We thank Dr. Burton Yang for helpful suggestions, Ms. Lee Wong for performing sequencing, and Mr. Chia-Ching Lien for technical assistance.
FOOTNOTES
1 Supported by a CIHR grant (MOP-53174) to C.P. P.C.K.L. is a Distinguished Scholar of the Michael Smith Foundation for Health Research. 
2 Correspondence: Chun Peng, Department of Biology, York University, 4700 Keele St., Toronto, ON, Canada M3J 1P3. FAX: 416 736 5698; cpeng{at}yorku.ca
Received: 6 November 2002.
First decision: 19 November 2002.
Accepted: 27 November 2002.
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