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Regular Article |
a Division of Molecular Biology and Biochemistry, School of Biological Sciences, University of Missouri–Kansas City, Kansas City, Missouri 64110
b Laboratory of Animal Breeding, Faculty of Agriculture, University of Tokyo, Tokyo 113, Japan
ABSTRACT
Cloning and sequencing of a cDNA amplified by RNA fingerprinting at the implantation site of pregnant rats revealed 80% similarity with Hß58, previously shown to be essential for formation of the chorioallantoic placenta in the mouse. Hß58 mRNA was detected in the endometrium of hormonally sensitized rats stimulated to undergo decidualization and in the contralateral uterine horns lacking a decidual stimulus, indicating that uterine expression of Hß58 mRNA did not require decidualization or the presence of a blastocyst. Immunodetection in the early postimplantation uterus (Days 6–8 of pregnancy) showed Hß58 localized in the luminal and glandular epithelia and some stromal cells. Decidual cells at Day 6 of pregnancy expressed Hß58, and by Day 9 of pregnancy, the protein localized throughout the maternal decidua. The temporal and spatial distribution of Hß58 in the developing chorioallantoic placenta was assessed at Days 10, 12, and 14 of pregnancy. Immunoreactive Hß58 localized to erythroid cells within the developing fetal vasculature of the chorioallantoic primordia at Day 10 of pregnancy. By Day 12, the fetal vasculature extended into the placental labyrinth, and the erythroid stem cells continued to strongly express Hß58. At Day 14 of pregnancy, immunoreactivity became evident in the trophoblast giant cells and syncytiotrophoblast of the fetal placenta. As the chorioallantoic placenta matured (Day 18), Hß58 mRNA was 3.6-fold higher in the labyrinth compared with the junctional region. Stable cell lines (HRP/LRP) isolated from the rat labyrinthine placenta expressed Hß58 mRNA and protein. The expression pattern of Hß58 in maternal and fetal placental tissues and its early expression in fetal erythroid stem cells during formation and maturation of the chorioallantoic placenta suggest that Hß58 plays key roles in the regulatory networks that control hematopoietic development and placentation.
decidua, implantation/early development, trophoblast, uterus
INTRODUCTION
The emergence of the chorioallantoic placenta in eutherian mammals is a critical developmental process that brings the maternal and fetal vascular systems into close proximity thereby providing the fetus with the means to obtain nutrients, respire, and eliminate wastes [1, 2]. The failure to establish and maintain vascular circulation and the lack of transition from yolk-sac-based to liver-based hematopoiesis are lethal events [1–3]. The fetal blood vessels constituting the chorioallantoic placenta are derived from the allantois [4], a structure that originates, in part, from the extraembryonic mesoderm that also gives rise to the visceral yolk sac, the chorionic mesoderm, and the amniotic mesoderm [5, 6]. As the allantois enlarges during development, it fuses with the chorion [7, 8], and shortly thereafter, the allantois becomes visibly vascularized throughout [9]. Although vasculogenesis and hematopoiesis are developmentally associated [10], vasculogenesis may be independent of erythropoiesis in the allantois [9, 10].
Red blood cells are formed from hematopoietic stem cells that reside in the adult bone marrow and are capable of differentiating into at least eight morphologically and functionally distinct types of mature blood cells [11, 12]. In the mouse, hematopoietic events begin in the yolk sac at Day 7 of pregnancy, shift to the fetal liver at midgestation, and then to the bone marrow shortly before birth [13]. Extraembryonic blood islands develop from groups of mesodermal cell aggregates in the developing yolk sac [14], and the cells at the interior of these aggregates become primitive blood cells [15]. As the yolk sac becomes vascularized, the extraembryonic circulation is directly linked to that of the embryo [16], and the hematopoietic cells can circulate within the vasculature between intraembryonic and extraembryonic sites [13].
In the rodent, the chorioallantoic placenta forms from the ectoplacental cone that differentiates into two regions that are morphologically distinguishable shortly after midgestation [17–19]. These regions of the fetal placenta, termed the junctional and labyrinth zones, physically separate as the allantoic mesoderm invades the ectoplacental cone [4]. Cells in the junctional zone contain the trophoblast giant cells, spongiotrophoblast, and glycogen cells. As differentiation ensues, the junctional cells develop an invasive phenotype and become responsible for the production of placental hormones. The labyrinth zone is comprised of the trophoblast giant cells, syncytial trophoblast, and the fetal mesenchyme and vasculature. The labyrinth region comprises the site of fetal-maternal exchange and it is distinguished by the extent of penetration of the fetal vasculature.
In the present study, we have used RNA fingerprinting to search for candidate gene products that function in implantation and early placentation. Examination of the sequence from one of the cDNAs amplified from the implantation site identified the putative rat homolog of the mouse Hß58 gene, shown to be essential for embryogenesis [20, 21], and yeast pep8, a gene involved in vacuole protein trafficking [22]. Transgenic mice homozygous for the insertional mutation Hß58 were developmentally abnormal at Day 7.5 and exhibited irregularities in the amnion and chorion of the placenta [20]. The allantois developed normally through Day 8.5, but it failed to fuse with the chorion to form the chorioallantoic placenta. In order to gain new insight into the function of Hß58 in the development and maturation of the chorioallantoic placenta, in this report we determined the temporal and spatial distribution of Hß58 in the early postimplantation uterus and fetal placenta. The temporal and spatial changes in Hß58 expression in these tissues are consistent with its performing multiple functions during placental development. In particular, the immunolocalization of Hß58 to the fetal erythroid cells during the emergence and maturation of the chorioallantoic placenta suggest that this protein may perform essential functions in the regulatory networks that control hematopoietic development.
MATERIALS AND METHODS
Animals
Mated female Sprague-Dawley rats were housed on a 14L:10D cycle at the University of Missouri-Kansas City and provided rat chow and water ad libitum. The presence of sperm in the vagina was determined by the vendor (Taconic, Germantown, NY) and was counted as Day 1 of pregnancy. Animals were treated in accordance with the principles and procedures outlined in the NIH Guidelines for the Care and Use of Experimental Animals. Protocols for the care and use of animals were approved by the University of Missouri Animal Care and Use Committee. Rats were injected in the lateral tail vein 15 min prior to euthanasia and necropsy with 1% (w/v) Evans blue dye (Sigma Chemical Co., St. Louis, MO) [23] to visualize implantation sites. The endometrium was decidualized in ovariectomized hormone-treated rats by injecting 50 µl of sesame oil per uterine horn as described by us in detail elsewhere [24]. The contralateral uterine horns from the same animals were used as control uterine tissues for the hormone treatments. Placentas from pregnant (Days 10, 12, and 14) Sprague-Dawley rats were a gift from Michael J. Soares (University of Kansas Medical Center).
RNA Fingerprinting
Isolated implantation sites (Evans blue dye positive) and intersite regions were pooled from the uterus of three rats at Day 5 of pregnancy. Total RNA was isolated by homogenization of the tissues in guanidine thiocyanate [25]. Complementary DNAs were synthesized from RNA samples using primers and reagents provided in an RNA fingerprinting kit (Delta RNA Fingerprinting Kit K1810–1, Clontech, Palo Alto, CA). Select cDNA populations were amplified by fingerprinting using combinations of one oligo-dT (T) primer and one 5' (P) primer and 10 µCi of [
-35S]dATP. Polymerase chain reaction (PCR) conditions were 40 cycles at 94°C for 30 sec, 40°C for 2 min, and 72°C for 5 min. Amplified products were separated by electrophoresis through 6% polyacrylamide sequencing gels at 1700 V. Gels were exposed to x-ray film for 3–6 days. A cDNA unique to the implantation site was excised from the gel and reamplified with PCR using the same primer pair corresponding to that used for the original display. The PCR product was subcloned into Bluescript T vector [26], and the cDNA was sequenced using an automated sequencer (model 377; Perkin Elmer, Foster City, CA) with the ABI Prism Dye Terminator Cycle Sequencing Ready Reaction System with AmpliTaq DNA Polymerase, FS (Perkin Elmer). Sequences were examined in the GenBank and EMBL data bases, using the BLAST program.
Northern Blot Analysis
Samples of total RNA were dried under vacuum centrifugation, suspended in denaturing solution, and loaded onto 2.2 M formaldehyde-1% agarose gels [27]. The RNA size markers (RNA ladder; Life Technologies, Gaithersburg, MD) were electrophoresed on the same gel to determine transcript size. The RNA was transferred by diffusion onto nylon membranes (Micron Separation, Inc., Westboro, MA) for 12 h, then the filter was removed and baked in a vacuum oven at 65°C for 2 h. Hybridization probes were prepared by random prime labeling the rat Hß58 cDNA previously isolated and cloned from the RNA fingerprinting analysis with [- 32P]deoxy-CTP (3000 Ci/mmol; ICN Biomedicals, Costa Mesa, CA) according to the manufacturer's protocol (Life Technologies). Hybridization conditions and blot washes were carried out as described in detail elsewhere [27].
Generation of Hß58 Antibody
Computer analysis was used to search for potential antigenic regions in the mouse Hß58 protein. One region of hydrophilicity was identified from amino acids 280 to 285 (DEEDRR). Eight copies of the sequence (DEEDRRYFKQQEILLWRK, Map-peptide) were synthesized, and the synthetic peptide was emulsified in Freund's complete adjuvant. Rabbits were immunized by standard methods, and the Hß58 titer was determined from blood samples collected over a 10-wk period. An ELISA was developed using the Map peptide attached to plates as the solid phase (1 µg/well), and a goat anti-rabbit IgG-horse radish peroxidase conjugate with a peroxidase dye as the detection system. The reciprocal of the serum dilution that resulted in an optical density at 492 nm of 0.2 was used to determine the titer. The sera from one rabbit provided a titer of 214 500 10 wk after the first injection. The preimmune serum and the Hß58 antiserum were collected from this rabbit and affinity purified by passage over protein A columns. Bound immunoglobulin proteins were eluted with acetic acid. The eluants were concentrated against polyethylene glycol 20 000. Total protein concentration in the preimmune serum (3.2 mg/ml) and the Hß58 antiserum (1.2 mg/ml) was determined using the BioRad reagent.
Antibody Specificity
To assess antibody specificity, the rat Hß58 cDNA was expressed as a fusion protein and reacted with the Hß58 antibody on Western blots. The Hß58 cDNA that had been isolated and cloned from the RNA fingerprinting analysis was amplified by PCR using Hß58 gene-specific primers. The resulting PCR product was subcloned in frame into the BamHI and EcoRI sites of the pGex-2T expression vector (Pharmacia, Piscataway, NJ), and XL-1 bacteria were transformed with pGex-Hß58 and pGex vector alone. Positive colonies were isolated and the DNA was sequenced to verify the Hß58 open reading frame. Fresh overnight cultures of wild-type vector and vector containing the Hß58 cDNA were diluted 1:50 in Luria broth medium. Expression of glutathione-S-transferase (GST) and GST-Hß58 fusion proteins were induced by addition of isopropyl-ß-D-thiogalactopyranoside (IPTG: 1 mM) for 2 h. Samples from noninduced and induced XL-1 bacterial cells were centrifuged for 2 min, and the bacterial pellet was suspended in sample buffer (10% glycerol, 4% SDS, 50 mM Tris-HCl, pH 6.8, 1 mM EDTA, 0.05% bromphenol blue, and 4% mercaptoethanol). The samples were boiled for 2 min, and the supernatants were loaded on 10% SDS polyacrylamide gels. One of the gels was stained with Coomassie brilliant blue to visualize the proteins. Proteins on the other gel were transferred to a nitrocellulose membrane using methods standard in our laboratory [28]. The blot was reacted with Hß58 antibody (50 µg/ml), and immunoreactive proteins were visualized after treatment of the blot with the TMB substrate kit for horse radish peroxidase (Vector Laboratory, Burlingame, CA).
Localization of Hß58 in the Postimplantation Uterus and the Maternal and Fetal Placenta
Implantation sites from pregnant rats at Days 6–9 of pregnancy were fixed in 4% (w/v) paraformaldehyde (Fisher Scientific, Hanover Park, IL) in PBS and embedded in paraffin using methods standard in our laboratory [24]. Placental tissues (Days 10, 12, and 14) were fixed in freshly prepared cold Bouin fixative for 24 h essentially as described elsewhere [29] and then embedded in paraffin as detailed by us [24]. Sections (8 µm) were rehydrated through a series of ethanols, washed in PBS, and treated with 0.1 M glycine in PBS at 22°C for 2 h. To remove endogenous peroxidase activity, tissue sections were quenched in 0.3% (v/v) hydrogen peroxide (Sigma) in methanol at 22°C for 30 min. Samples were blocked for 18 h in a blocking buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.2% gelatin, 0.05% Tween-20, 0.5% [w/v] powdered milk) at 4°C. The slides were washed in PBS and blocked in normal goat serum (diluted 1:5) for 30 min. The sections were reacted with HBß58 antibody (50 µg/ml) at 4°C for 18 h and washed three times in PBS. To evaluate specificity of the reaction, some sections were incubated with an equal amount (50 µg/ml) of preimmune serum. Primary antibody was reacted with biotinylated affinity-purified anti-rabbit secondary antibody (Vector Laboratory) for 30 min at 22°C. Slides were exposed to the Vectastain ABC reagent, washed in PBS, and reacted for 2 min with equal volumes of 1 µg/ml diaminobenzidine (Aldrich, Milwaukee, WI) dissolved in 0.1 M Tris, pH 7.2 and 0.1% (v/v) hydrogen peroxide diluted in PBS. Slides were counterstained with 1% (v/v) methyl green dye in deionized water. At least three separate implantation sites (Days 6–9) and placentas with conceptuses (Days 10, 12) or placentas without conceptuses (Day 14) were examined. Representative sections were photographed using brightfield optics on an Olympus microscope with Kodak Gold (ASA 200) film.
Ribonuclease Protection Assays
The rat Hß58 cDNA that was isolated and cloned from the RNA fingerprinting analysis was restricted with HindIII and transcribed in vitro using T3 polymerase to produce single-stranded RNA probes using the Riboprobe kit (Promega Biotech, Madison, WI) and [-32P]CTP (800 Ci/mmol; ICN). To adjust for assay variability a rat glyceraldehyde 3-phosphate dehydrogenase (G3PDH) riboprobe (Ambion, Inc., Austin, TX) was synthesized as a control mRNA using SP6 polymerase. The transcripts were gel purified, eluted from the gel overnight, and added to the RNA samples. Total placental or labyrinthine cell line RNA (10 µg) was combined with the single-stranded probes (
20 000 cpm, Hß58 and 1000 cpm, G3PDH), and reactions were adjusted to contain 50 µg total RNA using yeast RNA. Ribonuclease protection assays were performed using the HybSpeed RPA kits (Ambion) as described by us previously [30, 31]. Briefly, samples were hybridized according to the manufacturer's protocol, and free riboprobe was removed by digestion with a 1:100 dilution of a mixture of RNase A (500 units/ml) and RNase T1 (20 000 units/ml) for 30 min at 37°C. Protected fragments were separated on polyacrylamide (5% w/v) urea (8 M) gels. The RNA size markers (Ambion) were run on the same gel to determine product size. The gels were exposed to x-ray film (Fuji; Fisher Scientific) for 3 days using intensifying screens. The amount of Hß58 mRNA was measured from the optical density (NIH image software) for each sample, and values were divided by the optical density of the corresponding G3PDH standard in the same reaction. Adjusted mean values were calculated from triplicate samples on the same autoradiograph. Mean differences between steady-state mRNA levels were considered to be significant if the Mann-Whitney U-test for nonparametric data gave a P value of <0.05 [30, 31].
Cell Culture
The cell lines (HRP/LRP) used in this study were isolated from the labyrinth region of the rat placenta at midgestation [32, 33]. The cells were grown in RPMI medium containing 10% fetal bovine serum and supplements as described [32]. Confluent cells were collected in guanidine thiocyanate, and total RNA was extracted with acid phenol:chloroform [34].
Localization of Hß58 in Placental Cell Lines
The placental cell lines were grown on gelatin-coated coverslips. Hß58 immunoreactivity was assessed using standard methods [35] with some modifications. Briefly, cells on coverslips were fixed with 2% paraformaldehyde for 30 min at 4°C, washed, and nonspecific binding sites were blocked with Dulbecco-PBS containing powdered milk, gelatin, and Tween-20. Primary antibody (120 µg/ml) and preimmune serum (122 µg/ml) were applied to coverslips for 90 min at 22°C. The coverslips were washed in Dulbecco PBS and blocked with 5% goat serum for 30 min. Goat antirabbit-Oregon green secondary antibody (Molecular Probes, Eugene, OR) was diluted 1:100 and applied to coverslips for 60 min at 4°C. Random fields of cells were photographed using identical conditions for cells treated with preimmune serum and Hß58 antibody. Cells were photographed using an Olympus microscope equipped with an appropriate fluorescent filter and Tmax film [36].
RESULTS
Preferential Amplification of the Rat Hß58 cDNA from the Implantation Site
Complementary DNAs prepared from total RNA that was isolated from the implantation sites (Evans blue dye positive) and the intersite regions of the uterus at Day 5 of pregnancy were amplified using RNA fingerprinting. One PCR product unique to the implantation site was excised and reamplified by PCR using the same primer pairs (5' primer, P4; 3' primer, T2). This amplified product was subcloned into the EcoRV site of a pBluescript SK(+) cloning vector (Stratagene, La Jolla, CA) prepared for direct cloning of PCR products. Examination of the sequence (Fig. 1A) suggested this cDNA to be the rat homolog of the mouse Hß58 gene shown to be essential for embryogenesis [20, 21], and the yeast pep8 gene, a gene coding for a protein involved in vacuole protein trafficking [22]. Alignment of the predicted amino acid sequences of the rat with reported sequences from yeast [22], chicken, and mouse [21] sequences showed a high degree of identity and conservative substitutions of amino acid residues among those species (Fig. 1B). The rat Hß58 open reading frame exhibited 17 out of 20 amino acids identity (85%) to the mouse and chicken proteins. Over the region compared, 15 of 20 amino acids were identical (75%) to the yeast vacuolar protein.
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Endometrial Expression of Hß58 mRNA Does Not Require Decidualization
Previous experiments in the mouse showed Hß58 transcripts were present in the embryo, fetal placenta, and maternal placenta (decidua). To investigate whether uterine expression of Hß58 was induced by decidualization of the endometrium, RNA samples pooled from artificially decidualized endometria, from the control contralateral nondecidualized uterine horns, and from implantation sites at Day 5 of pregnancy were analyzed by Northern blotting (Fig. 2). Results from Northern blot analysis revealed a single-sized Hß58 transcript of approximately 2.1 kilobases (kb) in the endometrium (Fig. 2). The mRNA was evident in the uterus of hormonally sensitized rats stimulated to undergo decidualization (lane 1), in the contralateral uterine horns exposed to the same hormonal treatment but lacking a decidual stimulus (lane 2), and in the isolated implantation sites from Day 5 pregnant rats (lane 3). Taken together, those results indicated that Hß58 transcripts were expressed in the endometrium of rats hormonally prepared for decidualization, but decidualization and the presence of a blastocyst was not required for endometrial expression.
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Characterization of Hß58 Antibody by Western Blotting
We tested the specificity of the Hß58 antibody by Western blotting. Induction of protein expression was monitored by SDS PAGE (Fig. 3A). The induced GST-Hß58 fusion protein migrated at a molecular mass of 35 kDa consistent with the predicted amino acid sequence of the coding region of the rat cDNA isolated and cloned from the RNA fingerprinting (see Fig. 1A). The GST protein alone migrated at the smaller expected mass of 27.5 kDa (Fig. 3A). Aliquots from those same protein samples were analyzed concurrently by immunoblotting (Fig. 3B). Immunoreactivity to the induced GST-Hß58 fusion protein was strongly evident (lane 2), while in the absence of IPTG induction, Hß58 immunoreactivity was barely detectable (lane 3). There was slight nonspecific binding of the antibody to the GST protein alone (lanes 4 and 5) owing to the excessive amount of GST protein in these lanes (compare Fig. 3, A and B). Taken together, those results confirmed that the antibody reacted specifically with Hß58.
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Localization of Hß58 Protein in the Postimplantation Endometrium
We next investigated the distribution of Hß58 in the postimplantation endometrium between Days 6–9 of pregnancy. The distribution of Hß58 transcripts in the mouse conceptus and placenta had been previously reported [21]. However, no information about the cell-specific expression or the distribution of Hß58 protein in the maternal cells of the early postimplantation uterus was known. At Day 6 of pregnancy, when morphological evidence for decidualization at the antimesometrial aspect of the endometrium was distinguishable, Hß58 was strongly expressed in the nuclei of the luminal (Fig. 4A) and glandular epithelium (not shown). Some stromal cell nuclei were also positive at Day 6 of pregnancy. Immunoreactive Hß58 localized in the nucleus in the stromal cells, while in decidual cells both the nuclei and the cytoplasm were immunopositive. There was no specific immunoreactivity when sections were treated with preimmune serum (Fig. 6, B and F). At Day 7 of pregnancy, the luminal (Fig. 4C) and glandular (not shown) epithelial cells expressed Hß58. In the stromal compartment, decidual cells were immunopositive in both the nucleus and cytoplasm (Fig. 4C). The luminal and glandular epithelia were immunopositive at Day 8 of pregnancy (Fig. 4D). The immunoreactivity shown in Figure 4D was characteristic of that observed in the glandular epithelial cells at Days 6–8 of pregnancy. Decidualization had expanded through the stromal compartment at Day 9 of pregnancy, and Hß58 immunoreactivity was evident in the decidual nuclei and cytoplasm (Fig. 4E).
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Localization of Hß58 Protein in the Fetal Placenta
Partitioning of the fetal rat placenta into two morphologically distinct regions termed the labyrinth and junctional zones becomes evident early in chorioallantoic placental development [17]. Because mice lacking functional Hß58 failed to form a chorioallantoic placenta [20, 21], it was essential to investigate the distribution of Hß58 in the different placental cell types during the transition from the choriovitelline placenta, which is comprised of a single trophoblast cell phenotype, to formation of the chorioallantoic placenta that contains at least four differentiated trophoblast cell phenotypes [17]. Paraffin sections from placentas at Days 10, 12, and 14 of pregnancy were reacted with Hß58 antibody (Figs. 5 and 6). At Day 10 of pregnancy, the junctional and labyrinth regions of the placenta were morphologically indistinguishable, and fetal trophoblast giant cells and erythroid stem cells were evident in the chorioallantoic primordia (Fig. 5, A and B). No other fetal cell types characteristic of the more mature chorioallantoic placenta were identified. At this early stage, trophoblast giant cells in the chorioallantoic primordia did not react with Hß58 antibody. However, comparison of sections reacted with Hß58 antibody (Fig. 5A) versus those treated with preimmune serum (Fig. 5B) clearly showed that the small, nucleated erythroid cells within the developing fetal vasculature of the chorioallantoic primordial region were strongly immunopositive. Blood spaces were evident adjacent to the trophoblast giant cells and the maternal decidua (Fig. 5, A and B). At higher magnification of the cells in the chorioallantoic primordia (Fig. 5C), it was clear that Hß58 immunoreactivity localized to erythroid cells within the developing fetal vasculature. The immunoreactivity appeared to be both nuclear and cytoplasmic in those small fetal erythroid cells when the sections were observed under oil immersion at x100 (data not shown). Erythroid cells in sections reacted with preimmune serum did not show Hß58 immunoreactivity (Fig. 5D).
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At Day 12 of pregnancy two separate populations of trophoblast giant cells were identified; however, Hß58 expression was not detected in trophoblast cells at either of these stages. As maturation of the chorioallantoic placenta progressed to Day 14 of pregnancy, Hß58 was detected in the trophoblast giant cells, and it was particularly evident in the trophoblast giant cells located at the maternal interface (Fig. 5E). The fetal erythroid cells strongly expressed Hß58 at Day 14 of pregnancy (Fig. 5E), while in sections reacted with preimmune serum the fetal erythroid and trophoblast giant cells were negative (Fig. 5F).
As pregnancy progressed from Day 10 to Day 12, demarcation of the junctional and labyrinth regions in the fetal placenta were unquestionably distinguishable (Fig. 6, A and B). The fetal vasculature now extended throughout the labyrinth region, and it was clearly developed and more extensive than that at Day 10. The Hß58-positive fetal erythroid cells were evident throughout the labyrinth zone within the fetal vasculature (Fig. 6A), while erythroid cells in sections treated with preimmune serum showed no immunoreactivity (Fig. 6B). The junctional zone itself was poorly vascularized compared with the labyrinth region except at the interface between the maternal decidua and the trophoblast giant cells (Fig. 6, C and D). Nucleated fetal erythroid cells were evident within the developing vascular spaces between the trophoblast giant cells at the junctional zone juxtaposed to the maternal decidua (Fig. 6, C and D), and those cells were immunopositive for Hß58 (Fig. 6C).
Hß58 mRNA Is Preferentially Expressed in the Labyrinth Region of the Mature Chorioallantoic Placenta
As the chorioallantoic placenta matured between Days 10 through 14, the labyrinth zone became highly vascularized and contained a large number of Hß58-positive fetal erythroid cells. In addition, at Day 14 of pregnancy Hß58 was detected in the syncytiotrophoblast of the labyrinthine placenta (data not shown). We hypothesized that if Hß58 was synthesized in cells preferentially located in the labyrinthine placenta, then the amount of Hß58 mRNA would be higher in that region compared to the junctional zone. To test whether Hß58 mRNA was expressed in the mature chorioallantoic placenta (Day 18) and if there were differences in the amount of transcripts expressed between the labyrinth and junctional regions, we measured Hß58 mRNA. Total placental RNA (10 µg) from the labyrinth and junctional zones of the placenta at Day 18 of pregnancy were combined with single-stranded riboprobes and analyzed by RNase protection. A representative RNase protection assay is shown in Figure 7A. The full-length, single-stranded transcript from the Hß58 template migrated at the predicted size (350 bases) in the absence of placental RNA and RNase digestion (lane 1). In the absence of target RNA and RNase digestion (lane 2) the full-length G3PDH transcript migrated at the expected size (400 bases). No bands were evident in reactions digested with RNase that contained both riboprobes and lacked placental RNA (lane 3, control). When placental RNA from the labyrinth (lane 4) and junctional (lane 5) regions of the placenta were hybridized with the Hß58 riboprobe, the smaller protected fragment corresponding to placental Hß58 was evident. The protected fragment for placental rat G3PDH was the expected size of 316 base pairs (lanes 4 and 5). Those protected fragments were absent from reactions lacking placental RNA (lanes 1–3). The amount of Hß58 mRNA in the two different regions of the placenta was measured by densitometric scanning of an autoradiograph from samples analyzed in triplicate and adjusted to G3PDH as described by us previously [30, 31]. There was a significant (P < 0.05, 3.6-fold) difference in Hß58 mRNA in the labyrinth compared with the junctional zone of the placenta (Fig. 7B).
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Expression of Hß58 in Cultured Placental Cell Lines
The HRP/LRP trophoblast stem cells provide an important source of labyrinthine trophoblast cell precursors from which to obtain additional information about placental cell lineages [37]. We therefore determined whether cell lines isolated from the labyrinthine placenta (HRP/LRP) and postulated to contain trophoblast stem cells [37] expressed Hß58. The amount of Hß58 mRNA was similar (Fig. 8A) between the two cell lines in independent RNase protection assays and in cells from different cell passages (data not shown). We determined the spatial distribution of Hß58 in HRP placental cells processed for immunocytochemical detection of the protein (Fig. 8B). The Hß58 protein was distributed in the nucleus and cytoplasm of positive cells (Fig. 8B, panels A and C). Immunoreactive protein was absent from cells reacted with an equal concentration of preimmune sera (Fig. 8B, panels B and D). Comparison of phase-contrast images with immunoreactive cells showed that some HRP cells were negative (Fig. 8B, compare A and E). Because negative cells were present in HRP cell lines from different passages and experiments, the results suggest that Hß58 expression is restricted to particular placental cell types. That interpretation is consistent with our results from the immunolocalization studies in the fetal placenta (Figs. 5 and 6). Similar results of cell-specific expression were obtained from immunocytochemical analysis of LRP placental cell lines (data not shown).
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DISCUSSION
The data reported here indicate that the expression of a protein with sequence similarity to Hß58 is spatially and temporally regulated during the formation and maturation of the chorioallantoic placenta in the rat. During pregnancy, Hß58 is differentially expressed in the maternal endometrium, decidua, and fetal placental cells. The Hß58 in the fetal placenta was regulated temporally and in a cell-specific fashion. During the early development of the chorioallantoic placenta at Day 10 of pregnancy, Hß58 expression was evident in nucleated fetal erythroid cells within the developing vasculature located in the chorioallantoic primordia. Nucleated erythroid cells continued to express Hß58 throughout chorioallantoic placental maturation (Days 10–14). At Day 14 of pregnancy, expression of Hß58 expanded into other cell types including the trophoblast giant cells and the syncytiotrophoblast of the labyrinth region of the placenta. Taken together, our data suggest that Hß58 may function in both maternal and fetal tissues during the formation and maturation of the chorioallantoic placenta. Importantly, the distribution of Hß58 in fetal erythroid stem cells raises important implications for this protein as a participant in the regulatory networks involved in hematopoietic development.
Previous studies showed that mutation of the Hß58 gene in the mouse caused embryonic death shortly after implantation [20, 21]. Development proceeded until the primitive streak stage when abnormalities in the embryonic ectoderm, amnion, and chorion were detected. Although Hß58 was expressed in the oocyte and early embryo, the lack of the gene product did not inhibit preimplantation development or the ability of embryos to implant [20, 21]. Instead there was a failure of the chorion and allantois to fuse and form the chorioallantoic placenta. To ensure developmental success, an extensive and intimate relationship between the mother and conceptus develops in eutherian mammals. Mice lacking the Hß58 gene product presumably die because the embryo is completely dependent on the maternal vasculature for its source of nutrients, gas exchange, and elimination of toxic wastes. Failure to establish and maintain vascular circulation, exemplified by the Hß58 mutation, and lack of transition from yolk-sac-based to liver-based hematopoiesis are lethal events in eutherian mammals [38, 39].
Placental defects that impair further development of the conceptus can be due to developmental errors in particular cell lineages or to faulty vascularization in the placenta. Because the Hß58 mutation was lethal in early postimplantation development, any requirement for its function in later pregnancy could not be assessed in the mutant mice. The present study however, reveals that Hß58 is expressed in the nucleated erythroid cells of the developing chorioallantoic placenta suggesting that this protein functions in the cell population destined to establish extraembryonic hematopoiesis. It is now important to investigate the expression of Hß58 in the mesodermally derived site comprising the aorta, genital ridge, and mesonephros region (AGM) of the mouse embryo. This AGM region is postulated to be the intraembryonic site of definitive hematopoietic activity in the mouse. The existence of intraembryonic sites for hematopoietic development has been shown in amphibians and birds [40]. It will be interesting to test for the expression pattern of Hß58 at the AGM site because differences may exist in the regulation of extraembryonic hematopoiesis and definitive hematopoiesis in which the stem cells reside in the fetal liver but may originate in the AGM [14]. The possibility that differential regulation involving Hß58 could occur is suggested from previous analysis of chimeric mouse conceptuses generated from two independent Hß58 mutant homozygous cell lines [41]. There was a high level of chimerism in the fetus. However, low level or sporadic contributions were found in the extraembryonic tissues, even in chimeras where mutant cells were the predominant cell population in the embryo [41]. Further understanding the function of Hß58 in the regulation of the extraembryonic hematopoietic system should provide new insight into the formation and differentiation of hematopoietic cells during embryogenesis and placentation.
Isolation of the PEP8 (peptidase-deficient) mutant in yeast revealed 44% identity with the Hß58 gene in mouse [22]. The defect in yeast was not lethal and overexpression of PEP8 did not affect cell viability. Analysis of the protein distribution showed immunolocalization to the yeast vacuole (equivalent of the mammalian lysosome). Domain swap experiments between the carboxyl terminus of PEP8 and Hß58 complemented the pep8 mutation [22], suggesting that Hß58 is associated with the lysosome, either as a hydrolytic enzyme or in the trafficking of proteins to and from the organelle. However, our immunolocalization studies show that rat Hß58 localizes to the nucleus as well as the cytoplasm, suggesting that the mammalian protein may perform additional or alternative functions to the yeast equivalent. This interpretation is consistent with the failure of chorioallantoic placental development and death in Hß58 null mice, while in Hß58 mutant yeast, there were no effects on viability. Further studies on the role of Hß58 in the placenta, and in particular its potential importance in regulating events necessary for erythropoiesis, are required to obtain additional insight into its specific function in chorioallantoic placental development.
In the current study, expression of Hß58 was similar when HRP/LRP placental cell lines were maintained under conditions that promote proliferation. The labyrinthine cell lines utilized in this study are postulated to contain trophoblast stem cells capable of differentiating into syncytial trophoblast and labyrinthine trophoblast giant cells [37]. Our results suggest that HRP/LRP cell lines may provide a valuable system for studying the steps involved in trophoblast differentiation and for identifying the factors that promote stem cell specification along particular cell lineage pathways. Taken together, the results obtained from this study indicate that erythroid stem cells express a protein that has sequence similarity to a yeast vacuolar protein. Further characterization of Hß58 and its function in fetal erythroid and placental cells will provide new insight into the regulatory networks that control hematopoietic development. Improved understanding about the regulation of circulatory development and hematopoiesis in the fetal placenta should lead to novel approaches to reduce perinatal mortality and intrauterine growth retardation.
ACKNOWLEDGMENTS
The authors are grateful to Michael J. Soares (University of Kansas Medial Center) for donating the placental cell lines and placental tissues used in this study. We thank our colleagues L. Hutt-Fletcher and S. Turk (UMKC) for assistance with affinity purification of the antibody. We thank J. Swafford (UMKC) for help with the figures. The authors are grateful to the reviewers for their excellent comments.
FOOTNOTES
1 Supported in part by grants from the University of Missouri Research Board and the Sarah Morrison Fund. 
2 Correspondence: Virginia Rider, Department of Biology, Pittsburg State University, 1701 South Broadway, Pittsburg, KS 66762. FAX: 316 235 4194; vrider{at}pittstate.edu
Accepted: July 7, 2000.
Received: April 3, 2000.
REFERENCES
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