ARTICLE |
Correspondence to: Robert A. Lazzarini, Mount School of Medicine, Dept. of Biochemistry and Molecular Biology, Box 1020, 1 Gustave Levy Place, New York, NY 10029. E-mail: robert.lazzarini@mssm.edu
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Summary |
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Although gcm was first recognized for its role in specifying glial cell fate in Drosophila melanogaster, its mammalian counterparts are expressed predominantly in non-neural tissues. Here we demonstrate expression of the mouse and human GCM 1 proteins in placenta. We have prepared a highly specific antibody that recognizes the GCM 1 protein and have used it to assess the temporal and spatial expression profile of the protein. In both mouse and human placenta, the protein is associated with cells that are involved with exchange between maternal and fetal blood supplies: the labyrinthine cells of the mouse placenta and the syncytio- and cytotrophoblasts of the human placenta. Using the full-length hGcm 1 cDNA as a probe, we have mapped the gene on human chromosome 6p12 by fluorescent in situ hybridization. (J Histochem Cytochem 48:915922, 2000)
Key Words: glial cell missing, human placenta, trophoblast development
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Introduction |
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Early mammalian embryonic development is characterized by a sequence of programmed events in which trophoblast cell specification and differentiation are among the earliest. In human conceptuses, trophoblast stem cells differentiate and begin to invade the endometrial epithelial stroma by the end of the first week. Trophoblast stem cell differentiation is an unusual process that has some similarities to tumorigenesis. The attachment of the embryo to the uterus begins with a vigorous invasion of the decidualized endometrium (-integrins (
First identified in D. melanogaster, the gcm gene (also called glide for glial cell deficient) encodes a transcription factor that is expressed early in development and is pivotal in specification of glial cell fate (
Surprisingly, the mammalian Gcm 1 mRNA is more highly expressed in non-neural tissue and is only weakly expressed in brain (
Our analysis has revealed that although Gcm 1 expression in mouse placenta intersects that of several other transcription factors, its precise pattern is spatially and temporally distinct (
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Materials and Methods |
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Isolation of Human Gcm 1 cDNA
An anonymous human cDNA clone was identified in the expressed sequence tags database (dbEST, Genbank,
R62635; Washington University, St Louis, MO and Merck, Darmstadt, Germany) that exhibited striking sequence similarity to coding regions of the Drosophila gcm gene. This EST clone was used as a probe to isolate a 2.8-kb cDNA from a human placental library (Stratagene; La Jolla, CA). Both strands of cDNA were sequenced by dideoxy method using 35S-labeled nucleotides. Our nucleotide sequence agrees perfectly with that deposited in GenBank by
Chromosomal Localization of hGcm 1 Gene by FISH
FISH experiments were done on lymphocytes isolated from human blood and cultured in -minimal essential medium (
-MEM) supplemented with 10% fetal calf serum and phytohemagglutinin at 37C for 6872 hr. The lymphocyte cultures were then treated with BrdU (0.18 mg/ml) (Sigma; St Louis, MO) to synchronize the cell population. Synchronized cells were washed three times with serum-free medium to release the block and cultured at 37C for 6 hr in
-MEM with thymidine (2.5 µg/ml; Sigma). Cells were harvested and slides were made by using standard procedures including hypotonic treatment, fixation, and air-drying. FISH experiments were performed using a biotinylated 2.8-kb Gcm 1 cDNA as probe. The cDNA probe was labeled with biotindATP using a Bio-Nick labeling kit at 15C for 1 hr (Gibco-BRL; Gaithersburg, MD) (
Preparation of GCM 1 Antiserum and Western Blot Verification of Its Specificity
The portion of the mouse Gcm 1 cDNA encoding the carboxyl-terminal region of the protein (amino acids 169 to 437;
On the basis of the sequence homology between the mGcm 1 and the hGcm 1 cDNA, the anti-mouse GCM 1 antibody should also crossreact with the human protein. To verify this conclusion, we used the antisera in Western blotting analyses of samples of placental extracts. Soluble proteins were extracted from third-trimester human and E 15.5 mouse placenta by desegregating and sonicating the tissue in extraction buffer (0.1 M PBS, 10 mM DTT, 1% Triton X-100, 1 mM PMSF, 10 µg/ml aprotin, 10 mM pepstatin). Particulate material was removed by centrifugation. Samples were adjusted to 1 x SDS loading buffer and separated on a 10% denaturing polyacrylamide gel as previously reported (
Immunohistochemistry
Sections of human first- and third-trimester and mouse E 8.5 to E15.5 placentas were used to investigate the expression of GCM 1 protein by immunohistochemistry. Briefly, paraffin sections (10-µm thickness) were deparaffinized in xylene and rehydrated via a graded series of ethyl alcohol. Sections were then incubated for 20 min with 1% hydrogen peroxide in 10% methanol to inhibit endogenous peroxidase activity. After saturation with 10% normal goat serum in 0.1 M PBS/0.2% Triton, sections were incubated overnight at 4C with the affinity-purified anti-GCM 1 antibody diluted 1:100 in the case of mouse and 1:40 in the case of human tissue. After incubation with the primary antibody, sections were washed in PBS/0.2% Triton and the secondary antibody, peroxidase-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories; West Grove, PA) was then applied for 1 hr. After washing with PBS, the bound antibody was visualized by detection of the peroxidase activity using diaminobenzidine as substrate (Sigma). The reaction was monitored for 5 min and the sections were then rinsed with distilled water and mounted in xylene-based mounting medium. Sections were analyzed by brightfield microscopy (Axiophot; Zeiss, Oberkochen, Germany). For the above immunohistochemical procedures, controls were performed by replacing the primary antibody with preimmune serum. Further controls were also performed by omitting the primary antibody. Controls were always free of labeling.
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Results |
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Sequence Homology Between mGcm 1 and hGcm 1
The sequence of the 2.8-kb cDNA clone that we isolated revealed a large open reading frame (nucleotides 1991506) that encodes a 436-amino-acid protein identical to the hGCM 1/a protein sequence previously reported (
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Specificity of the GCM 1 Antiserum
The specificity of the crude and affinity-purified anti-mouse GCM 1 antibodies was analyzed by Western blotting, as illustrated in Fig 2. After removal of the GST-specific antibodies by repeated adsorption to matrix-bound GST, the rabbit antiserum recognized the GSTGCM 1 fusion protein used as positive control but not GST itself (Fig 2, Lanes 1 and 2). The antiserum recognized a protein of approximately 50 kD, the expected size of GCM 1 protein in mouse placenta (Fig 2, Lane 3). A faint band at approximately 90 kD was occasionally seen but only with the partially purified antibodies. The affinity-purified antibodies recognized a single antigen in extracts of mouse E10 and human third-trimester placenta (Fig 2, Lanes 4 and 5). The human protein has an apparent molecular weight that is slightly larger than predicted. Discrepancies between predicted size and that estimated from SDS gel electrophoresis are commonplace and may result from the residual secondary structure of the protein or from post-translational modifications. The immunochemically identified bands obtained with both mouse and human placental extracts were completely abolished after preabsorption of the GCM 1 antiserum on the matrix-bound GCM 1 peptide used in the affinity-purification step (data not shown).
Cellular Localization of GCM 1 in Human and Mouse Placenta
Earlier attempts by in situ hybridization to determine the cell type expressing the mouse Gcm 1 gene identified the chorionic and, in later development, the labyrinthine trophoblast cells of the mouse placenta (
Chromosomal Location of the hGcm 1 Gene
The full-length hGcm 1 cDNA was used to map the Gcm 1 gene by FISH. We mapped the chromosomal position of Gcm 1 by FISH analyses on metaphase chromosome spreads. Under the conditions used, the FISH detection efficiency was approximately 53% for the hGcm 1 probe (among 100 checked mitotic figures, 53 showed signals). In all cases, the signal was on both chromatid pairs at the same position on the chromosome. The DAPI banding pattern was used to identify the specific chromosome and to estimate the position of the fluorescent signal. The FISH signals (Fig 4A) all mapped to the short arm of chromosome 6 (Fig 4B). Fig 4C shows a histogram summarizing the results of 10 spreads. We conclude from these data that the human Gcm 1 maps to human chromosome 6, region p12. This region of chromosome 6 is known to be syntenic to parts of mouse chromosome 9 (http://www.ncbi.nlm.nih.gov/Omim/Homology/human6.html) where we previously mapped the mouse gene (
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Discussion |
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Trophoblast invasion of the maternal uterus during human placentation is a highly complex process that involves cell proliferation, migration, and cell differentiation. Several known factors have been described in human syncytiotrophoblasts, including the Wnt genes (
The mammalian GCM 1 and GCM 2 proteins show striking sequence similarities at their amino termini but no detectable similarities at their carboxyl termini (
In this communication we present the characterization of the GCM 1 immunoreagent and show that it crossreacts with the cognate human protein. Moreover, we show that the population of cells expressing this protein in placental sections appears larger than that expressing the Gcm 1 mRNA, as revealed by previous in situ hybridization in mouse and rat (
Successful development of the human embryo is largely dependent on the proper specialization of the extraembryonic tissue and the formation of a functional placenta. The individual roles of transcription factors such as Gcm 1 in placental development are largely unknown, and only recently have efforts been made to identify the key players in the transcriptional cascades that control trophoblastic differentiation (
In this study we have mapped the Gcm 1 gene to human chromosome 6p12 but have not identified any known genetic diseases of placentation that map to this region. The hGcm 1 was previously mapped to chromosome 6 by physical mapping of ESTs to the interval D6S427D6S294. This broad interval of chromosome 6 appears to include the band 6p12, the position to which we mapped the gene. Therefore, the FISH mapping data reported here are a higher resolution and refine the chromosomal localization of Gcm 1. These data also are consistent with the mapping of the mouse gene to mouse chromosome 9, band E1E3 (
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Acknowledgments |
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We gratefully acknowledge the participation of Eugenia Basyuk and Christerfer Demarco in the early phases of the work. We also thank Nancy Amalbert for assistance in assembling and editing this manuscript.
Received for publication December 7, 1999; accepted March 8, 2000.
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