BOR - Papers in Press, published online ahead of print
October 14, 2002.
Biol Reprod 2002, 10.1095/biolreprod.102.004945
BIOLOGY OF REPRODUCTION 68, 40–44 (2003)
DOI: 10.1095/biolreprod.102.004945
© 2003 by the Society for the Study of Reproduction, Inc.
Structure and Regulation of the Murine Mash2 Gene
Holger Stepana,
Wiebke Marqwardta,
Yvonne Kuhna,
Michael Höckela,
Heinz-Peter Schultheissb, and
Thomas Walther1,b
a Department of Obstetrics and Gynecology, University of Leipzig, D-04103 Leipzig, Germany
b Department of Cardiology, University Hospital Benjamin Franklin, Free University of Berlin, D-12200 Berlin, Germany
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ABSTRACT
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Transcription factors of the basic helix-loop-helix family such as Mash2 are essential for adequate differentiation of the trophoblast. Disruption of the Mash2 gene leads to early intrauterine death caused by placental insufficiency with an absent spongiotrophoblast and an underdeveloped chorion. The aim of the present study was to analyze the structure of the murine Mash2 gene, to screen a broad spectrum of organs for its expression, and to investigate placental Mash2 expression at different gestational ages. The RNase protection assay identified, in addition to the postulated Mash2 mRNA, two unexpected Mash2 transcripts that could be confirmed by a 5' rapid amplification of cDNA ends. However, all three transcripts were detectable exclusively in murine placenta and not in other organs, such as the ovary, uterus, skin, lung, femur, skeletal muscle, kidney, skull, adrenal gland, tongue, stomach, spleen, skin, testis, or pancreas. Sequence analysis disclosed an additional transcription start site upstream of exon 2. Placental Mash2 mRNA is measurable at all investigated stages of gestation. After its initial detection on Day 8.5 postcoitum (p.c.; set to 100%; 100.0% ± 28.4%), the Mash2 mRNA concentration increases significantly and reaches a maximum of 812.0% ± 69.7% on Day 12.5 p.c. The second half of gestation is marked by a more than 8-fold Mash2 decrease by Day 18.5 p.c. (77.0% ± 28.4%). A 36.9% ± 4.7% level of placental Mash2 mRNA is measurable at term.
embryo, placenta, pregnancy
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INTRODUCTION
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Early trophoblast development and placental function are regulated by a precisely tuned interaction of numerous factors. The trophoblast cell line that constitutes the outer placental epithelium is the first to differentiate in the early mammalian placenta, and it plays a pivotal role in development of the fetomaternal interface [1]. Normal placentation and specialization of the trophoblast into distinctive cell structures of the mature placenta are essential for its endocrine, nutritive, and immunological function, and disturbance of this orchestrated process seems to be the key event in diseases such as preeclampsia and intrauterine growth retardation [2].
Increasing knowledge regarding the genetic control of early extraembryonic tissue has provided the basis for demonstrating that transcription factors of the basic helix-loop-helix family are of vital importance for adequate differentiation of the trophoblast [3, 4]. The Mash2 gene (mammalian Achaete-Scute homologues), a conserved cognate of the Drosophila Achaete-Scute complex, is highly expressed in diploid trophoblast cells and is essential for the morphogenesis and function of the spongiotrophoblast [5, 6]. Localized on the distal portion of mouse chromosome 7, Mash2 constitutes, together with H19, Igf2, and Ins2, a cluster of imprinted genes that are differentially expressed by paternally and maternally inherited chromosomes [7]. The paternal Mash2 allele is expressed by trophoblast cells until Day 7.5 postcoitum (p.c.) and is then repressed [7]. Mouse embryos with two uniparental copies of this region show growth retardation and die in utero [8]. Disruption of the Mash2 gene leads to early intrauterine death on Day 10 p.c. because of placental insufficiency with an absent spongiotrophoblast and an underdeveloped chorion [6]. Interestingly, Mash2+/- embryos expressing only the paternal allele die at the same stage with a similar placental phenotype [8]. All these observations illustrate the paternal imprinting of the murine Mash2 gene. That hypoxia induces Mash2 expression [9] indicates this transcription factor at least partly mediates the regulation of trophoblast proliferation and differentiation by oxygen tension [10].
A recent study showed that the human Achaete-Scute homologue 2 (HASH2) gene contains two promoters. The two overlapping transcripts encode two different proteins (HASAP [human Achaete-Scute associated protein] and HASH2) with different nuclear localizations. Interestingly, HASH2 seems to be expressed exclusively in the placenta, whereas generation of HASAP does not seem to be restricted to this organ [11]. Neither a second promoter nor an independent transcript/protein not restricted to placenta have been described in mouse.
Therefore, the aim of the present study was to investigate the structure of the murine Mash2 gene in comparison to the human homologue, the Mash2 expression in different organs, and the regulation of placental Mash2 expression throughout the entire course of gestation.
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MATERIALS AND METHODS
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Animals
All investigations were performed using mice of the inbred strain C57Bl6. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH Publication 85-23, revised 1985). Female mice aged 3–4 mo were kept under standardized conditions with an artificial 12L:12D photoperiod and access to food and water ad libitum. The daily plug check was performed between 0800 and 1000 h. Plugged animals were set on Day 0.5 p.c. Pregnant mice were killed by cervical dislocation, and placentas were isolated on Days 8.5, 9.5, 10.5, 11.5, 12.5, 15.5, and 18.5 p.c. and immediately after delivery. All tissue samples were collected between 0800 and 1200 h and immediately snap-frozen in liquid nitrogen (for each experiment, probes of six different animals were sampled). In addition, male and female mice aged 3–4 mo were killed and dissected to isolate organs and tissues for expression screening.
RNase Protection Assay
Total RNA was isolated from tissues using the TRIzol reagent (Life Technologies, Eggenstein, Germany) with subsequent chloroform-isopropanol extraction according to the manufacturer's instructions. The polymerase chain reaction (PCR) amplified a 552-base pair (bp) chromosomal fragment (probe: MASH2) from mouse placenta DNA (Fig. 1) using the 5'-primer GCCCGGAGCATGGAAGCAC (P1) and the 3'-primer CCGAGCAGCGCAGGCTCC (P2) that was subcloned in a T-vector (Promega, Mannheim, Germany). A T7 polymerase transcribed a 604-bp radioactive probe complementary to 552-bp Mash2 mRNA, and a 197-nucleotide RNA complementary to 130 nucleotides of the glyceraldehyde phosphate dehydrogenase (GAPDH) mRNA or a 144-nucleotide RNA complementary to 127 nucleotides of the rL32 mRNA were used as positive controls. Murine Mash2-specific mRNA was identified by RNase protection assay (RPA) using the Ambion RPA II kit (AMS Biotechnology, Whitney, U.K.). A 30-µg fraction of each sample was hybridized with approximately 60 000 cpm for Mash2 and 35 000 cpm for GAPDH of the radiolabeled antisense probe in the same assay. The hybridized fragments protected from RNase A + T1 digestion were separated by electrophoresis on a denaturing gel (5% [w/v] polyacrylamide, 8 M urea) and analyzed using a FUJIX BAS 2000 Phospho-Imager system (Raytest GmbH, Straubenhardt, Germany). Quantitative analysis was performed by measuring the intensity of the Mash2 bands normalized by the intensity of GAPDH or rL32.
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FIG. 1. Structural organization of the murine Mash2 gene [7, 11] (upper part), generation of the RPA probe and detected Mash2 transcripts (middle part), and actual gene structure of the murine Mash2 (lower part)
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5' Rapid Amplification of cDNA Ends
The 5' rapid amplification of cDNA ends (RACE) was performed using two gene-specific reverse primers [12] and the 5' RACE kit from Gibco (Invitrogen GmbH, Karlsruhe, Germany) according to the protocol of the manufacturer. The PCR fragments were separated by gel electrophoresis and subcloned in a T-vector system (Promega). Cloned fragments have been sequenced, and sequences have been compared to the previously published murine Mash2 sequence (GenBank accession no. AF139595).
Sequence Analysis
For sequence analysis, the program BLAST (National Center for Biotechnology Information) was used. The program PROMOTER (available at http://www.fruitfly.org/seq_tools/promoter.html) has been used for the prediction of a new transcription start and the program HCtata (available at http://123genomics.homestead.com/files/analysis.html; link: TATA prediction) to identify TATA boxes.
Statistical Analysis
Data analysis was performed using the Statistical Package for Social Sciences, Version 6.1 software (SPSS, Inc., Chicago, IL). We used one-way analysis of variance and the Tukey-HSD (honestly significantly different) test to assess the statistical significance of differences between the groups. Significance was established at P < 0.05.
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RESULTS
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Gene Structure
Regarding the postulated gene structure of Mash2 [11], a protection probe has been designed to identify and quantify the expected transcript (Fig. 1). Because Mash2-deficient mice die on Day 10 p.c. [6], we used murine placentas from Day 12.5 p.c. as well as negative controls (embryos from Day 15.5 p.c. and myometrium). The quality of RNA was visualized by a signal for rL32 mRNA (Fig. 2). Though no Mash2 mRNA was detectable in the ovaries and myometrial probes, both placentas showed a band of expected length (transcript 1) (Figs. 1 and 2). Surprisingly, both placentas expressed two additional Mash2 mRNAs (transcripts 2 and 3). Therefore, we analyzed the 1600 bp of the Mash2 gene in front of the stop codon using the program PROMOTER to find possible additional starts of transcription. The program identified a start of transcription with a score of 1.00 (maximal score) that is 69 bp in front of the splice-acceptor site of exon 2 (Fig. 3A). This prediction was strengthened using the program HCtata that identified a TATA box 35 bp upstream of the postulated transcription start (Fig. 3B). This start point would elongate the detected transcript 1 to a length of 331 bp, which correlates with transcript 2 (Fig. 2). To determine additionally the start site for transcript 3, we used 5' RACE. Two fragments could be isolated, the first starting 2 bp in front of the computer-predicted transcription start (Fig. 3A) and the second 19 bp downstream of the first (Fig. 3B). Summarizing these data, the two unexpected RPA bands have a length of 333 bp (band for transcript 2) and 314 bp (band for transcript 3).
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FIG. 2. RPA showing placental expression of Mash2 mRNA (transcripts 1–3). None of the transcripts is expressed in embryos from Day 15.5 p.c. and in myometrium. The rL32 probe has been used to visualize the quantity of RNA. The first three left lanes show length markers
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FIG. 3. A) Computer-based analysis of an additional transcription start using 1600 bp in front of the murine Mash2 stop codon. B) Sequence of the murine Mash2 gene. Known exons are marked in bold, computer-predicted transcription start in italic, TATA box in inverted colors, transcription start points by 5' RACE framed, intron sequences in small letters, and transcribed sequences in capital letters. Translation start points and stop codon are underlined
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Gene Regulation
Screening various organs of adult mice showed that Mash2 is expressed only in the placenta (Fig. 4). As a positive control, GAPDH was detectable in all tissue probes, whereas no Mash2 expression could be measured in the testis, skin, spleen, stomach, tongue, lung, kidney, femur, skeletal muscle, uterus, ovary (Fig. 4), skull, adrenal gland, or pancreas (data not shown). To analyze whether Mash2 expression is regulated during murine gestation, placental probes were analyzed by RPA at different time points. Mash2 mRNA was detectable in all placental samples (Fig. 5A). The Mash2 mRNA concentration on Day 8.5 p.c. (set to 100%; 100.0% ± 28.4%) increases significantly (Day 9.5 p.c., 354.5% ± 81.6%; Day 10.5 p.c., 401.8% ± 80.9%; Day 11.5 p.c., 674.3% ± 150.7%) and reaches its maximum on Day 12.5 p.c., with 812.0% ± 69.7 %. Mash2 decreases during the second half of gestation (Day 15.5 p.c., 160.2% ± 34.6%; Day 18.5 p.c., 77.0% ± 28.4%). At term, placental Mash2 mRNA is detectable at a level of 36.9% ± 4.7% (Fig. 5B).
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FIG. 4. Representative RPA using adult murine organs showing Mash2 mRNA (552 bp) versus GAPDH (130 bp) expression. 1, Testis; 2, skin; 3, spleen; 4, stomach; 5, tongue; 6, lung; 7, kidney; 8, femur; 9, skeletal muscle; 10/11, uterus; 12, ovary; 13/14, placenta; Y+, cDNA probes incubated with yeast RNA and RNase; Y-, cDNA probes incubated with yeast RNA but without RNase
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FIG. 5. A) Representative analysis of an RPA from murine placentas showing Mash2 mRNA (552 bp) versus GAPDH (130 bp) expression. B) Quantification of placental Mash2 mRNA expression after autoradiographic signal analysis. Data are shown as the mean ± SD as percentage after normalization to GAPDH mRNA levels (n = 6 per group). Probes on Day 8.5 p.c. are arbitrarily set at 100%. *P < 0.05 and **P < .01 versus Day 8.5 p.c
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DISCUSSION
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This present paper is, to our knowledge, the first to describe the regulation of murine Mash2 expression, and it contributes to a more comprehensive knowledge regarding its gene structure. Despite the detailed study examining the structure of the human Achaete-Scute homologue by Westerman et al. [11], knowledge regarding the structure and regulation of the murine Mash2 gene is still limited. Thus, it was the aim of the present study to analyze the gene activity of the murine Mash2.
The postulated structure of the murine Mash2 included one promoter and three exons, with the translation start at the end of exon one, but our data suggest that a second promoter may be in intron 1. Thus, we identified different transcripts, and the activity of the known promoter was found to exceed that of the new one (transcript 1:2:3, 59%:21%:20%). The start of transcript 2 was confirmed by computer analysis. However, it is still uncertain whether the first base pairs of transcript 3 are a transcription start or a splice-acceptor site. Consequently, the use of promoter 2 would lead to a shortened Mash2 protein, because the postulated translation start of promoter 1 is not part of the newly identified transcripts. However, in a set of 15 screened organs, all three transcripts are expressed exclusively in placental tissue. Obviously, our transcripts 2 and 3 do not correspond to the second transcript of the human HASH2 gene HASAP, because that transcript is detectable in a variety of other organs whereas our newly identified transcripts are strictly confined to the placenta. This idea is also confirmed by the human HASAP not showing any significant sequence similarities to known mouse genome sequences. We conclude that, despite the similar chromosomal organization of Mash2 and HASH2, the posttranslational processing and gene products differ between mammalian species.
A previous study showed that Mash2 expression is regulated in the earliest stages of embryonic development, with an increase at the morula stage [13]. Thus, Mash2 seems to have a function during oogenesis and preimplantation and, later, becomes specific for spongiotrophoblast development [14]. Our investigation of the Mash2 expression profile during gestation until term demonstrated that Mash2 mRNA expression is not restricted to the early trophoblast, because the transcripts could be detected throughout the entire course of gestation. The bell-shaped expression curve reaches its maximum (Day 12.5 p.c.) around the time when Mash2-deficient embryos die because of placental insufficiency. Thus, our data confirm the essential role of this transcription factor in spongiotrophoblast development and differentiation. However, the measurable Mash2 mRNA concentration until delivery implies an additional role of Mash2 in the maintenance of placental function. Previous studies have shown that human placentas of all gestational ages also contain other transcription factors essential for the control of early trophoblast development, such as hypoxia-inducible transcription factors 1
and 2 [15]. The biological function of these transcription factors in the advanced stage of trophoblast differentiation should thus be elucidated. Moreover, it could be shown that human third-trimester placentas from patients with preeclampsia are characterized by selective overexpression of hypoxia-inducible transcription factor 2 [16]. Further studies are needed to clarify whether abnormal placental development alters the expression of Mash2 or HASH2.
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FOOTNOTES
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1 Correspondence: Thomas Walther, Department of Cardiology and Pneumology, University Hospital Benjamin Franklin, Hindenburgdamm 30, D-12200 Berlin, Germany. FAX: 49 30 8445 4648; thomas.walther{at}ukbf.fu-berlin.de 
Received: 25 February 2002.
First decision: 15 March 2002.
Accepted: 12 July 2002.
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REFERENCES
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-
Cross JC. Genetic insights into trophoblast differentiation and placental morphogenesis. Semin Cell Dev Biol 2000 11:105-113[CrossRef][Medline]
-
Dekker GA, Sibai BM. Etiology and pathogenesis of preeclampsia: current concepts. Am J Obstet Gynecol 1998 179:1359-1375[CrossRef][Medline]
-
Soares MJ, Chapman BM, Rasmussen CA, Dai G, Kamei T, Orwig KE. Differentiation of trophoblast endocrine cells. Placenta 1996 17:277-289[CrossRef][Medline]
-
Knöfler M, Kalionis B, Huelseweh B, Bilban M, Morrish DW. Novel genes and transcription factors in placental development—a workshop report. Placenta 2000 21:71-73[CrossRef]
-
Johnson JE, Birren SJ, Anderson DJ. Two rat homologues of Drosophila Achaete-Scute specifically expressed in neuronal precursors. Nature 1990 346:858-861[CrossRef][Medline]
-
Guillemot F, Nagy A, Auerbach A, Rossant J, Joyner AL. Essential role of Mash2 in extraembryonic development. Nature 1994 371:333-336[CrossRef][Medline]
-
Guillemot F, Caspary T, Tighman SM, Copeland NG, Gilbert DJ, Jenkins NA, Anderson DJ, Joyner AL, Rossant J, Nagy A. Genomic imprinting of Mash2, a mouse gene required for trophoblast development. Nat Genet 1995 9:235-242[CrossRef][Medline]
-
McLaughlin , Szabo P, Haegel H, Mann JR. Mouse embryos with paternal duplication of an imprinted chromosome 7 region die at midgestation and lack placental spongiotrophoblast. Development 1996 122:265-270[Abstract]
-
Jiang B, Kamat A, Mendelson CR. Hypoxia prevents induction of aromatase expression in human trophoblast cells in culture: potential inhibitory role of the hypoxia-inducible transcription factor Mash-2 (mammalian Achaete-Scute homologous protein-2). Mol Endocrinol 2000 14:1661-1673[Abstract/Free Full Text]
-
Genbacev O, Zhou Y, Ludlow JW, Fisher SJ. Regulation of human placental development by oxygen tension. Science 1997 277:1669-1672[Abstract/Free Full Text]
-
Westerman BA, Poutsma A, Looijenga LHJ, Wouters D, van Wijk IJ, Oudejans CBM. The human Achaete-Scute homologue 2 gene contains two promoters, generating overlapping transcripts and encoding two proteins with different nuclear localization. Placenta 2001 22:511-518[CrossRef][Medline]
-
Hug AM, Globeck HG. An improved 5' RACE technique facilitates the detection of multiple transcript initiation sites. Elsevier Trends Journals Technical Tips online 22.
-
Rossant J, Guillemont F, Tanaka M, Latham K, Gertenstein M, Nagy A. Mash2 is expressed in oogenesis and preimplantation development but is not required for blastocyst formation. Mech Dev 1998 73:183-191[CrossRef][Medline]
-
Tanaka M, Gertenstein M, Rossant J, Nagy N. Mash2 acts cell autonomously in mouse spongiotrophoblast development. Dev Biol 1997 190:55-65[CrossRef][Medline]
-
Rajakumar A, Conrad KP. Expression, ontogeny, and regulation of hypoxia-inducible transcription factors in the human placenta. Biol Reprod 2000 63:559-569[Abstract/Free Full Text]
-
Rajakumar A, Whitelock KA, Weissfeld LA, Daftary AR, Markovic , Conrad KP. Selective overexpression of the hypoxia-inducible transcription factor, HIF-2, in placentas from women with preeclampsia. Biol Reprod 2001 64:499-506[Abstract/Free Full Text]
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ABSTRACT
INTRODUCTION
