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Esx1, a Novel X Chromosome-Linked Homeobox Gene Expressed In

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Esx1, a Novel X Chromosome-Linked Homeobox Gene Expressed In DEVELOPMENTAL BIOLOGY 188, 85±95 (1997) ARTICLE NO. DB978640 Esx1, a Novel X Chromosome-Linked Homeobox View metadata,Gene citation and Expressed similar papers at core.ac.uk in Mouse Extraembryonic brought to you by CORE provided by Elsevier - Publisher Connector Tissues and Male Germ Cells Yuanhao Li,* Patrick Lemaire,² and Richard R. Behringer* *Department of Molecular Genetics, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, Texas 77030; and ²Laboratoire de Genetique et Physiologie de DeÂveloppement, Institut de Biologie du DeÂveloppement de Marseille, Campus de Luminy, F-13288 Marseille Cedex 09, France A novel paired-like homeobox gene, designated Esx1, was isolated in a screen for homeobox genes that regulate mouse embryogenesis. Analysis of a mouse interspeci®c backcross panel demonstrated that Esx1 mapped to the distal arm of the X chromosome. During embryogenesis, Esx1 expression was restricted to extraembryonic tissues, including the endoderm of the visceral yolk sac, the ectoderm of the chorion, and subsequently the labyrinthine trophoblast of the chorioallantoic placenta. In adult tissues, Esx1 expression was detected only in testes. However, Esx1 transcripts were not detected in the testes of sterile W/W v mice, suggesting that Esx1 expression is restricted to male germ cells. In situ hybridization experi- ments of testes indicated that Esx1 transcripts were most abundant in pre- and postmeiotic germ cells. Hybridization experiments suggested that Esx1 was conserved among vertebrates, including amphibians, birds, and mammals. During mouse development, the paternally derived X chromosome is preferentially inactivated in extraembryonic tissues of XX embryos, including the trophoblast, visceral endoderm, and parietal endoderm. In addition, the X chromosome is transiently inactivated during the meiotic stages of spermatogenesis. Thus, the identi®cation of Esx1 provides a molecular entry point into a genetic pathway to understand X chromosome-regulated fetal±maternal interactions and male germ cell development. q 1997 Academic Press INTRODUCTION (ICM) of the blastocyst (Pederson, 1986). The trophectoderm cells distal to the ICM that surround the blastocoel cavity The oocytes of therian mammals do not contain suf®cient (mural trophectoderm) stop proliferating and differentiate nutrition to support embryogenesis to birth. Therefore, nu- into giant cells. In contrast, the trophectoderm cells adja- trition must be provided by the mother from the uterine cent to the ICM (polar trophectoderm) continue proliferat- environment and subsequently through the placenta. The ing to form the placental precursor, the ectoplacental cone. placenta provides maternal nutrition, facilitates fetal respi- ICM cells adjacent to the blastocoel cavity form the primi- ration, eliminates fetal waste, and protects the fetus. This tive endoderm, whereas the other cells of the ICM form the transitional organ is also an important source of hormones primitive ectoderm. The primitive endoderm gives rise to and enzymes that are essential for normal embryonic devel- the visceral endoderm and the parietal endoderm (Gardner opment. Although embryos with organ defects can survive and Rossant, 1979; Gardner, 1982). The parietal endoderm to term, defects in placentation usually result in embryonic and the trophoblast giant cells form an early placental struc- death, which re¯ects the importance of the placenta during ture, the parietal yolk sac. Gastrulation of the mouse em- embryogenesis (Guillemot et al., 1994; Copp, 1995; Schmidt bryo results in the formation of extraembryonic mesoderm et al., 1995; Uehara et al., 1995). that, together with the visceral endoderm, forms another In eutherian mammals such as the mouse, the ®rst em- placental structure, the visceral yolk sac. In the mouse, the bryonic cell lineages to develop are those that contribute to function of the yolk sac placenta is subsequently replaced extraembryonic tissues with placental function. In morula- by the chorioallantoic placenta at Embryonic Day 10.0 stage embryos, peripheral cells are allocated to form troph- (E10.0) (Kaufman, 1992). ectoderm and more central cells to form the inner cell mass There are diverse forms of placentation among therian 0012-1606/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved. 85 AID DB 8640 / 6x29$$$101 07-07-97 13:31:50 dba 86 Li, Lemaire, and Behringer mammals. In marsupials, implantation and placentation By using the longest cDNA insert as a probe, we screened a mouse within the uterus occur relatively late in embryogenesis, E7.5 cDNA library (generously provided by John Gearhart, Johns which is coincident with organogenesis (Tyndale-Biscoe Hopkins) and isolated two identical cDNA phage clones. The and Renfree, 1987). In most marsupials, the trophoblast does cDNA inserts were subcloned into pBluescript II KS(0) (Stratagene) and sequenced by automated DNA sequencing. not invade the uterus and an apposed placentation leads to the formation of a yolk sac placenta. In eutherian mammals, the embryo implants relatively early in development, and Northern Blot and RT-PCR Analysis then the trophoblast invades the uterus to ultimately form a chorioallantoic placenta (Cross et al., 1994). Chorioallan- For Northern blot analysis, total RNA was prepared with toic placentas are generally divided into ®ve types based RNAzol B (Biotecx Laboratories, Inc.) according to the manufac- turer's instructions. Northern blots of total RNA (15 mg/lane) on the extent of fetal trophoblast invasion into the uterus were performed according to standard methods and hybridized (Billington, 1971). The biological reason for this diversity is with a 972-bp cDNA fragment (nt 465±1440, Fig. 1) in Rapid- unknown, although there is speculation that the various hyb buffer (Amersham Life Science) at 657C. Uniform loading of types of placentas evolved to better facilitate fetal develop- RNA was monitored by ethidium bromide staining of ribosomal ment or to drive speciation by establishing a hybridization RNA. The size of the Esx1 transcripts was determined by com- barrier (Benirschke, 1983). parison with RNA size markers (GIBCO-BRL). For RT-PCR, total Failures of the embryo to implant or the placenta to form RNA(1±3 mg) was reversed transcribed using an oligo(dT) primer account for most prenatal lethality in humans and domesti- and ampli®ed for 30 cycles using two Esx1 primers (shown in cated animal species (Cross et al., 1994). Recent analyses of Fig. 1). The integrity of the RNA and the ef®ciency of the reverse mouse gene knockouts, transgenic mice, and spontaneous transcription were monitored by ampli®cation of hypoxanthine phosphoribosyltransferase (HPRT). The sequences of the HPRT mutations also suggested that there are important develop- primers were 5*-GTTGAGAGATCATCTCCACC-3* and 5*- mental checkpoints around the time of implantation and AGCTATGATGAACCAGGTTA-3*. placentation (Copp, 1995). These observations suggest that extraembryonic development may be complex and subject to precise genetic control that ensures the normal develop- In Situ Hybridization ment of mammalian embryos. However, relatively few fac- In situ hybridization was performed on embryo sections with an tors speci®cally expressed in the extraembryonic lineages [a-35S]UTP-labeled antisense RNA probe, corresponding to nt 465± have been identi®ed, perhaps because much more attention 1440. Pregnant Swiss random-bred mice were killed, and their em- has been given to the embryo proper than to extraembryonic bryos were ®xed in 4% paraformaldehyde in phosphate-buffered tissues. Thus, to begin to de®ne the genetic pathways that saline for 16 hr at 47C. The embryos were then dehydrated with are essential for the development and function of the extra- a series of increasing concentrations of ethanol. The dehydrated embryonic lineages in mammals, more genes expressed in embryos were cleared in xylene before embedding in paraf®n. extraembryonic tissues must be identi®ed. Seven-micrometer-thick sections were mounted onto silane-coated In a search for homeobox genes that regulate early mouse slides (Histology Control Systems, Glen Head, NY). In situ hybrid- embryogenesis, we identi®ed a new member of the paired- ization was then performed as described by Wilkinson et al. (1987) and Sassoon et al. (1988). Hybridization was performed at 63 C for like subfamily of homeobox genes. The gene was designated 7 16 hr. A high-stringency wash was performed with 21 standard Esx1 because it was expressed during embryonic develop- saline citrate (SSC) and 50% formamide with 1.0 mM b-mercapto- ment in extraembryonic tissues (including the endoderm of ethanol at 657C for 30 min. Nonhybridized RNA probe was digested the visceral yolk sac, ectoderm of the chorion, and labyrin- with an RNase A solution (20 mg/ml RNase A in 10 mM Tris, pH thine trophoblast of the chorioallantoic placenta) and in 7.4, 500 mM NaCl, and 1 mM EDTA) at 377C for 30 min. High- adult tissues in the testis. In the testis, Esx1 expression was stringency washes with 21 SSC and 50% formamide with 0.5 mM restricted to pre- and postmeiotic germ cells. Esx1 mapped b-mercaptoethanol, then 21 SSC, and ®nally 0.11 SSC were per- to the distal end of the X chromosome. The established formed at 657C for 30, 15, and 12 min, respectively. Autoradiogra- role of, as yet unidenti®ed, X chromosome-linked genes in phy was performed with NTB-2 Kodak emulsion, and the slides placental development suggests that Esx1 may have an im- were exposed for 6±10 days at 47C. The developed slides were coun- terstained with hematoxylin. Photographs were taken with both portant role in the differentiation of speci®c extraembry- light- and dark-®eld optics. onic tissues and also in male germ cell development. Mouse Interspeci®c Backcross Mapping MATERIALS AND METHODS A mouse interspeci®c mapping panel was obtained from the Jackson Laboratory (Bar Harbor, ME). The mapping panel was com- Isolation of Mouse Esx1 cDNA Clones posed of genomic DNA from 94 backcross progeny from an inter- speci®c cross between (C57BL/6J 1 SPRET/Ei)F1 hybrid female and A mouse E8.5 cDNA library (generously provided by Brigid Ho- SPRET/Ei male mice.
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