Dnaj-Related Protein Essential for Placentation 1249

Development 126, 1247-1258 (1999) 1247 Printed in Great Britain © The Company of Biologists Limited 1999 DEV3915 Mrj encodes a DnaJ-related co-chaperone that is essential for murine placental development Patricia J. Hunter1, Bradley J. Swanson2, Melissa A. Haendel3, Gary E. Lyons4 and James C. Cross1,* 1Samuel Lunenfeld Research Institute, Mount Sinai Hospital, and the Departments of Obstetrics and Gynaecology, and Molecular and Medical Genetics, University of Toronto, 600 University Avenue, Toronto, Ontario M5G 1X5, Canada 2Program in Cellular and Molecular Biology, 3Neuroscience Training Program and 4Department of Anatomy, University of Wisconsin, Madison WI 53706, USA *Author for correspondence (e-mail: [email protected]) Accepted 28 December 1998; published on WWW 15 February 1999 SUMMARY We have identified a novel gene in a gene trap screen that chorionic trophoblast-specific transcription factor genes encodes a protein related to the DnaJ co-chaperone in E. Err2 and Gcm1 was significantly reduced. The mutants coli. The gene, named Mrj (mammalian relative of DnaJ) showed no abnormal phenotypes in other trophoblast cell was expressed throughout development in both the embryo types or in the embryo proper. This study indicates a and placenta. Within the placenta, expression was previously unsuspected role for chaperone proteins in particularly high in trophoblast giant cells but moderate placental development and represents the first genetic levels were also observed in trophoblast cells of the chorion analysis of DnaJ-related protein function in higher at embryonic day 8.5, and later in the labyrinth which eukaryotes. Based on a survey of EST databases arises from the attachment of the chorion to the allantois representing different mouse tissues and embryonic stages, (a process called chorioallantoic fusion). Insertion of the there are 40 or more DnaJ-related genes in mammals. In ROSAβgeo gene trap vector into the Mrj gene created a null addition to Mrj, at least two of these genes are also allele. Homozygous Mrj mutants died at mid-gestation due expressed in the developing mouse placenta. The specificity to a failure of chorioallantoic fusion at embryonic day 8.5, of the developmental defect in Mrj mutants suggests that which precluded formation of the mature placenta. At each of these genes may have unique tissue and cellular embryonic day 8.5, the chorion in mutants was activities. morphologically normal and expressed the cell adhesion molecule α4 integrin that is known to be required for Key words: Chaperone, Chorioallantoic fusion, DnaJ, Gene trap chorioallantoic fusion. However, expression of the screen, Placenta, Mouse INTRODUCTION A number of genes that are essential for development and early morphogenesis of the chorioallantoic placenta have been Implantation and formation of the placenta are critical for identified (Copp, 1995; Cross et al., 1994; Rinkenberger et al., embryonic survival in eutherian mammals. Indeed much of 1997). These include transcription factor genes that are early embryonic development is devoted to establishing essential for formation and/or maintenance of different extraembryonic cell types which make up the placenta (Copp, trophoblast cell subtypes; Err2 of the chorion (Luo et al., 1995; Cross et al., 1994; Rossant, 1995). A critical point in 1997), Mash2 of the spongiotrophoblast (Guillemot et al., gestation occurs when simple diffusion of gases and nutrients 1994; Tanaka et al., 1997) and Hand1 (formerly called from the mother is no longer sufficient to maintain embryo Hxt/eHAND) of trophoblast giant cells (Riley et al., 1998). viability and a transformation in placental structure must occur Err2 mouse mutants fail to form a chorioallantoic placenta (Copp, 1995; Cross et al., 1994). In the mouse, this occurs at because they lack chorionic trophoblast cells. Lack of mid-gestation with the formation of the labyrinth, a chorioallantoic placentae can also be due to primary defects in vascularized placenta. The labyrinth is a ‘chorioallantoic the allantois. This is observed in conceptuses with mutations placenta’ in that it forms after attachment of the allantois to the in genes such as brachyury (Glueksohn-Shoenheimer, 1944), chorionic plate (chorioallantoic fusion). Thereafter, extensive DNA methyltransferase (Li et al., 1992), Lim1 (Shawlot and morphogenesis produces the three-dimensional labyrinthine Behringer, 1995) and Csk1 (Thomas et al., 1995). The structure which consists of narrow maternal blood sinuses lined attachment of the allantois to the chorion depends on specific by trophoblast cells. In this way trophoblast cells act as a cell adhesion molecules. Vascular cell adhesion molecule-1 barrier between the maternal and fetal blood compartments. (VCAM1) is expressed on the distal tip of the allantois in 1248 P. J. Hunter and others anticipation of binding to its receptor, α4 integrin, which is To clone genomic DNA 5′ to the βgeo insertion site (from intron expressed on the basal surface of the chorion (Gurtner et al., one), inverse PCR was performed as previously described (Jonsson et 1995; Kwee et al., 1995; Yang et al., 1995). Deficiencies in al., 1996) using primers, oriented in divergent directions, which either VCAM1 or α4 integrin result in failure of anneal to sequences in the 3′ LTR (5′- TGGGAGGGTCTCCTCTGAGT-3′) and β-galactosidase (5′- chorioallantoic fusion in mice (Gurtner et al., 1995; Kwee et ′ β al., 1995; Yang et al., 1995). However, this phenotype occurs CACATGGCTGAATATCGACGGTT-3 ) regions of the geo insertion. To prepare the template DNA, genomic DNA isolated from in only a portion of mutant conceptuses indicating that the α 6AD1 cells was digested with EcoRI, diluted, ligated to form circular VCAM1/ 4 integrin interaction is not the only mechanism DNAs and then linearized with EcoRV. A single band of mediating chorioallantoic fusion. FGF signaling also plays a approximately 700 bp was produced after PCR amplification. It was role in placental development since a hypomorphic mutation ligated into pBluescript to produce the plasmid pE5G and sequenced. in the FGFR2 gene causes either defects in chorioallantoic Southern blots made from 6AD1 cell genomic DNA confirmed fusion or labyrinthine morphogenesis (Xu et al., 1998). linkage between the cloned intronic DNA and βgeo. Gene trapping in murine embryonic stem (ES) cells has been widely used to identify new developmentally important ES cell aggregation and mouse breeding genes. We have made use of the ROSAβgeo retroviral vector Aggregation chimeras were generated with 6AD1 ES cells using wild- (Friedrich and Soriano, 1991) which contains a promoterless type CD-1 morulae as previously described (Nagy et al., 1993). Two founder male chimeras were backcrossed to wild-type 129Sv and βgeo gene, a fusion of β-galactosidase and neomycin ′ outcrossed to CD-1 females to produce progeny which were resistance genes, flanked by a splice acceptor at the 5 end and heterozygous for the 6AD1βgeo allele. Heterozygous mice were a polyadenylation signal at the 3′ end. If the βgeo cassette intercrossed to produce homozygotes. inserts into a transcriptionally active gene, the βgeo protein will be expressed, thus conferring neomycin resistance. In Embryo genotyping, Southern and northern blot addition, the expression pattern of the trapped gene can be hybridization observed by staining specimens for β-galactosidase activity. Southern blot analysis of genomic DNA isolated from tail samples, In about 30% of the cases, the vector insertion disrupts gene yolk sacs or embryos (Riley et al., 1998) and northern blot analysis function thus producing a mutant phenotype (Friedrich and of tissue total RNA (Cross et al., 1995) was performed as previously Soriano, 1991). In our screen, expression patterns of ‘trapped’ described. The EcoRI fragment of pE5G (genomic sequence from intron one) was used as a probe for genotyping specimens by Southern genes were studied by in situ hybridization using probes from ′ blot analysis because it detects a polymorphism in the Mrj locus endogenous sequences that were cloned by 5 RACE (Baker caused by the insertion of βgeo (see Fig. 5). Since the Mrj coding et al., 1997). One ES cell line (6AD1) was selected for further region is similar to several other DnaJ-related genes in mice, we study and subsequent analysis revealed that this line carries generated a 3′ untranslated region probe that was Mrj-specific. The the βgeo insertion in a novel gene, named Mrj, that we show pC1200 plasmid containing part of the Mrj cDNA was digested with here is essential for chorioallantoic fusion. Mrj is a member Eco01091 and re-closed to produce the plasmid pE3 which contained of a large gene family related to the DnaJ gene in E. coli. only the distal 3′ untranslated region. This fragment was use to DnaJ-related proteins in other organisms function as adaptors generate probes for northern blot and in situ hybridization experiments. The XhoI/EcoRV fragment of pSAβgeo (Friedrich and and activators for HSP70-type chaperones (Hartl, 1996). The β specific nature of the Mrj mutant phenotype, despite the fact Soriano, 1991) was used as a probe to detect geo sequences. The mouse GAPDH cDNA (Piechaczyk et al., 1984) was used as a probe that several other DnaJ-related genes are expressed in the to show loaded amounts of RNA (Fig. 2). placenta, suggests that these proteins do not have redundant functions. Conceptus dissections and X-gal staining Conceptuses were dissected at various gestational ages: noon of the day that a vaginal plug was detected was defined as embryonic day MATERIALS AND METHODS (E) 0.5. For routine histology, conceptuses were fixed in 4% paraformaldehyde and paraffin embedded. For X-gal staining, Cloning of the Mrj gene specimens were fixed for 15 to 30 minutes in 1% formaldehyde, 0.2% The 6AD1 cell line was identified in a previously described gene trap glutaraldehyde, 0.02% NP-40, 5 mM EGTA, 2 mM MgCl2, 0.1 M screen (Baker et al., 1997) using the ROSAβgeo retrovirus (Friedrich sodium phosphate (pH 7.3). Specimens were stained whole, or as and Soriano, 1991) to infect R1 ES cells.

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