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. The allele created by the cryosections, for 4-24 hours at 37°C in 0.1% 4-chloro-5-bromo-3- proviral integration was called 6AD1βgeo. ES cells were grown with idolyl-β-D-galactopyranoside (X-gal; Nova Biochem), 5 mM or without STO feeder cells (ATCC) in medium consisting of 15% K3Fe(CN)6, 5 mM K4Fe(CN)6 in buffer (0.02% NP-40, 0.01% fetal bovine serum (HyClone), 0.1 mM β-mercaptoethanol, 2 mM L- deoxycholate, 2 mM MgCl2, 0.1 M sodium phosphate (pH 7.3). Some glutamine, 0.1 mM MEM nonessential amino acids, 150 µg/ml G418 X-gal-stained conceptuses were paraffin embedded and cut into 8 µm and 1000 U/ml leukemia inhibitory factor in DMEM. mRNA was histological sections. For cryosections, fixed conceptuses were harvested from the 6AD1 cell line and 5′ rapid amplification of cDNA equilibrated in 15% sucrose followed by 30% sucrose in PBS for 12 ends (RACE) was performed as previously described (Baker et al., hours each at 4°C. Conceptuses were then embedded in OCT medium 1997) in order to clone DNA adjacent to the βgeo insertion. A 90 bp (Miles) and stored at −80°C prior to cutting into 10 µm sections with 5′ RACE product, which represented the 5′ end of the Mrj cDNA, was a cryostat. Following X-gal staining, sections were counterstained cloned and used to screen a λgt10 cDNA library made from E8.5 with eosin (Sigma). embryos (kindly provided by Dr Brigid Hogan). A cDNA of approximately 1.6 kb was recovered and was cloned as two EcoRI In situ hybridization fragments of approximately 400 (pC400) and 1200 bp (pC1200) In situ hybridization data presented in Fig. 1 were prepared as encompassing the entire mRNA. cDNA fragments were ligated into described by Baker et al. (1997). Otherwise, conceptuses were fixed pBluescript (KS−) (Stratagene) and sequenced (GenBank accession in 4% paraformaldehyde, 0.02% glutaraldehyde in PBS and paraffin no. AF035962). embedded. Serial histological sections (5 µm) were either stained with DnaJ-related protein essential for placentation 1249

Harris’ haematoxylin and eosin (Sigma) or subjected to in situ notably higher levels in a few tissues. Specifically, Mrj hybridization (Millen and Hui, 1996). Antisense 33P-labeled expression was detected in the ganglion neural layer of the riboprobes were prepared using an RNA transcription kit (Stratagene). developing retina (Fig. 1C,F). Beginning at E12.5, Mrj Probes specific for Gcm1 (Altshuller et al., 1996), Err2 (Pettersson et expression in the brain was consistently higher in the al., 1996), 4311 (Lescisin et al., 1988), and Pl1 (Colosi et al., 1987) trigeminal ganglia, diencephalon and midbrain (Fig. 1G,H). have been described previously. A Mrj-specific riboprobe was Other prominently expressing tissues included the dorsal root prepared from pE3 which was linearized with Asp718. A βgeo β ganglia (Fig. 1D,E), thymus (Fig. 1D,E), nasal epithelium (Fig. riboprobe was prepared from pSA geo which was linearized with β PstI. After development, the sections were counterstained with 1G,H) and testis (not shown). Expression of Mrj and geo in Carazzi’s haematoxylin. adult organs of +/6AD1βgeo mice was assessed by northern blot analysis. Mrj mRNA was readily detected in the testis, Immunohistochemistry uterus, liver and brain with somewhat weaker expression in the Conceptuses were fixed in 2% paraformaldehyde for 2 hours at 4°C, eye, heart and gut. The mRNA was not detected in muscle or equilibrated in 8% sucrose followed by 18% sucrose in PBS for 4 and kidney (Fig. 2). The βgeo transcript showed a similar tissue 12 hours, respectively, at 4°C and finally embedded in OCT medium (Miles) and stored at −80°C. Cryosections were air-dried and post-fixed in acetone at −20°C for 5 minutes. They were then subjected to immunoperoxidase staining for VCAM1 using the MK-2 monoclonal antibody (Gurtner et al., 1995) (generously provided by Dr Myron Cybulsky), α4 integrin using the PS-2 monoclonal antibody (Yang et al., 1995) (Chemicon), and E-cadherin using the DECMA-1 monoclonal antibody (Sigma). Horseradish peroxidase-conjugated secondary antibodies (Amersham) were used at a 1:50 dilution.

RESULTS Identification and expression of the Mrj gene A gene trap screen was previously performed by infecting R1 ES cells with the ROSAβgeo retrovirus vector (Baker et al., 1997). The 6AD1 ES cell line, from which the Mrj gene was identified, was selected for further study. The expression of Mrj during mouse development was studied by following β-galactosidase expression in conceptuses carrying the 6AD1βgeo allele. Mice carrying this allele were produced by first generating chimeric males from the 6AD1 ES cells. The chimeras were bred to wild-type females in order to produce progeny that were heterozygous for the 6AD1βgeo allele (+/6AD1βgeo). Conceptuses were dissected at embryonic days (E) 7.5 to 15.5 and stained with X-gal to detect β- galactosidase activity. Positive (blue) staining was observed in approximately half of the embryos. In this strain, embryos that genotyped as +/+ failed to show any blue staining except for occasional staining in visceral endoderm after prolonged incubation (Fig. 3E; data not shown). Among the β-galactosidase-positive embryos, activity was detected in the embryo proper at all Fig. 1. Expression of Mrj during embryonic development revealed by (A-D,G) X- stages (Fig. 1A-C). Positive staining was observed gal staining of heterozygous embryos (+/6AD1βgeo) and (E,F,H) in situ in the egg cylinder at E7.5, and was fairly hybridization on sections of wild-type embryos. (A) E7.5. (B) E8.5. (C) E12.5. widespread in the embryo at subsequent stages. In (D) E15.5 embryo cut in the coronal plane at the C6 vertebra to reveal high situ hybridization experiments using conceptuses expression in the thymus and dorsal root ganglia. (E) E17.5 embryo section from aged between E8.5 and 17.5 revealed that the β- mid-C6 vertebra. (F) Coronal section through the head at E15.5. Note the pigmented layer of the retina is refractile in dark-field illumination and does not galactosidase activity in the embryo essentially represent hybridization signal. (G) Mid-sagittal view of E15.5 embryo to reveal β- replicated the pattern of the wild-type Mrj gene galactosidase activity in brain and nasal epithelium. (H) E15.5 sagittal section. de, (Fig. 1, compare D with E, G with H). Although diencephalon; drg, dorsal root ganglia; mb, midbrain; nc, neopallial cortex; ne, Mrj appeared to be widely expressed at low levels nasal epithelium; rn, ganglion layer of neural retina; th, thymus; tgg, trigeminal throughout most of the embryo, it was expressed at ganglia; s, somites. 1250 P. J. Hunter and others

cDNA was recovered which was similar to the predicted size of the full length mRNA based on northern blot analysis of mouse placental mRNA (Fig. 5D). The cDNA sequence predicted an open reading frame encoding a 242-amino acid protein (Fig. 4A). Several cDNAs were identified in the NCBI database of expressed sequence tags (ESTs) that together represent the complete human Mrj cDNA. The open reading frames of the mouse and human cDNAs were 96% and 90% identical in nucleotide and amino acid sequence, respectively (Fig. 4A). Although the MRJ protein was unique when compared to sequences in GenBank, the N-terminal 74 amino acids were similar to the J domain present in the E. coli DnaJ protein, as well Fig. 2. Expression of Mrj mRNA in adult mouse tissues. Total RNA as in several proteins in yeast, Drosophila, C. elegans and (10 µg) harvested from organs of +/6AD1βgeo mice was used to mammals. DnaJ-related proteins interact with HSP70 chaperones make two equally loaded northern blots. The blots were hybridized via the J domain and stimulate their ATPase activity (Hartl, 1996). with βgeo, Mrj 3′ untranslated region and GAPDH probes. The Mrj In searching GenBank and EST databases, we found more than and βgeo blots were exposed to the phosphorimager cassette for 48 40 unique cDNA sequences which encode J domain proteins in hours compared to 6 hours for GAPDH. Single bands were observed both humans and mice. Each of these sequences was predicted and their estimated sizes are shown. In, intestine; Ki, kidney; Br, to encode the His-Pro-Asp tripeptide within the J domain, which brain; Li, liver; He, heart; Mu, muscle; Ut, uterus; Te, testis; Ey, eye. is essential for interaction with HSP70, as well as flanking α- helices which are conserved among family members. Twenty cDNAs encoded J domains which shared greater than 50% amino distribution except that expression was not detected in the eye acid sequence identity with MRJ (Fig. 4B shows a dendrogram). and heart. Within this group were five previously identified proteins called MSJ1 (Berruti et al., 1998), HSJ1 (Cheetham et al., 1992), HSP40 Mrj is expressed in the trophoblast lineage of the (HDJ1) (Ohtsuka, 1993; Raabe and Manley, 1991), HDJ2 placenta (Chellaiah et al., 1993) and MTJ1 (Brightman et al., 1995). C- At all embryonic stages examined, the highest β-galactosidase terminal to the J domain, the MRJ protein had three other regions activity in the conceptus was observed in trophoblast giant cells of sequence similarity to three of these family members (Fig. 4A, of the placenta (Fig. 3). Secondary giant cells (which form regions II-IV). Region II is a Gly- or Gly/Phe-rich sequence around the ectoplacental cone) showed weaker staining before which is also present in E. coli DnaJ. The significance of regions E9.5 compared to primary giant cells. β-galactosidase activity III and IV which are conserved in MRJ, MSJ1, HSJ1 and HSP40 was also evident in trophoblast cells of the chorion but not the is unknown. ectoplacental cone at E8.5, and in the labyrinth but not spongiotrophoblast at E10.5 (Fig. 3A,C,F). In situ hybridization The βgeo insertion maps to the first intron of the Mrj was performed on histological sections from placentae of similar locus stages. Nine sections for each tissue and probe combination were Southern blot analysis of genomic DNA extracted from 6AD1 examined and typical results shown (Fig. 3G,H). Mrj and βgeo mRNAs were detectable in trophoblast cells of the chorion and ectoplacental cone, and in giant cells. However, a subset of giant Table 1. Genotype of offspring from 6AD1βgeo cells expressed these transcripts at strikingly higher levels. The heterozygous mice (number of progeny shown) latter result differed from the β-galactosidase staining which was Number +/ 6AD1bgeo/ uniformly high in all giant cells. Another difference between Mating ((×&) of litters +/+ 6AD1bgeo 6AD1bgeo enzyme activity and transcript levels was apparent in the +/6AD1βgeo × +/+ ectoplacental cone and spongiotrophoblast layer. Both Mrj and expected 50% 50% − βgeo mRNAs were detected by in situ hybridization whereas β- observed galactosidase activity was never observed (Fig. 3A-C). β- newborn n=5 27 26 − galactosidase was extremely weak or undetectable in the +/+ × +/6AD1βgeo allantois and the mesothelial cell layer of the chorion (Fig. 3C), expected 50% 50% − tissues which provide mesodermally derived components of the observed newborn n=5 30 28 − chorioallantoic placenta. Likewise, Mrj and βgeo mRNAs were undetectable by in situ hybridization (Fig. 3G,H; data not +/6AD1βgeo × +/6AD1βgeo expected 25% 50% 25% shown). After chorioallantoic fusion and subsequent formation observed of the labyrinth, β-galactosidase activity was detected in the E8.5 n=6 12 25 11 trophoblast component of the labyrinth (Fig. 3F), a pattern which E9.5 n=7 17 46 20* resembled the Mrj mRNA expression (data not shown). E10.5 n=7 12 43 18* E11.5 n=4 10 20 12*‡ Mrj encodes a novel member of a large family of E12.5 n=3 4 13 11‡ E14.5 n=2 6 11 6‡ DnaJ-related proteins newborn n=7 22 36 0 The Mrj cDNA was cloned from an E8.5 mouse embryo cDNA library using a 5′ RACE product as the initial probe. A 1.6 kb *Small embryos, ‡dead embryos, resorptions. DnaJ-related protein essential for placentation 1251

Table 2. Incidence of placental phenotype in offspring from intercrosses of 6AD1βgeo heterozygous mice No chorio- Labyrinth Age Genotype Total Normal allantoic fusion defects Resorption E8.5 +/+ 12 12 0 - 0 +/6AD1βgeo 25 24 1 - 0 6AD1βgeo/6AD1βgeo 11 0 11 - 0 (n=6 litters) E9.5 +/+ 17 17 0 0 0 +/6AD1βgeo 46 46 0 0 0 6AD1βgeo/6AD1βgeo 20 1 17 2 0 (n=7 litters) E10.5 +/+ 8 8 0 0 0 +/6AD1βgeo 18 17 0 0 1 6AD1βgeo/6AD1βgeo 70610 (n=3 litters) E11.5 +/+ 10 10 0 0 0 +/6AD1βgeo 20 20 0 0 0 6AD1βgeo/6AD1βgeo 12 0 4 4 4 (n=4 litters) cells indicated that a complete copy of ROSAβgeo cassette, insertion indicating that the two were separated by some including full length LTR sequences, had inserted into the Mrj distance (>12 kb). Because of this distance, we were unable to locus. Since the cDNA cloned by 5′ RACE represented the ﬁrst detect any restriction enzyme polymorphisms on Southern 90 bases of 5′ untranslated region in the full length mRNA, this blots caused by the insertion of βgeo into the Mrj locus when region was assumed to be exon 1. Therefore, βgeo had inserted using exon 1 as the probe (Fig. 5B). To determine if the either into exon one or downstream within an intron. To insertion had disrupted exon 2 or sequence further 3′, we distinguish these possibilities, Southern blot analysis was used probed Southern blots using distal 3′ cDNA probes (plasmids to generate a restriction map around exon 1 and the βgeo pC400 and pC1200). However, we were unable to detect insertion (data summarized in Fig. 5A). There was no overlap restriction enzyme polymorphisms (data not shown). We between the restriction maps around exon 1 and the βgeo concluded, therefore, that Mrj exon sequences were not

Fig. 3. Mrj expression in the placenta of heterozygous conceptuses (+/6AD1βgeo) (except as noted) revealed by X-gal staining (A-F) and in situ hybridization (G-I). (A) Bissected implantation site at E8.5 (embryo removed) showing β-galactosidase activity in trophoblast giant cells (arrowheads) and the chorion but not the ectoplacental cone. (B) E8.5 embryo with yolk sac, chorion and ectoplacental cone attached. (C) Histological section of stained E8.5 conceptuses; boxed area is shown at higher magnification in D. Note that β- galactosidase activity is very low or undetectable in the allantois and mesothelium. (E) Histological section of stained E8.5 wild-type conceptus, showing the absence of β-galactosidase activity. (F) Section of a mature placenta at E11.5. Arrowheads indicate giant cells. The dotted line demarcates the border between the spongiotrophoblast and labyrinthine layers. (G,H) Serial histological sections of E8.5 conceptus (+/6AD1βgeo) subjected to in situ hybridization using Mrj (G) and βgeo (H) riboprobes and visualized using dark field microscopy. Signals for Mrj and βgeo were detected in the chorion, ectoplacental cone and in giant cells. Arrowheads indicate two giant cells with high expression. (I) Bright-field microscopy of toluidine blue stained section shown in H. epc, ectoplacental cone; ch, chorion; al, allantois; me, mesothelium; sp, spongiotrophoblast; lab, labyrinth; ec, exocoelomic cavity; dec, decidua. Bar represents 100 µm. 1252 P. J. Hunter and others A

M.m. MRJ M VDYYEVLGVQRHASPEDIKKAYRKQALKWHPDKNPENKEEAERKFKQVAEAYEVLSDAKKRDIYDKYGKEGLNGGG GGGIHFD SP H.s. MRJ M VDYYEVLGVQRHASPEDIKKAYRKLALKWHPDKNPENKEEAERKFKQVAEAYEVLSDAKKRDIYDKYGKEGLNGGG GGGSHFD SP M.m. MSJ-1 M VDYYEVLGVPRQASAEAIRKAYRKLALKWHPDKNPEHKEEAERRFKQVAQAYEVLSDV KREVYDRCGEVGEVGGGGAAGSPFHDA H.s. HSJ1 M ASYYEILDVPRSASADDIKKAYRRKALQWHPDKNPDNKEFAEKKFKEVAEAYEVLSDKHKREIYDRYGRERLTGTG TGPSRAEAGSGG H.s. HSP40 MGKDYYQTLGLARGASDEEIKRAYRRQALRYHPDKNKE PGAEEKFKEIAEAYDVLSDPRKREIFDRYGEEGLKGSGPSGGSGGGANGTS Region I J Domain Region II M.m. MRJ FEFGFTFRNPDDVFREFFGGRDPFSFDFFE DPFDDFFGNRRGPRGNRSRGAAPFFSTFSGFPSFGSGFPAFDTGFTPFGSLGHGGLTSFS H.s. MRJ FEFGFTFRNPDDVFREFFGGRDPFSFDFFE DPFEDFFGNRRGPRGSRSRGTGSFFSAFSGFPSFGSGFSSFDTGFTSFGSLGHGGLTSFS M.m. MSJ-1 FQYVFSFRDPAEVFREFFGGHDPFSFDFFGGDPLENFFGDRRSTRGSRSRGAVPFSTSFTEFPGFGGGFASLDTGFTSFGSPGNSGLSSFS H.s. HSJ1 PGFTFTFRSPEEVFREFFGSGDPFAELFDLG PFSELQ NR GSRHS GPFFTFSSSFP GH SDFS H.s. HSP40 FSTYFHGD PHAMFAEFFGGRNPFDTFFGQRNGEEGMDIDDPFS GFPMGMGGFTNVNFGRSRSAQEPARKKQDPPVTHDLRVSLEEIYSG Region III M.m. MRJ STSFGGS GMGNFKSISTSTKIVNGKKITTKRIVENGQERVEVEEDGQLKPLTINGKEHLLRLDNK H.s. MRJ STSFGGS GMGNFKSISTSTKMVNGRKITTKRIVENGQERVEVEEDGQLKSGTINGKEQLLRLDNK M.m. MSJ-1 M SCGGGAA GNYKSVSTSTEIINGKKITTKRIVENGQERVEVEEDGELKSLIINGREQLLRINTQ H.s. HSJ1 SSSFSFSPGAGAFRSVSTSTTFVQGRRITTRRIMENGQERVEVEEDGQLKSVTINGVPDDLARGLE... 133 amino acids H.s. HSP40 CTKKMKISHKRLNPDGKSIRNEDKILTIEVKKGWKEGTKITFPKEGDQTSNNIPADIVFVLKDKPH... 96 amino acids Region IV HDJ-2 AA068317 B AA118344 MRJ MSJ-1 AA097630 AA072835 AA105758 HSJ1 AA059999 AA172971 AA497706 MTJ1 AA000210 W75056 AA426920 AA023589 AA237153 HSP40 AA144155 AA545701

50 60 70 80 90 100 % identity Fig. 4. Sequence comparison of MRJ with other mammalian DnaJ-related proteins. (A) Amino acid sequence alignment of human and mouse MRJ protein with MSJ1, HSJ1 and HSP40. Amino acids identical to human MRJ are in bold. The gaps in sequence were introduced in order to maximize the sequence alignments. The J domain and three other regions of similarity are shown. H.s., Homo sapiens; M.m., Mus musculus. (B) Evolutionary tree analysis of the mouse DNA sequences in GenBank and dbEST encoding J domains. disrupted by the insertion and that βgeo had inserted into intron the progeny at birth (Table 1). Progeny from heterozygous one. In order to detect polymorphisms associated with the matings were then dissected at E8.5 to E14.5. Conceptuses that 6AD1βgeo allele that were required to genotype mice by were homozygous for the 6AD1βgeo allele were viable only Southern blotting, we cloned a fragment of genomic DNA up to about E11.5 (Table 1). The matings summarized in Table ﬂanking the 5′ end of βgeo by using inverse PCR. The 1 represent mice produced by outcrossing the founder chimeras sequence of this fragment was unique and, when used as a to an outbred background. However, the same phenotype was probe, revealed restriction site polymorphisms between DNA observed on a 129Sv inbred background. from wild-type and +/6AD1βgeo mice (Fig. 5C). To investigate the embryonic lethal phenotype of conceptuses that were homozygous for the 6AD1βgeo allele, Disruption of Mrj expression from the 6AD1βgeo we determined if Mrj mRNA expression was reduced. allele Northern blots of E10.5 placental RNA from conceptuses of The mapping data indicated that the βgeo insertion had not wild-type (+/+), heterozygous (+/6AD1βgeo) and disrupted the Mrj coding sequence. To determine if the βgeo homozygous (6AD1βgeo/6AD1βgeo) mutant genotypes were insertion had disrupted the function of the Mrj gene, we probed with a fragment of Mrj gene which lies downstream of examined mice carrying the mutant gene for an abnormal the βgeo insertion (3′ UTR fragment, see Materials and phenotype. Heterozygous mice appeared normal and Methods). We were unable to detect any Mrj mRNA in transmitted the 6AD1βgeo allele at roughly the predicted homozygous mutant placentae (Fig. 5D). Furthermore, Mrj Mendelian frequency of 50% (Table 1), but in intercrosses of transcript levels appeared to be reduced (by about one-half) in heterozygous animals, no homozygotes were detected among samples from heterozygous conceptuses. To conﬁrm the DnaJ-related protein essential for placentation 1253 northern blot results, mRNA in situ hybridization analysis was from a heterozygous mating were analyzed, in which nine performed on E8.5 conceptuses using a riboprobe generated histological sections from each conceptus were assessed. We from the same 3′ fragment. Fourteen conceptuses produced failed to observe Mrj hybridization signals above background

Fig. 5. The βgeo insertion disrupts the Mrj gene and reduces mRNA expression. (A) Schematic representation of the 5′ region of the Mrj locus. The line indicates the genomic DNA with restriction enzyme sites indicated. The position of the ROSAβgeo insertion is indicated. Exons, indicated by the lightly shaded rectangles, are not drawn to scale. Bars represent probes for Southern blot analysis. E, EcoRI; EV, EcoRV; H, HindIII; A, Asp718; B, BamHI. (B) Wild-type and 6AD1 ES cell DNA digested with indicated enzymes, Southern blotted and hybridized with probe A. Note that no restriction site polymorphisms are associated with the 6AD1βgeo allele using this probe. (C) DNA from wild-type, 6AD1βgeo heterozygous and homozygous mice digested with EcoRV, Southern blotted and hybridized to probe B (from intron one). (D) Northern blot of total RNA isolated from placentae of wild-type, 6AD1βgeo heterozygous and homozygous conceptuses at E10.5. A Mrj probe representing 3′ UTR sequence downstream of the βgeo insertion was used. (E) Bright-field (top) and dark-field (bottom) views of in situ hybridization using an antisense probe specific to Mrj on sections from a wild-type and a homozygous Mrj mutant embryo at E8.5. Giant cells expressing Mrj are indicated with arrowheads. ch, chorion. Bar represents 100 µm. 1254 P. J. Hunter and others in trophoblast cells of the three homozygous mutant embryos allantois remained unattached to the chorion in the vast (Fig. 5E). Based on these data, the 6AD1 mutation appeared majority of homozygous mutant conceptuses at E9.5 and later to be a null allele of Mrj. (Fig. 6; Table 2). However, at E9.5, 10.5 and 11.5, we found a few conceptuses in which the allantois had formed a loose Failure of chorioallantoic fusion in Mrj homozygous attachment to the chorion (Table 2). Notably, placental mutants labyrinth morphogenesis never proceeded in these cases and At E8.25, all embryos produced from crosses between all homozygous mutants were undergoing resorption by E12.5. heterozygous mice had reached the three somite stage and were We looked for abnormalities in placental histology in indistinguishable (Fig. 6A). At E8.5, the homozygous mutant homozygous Mrj mutants. At E8.25-8.5, no differences in size embryos appeared to be of normal size and had developed 6-7 or histological appearance of the allantois were apparent in somites, similar to their wild-type and heterozygous mutants except that it remained unattached. The mesothelium littermates. However, chorioallantoic fusion was never lining the basal surface of the chorion (Fig. 6B) and all observed in the homozygous mutants in contrast to the wild- trophoblast cell subtypes appeared morphologically normal in type and heterozygous littermates (Table 2; Fig. 6). For other the homozygous mutant conceptuses (Fig. 6B). As development mouse strains, chorioallantoic fusion occurs during a narrow proceeded, the chorionic plate remained intact in the placentae window between E8.25 and 8.5 when embryos are at the 5-6 of Mrj mutants (Fig. 6B) although, starting at E9.5, vacuolated somite stage (Downs and Gardner, 1995). Dissection of cells and pyknotic nuclei were observed at high magnification embryos between E8.25 and 8.5 revealed that this is also true (data not shown). Marker analysis was performed to detect of the 129/CD-1 background as well (data not shown). It is changes in gene expression of trophoblast cell types, using significant therefore that while development of the embryo serial histological sections. Probes were hybridized to nine proper occurred with normal timing in homozygous mutants sections of 3-4 mutants at each developmental stage and typical up to E8.5, the process of chorioallantoic fusion did not. For results are shown in Fig. 7. Expression patterns for most embryos dissected at E9.5, although they had turned, the markers appeared to be normal. For example, Pl1, a trophoblast homozygous mutant embryos were smaller than their giant cell-specific gene (Colosi et al., 1987), and 4311, an littermates and had arrested at the 18 somite stage (E9.25). The ectoplacental cone and spongiotrophoblast-specific gene

Fig. 6. Placental phenotype in homozygous Mrj mutants. (A) Whole mount views of conceptuses dissected and removed from the parietal yolk sac at E8.25, and partially dissected feto-placental units at E9.5. Notice that in the heterozygous conceptus at E9.5, the allantois has attached to the chorion. In homozygous Mrj mutants, the allantois does not fuse to the chorion and appears as a bud. (B) Histology of the placenta in Mrj mutant conceptuses. Histological sections of wild-type and Mrj mutant placentae at E8.5 (low and high magniﬁcation), 9.5 and 10.5. Dotted lines mark the interface between trophoblast giant cells and the decidua. al, allantois; ch, chorion; epc, ectoplacental cone; lab, labyrinth; me, chorionic mesothelium; pl, placenta; sp, spongiotrophoblast; uc, umbilical cord. Bars in B represents 100 µm. DnaJ-related protein essential for placentation 1255

Fig. 7. Trophoblast marker analysis of homozygous Mrj mutants at E8.5 and 10.5. Serial sections of wild-type and mutant (6AD1βgeo/6AD1βgeo) conceptuses were probed with antisense riboprobes for Gcm1, 4311 and Pl1. Pl1 is expressed in trophoblast giant cells, 4311 is expressed in the ectoplacental cone and spongiotrophoblast layer, and Gcm1 is expressed in chorionic and labyrinthine trophoblast cells. Note that Gcm1 expression is reduced in Mrj mutant placentae. epc, ectoplacental cone; ch, chorion; sp, spongiotrophoblast; lab, labyrinth. Bar represents 100 µm.

(Lescisin et al., 1988), were both expressed normally in Mrj (5-6 somite stage). We found that both VCAM1 and α4 integrin mutant placentae (Fig. 7). E-cadherin is expressed by basal cells were expressed in the mutants similar to wild-type and in the chorionic plate prior to allantoic fusion (Reuss et al., heterozygous conceptuses (Fig. 8; data not shown). The timing 1996), a pattern which was unaltered in Mrj mutants (data not of receptivity for chorioallantoic fusion is thought to be tightly shown). However, in situ hybridization analysis showed that regulated (Downs, 1998). It was notable, therefore, that we saw signals for Gcm1 (Altshuller et al., 1996), a gene whose persistent expression of α4 integrin and VCAM1 to at least E9.5 expression is restricted to the chorion at E8.5 and the in Mrj mutants in which chorioallantoic fusion had not occurred trophoblast component of the labyrinth at later stages (J. C. C., (Fig. 8). unpublished data), were dramatically reduced and difficult to discern above background at E8.5 coincident, therefore, with the ﬁrst observed defects in Mrj mutants. Hybridization signals DISCUSSION for another chorionic trophoblast marker, Err2 (Pettersson et al., 1996), were also reduced in mutants compared to wild-type and Chaperone and co-chaperone proteins have been highly heterozygous conceptuses (data not shown). conserved throughout evolution and are implicated in a number of cellular processes. We have described here the identiﬁcation Normal VCAM1 and α4 integrin expression in Mrj of Mrj, a gene encoding a new member of the DnaJ-related co- mutants chaperone family, and its expression pattern during To investigate the molecular basis of the chorioallantoic fusion development. We found that Mrj is expressed in the placenta, defect, we looked for abnormalities in expression of cell adhesion several regions of the embryo and in some tissues into molecules which are known to be involved. Around the time of adulthood. Mrj is essential for formation of the chorioallantoic chorioallantoic fusion (E8.25-8.5), α4 integrin is normally placenta, implying a previously unsuspected requirement for expressed on the surface of the chorion (Yang et al., 1995) and chaperone activity in this developmental process. VCAM1 is expressed on the distal two thirds of the allantois (Gurtner et al., 1995; Kwee et al., 1995). The expression of these Mrj expression is developmentally regulated proteins was assessed using immunohistochemistry on serial Mrj expression occurs broadly in several organs during histological sections (10 sections for each antibody) of three development and into postnatal life. We studied its expression homozygous mutant conceptuses that had been dissected at E8.5 during placental development in detail because the phenotype 1256 P. J. Hunter and others

albeit at low levels, but βgeo transcripts were not. It is possible that the βgeo insertion disrupted intronic sequences which regulate tissue-specific transcription or splicing. However, in all other tissues we observed a good correlation between Mrj and βgeo mRNA expression. Two other interesting features of expression were apparent in the trophoblast lineage. In the trophoblast giant cell population, while expression was detectable in all cells by mRNA in situ hybridization, a much higher expression level was observed in a subset of cells. The same pattern was observed when using the βgeo probe. These strongly expressing cells were randomly distributed around the conceptus in a pattern unlike any other gene expression pattern in giant cells that is known to us. Notably, the variable expression level was not apparent from the β-galactosidase staining, which was uniformly strong in every giant cell. An explanation for this difference is that the βgeo protein is stable and, therefore, persists in the cell even though Mrj mRNA expression may be variable. Another difference between Mrj and βgeo mRNA expression and β-galactosidase enzymatic activity was apparent in the spongiotrophoblast layer and its precursor, the ectoplacental cone. β-galactosidase activity was never observed in these trophoblast cells despite the presence of Mrj transcripts. Importantly, we detected βgeo transcripts in these cells indicating that splicing to the βgeo cassette occurred Fig. 8. VCAM1 and α4 integrin expression in the developing properly. It is unlikely that protein instability accounts for the chorioallantoic region at E8.5 and 9.5. Histological sections of wild- absence of β-galactosidase enzymatic activity, since it can be type and homozygous Mrj mutant (6AD1βgeo/6AD1βgeo) placentae detected in the ectoplacental cone and spongiotrophoblast of were subjected to immunostaining. Note that both VCAM1 and α4 ROSA26 conceptuses (Tanaka et al., 1997). It is possible, integrin continue to be expressed in Mrj mutants at E9.5 even though though, that a βgeo transcript with the Mrj 5′ untranslated fusion between the chorion and the allantois (al) has not occurred. region is not efficiently translated in ectoplacental cone or The allantois is not present in the E8.5 mutant conceptus shown spongiotrophoblast. It will be important to study MRJ protein because the embryo had been removed for genotyping. Bar expression in order to clarify these issues. represents 100 µm. Mrj is essential for chorioallantoic fusion at mid- of Mrj-deficient conceptuses indicated an essential function in gestation development of the chorioallantoic placenta. We readily Although the βgeo insertion in the 6AD1 cell line did not detected Mrj expression in trophoblast cells of the chorion but disrupt coding exons, it apparently created a null allele of the not in the chorionic mesothelium or the allantois. The Mrj gene because we were unable to detect Mrj mRNA in trophoblast lineage arises first as the trophectoderm at the conceptuses that were homozygous for the 6AD1βgeo allele. blastocyst stage (E3.5 in mice) (Cross et al., 1994; Rossant, This probably resulted from failure to splice around the βgeo 1995). By the early postimplantation period (E6.5-7.5), three cassette and truncation of the transcript by the polyadenylation anatomically and functionally distinct trophoblast cell types signal at the 3′ end of the βgeo sequence. We also noted that are apparent. Chorionic trophoblasts (also called there was a reduction in Mrj mRNA levels in heterozygotes extraembryonic ectoderm) lie next to the embryo; ectoplacental indicating that there was no compensation for loss of one allele. cone trophoblasts sit as a cap of tissue between the chorion and Despite this, heterozygotes had no obvious phenotypic defects the outer layer of trophoblast giant cells. Chorionic trophoblast though have not been examined in detail. cells, in addition to contributing to the labyrinth after contact It seems likely that the phenotype of Mrj-deficient with the allantois, are thought to be the proliferating conceptuses is due to the defect in placentation. The formation trophoblast stem cells (Rossant, 1995; Rossant and Ofer, of a chorioallantoic placenta is a critical ‘checkpoint’ that must 1977). In culture, chorionic trophoblast cells differentiate first be achieved by mid-gestation (Copp, 1995; Cross et al., 1994). into ectoplacental cone-type and subsequently to trophoblast We observed no obvious developmental defects in the embryo giant cells (Carney et al., 1993), suggesting that these three cell proper of Mrj mutants up to E9.25 as they were able to develop types represent stages in a differentiation pathway. Mrj mRNA to the 18 somite stage on schedule. Arrest of the mutant is, therefore, expressed throughout the trophoblast lineage embryos at the 18 somite stage (approximately E9.25) is also since we detected it in chorion, ectoplacental cone and giant observed in mouse embryos which are deficient for VCAM1 cells. Nonetheless, we have observed a mutant phenotype (Gurtner et al., 1995; Kwee et al., 1995) and α4 integrin (Yang associated with only the chorion of Mrj-deficient conceptuses. et al., 1995). For several reasons we favour the hypothesis that Expression studies revealed some potentially interesting the failure of chorioallantoic fusion is specifically due to features of Mrj regulation. First, there were differences defects in chorionic trophoblast function. First, Mrj expression between Mrj and βgeo transcript levels in some tissues. For appeared to be limited to the chorionic trophoblast cells and example, Mrj transcripts were detectable in the heart and eye, was not appreciably detected in the allantois or the chorionic DnaJ-related protein essential for placentation 1257 mesothelium. Second, at E8.25-8.5 (the period when binding and release of unfolded proteins (Hartl, 1996). Distal chorioallantoic fusion normally occurs) the allantois of Mrj to the J domain of MRJ, there are 6 Gly residues which are mutants appeared to be of normal size, showed no histological conserved with E. coli DnaJ; in other DnaJ-related proteins, a abnormalities and expressed the cell adhesion molecule Gly/Phe-rich region occurs at the same position. This region VCAM1. Third, we detected changes in two chorionic may form a flexible linker between the J domain and the rest trophoblast-specific transcription factor genes; downregulation of the protein (Pellecchia et al., 1996; Qian et al., 1996). The of Err2 and an apparent absence of Gcm1 expression. Err2- remainder of MRJ protein differs from E. coli DnaJ but shares deficient mouse mutants lack chorionic structures, thus regions of similarity with three mammalian DnaJ-like proteins, implicating Err2 in chorion cell proliferation (Luo et al., 1997) MSJ1, HSJ1 and HSP40. Chaperone function has been but the function of Gcm1 is unknown. Whether the reduction implicated in a number of cellular processes including protein of Err2 and Gcm1 expression in Mrj mutants is a primary cause folding and re-folding after cell stress (e.g., the heat shock of the phenotype or is secondary to other events is not clear. response), intracellular protein trafficking and protein-protein The fact that the chorion formed at the normal time and interactions (Hartl, 1996). persisted in Mrj mutants implies that the failure probably In contrast to prokaryotes, eukaryotic genomes appear to resulted from a lack of receptivity of the chorion. An encode multiple Hsp70- and DnaJ-related proteins. This raises alternative hypothesis is that chorionic trophoblast cells the question as to whether the multiple members have unique produce something which affects either the mesothelium or the or overlapping functions. In the budding yeast S. cerevisiae, allantois. The precise receptivity mechanism which is affected there are eight DnaJ-like genes and mutations in each produce in Mrj mutants is unresolved since the expression of α4 and distinct phenotypes (Cyr et al., 1994), indicating that they have VCAM1 was normal. non-overlapping functions. The J domain protein specificity is Chorioallantoic fusion was never observed in Mrj thought to reflect a restricted interaction with different homozygous mutants dissected at E8.5, in contrast to nearly all HSP70s, of which there are 14 in yeast (James et al., 1997), as of the wild-type and heterozygous littermates. Examination of well as different substrate binding abilities. In mammals, 11 conceptuses between E9.5 and 11.5 revealed that a loose Hsp70-related genes have been identified thus far (Tavaria et chorioallantoic attachment had occurred in a few homozygous al., 1996) compared to over 40 different J domain proteins (this mutants (8/50) (Table 2). Although the numbers of embryos study). There are only a few examples in higher eukaryotes of are too few to make a firm conclusion, there was a trend for chaperones whose essential functions have been identified by the frequency to increase with gestational age. Therefore, the loss-of-function gene mutations. In Drosophila, the lethal(2) most conservative interpretation of these data is that Mrj- tumorous imaginal discs (l(2)tid) gene encodes a J domain deficiency results in a significant delay in chorioallantoic protein which is involved in imaginal disc cell differentiation fusion. It is clear though that even if chorioallantoic fusion does (Kurzik-Dumke et al., 1995). In mice, Hsp70-2 deficiency occur by E9.5 or 10.5 in Mrj mutants, a functional results in arrest of spermatogenesis due to failure of cdc2 to chorioallantoic placenta does not form and the mutants die at associate with cyclin B1 despite the fact that other Hsp70s are a similar time in development. FGFR2, VCAM1 and α4 expressed in testis (Zhu et al., 1997). It is clear from these integrin are the only other molecules which have been examples that individual chaperones and co-chaperones can implicated, to date, in the process of chorioallantoic fusion have very specific functions during development, similar to our (Gurtner et al., 1995; Kwee et al., 1995; Xu et al., 1998; Yang observations with Mrj mutants. A role for chaperone activity et al., 1995). Notably, mouse mutants for all of these factors during development of the placenta was previously show only variably penetrant defects in chorioallantoic fusion unsuspected. In addition to our insights from Mrj mutants (Gurtner et al., 1995; Kwee et al., 1995; Xu et al., 1998; Yang though, it has recently been discovered that the chaperone et al., 1995). Chorioallantoic fusion was observed in two-thirds Hsp90β is also required for placental development in mice (A. of FGFR2 mutants (Xu et al., 1998), one-half of α4 integrin K. Voss, T. Thomas and P. Gruss, personal communication). mutants (Yang et al., 1995) and 9-50% of VCAM1 mutants Importantly by searching UniGene and EST databases, we (depending on genetic background) (Gurtner et al., 1995; Kwee have found that several DnaJ-related genes other than Mrj are et al., 1995). Given the incomplete penetrance, these studies expressed in the developing placenta: ESTs representing suggest that there may be independent pathways for several DnaJ-related genes have been identified in human chorioallantoic attachment. It would be interesting to placental libraries (7 different genes) as well as murine determine if inactivating both the FGF and α4 placental (1 gene) and ectoplacental cone libraries (2 genes) (J. integrin/VCAM1 pathways produces a more penetrant C. C., unpublished data). The occurrence of the placentation phenotype. defect in Mrj-deficient conceptuses, despite the fact that other DnaJ-related proteins are expressed in the placenta, supports Non-overlapping roles of chaperones during the idea that individual members of this gene family may have development non-overlapping cellular and molecular functions. The only recognizable motif in the MRJ sequence was the J domain at the N terminus of the protein. Based on the We thank M. Cybulsky, B. Hogan, D. Linzer, J. Rossant, P. Soriano conserved function of J domains, it is likely, though not yet for reagents, Z. Chen and R. Han for technical advice, A. Voss and P. Gruss for sharing information prior to publication, C. C. Hui, J. proved, that MRJ functions as a co-chaperone for an HSP70. Rossant and members of the Cross lab for discussions, and J. The J domain of DnaJ-like proteins binds to HSP70s and Copeman and P. Riley for comments on the manuscript. The work was thereby stimulates their ATPase activity (Burston and Clarke, supported by a grants from the MRC of Canada (to J. C. C.) and the 1995; Caplan et al., 1993). ATP hydrolysis allows NIH (HD29471 to G. E. L. and GMO 7507-18 to M. A. H.). P. H. conformational changes in the HSP70 necessary for the was supported by a studentship from the Genesis Foundation. G. E. 1258 P. J. Hunter and others

L. is an Established Investigator of the American Heart Association lethal(2)tumorous imaginal discs gene, a dnaJ homolog. Dev. Genetics 16, and J. C. C. is an MRC Scholar. 64-76. Kwee, L., Baldwin, H. S., Shen, H. M., Stewart, C. L., Buch, C., Buck, C. A. and Labow, M. A. (1995). Defective development of the embryonic and extraembryonic circulatory systems in vascular cell adhesion molecule REFERENCES (VCAM-1) deficient mice. Development 121, 489-503. Lescisin, K. R., Varmuza, S. and Rossant, J. (1988). Isolation and Altshuller, Y., Copeland, N. G., Gilbert, D. J., Jenkins, N. A. and Frohman, characterization of a novel trophoblast-specific cDNA in the mouse. Genes M. A. (1996). Gcm1, a mammalian homolog of Drosophila Glial Cells Dev. 2, 1639-1646. Missing. FEBS Lett. 393, 201-204. Li, E., Bestor, T. H. and Jaenisch, R. (1992). Targeted mutation of the DNA Baker, R. K., Haendel, M. A., Swanson, B. J., Shambaugh, J. C., Micales, methyltransferase gene results in embryonic lethality. Cell 69, 915-926. B. D. and Lyons, G. E. (1997). In vitro preselection of gene-trapped Luo, J., Sladek, R., Bader, J.-A., Matthyssen, A., Rossant, J. and Giguere, embryonic stem cell clones for characterizing novel developmentally V. (1997). Placental abnormalities in mouse embryos lacking the orphan regulated genes in the mouse. Dev. Biol. 185, 201-214. nuclear receptor ERR-β. Nature 388, 778-782. Berruti, G., Perego, L., Borgonovo, B. and Martegani, E. (1998). MSJ-1, a Millen, K. and Hui, C. (1996). Radioactive hybridization of tissue sections. new member of the DnaJ family of proteins, is a male germ cell-specific In A Laboratory Guide to RNA: Isolation, Analysis and Synthesis, (ed. P. gene product. Exp. Cell Res. 239, 430-441. Krieg), pp. 339-355. New York: Wiley-Liss. Brightman, S. E., Blatch, G. L. and Zetter, B. R. (1995). Isolation of a mouse Nagy, A., Rossant, J., Nagy, R., Abramow-Newerly, W. and Roder, J. C. cDNA encoding MTJ1, a new murine member of the DnaJ family of (1993). Derivation of completely cell culture-derived mice from early- proteins. Gene 153, 249-254. passage embryonic stem cells. Proc. Natl. Acad. Sci. USA 90, 8424-8428. Burston, S. and Clarke, A. R. (1995). Molecular chaperones: physical and Ohtsuka, K. (1993). Cloning of a cDNA for heat-shock protein hsp40, a mechanistic properties. Essays Biochem. 29, 125-136. human homologue of bacterial DnaJ. Biochem. Biophys. Res. Commun. 197, Caplan, A. J., Cyr, D. M. and Douglas, M. G. (1993). Eukaryotic 235-240. homologues of Escherichia coli dnaJ: a diverse family that functions with Pellecchia, M., Szyperski, T., Wall, D., Georgopoulos, C. and Wuthrich, hsp70 stress proteins. Mol. Biol. Cell 4, 555-563. K. (1996). NMR structure of the J-domain and the Gly/Phe-rich region of Carney, E. W., Prideaux, V., Lye, S. J. and Rossant, J. (1993). Progressive the Escherichia coli DnaJ chaperone. J. Mol. Biol. 260, 236-250. expression of trophoblast-specific genes during formation of mouse Pettersson, K., Svensson, K., Mattsson, R., Carlsson, B., Ohlsson, R. and trophoblast giant cells in vitro. Mol. Reprod. Dev. 34, 357-368. Berkenstam, A. (1996). Expression of a novel member of estrogen response Cheetham, M. E., Brion, J. P. and Anderton, B. H. (1992). Human element-binding nuclear receptors is restricted to the early stages of chorion homologues of the bacterial heat-shock protein DnaJ are preferentially formation during mouse embryogenesis. Mech. Dev. 54, 211-223. expressed in neurons. Biochem. J. 284, 469-476. Piechaczyk, M., Blanchard, J. M., Marty, L., Dani, C., Panabieres, F., El Chellaiah, A., Davis, A. and Mohanakumar, T. (1993). Cloning of a unique Sabouty, S., Fort, P. and Jeanteur, P. (1984). Post-transcriptional human homologue of the Escherichia coli DNAJ heat shock protein. regulation of glyceraldehyde-3-phosphate-dehydrogenase gene expression Biochim. Biophys. Acta 1174, 111-113. in rat tissues. Nucleic Acids Res. 12, 6951-6963. Colosi, P., Talamantes, F. and Linzer, D. I. (1987). Molecular cloning and Qian, Y. Q., Patel, D., Hartl, F. U. and McColl, D. J. (1996). Nuclear expression of mouse placental lactogen I complementary deoxyribonucleic magnetic resonance solution structure of the human Hsp40 (HDJ-1) J- acid. Mol. Endocrinol. 1, 767-776. domain. J. Mol. Biol. 260, 224-235. Copp, A. J. (1995). Death before birth: clues from gene knockouts and Raabe, T. and Manley, J. L. (1991). A human homologue of the Escherichia mutations. Trends Genet. 11, 87-93. coli DnaJ heat-shock protein. Nucleic Acids Res. 19, 6645. Cross, J. C., Flannery, M. L., Blanar, M. A., Steingrimsson, E., Jenkins, Reuss, B., Hellmann, P., Dahl, E., Traub, O., Butterweck, A., Grummer, N. A., Copeland, N. G., Rutter, W. J. and Werb, Z. (1995). Hxt encodes a basic helix-loop-helix transcription factor that regulates trophoblast cell R. and Winterhager, E. (1996). Connexins and E-cadherin are development. Development 121, 2513-2523. differentially expressed during trophoblast invasion and placenta Cross, J. C., Werb, Z. and Fisher, S. J. (1994). Implantation and the placenta: differentiation in the rat. Dev. Dyn. 205, 172-182. key pieces of the development puzzle. Science 266, 1508-1518. Riley, P., Anson-Cartwright, L. and Cross, J. C. (1998). The Hand1 helix- Cyr, D. M., Langer, T. and Douglas, M. G. (1994). DnaJ-like proteins: loop-helix transcription factor is essential for placentation and cardiac molecular chaperones and specific regulators of Hsp70. Trends Biochem. morphogenesis. Nature Genetics 18, 271-275. Sci. 19, 176-181. Rinkenberger, J., Cross, J. C. and Werb, Z. (1997). Molecular genetics of Downs, K. (1998). The murine allantois. Curr. Topics Dev. Biol. 39, 1-33. implantation in the mouse. Dev. Genetics 21, 6-20. Downs, K. M. and Gardner, R. L. (1995). An investigation into early Rossant, J. (1995). Development of the extraembryonic lineages. Semin. Dev. placental ontogeny: allantoic attachment to the chorion is selective and Biol. 6, 237-247. developmentally regulated. Development 121, 407-416. Rossant, J. and Ofer, L. (1977). Properties of extra-embryonic ectoderm Friedrich, G. and Soriano, P. (1991). Promoter traps in embryonic stem cells: isolated from postimplantation mouse embryos. J. Embryol. Exp. Morphol. a genetic screen to identify and mutate developmental genes in mice. Genes 39, 183-194. Dev. 5, 1513-1523. Shawlot, W. and Behringer, R. R. (1995). Requirement for Lim1 in head- Glueksohn-Shoenheimer, S. (1944). The development of normal and organizer function. Nature 347, 425-430. homozygous brachy (T/T) mouse embryos in the extraembryonic coelom of Tanaka, M., Gertsenstein, M., Rossant, J. and Nagy, A. (1997). Mash2 acts the chick. Proc. Natl. Acad. Sci. USA 30, 134-140. cell autonomously in mouse spongiotrophoblast development. Dev. Biol. Guillemot, F., Nagy, A., Auerbach, A., Rossant, J. and Joyner, A. L. (1994). 190, 55-65. Essential role of Mash-2 in extraembryonic development. Nature 371, 333- Tavaria, M., Gabriele, T., Kola, I. and Anderson, R. L. (1996). A 336. hitchhiker’s guide to the human Hsp70 family. Cell Stress Chaperones 1, Gurtner, G. C., Davis, V., Li, H., McCoy, M. J., Sharpe, A. and Cybulsky, 23-28. M. I. (1995). Targeted disruption of the murine VCAM1 gene: essential role Thomas, S. M., Soriano, P. and Imamoto, A. (1995). Specific and redundant of VCAM-1 in chorioallantoic fusion and placentation. Genes Dev. 9, 1-14. roles of Src and Fyn in organizing the cytoskeleton. Nature 373, 702-705. Hartl, F. U. (1996). Molecular chaperones in cellular protein folding. Nature Xu, X., Weinstein, M., Li, C., Naski, M., Cohen, R. I., Ornitz, D. M., Leder, 381, 571-580. P. and Deng, C. (1998). Fibroblast growth factor receptor 2 (FGFR2)- James, P., Pfund, C. and Craig, E. (1997). Functional specificity among mediated reciprocal regulation loop between FGF8 and FGF10 is essential Hsp70 molecular chaperones. Science 275, 387-389. for limb induction. Development 125, 753-765. Jonsson, J. I., Wu, Q., Nilsson, K. and Phillips, R. A. (1996). Use of a Yang, J. T., Rayburn, H. and Hynes, R. O. (1995). Cell adhesion events promoter-trap retrovirus to identify and isolate genes involved in mediated by alpha-4 integrins are essential in placental and cardiac differentiation of a myeloid progenitor cell line in vitro. Blood 87, 1771- development. Development 121, 549-560. 1779. Zhu, D., Dix, D. J. and Eddy, E. M. (1997). HSP70-2 is required for CDC2 Kurzik-Dumke, U., Gundacker, D., Rentrop, M. and Gateff, E. (1995). kinase activity in meiosis I of mouse spermatocytes. Development 124, Tumor suppression in Drosophila is causally related to the function of the 3007-3014.