cited1 is required in trophoblasts for placental development and

MOLECULAR AND CELLULAR BIOLOGY, Jan. 2004, p. 228–244 Vol. 24, No. 10270-7306/04/$08.00�0 DOI: 10.1128/MCB.24.1.228–244.2004Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Cited1 Is Required in Trophoblasts for Placental Development and forEmbryo Growth and Survival†

Tristan A. Rodriguez,1‡ Duncan B. Sparrow,2 Annabelle N. Scott,2 Sarah L. Withington,2 Jost I. Preis,2Jan Michalicek,3 Melanie Clements,1 Tania E. Tsang,2 Toshi Shioda,4 Rosa S. P. Beddington,1§ and

Sally L. Dunwoodie1,2,5*Mammalian Development Division, National Institute for Medical Research, London, United Kingdom1; Developmental BiologyProgram2 and Molecular Cardiology Program,3 Victor Chang Cardiac Research Institute, and Department of Biotechnology and

Biomolecular Sciences, University of New South Wales,5 Sydney, Australia; and Laboratory of Tumor Biology, MassachusettsGeneral Hospital Cancer Center, Charlestown, Massachusetts4

Received 4 November 2002/Returned for modification 13 December 2002/Accepted 18 September 2003

Cited1 is a transcriptional cofactor that interacts with Smad4, estrogen receptors � and �, TFAP2, andCBP/p300. It is expressed in a restricted manner in the embryo as well as in extraembryonic tissues duringembryonic development. In this study we report the engineering of a loss-of-function Cited1 mutation in themouse. Cited1 null mutants show growth restriction at 18.5 days postcoitum, and most of them die shortly afterbirth. Half the heterozygous females, i.e., those that carry a paternally inherited wild-type Cited1 allele, aresimilarly affected. Cited1 is normally expressed in trophectoderm-derived cells of the placenta; however, inthese heterozygous females, Cited1 is not expressed in these cells. This occurs because Cited1 is located on theX chromosome, and thus the wild-type Cited1 allele is not expressed because the paternal X chromosome ispreferentially inactivated. Loss of Cited1 resulted in abnormal placental development. In mutants, the spon-giotrophoblast layer is irregular in shape and enlarged while the labyrinthine layer is reduced in size. Inaddition, the blood spaces within the labyrinthine layer are disrupted; the maternal sinusoids are considerablylarger in mutants, leading to a reduction in the surface area available for nutrient exchange. We conclude thatCited1 is required in trophoblasts for normal placental development and subsequently for embryo viability.

The CITED (for CBP/p300-interacting transactivators withglutamic acid [E]/aspartic acid [D]-rich carboxyl-terminal do-main) gene family is represented by four genes: Cited1 (for-merly Msg1), Cited2 (formerly Mrg1), Cited3, and Cited4 (2, 12,17, 59, 60, 75). CITED proteins share three conserved regionsof homology (CR1, CR2, and CR3), outside of which they aredivergent. Given that they lack sequence identity to knownprotein domains, they therefore represent a novel family ofproteins. When tethered to heterologous DNA-binding do-mains, CITED proteins activate transcription, a function thatis dependent on the CR2 domain that binds the transcriptionalcoactivators CBP/p300 (11, 73).

No evidence exists that CITED proteins bind DNA; how-ever, they can interact with DNA-binding proteins and poten-tiate the activation of reporter constructs in vitro. For example,Cited1 binds Smad4, and estrogen receptors � and � (ER�/�)(61, 74); Cited2 binds the LIM domain of Lhx2; and Cited2and Cited4 bind TFAP2, as does Cited1 but weakly (6, 12, 24,75).

Embryonic expression of the CITED family during variousstages of vertebrate development has been analyzed (2, 17, 55,

75). In the mouse, Cited1, Cited2, and Cited4 (Cited3 is notpresent in mammals) each display restricted and distinct ex-pression profiles; however, there is some overlap in the expres-sion of Cited1 and Cited2. We have previously shown thatCited1 transcripts are localized to progenitors of the heart,limb, axial skeleton, and placenta (17). Cited1 is also expressedin the heart and mammary gland in adults, as well as melano-cytes, melanoma cells, and papillary thyroid carcinoma (17, 28,34, 60, 73).

To investigate the function of Cited1 during embryonic de-velopment, we generated a Cited1 null mutant mouse. Here wereport that the penetrance of the Cited1 null phenotype isdependent on the genetic background and that the placentadevelops abnormally in Cited1 mutants. We show that Cited1(an X-linked gene) is expressed in placental trophoblasts andthat it is required in these cells for normal placental develop-ment. In addition, we demonstrate that the spongiotrophoblastlayer is irregular in shape and that the maternal blood sinu-soids of the labyrinth are greatly enlarged in Cited1 mutants.Consistent with this, Cited1 mutants are small in the perinatalperiod, with the mutation resulting in death on the day of birthor shortly after. The exact cause of death is unknown; however,it appears to be a consequence of aberrant placental functionlate in gestation.

MATERIALS AND METHODS

Targeting vectors and generation of the Cited1flox and Cited1neo mutant mouselines. Two genomic clones spanning the Cited1 locus were isolated using Cited1cDNA (17). A 20-kb Cited1 genomic clone (Cited1/sv) was isolated from a 129svlibrary (Stratagene), and a 17-kb Cited1 genomic clone (Cited1/Ola) was isolatedfrom a 129Ola genomic library. The targeting construct contained a 6.5-kb 5�

* Corresponding author. Mailing address: Victor Chang CardiacResearch Institute, 384 Victoria St., Darlinghurst, NSW 2010, Austra-lia. Phone: 612 9295 8513. Fax: 612 9295 8501. E-mail: [email protected].

† Dedicated to the memory of Rosa Beddington (23 March 1956 to18 May 2001).

‡ Present address: MRC Clinical Sciences Centre, Imperial College,London, United Kingdom.

§ Deceased.

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FIG. 1. Generation of Cited1 null mutant mice. (A) Schematic representation of the Cited1 locus, targeting vector, and Cited1flox and Cited1neo

alleles. The exon (black box), translation start site (ATG), LoxP site (empty box), GFP, flip recombinase target (FRT), phosphoglycerate kinaseI promoter (PGK), neomycin gene (neo) are shown. (B) Southern blot analysis distinguishing between wild-type ES cells (XY) and those carryingthe targeted allele (XfloxY). The 5� probe hybridizes with a 9.5-kb fragment following BamHI restriction in wild-type (XY) ES cells and an 8.5-kbfragment in correctly targeted XfloxY ES cells. (C) Southern blot analysis distinguishes between the Cited1 targeted (Xflox) and null (Xneo) allelesin mice. A neo-derived probe hybridizes with a 9.5-kb fragment identifying the XfloxX� progeny and a 4-kb fragment identifying the XneoX�

progeny. (D) Genotype and sex of embryos and mice as determined using PCR. Three primers distinguish between the Cited1 wild-type (Cited1�)and null (Cited1neo) alleles. Males possess the Sry gene located on the Y chromosome. Heterozygous female, Cited1�/neo, wild-type male, Cited1�/Y;null male, Cited1neo/Y; null female, Cited1neo/neo.

VOL. 24, 2004 Cited1 IS REQUIRED FOR NORMAL PLACENTAL DEVELOPMENT 229

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homology arm (derived from Cited1/Ola) interrupted by a LoxP sequence 51 bp5� of the Cited1 ATG. A second LoxP sequence, preceded by translation stopcodons in all reading frames, was placed 5� of the coding sequence for the greenfluorescent protein (GFP), the Pgk-neo expression cassette flanked by FRT sites,and a 2.5-kb 3� homology arm (derived from Cited1/sv) (18, 79) (Fig. 1A). ThePgk-Tk expression cassette was used for negative selection. This vector waslinearized with XhoI and electroporated into E14.2 embryonic stem (ES) cells asdescribed previously (62). Following double selection with G418 and ganciclovir,209 ES cell clones were picked, expanded, and frozen by standard methods (27).Homologous recombinants were identified following BamHI restriction and hy-bridization with sequences located 5� and external to the homology arms (Fig. 1Aand B). A total of 13 targeted clones were identified, and chimeric males weregenerated from 1 of these clones following blastocyst injection by standardmethods (27). These mice were mated with C57BL/6 females to establish F0

heterozygous females. Animals carrying this conditional allele of Cited1 weredesignated Cited1flox. The Cited1 coding region was deleted in vivo to generatethe Cited1neo mouse line as follows: CBAxC57BL/10 F1 females were mated to F1

Cited1flox males generated from the cross between F0 Cited1flox females andC57BL/6 males. A CMV-Cre plasmid was introduced by pronuclear injection intofertilized eggs from these mice by standard procedures (27, 43). Digestion withBamHI and hybridization with a probe specific for the neo gene allowed identi-fication of mice carrying the null Cited1 allele (Cited1neo) (Fig. 1C).

Genotyping of the wild-type and modified Cited1 alleles. Mice and embryoswere genotyped by PCR. DNA samples were prepared from tails, yolk sacs, orwhole embryos as described previously (27). The PCR primers used to distin-guish between the Cited1� and Cited1neo alleles were Cited1-F (5�-TTACTTGCAGACCAACAGGC-3�), Cited1-R (5�-TGCTTCTTTGACCCATTTCC-3�),and GFP-R (5�-TGTTGCATCACCTTCACCCT-3�). The fragment sizes gener-ated were 367 bp for the Cited1� allele and 206 bp for the Cited1neo allele. Theprimers were used at a ratio of 1:2:1, respectively. Embryos were sexed by usingSry primers: Sry F (5�-TTCAGCCCTACAGCCACATGA-3�) and Sry R (5�-ATGTGGGTTCCTGTCCCACTG-3�) (63).

Histological analysis. In all cases, the central region of the placenta wasexamined since this is where the morphology is well defined and most consistent.For histological analysis, embryos were fixed in Bouin’s fixative, dehydrated,embedded in paraffin wax, sectioned (7-�m-thick sections), and stained withhematoxylin-eosin (29).

Placentas for cryosection were dissected at 14.5 days postcoitum (p.c.), fixed in4% paraformaldehyde (in phosphate-buffered saline [PBS]) overnight at 4°C,incubated at 4°C overnight in 30% sucrose in PBS, incubated in OCT (BDH) for10 min at room temperature, and frozen at �80°C. Sections (10 to 12 �m thick)were cut, allowed to dry, and either refrozen (at �80°C) or immediately pro-cessed for RNA in situ hybridization (16). RNA probes were generated aspreviously described: Cited1 (17), 4311 (Tpbp) (14), Mest (Peg1) (38), proliferin(PLF) (33), and mouse placental lactogen II (mPLII) (58). The sections werecounterstained with eosin.

For immunohistochemistry, placentas were dissected on day 14.5 or 16.5 p.c.,fixed in 4% paraformaldehyde (in PBS), paraffin embedded, and serially sec-tioned. Dewaxed sections were incubated with an affinity-purified rabbit anti-CITED1 antibody raised against residues 188 to 202 at the carboxy terminus ofCITED1 (60), and the antigen was visualized with a Vectastain ABC kit asspecified by the manufacturer (Vector Labs). Alkaline phosphatase activity wasdetected by dewaxing sections and incubating them in nitroblue tetrazolium–5-

bromo-4-chloro-3-indolyl phosphate (NBT/BCIP) substrate (Roche) for 15 minat room temperature in the dark. The sections were subsequently counterstainedwith nuclear fast red.

Vascular casts. To make fetal vascular casts, resin injections were performedusing Batson’s no. 17 plastic replica and corrosion kit (containing methyl methac-rylate casting resin) (Polysciences Inc.). Aliquots (2 ml) of base solution con-taining either red or blue pigment were prepared, and either 1 ml of catalyst or10 �l of promoter was added. Embryos aged 16.5 days p.c. were dissected intoM2 medium (27) plus 10% fetal calf serum, taking care to remove all membranessurrounding the umbilical vessels. At this point, the aliquots of blue base solutionwere mixed, drawn into a 1-ml syringe, attached to a 30.5-gauge needle, andplaced on ice. Each embryo was placed into a 3-cm petri dish containing M2medium, and the placenta was pulled over the side of the dish so that theumbilical vessels were dry and under tension. Chilled heparinized xylocaine (1%xylocaine in 0.9% NaCl with 1 IU of heparin/ml) was first injected into theumbilical artery, to dilate the vasculature and clear the placenta of blood. Blueresin was then also injected into the umbilical artery; this was repeated for everyplacenta in the litter. The aliquots of red base solution were then mixed, drawnup into a syringe, and injected into each umbilical vein, also using a 30.5-gaugeneedle. Following the injections, the placentas were washed in PBS and left at4°C at least overnight to allow the resin to set. Fetal vascular casts were gener-ated and analyzed for 21 wild-type (Cited1�/Y or Cited1�/�), 3 heterozygousfemales (Cited1�p/neo), and 9 null male (Cited1neo/Y) placentas.

Maternal vascular casts were made by methods previously described by Ad-amson et. al. (1) and modified as follows. Female mice on day 16.5 of pregnancywere anesthetised with an intraperitoneal dose of ketamine (100 mg/kg of bodyweight) and xylazine (20 mg/kg of body weight). Each mouse was ventilated withO2 at 120 breaths/min and a volume of 0.5 ml. Occlusive catheters filled withheparinized saline were inserted into the right carotid and the femoral artery fordrug infusion and continuous pressure monitoring, respectively. The chest wasopened, and 0.2 ml of 10-IU/ml heparin at 0.1 ml/min was infused into the rightcarotid artery. This was then followed by infusion of 3 mM KCl solution (0.1 to0.3 ml) to stop the heart. For arterial casts, a polyethylene catheter (0.80 mm[outer diameter] and 0.50 mm [inner diameter]) was placed into the aortic arch.The right atrium was cut so that it could serve as an exit point. An infusion pump(sp200i syringe pump; KD Scientific) was used to perfuse the animal’s circulationwith 10 ml of warm (40 to 50°C) heparinized saline (0.9% NaCl, 1 IU ofheparin/ml, 1% xylocaine) at 2 ml/min, followed by 10 ml of the same perfusatechilled to 4°C. The prechilled mixed resin was infused at 0.2 ml/min. This ratewas reduced during infusion, so that the femoral arterial pressure never exceededthe physiologically comparable pressure of 100 mm Hg. A total volume of 3 mlof plastic mixture was infused, and then a ligature was tightened around theintrathoracic vena cava. The infusion syringe was kept under pressure (20 mmHg) until the plastic had set. For venous casts, a polyethylene catheter wasinserted into the intrathoracic vena cava in a retrograde direction and then theinfusion procedure described above was followed; however, only 2 ml of resinwas used. Maternal vascular casts were generated and analyzed for 15 wild-type(Cited1�/Y or Cited1�/�), 10 heterozygous female (Cited1�p/neo), and 10 nullmale (Cited1neo/Y) placentas.

After both fetal and maternal injections, the internal cast was visualized bydigesting the surrounding placental tissue with 20 to 30% KOH in H2O. Thecasts were washed thoroughly in distilled water, examined by light microscopy(Leica MZ8 stereo dissecting microscope), air dried, sputtered with gold, andanalyzed with a scanning electron microscope (Cambridge S360).

Morphometric and statistical analysis. Morphometric analysis was performedusing NIH Image 1.62 software. For comparison of the areas of the labyrinthineand spongiotrophoblast layers of the placentas, cryosections processed by RNAin situ hybridization for 4311 expression were analyzed. The cross-sectional areaof each of these layers was determined for each of 12 sections (taken every eighthsection, 12 �m wide, starting from the center of the placenta) from two wild-type(Cited1�/Y) and two null (Cited1neo/Y) placentas at 14.5 days p.c. These data werecompared using analysis of variance.

For comparison of the maternal sinusoid size within the labyrinthine layer,placental sections processed for alkaline phosphatase activity were analyzed. Thelength around the sinusoids and the cross-sectional area of the sinusoids weremeasured. The sinusoids were measured from three sections (720 by 530 �m),160 �m apart, taken from a wild-type (Cited1�/Y or Cited1�/�), a heterozygousfemale (Cited1�p/neo), and a null male (Cited1neo/Y) placenta. A total of 151 to255 measurements were made for each placenta. These data were not normallydistributed, either before or following transformation; therefore, the nonpara-metric Wilcoxon test was employed.

For comparison of the sizes of the fetal capillaries within the placenta, thediameter of the capillaries was measured from scanning electron micrographs.

TABLE 1. Genotype of offspring from Cited1�/Y � Cited1�/neo andCited1neo/Y � Cited1�/neo intercrosses with a mixed genetic

background (129Ola; C57BL/6; C57BL/10; CBA)a

IntercrossNo. of offspring with Cited1 genotype of:

P�m/Y neo/Y �p/�m �p/neo neo/neo

Cited1�/Y � Cited1�/neo 49 43 64 59 NAb 0.17Cited1neo/Y � Cited1�/neo 110 81 NA 115 71 0.002

a Crosses were performed as designated, and the genotype was established onpostnatal day 10. Cited1 is on the X chromosome; therefore, wild-type males areindicated by �/Y; hemizygous (null) males are indicated by neo/Y; wild-typefemales are indicated by �/�; heterozygous females are indicated by �/neo, andhomozygous null females are indicated by neo/neo. Ratios of genotypes weretested for goodness of fit to expected Mendelian segregation (1:1:1:1) by chi-square analysis, calculated with 3 degrees of freedom. �p, paternal wild-typeallele; �m, maternal wild-type allele.

b NA, not applicable.

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The diameter was measured 20 �m from the tip of the capillary in 16 to 72capillaries from two to six different capillary beds in three wild-type (Cited1�/Y orCited1�/�) and four null male (Cited1neo/Y) individuals. These data were com-pared using nested analysis of variance.

Primordial germ cell analysis. Embryos were collected on day 8.5 p.c., andalkaline phosphatase-expressing cells were identified by the method of Lawson etal. (30) with the following modifications. The number of somites was recordedbefore fixation in 4% paraformaldehyde (more than 2 h at 4°C). Embryos weredehydrated in 70% ethanol overnight and washed three times in PBS at roomtemperature and twice with NTMT (100 mM NaCl, 100 mM Tris-HCl [pH 9.5],50 mM MgCl2, 0.1% Tween 20) with 3 mM levamisole (Sigma). Primordial germcells were stained in NBT/BCIP (GIBCO BRL). Whole embryos were embeddedin paraffin, serially sectioned at 8 �m thick, and processed for histologicalanalysis. Primordial germs cells were counted from 6 wild-type (Cited1�/Y orCited1�/�), 5 heterozygous female (Cited1�p/neo), and 16 null (Cited1neo/Y orCited1neo/neo) embryos.

RESULTS

Targeted disruption of the Cited1 gene and generation ofnull mutant mice. The conditional Cited1flox allele was gener-ated by homologous recombination in E14.2 mouse ES cells.The Cited1 locus, targeting vector, targeted allele, and deletedallele are represented in Fig. 1A. Homologous recombinationin ES cell clones was identified by Southern blot analysis (Fig.1B). The entire Cited1 coding region was subsequently deletedvia Cre-mediated recombination in vivo following pronuclearinjection of a Cre-expressing plasmid. Founder mice carryingthe Cited1 null allele (Cited1neo) were identified by Southernblot analysis (Fig. 1C). Initial confirmation of genotype wasachieved using Southern blot analysis. After this, the genotypewas determined using PCR (Fig. 1D). The targeting vector wasdesigned so that Cre-mediated recombination would bring thecoding region of GFP under the transcriptional control of theCited1 locus. However, no expression of GFP could be de-tected in either ES cell-derived embryoid bodies or embryos(data not shown). To investigate this lack of expression,genomic DNA from mice carrying the Cited1neo allele wassequenced and a single nucleotide insertion was detected thatcaused a frameshift in the GFP coding region. This frameshift

would result in the production of a premature terminationcodon (data not shown). To establish that the Cre-mediatedrecombination had occurred as predicted, deletion of the Cit-ed1 locus (from 51 nucleotides upstream of the Cited1 trans-lation start site) was confirmed by sequencing the Cited1neo

allele from genomic DNA (data not shown).On a mixed genetic background Cited1neo affects viability

and growth. In the mouse, Cited1 maps to the X chromosomeat 40.1 centimorgans (cM) (20). Therefore, mice had to beintercrossed in two distinct ways to generate progeny that rep-resent each possible genotype. Crosses between wild-type Cit-ed1 males (Cited1�/Y) and heterozygous Cited1 females (Cit-ed1�/neo) were expected to produce the following offspringwith equal probability: Cited1�/Y, Cited1neo/Y, Cited1�/neo, andCited1�/� (Table 1). This intercross showed that although thegenotype of progeny (on postnatal day 10) did not significantlydeviate from the Mendelian ratio, 12% fewer Cited1 null males(Cited1neo/Y) survived compared to wild-type males. However,in the cross between Cited1 null males (Cited1neo/Y) and het-erozygous Cited1 females (Cited1�/neo), the ratio of progeny thatsurvived (Cited1�/Y, Cited1neo/Y, Cited1�/neo, and Cited1neo/neo) de-viated significantly from the Mendelian ratio; there were 20%fewer Cited1 null mice than wild-type mice (Table 1). Survivingnull mice were smaller than their littermates; therefore, micewere weighed at birth and at 32 regular time points until theyreached 160 days old. On the day of birth, Cited1neo/Y males were23% lighter than Cited1�/Y control littermates while Cited1neo/neo

females were 19% lighter than Cited1�/neo controls (Fig. 2A).After 160 days, Cited1 null mice were not significantly different inweight from control littermates (Fig. 2B). Taken together, thesedata demonstrate that some Cited1 null mice die prior to postna-tal day 10 and that those that do survive are smaller at birth thanthe controls are. Since the growth deficit is not apparent after 160days, this suggests that Cited1 null individuals do not suffer froma growth defect per se but, rather, that they are likely to besubjected to an impaired uterine environment.

FIG. 2. Loss of Cited1 results in reduced embryo weight at birth. Cited1neo/Y � Cited1�/neo crosses were performed. Weights are shown on PND0(birth) (A) and PND160 (B). Genotype annotation is as described for Table 1. The mean weight (and standard error of the mean) was plottedagainst genotype. Nested analysis of variance demonstrates that the weight of Cited1 mutants (Cited1neo/neo and Cited1neo/Y) was significantlydifferent from that of controls (Cited1neo/� and Cited1�/Y) at birth (P � 0.0022). At 160 days, although the weights are different between the sexes,there is no statistical difference between genotypes within each sex.


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Death due to the Cited1 null allele occurs at birth, is depen-dent on genetic background, and is due to a defect in tropho-blast-derived extraembryonic tissues. In combination with aninbred genetic background, death occurred in the majority ofCited1 null individuals and in a considerable proportion ofheterozygous females (Table 2). The loss of heterozygous fe-males represents a parent-of-origin effect since those that suc-cumbed to the mutation did so only when the Cited1 wild-typeallele was inherited from their father. Penetrance of the phe-notype associated with the Cited1 null allele was increased inthe presence of a C57BL/6 genetic background. The Cited1neo

allele was backcrossed six times with C57BL/6 individuals. Sub-sequently, when Cited1 null males (Cited1neo/Y) and heterozy-gous Cited1 females (Cited1�/neo) were crossed, null mutantswere underrepresented by 78% compared with controls (Cit-ed1�/Y) on postnatal day 28. The numbers of Cited1�/neo het-erozygous females were not different from the numbers ofcontrol mice derived from this cross. However, when wild-typeCited1 males (Cited1�/Y) and heterozygous Cited1 females (Cit-ed1�/neo) were crossed, there were 82% fewer Cited1 nullmales (Cited1neo/Y) than controls (Cited1�/Y) on postnatal day28. In addition, this cross revealed that there were 47% fewerheterozygous Cited1 females (Cited1�/neo) than control fe-males (Cited1�/�) (Table 2). Since viability is reduced in indi-viduals carrying the Cited1neo allele, we therefore determinedthe time when death occurred by identifying the genotype ofembryos at different stages of gestation. Table 2 demonstratesthat regardless of the type of parental cross, death did not

significantly occur during gestation. This was the case for bothCited1 null (Cited1neo/Y and Cited1neo/neo) and heterozygous(Cited1�P/neo) females. In fact, the majority of these individualsdied on the day of birth or shortly thereafter.

The underrepresentation of heterozygous Cited1 females(Cited1�/neo), derived from the Cited1�/neo � Cited1�/Y cross,demonstrates that there is a parent-of-origin effect on allelebehavior since the proportion of heterozygous females (Cit-ed1�/neo) on this background was not reduced when Cited1 nullmales (Cited1neo/Y) were crossed with heterozygous females(Cited1�/neo). Specifically, if a heterozygous Cited1 female (Cit-ed1�/neo) inherited a wild-type Cited1 allele from the mother,then the expected number of Cited1�/neo individuals survived;however, if the wild-type allele was inherited from the father,a significant proportion (47%) of heterozygous females died.This parent-of-origin effect affects embryo viability in heterozy-gous Cited1 females (Cited1�/neo) and is likely to be due to thedistinct regimes applied to X chromosome inactivation in spe-cific tissues of the conceptus. In females, X chromosome inac-tivation is random both in the embryo and in mesodermallyderived extraembryonic tissues. By contrast, in extraembryonicectoderm and endoderm, only the paternal X chromosome isinactivated (21, 47, 66). Therefore, heterozygous females thatinherit the Cited1 wild-type allele from their mother(Cited1�m/neo) are both genotypically heterozygous and phe-notypically wild type in extraembryonic ectoderm andendoderm. However, heterozygous females that inherit theCited1 wild-type allele from their father (Cited1�p/neo) are ge-

TABLE 2. Genotype of offspring from Cited1neo/Y � Cited1�/neo and Cited1�/Y � Cited1�/neo matings after six generations of backcrossingwith C57BL/6 wild-type animalsa

Backcross and dayNo. of offspring with Cited1 genotype of:

P�m/Y neo/Y �m/�p �m/neo �p/neo neo/neo

Cited1neo/Y � Cited1�/neo

Postnatal day 28 305 56 NAb 279 89 0.0001Postnatal day 0 27 18 (2 dead) 32 18 (2 dead) live 0.04Prenatal (total) 31 21 27 23 0.5118.5 days p.c. 1 1 3 0 0.2816.5 days p.c. 6 1 1 1 0.0415.5 days p.c. 2 3 2 1 0.9514.5 days p.c. 8 7 5 1 0.1412.5 days p.c. 5 2 6 8 0.319.5 days p.c. 3 4 6 6 0.708.5 days p.c. 6 3 4 5 0.77

Cited1�/Y � Cited1�/neo

Postnatal day 28 51 9 51 27 NA 0.0001Postnatal day 0 31 (1 dead) 15 (4 dead) 31 (1 dead) 17 (2 dead) live 0.003Prenatal (total) 124 136 121 125 0.8018.5 days p.c. 17 16 16 17 1.0016.5 days p.c. 67 79 64 66 0.5715.5 days p.c. 9 10 7 11 0.8114.5 days p.c. 9 11 12 13 0.8513.5 days p.c. 3 2 4 7 0.3212.5 days p.c. 8 7 9 4 0.5711.5 days p.c. 1 3 0 2 0.3410.5 days p.c. 6 4 5 3 0.779.5 days p.c. 4 4 4 2 0.84

a Crosses were performed as designated, with genotype annotation as described for Table 1. Wild-type males (�/Y) mated with heterozygous females (�/neo) produceCited1 heterozygous females that carry no active copy of the Cited1� allele in trophectoderm derivatives and are annotated �p/neo. Ratios of genotypes were testedfor goodness of fit to the expected Mendelian segregation (1:1:1:1) by chi-square analysis, calculated with 3 degrees of freedom. The paternal wild-type allele (�p), andmaternal wild-type allele (�m) are indicated. Postnatal day 0 is the day of birth.

b NA, not applicable.


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notypically heterozygous but phenotypically null in extraem-bryonic ectoderm and endoderm since the paternal Cited1�P

allele is inactivated. We conclude that Cited1 inactivation inextraembryonic ectoderm and/or endoderm is responsible forthe observed deaths.

Cited1 is required to maintain embryonic growth late ingestation. Since Cited1 mutants die shortly after birth, we de-termined embryo and placenta weight during the perinatalperiod (Fig. 3). Embryos and placentas were weighed on days14.5, 16.5, and 18.5 (day 18.5 p.c. is the day before birth). Inaddition, pups were weighed on the day of birth (postnatal day0 [PND0]). Placental weights were the same for all genotypesat all stages examined (Fig. 3A). The same held true for em-bryo weights on days 14.5 and 16.5 p.c. However on day18.5 p.c. and on PND0, both Cited1 null (Cited1neo/Y andCited1neo/neo) and heterozygous (Cited1�P/neo) femalesweighed significantly less than did the controls (Fig. 3B). Thesedata demonstrate that the Cited1 mutant phenotype is manifestin the embryo, late in gestation, but does not lead to death inutero (Table 2; Fig. 3).

Cited1 is expressed in trophectoderm-derived cells of theplacenta. The preceding genetic analysis demonstrates thatCited1 is required in extraembryonic ectoderm and/orendoderm for embryo survival. Using in situ analysis, we pre-viously reported that the Cited1 transcript is localized to ex-traembryonic ectoderm, visceral endoderm, and visceral yolksac endoderm (6.5 to 8.5 days p.c.); by Northern analysis, wefound that it is also localized to the placenta (11.5 days p.c.)(16, 54). It is most likely that loss of Cited1 in the extraembry-onic ectoderm-derived cells of the placenta (rather than inextraembryonic tissues during early development) is the causeof the late-onset death. To further examine Cited1 expression

in the placenta, we used in situ RNA hybridization and immu-nohistochemistry (Fig. 4). The mature placenta is establishedin mouse by day 10 p.c. and consists of trophoblasts (extraem-bryonic ectoderm) and endothelial and stromal cells (extraem-bryonic mesoderm). It is composed of three principal layers: anouter layer of secondary trophoblast giant cells, a middle spon-giotrophoblast layer, and the innermost labyrinth. The laby-rinth contains both trophoblasts and mesodermally derivedcells that are embryonic in origin. Cited1 was expressed in alltrophoblast-derived tissues of the placenta (Fig. 4). Cited1transcripts were localized to secondary giant cells and spon-giotrophoblasts (Fig. 4A to C). Expression was also detectedwithin the labyrinthine layer in cuboidal and elongated tropho-blasts (Fig. 4D) and in lining maternal blood spaces at the base(embryo side) of the labyrinth (Fig. 4E). Expression in cuboi-dal and elongated trophoblasts was particularly clear whennuclear �-galactosidase, targeted to the Cited1 locus, was an-alyzed (data not shown). Like the RNA, the Cited1 protein wasdetected in each of these trophoblast cell types (Fig. 4F). Ingiant cells and spongiotrophoblasts, the protein was localizedpredominantly to the nucleus but was also detected in thecytoplasm (Fig. 4G and H), while protein was localizedthroughout the cell in the labyrinthine layer (Fig. 4I and J).These data demonstrate that Cited1 is expressed in tropho-blast-derived placental cells but not in mesoderm-derived cells(such as the fetal vasculature). This supports the hypothesisthat Cited1 heterozygous females in which the Cited1� allele isinherited from the father (Cited1�P/neo) die due to inactivationof the paternally acquired Cited1�P allele in trophoblast-de-rived cells of the placenta.

Cited1 is required for normal placental morphology. Re-duced embryonic weight on day 18.5 p.c. indicated that pla-

FIG. 3. Loss of Cited1 results in reduced weight perinatally. Placenta (A) and embryo (B) weights are shown at various stages. All data werederived from the crossing of Cited1�/Y � Cited1�/neo individuals, except for embryo weights designated PND0b, which were taken from the progenyof a Cited1neo/Y � Cited1�/neo cross. Genotype annotation is as described for Table 1. For cross Cited1�/Y � Cited1�/neo, at 14.5, 16.5, and 18.5 daysp.c. and PND0a, wild-type males (n � 4, 12, 13, and 27) and females (n � 4, 12, 9, 31 [open squares]) were pooled since there was no statisticaldifference between these two groups. These were compared to heterozygous females (Cited1�p/neo, n � 6, 10, 12, and 17 [solid diamonds]) andhemizygous (null) males (n � 3, 14, 14, and 15 [solid squares]). For cross Cited1neo/Y � Cited1�/neo, wild-type males (n � 27) and heterozygousfemales (n � 32 [open squares]) were pooled since there was no statistical difference between the two groups. These were compared to null females(n � 18 [solid diamonds]) and hemizygous (null) males (n � 18; solid squares). The mean weight (and standard error of the mean) was plottedagainst genotype. Analysis of variance was used to identify differences between genotypes. Asterisks indicate a statistical difference betweenwild-type embryo weight and the weight of heterozygous females or hemizygous (null) males (P � 0.0001). Pound signs indicate statisticaldifference between control (wild-type males and heterozygous females [Cited1�m/neo]) and null females (P � 10�19) or hemizygous (null) males(P � 10�13).


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cental function was likely to be abnormal late in gestation;therefore, placentas were examined each day from days 14.5 to18.5 p.c. Hematoxylin-and-eosin staining of sections showedthat secondary giant cells, spongiotrophoblasts, and labyrin-thine trophoblasts were present in placentas carrying theCited1neo allele as well as in control placentas (data notshown). Gene expression analysis on day 14.5 p.c. demon-strated that the border between the spongiotrophoblast andlabyrinthine layers was irregular in Cited1 null males(Cited1neo/Y) and heterozygous females (Cited1�P/neo). Finger-like projections of spongiotrophoblasts (identified by 4311,PLF, and mPLII expression) often extended through the lab-yrinthine layer in mutants, unlike in controls (Fig. 5 and datanot shown). Serial sections showed that the fetal vasculature(identified by Mest expression) was disrupted (Fig. 5A to D)due to the projection of these spongiotrophoblasts (Fig. 5E toL). Although the total size of each placenta was the same,there was a significant increase in the spongiotrophoblast layerand a concomitant decrease in the labyrinthine layer in Cited1null males (Cited1neo/Y) compared with controls (Cited1�/Y)(Table 3). It was also clear that organization of the labyrinthwas disrupted in the mutant placentas. The size of trophoblast-lined maternal sinusoids (trophoblasts identified by alkalinephosphatase activity) was compared between Cited1 null males(Cited1neo/Y), heterozygous females (Cited1�P/neo), and con-trols (Cited1�/Y) (Fig. 6, Table 4, and data not shown). Thelength of the surface surrounding the maternal sinusoid and itscross-sectional area were measured from placental sections.The mean values of surface length and the cross-sectional areawere up to 62 and 262% greater in Cited1 mutants than incontrols. This defect is consistent with the fact that Cited1 isexpressed in labyrinthine trophoblasts (Fig. 4) and is requiredin the extraembryonic ectoderm, such as placental tropho-blasts, for neonate survival.

Cited1 is required for normal organization of the fetal andmaternal blood in the labyrinth. To determine the arrange-ment of fetal blood vessels and maternal blood sinusoids in thelabyrinthine layer of Cited1 mutants, we generated vascularcasts on day 16.5 p.c. since this was the earliest stage at whicha placental defect was detected using histological analysis. Thearterial and venous systems were filled with resin, and a com-bination of light microscopy and scanning electron microscopy(SEM) showed that placentas carrying the Cited1neo allele weredifferent from the controls. Maternal vascular casts were gen-erated following resin infusion from the arterial (Fig. 7) orvenous (data not shown) side. A lateral view of the vascularcast filled from the arterial side showed the arrangement of thematernal arteries and trophoblast-lined compartments of theplacenta. The organization of the radial arteries, spiral arter-ies, and central canals was similar to that described by Adam-son et al. (1) and appeared to be the same regardless of thegenotype. However, the labyrinth was irregular in shape, and

when it was viewed from the base of the placenta (proximal tothe embryo), it was clear that the arterial side casts differedbetween Cited1 mutants and controls. In all wild-type (Cit-ed1�/Y or Cited1�/� [n � 15]) placentas, canal branches andanastomosing sinusoids spread out producing an even circularbase, whereas in heterozygous females (Cited1�P/neo [n � 10])and null males (Cited1neo/Y [n � 10]) the base was very irreg-ular in shape. Although this was clearly evident by observingwith the naked eye, light microscopy and SEM revealed theextent of the irregularity (Fig. 7A to F). In addition, the sinu-soids were examined at several sites within the labyrinth (cen-trally, laterally, at the edges, and internally following breakingthe cast) using SEM. The degree of branching and the size ofthe sinusoids appeared to be uniform regardless of their posi-tion within the labyrinth (data not shown), and in the controlsthey were well branched and fairly regular in size. In contrast,the sinusoids in all mutants showed less branching and wereconsiderably larger (Fig. 7G to I). Due to the irregular shapeof the sinusoid casts, it was not possible to accurately quantifysize differences; however, mutant sinusoids were approxi-mately 2- to 3.5-fold greater in diameter than were those incontrol placentas. Sinusoids filled from the venous side weresimilarly large and poorly branched in mutant placentas (datanot shown). The increase in the size of the sinusoids shownhere in the resin casts was similar to the increases measuredfollowing histological analysis (Fig. 6).

Infusion of resin into the umbilical artery (blue) and vein(red) on day 16.5 p.c. generated casts of the fetal vasculature(Fig. 8). These dually filled casts showed that the umbilicalvessels extended into two planes of the placenta: within theplane of the base of the placenta, and through the labyrinthinelayer toward the spongiotrophoblast layer. Within the base ofthe placenta, the artery (blue) and vein (red) branched in aradial pattern; the artery branched a few times, stopping at theperiphery of the placenta, while the vein branched more ex-tensively and stopped before reaching the periphery (Fig. 8Aand B). The arterioles that expanded through the labyrinthinelayer extended toward the spongiotrophoblast layer, wherethey branched before anastomosing into a dense mass of cap-illaries that extended back toward the base of the placenta. Thevenuoles extended only a short distance into the labyrinthinelayer, without branching, and formed an equally dense capil-lary mass which extended up toward those derived from thearterioles (Fig. 8C to H and data not shown) (1). Althoughthere was a degree of variation in the casts generated, we wereunable to correlate this with the Cited1 genotype; therefore,the overall arrangement of fetal vasculature in both controland mutant placentas appeared to be essentially the same. Thewidth of the resin-filled arterioles that extended into the lab-yrinthine layer was measured, but no difference was observed(data not shown). Since fetal capillaries are surrounded bymaternal sinusoids, which are enlarged in Cited1 mutants, the

FIG. 4. Cited1 transcript and protein are expressed in trophectoderm-derived cells of the placenta. RNA in situ hybridization (A to E) andimmunohistochemistry (F to J) show Cited1 transcript and protein localization on day 14.5 p.c. Cited1 is expressed in secondary giant cells (arrowin panels B and G), spongiotrophoblasts (C and H), and cuboidal (arrow) and elongated (arrowhead) trophoblasts within the labyrinthine layer(D and I) and lining maternal blood spaces at the base (embryo side) of the labyrinthine layer (arrow in panels E and J). Fetal blood vessel (v),maternal sinus (s), and mesenchyme surrounding fetal blood vessel (m) are also shown. Bar, 1.2 mm (A and F), 310 �m (B, E, G, and J), and 150�m (C, D, H, and I).


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diameter of the fetal capillaries was determined. Those filledfrom the venous side were most similar to the controls, whilethose filled from the arterial side showed a small but significantincrease in diameter (Fig. 8 G and H; Table 5). The resin castsof wild-type placentas show that the maternal sinusoids andfetal capillaries are approximately the same size (Fig. 7G and8E and G). This presumably allows the efficient exchange ofgases and nutrients to occur between maternal and fetal blood.In contrast, the surface area available for exchange in Cited1mutants between the fetus and mother is greatly reduced dueto the increased size of the maternal sinusoids (Fig. 7H and Iand 8F and H).

Cited1 is not required for primordial germ cell productionor migration. In an effort to further analyze the Cited1 nullphenotype, we examined other tissues in which Cited1 is ex-pressed. Expression is detected in extraembryonic ectodermadjacent to embryonic ectoderm and posterior to the primitivestreak (17). These sites are significant with respect to the in-duction and collection of primordial germ cells (PGCs) respec-tively. PGCs are induced in the proximal embryonic ectoderm(6.5 days p.c.) due to inductive signals (BMP4 derived) fromthe adjacent extraembryonic ectoderm. Once induced, theymove through the primitive streak (30–32, 67). Having passedthrough the primitive streak, they reside in a cluster posteriorto the streak, in a position that will later become the base ofthe allantois (7.2 days p.c.) (23, 46). It is at this stage that PGCscan be identified as large cells possessing high levels of alkalinephosphatase (15, 23). The PGCs continue to express alkalinephosphatase as they proliferate and populate the developinghindgut (8.5 to 10.5 days p.c.) and as they migrate along thedorsal mesentery into the paired gonadal primordia (10.5 to13.5 days p.c.) (39). Due to the coincidence of Cited1 expres-sion with the site of induction and subsequent location ofPGCs (between 6.5 and 7.5 days p.c.) and the ability of Cited1to bind Smad4 (required for transforming growth factor �[TGF-�] and bone morphogenetic protein signaling) (5, 61),

we examined PGCs in embryos carrying the Cited1neo allele at8.5 days p.c. Large and readily discernible PGCs were residentin the hindgut endoderm of Cited1�/Y, Cited1�/neo, andCited1neo/neo embryos (Fig. 9). Although variation in the num-ber of PGCs consistent with previous reports was observed (30,69), there was no correlation between PGC number and thepresence of the Cited1neo allele. These findings demonstratethat Cited1 is not required for the induction or maintenance ofPGCs in mouse embryos up to day 8.5 p.c.

DISCUSSION

Cited1 gene function is affected by genetic background andmodifier loci. The Cited1 locus, like many other loci, is subjectto modification by elements within the genome that are yet tobe determined (42). This is clear since penetrance of the phe-notype (postnatal lethality) is influenced by the genetic back-ground. For example, on a mixed background (129Ola-C57BL/6-C57BL/10CBA) there was a 20% reduction in Cited1 nullmice compared to the wild type, while on a background derivedlargely from C57BL/6 (produced following six crosses back toC57BL/6), there was a 78% reduction in Cited1 null micecompared to the wild type (Tables 1 and 2).

Cited1 is required for normal development of the labyrin-thine layer. The Cited1 mutant placenta has an increased num-ber of spongiotrophoblasts that penetrate into the labyrinthinelayer, and late in gestation the maternal blood sinusoids aresignificantly larger than controls. In the mouse, the labyrin-thine layer of the placenta consists of fetal blood vessels andmaternal blood sinusoids enmeshed in trophoblasts. This layerbegins to function as a nutrient transport unit from about day10.5 p.c. It is here that gas, nutrient, and waste exchange occursbetween the mother and fetus, and so abnormal developmentor function of the labyrinth results in impaired fetal develop-ment.

Mutational analysis in the mouse has revealed that numer-ous genes are required for normal formation of the labyrin-thine layer (25, 53). In the majority of cases, labyrinth forma-tion is very limited and embryonic death occurs by day10.5 p.c.; this correlates well with the time when the labyrinthbegins to function as a nutrient transport unit (22). In somemutants, development of the labyrinthine layer is relativelyextensive, but fetal death occurs later in gestation (HGF; days13.5 to 15.5 p.c.) or during the perinatal period (Pdgfra, Pdgfb,Lifr, Wnt2, Esx1, and p185/Cul7) (4, 35, 41, 44, 45, 70, 71). TheCited1 mutant placenta associates most closely with this lattergroup since the labyrinth is well developed and death is post-natal. However, Cited1 mutants develop dilated maternal sinu-soids, in contrast to fetal vessel dilation (Pdgfra, Pdgfb, Esx1,and p185/Cul7) and maternal vascular lesions (Lifr and Wnt2).This indicates that the Cited1 mutant placental phenotype isunique among those studied.

FIG. 5. Placental structure is affected by the loss of Cited1. RNA in situ hybridization of serial placental sections on day 14.5 p.c. shows thelocalization of Mest (A to D), 4311 (E to H), and PLF (I to L) transcripts in Cited1�m/Y (A, C, E, G, I, and K) and Cited1neo/Y (B, D, F, H, J, andL) mice. Panel C and D (higher magnifications of panels A and B) show the extent of fetal vasculature in the labyrinthine layer, with arrowsindicating where the vasculature is interrupted. In panels G and H and panels K and L (higher magnifications of panels E and F and panels I andJ, respectively), arrows show where the fetal vasculature is interrupted by spongiotrophoblasts expressing 4311 and PLF. Bar, 1.4 mm (A, B, E,F, I, and J) and 454 �m (C, D, G, H, K, and L).

TABLE 3. Morphometric analysis of mouse placentas on day14.5 p.c. following RNA in situ hybridizationa

Location

Cross-sectional area (mm2) forCited1 genotype:

PWild type (�m/

Y, �p/�m) Null (neo/Y)

Total placenta 10.84 0.19 10.61 0.18 0.4151Spongiotrophoblasts 3.86 0.09 4.35 0.09 0.0004Labyrinthine layer 6.74 0.14 5.94 0.12 0.0001

a The cross-sectional area was measured from placentas that had been cryo-sectioned and hybridized with spongiotrophoblast gene marker 4311 (see Mate-rials and Methods). Mean areas are presented with standard error of the mean.Analysis of variance showed that wild-type (Cited1�p/�m, Cited1�m/Y) and null(Cited1neo/Y) placentas were always significantly different from each other, exceptwhen the total placental area was compared.


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The mouse placenta, like that of humans, is hemochorial;maternal blood is in direct contact with placental trophoblasts(72). The sinusoid spaces, which are filled with maternal blood,are formed as the chorioallantoic interface branches. The tran-scription factor Gcm1 is required for trophoblast branching; inits absence, the labyrinthine layer does not form and the ma-ternal sinusoids are large (1, 3, 56). It is unlikely that the largematernal sinusoids, apparent in Cited1 mutants, are associated

with impaired Gcm1 function because the Cited1 mutant phe-notype develops for up to 7 days after the Gcm1 phenotypebecomes apparent. If Gcm1 is involved, Cited1 must be re-quired late in gestation for Gcm1 expression or function. SinceCited1 mutants have an enlarged spongiotrophoblast layer andprojections of these cells extend into the labyrinthine layer, itis possible that the physical presence of spongiotrophoblastprojections into the labyrinthine layer interferes with tropho-

FIG. 6. Maternal blood sinusoids are enlarged in the absence of Cited1. (A to F) Alkaline phosphatase-expressing trophoblasts surroundmaternal blood sinusoids in the labyrinthine layer of Cited1�m/Y (A and B), Cited1�p/neo (C and D), and Cited1Y/neo (E and F) on day 16.5 p.c. Bar,1.26 mm (A, C, and E) and 100 �m (B, D, and F). (G) Length around sections of maternal blood sinusoids plotted against genotype shows thatthe sinusoids are larger in mutants (Cited1�p/neo and Cited1Y/neo) than in the control (Cited1�m/Y).

TABLE 4. Morphometric analysis of maternal blood sinusoids in placentas on day 14.5 p.c. following staining for alkalinephosphatase activitya

Measurement

Value in Cited1 genotype

PWild type (�m/Y) Heterozygous

(�p/neo) Null (neo/Y)

Surrounding length (�m) 52.22 1.86 84.69 5.16 63.06 2.53 0.0001Cross sectional area (�m2) 243.94 14.22 639.03 64.17 389.65 26.49 0.0001

a The surrounding length and cross-sectional area of maternal blood sinusoids was quantified from placental sections on day 14.5 p.c. (see Materials and Methods).Mean areas are presented with standard error of the mean. These data were not normally distributed, either before or following transformation, and so thenonparametric Wilcoxon test was used to compare these data. The surrounding length and cross-sectional area of maternal sinusoid sections were significantly differentin Cited1 mutants (Cited1�p/neo, Cited1neo/Y) compared with controls (Cited1�m/Y).


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blast branching and hence with the size and shape of thematernal blood sinusoids. This potentially also explains whythe overall shape of the labyrinthine layer in the maternal sideresin casts is highly irregular. Alternatively, the spongiotropho-blast projections, rather than having a physical impact on lab-yrinthine development, may secrete a factor(s) that influencestrophoblast behavior. Clues to how this phenotype developsmay come from examining the known molecular interactions ofCited1.

Cited1 is a transcriptional cofactor that associates withSmad4, estrogen receptors � and � (ER�/�), and TFAP2 (12,61, 74). Consequently, the number of genes whose expressioncould be affected in Cited1 mutants is potentially very large.Some of these genes include those encoding TGF-�1, TGF-�3,activin A, inhibin, follistatin and nodal (dependent on Smad4),TGF-� (dependent on ER�/�), and adenosine deaminase and3�-hydroxysteroid dehydrogenase VI (dependent on TFAP2)(36, 48, 51, 57). These factors have opposing effects on keyprocesses during placental development: trophoblast prolifer-ation and differentiation, and the promotion and inhibition ofhormone and vasoactive factor secretion (76). We examined

the expression of PLF and mPLII (which is potentially down-stream of ER�/�), both of which are vasoactive factors, butcould see no difference in the level of expression by RNA insitu hybridization. Since both of these factors are expressed byspongiotrophoblasts, a cell type overrepresented in Cited1 mu-tants, it is possible that absolute levels of PLF and mPLII areincreased. An in situ approach is not the most efficient way toshow the effect of the loss of Cited1 on placental gene expres-sion; microarray analysis provides a broader and more quan-tifiable result.

Effects of X chromosome inactivation on the Cited1 alleleand embryo viability. The X chromosome in placental mam-mals is subject to a unique system of developmental regulation,involving the coordinate activation and inactivation of thechromosome during female development. This mechanism hasevolved to make the X-linked gene dosage equivalent in malesand females. X chromosome inactivation is random in cellsderived from the inner cell mass. Therefore, all cells of theembryo, plus the mesodermal components of the extraembry-onic tissues, have either the paternally or maternally derived Xchromosome inactivated. This occurs approximately on day

FIG. 7. Resin casts of maternal blood spaces reveal enlarged sinusoids in the absence of Cited1. (A to C) Light micrographs show the lateralview of resin casts filled from the arterial side. (D to I) Scanning electron micrographs of the placental base (D to F) and sinusoids (G to I).Cited1�/� (A, D, and G), Cited1�p/neo (B, E, and H), and Cited1Y/neo (C, F, and I) at day 16.5 p.c. are shown. Bar, 1.6 mm (A to C), 1.8 mm (Dto F), and 36 �m (G to I).


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6 p.c. (40, 50), although it is not complete in some somatictissues until day 11.5 p.c. (68). In contrast, X inactivation isnonrandom in cells derived from the trophectoderm and oc-curs much earlier, between days 3.5 and 4.5 p.c. (19, 64–66).Thus, in the ectodermal and endodermal components of theextraembryonic tissues, only the paternal X is inactive.

Cited1 is located on the X chromosome; this allowed us todetermine, using heterozygous females in which the Cited1wild-type allele was derived from the father (Cited1�p/neo), thatthe function of Cited1 function in trophectoderm-derived com-ponents of the conceptus is essential for survival. Cited1 isexpressed in several of these components: visceral/yolk sacendoderm, extraembryonic ectoderm, and the placenta (17)(Fig. 3). Despite the expression in various extraembryonic tis-sues, we consider that embryo survival requires Cited1 in theplacenta, since defects in this organ are apparent late in ges-tation and at a time when Cited1 expression in other trophec-toderm-derived tissues would be expected to have no effect.The labyrinthine layer of the placenta is composed of cells withdistinct origins: those derived from the trophectoderm andfetal blood vessels derived from the embryo. The trophecto-derm-derived cells (chorionic trophoblasts) normally expressCited1, but in Cited1�p/neo females the paternally derived wild-type Cited1 allele is inactive; therefore, despite the presence ofa wild-type Cited1 allele, we see a defect in the size of thetrophoblast-lined maternal sinusoids. In addition to the par-ent-of-origin effects in extraembryonic cells, heterozygous fe-males are mosaic, with random X inactivation causing approx-imately half of the somatic cells to lack Cited1 expression.However, this does not appear to affect embryo viability sinceno detectable loss of heterozygous females occurs when thewild-type allele is maternally derived (Table 1).

Cited1 is not the only X chromosome-linked gene to affectplacental development. Abnormal placental growth is ob-served when crosses and backcrosses between different mouse(Mus) species are performed. Placental dysplasia is X chromo-

some linked and is likely to be due to multiple loci (78). Themain placental cell type to be affected in these dysplastic pla-centas is the spongiotrophoblast (52, 78). On the X chromo-some, the genetic interval associated with hyperplastic placen-tas is large (between the centromere and 50 cM) (26). Esx1 (57cM) may represent one of the genes associated with this phe-notype since Esx1 mutants have a hyperplastic placenta and theexpression of Esx1 is reduced in the placentas of interspecifichybrids (35, 77). It would be interesting to examine the expres-sion of Cited1 in the placentas of interspecific hybrids since thegene is located at 40.1 cM and there is an increase in spongio-trophoblasts in the Cited1 mutant placenta.

Could the function of Cited1 in the embryo be masked byfunctional redundancy? The only phenotypic effect of the Cit-ed1 loss-of-function mutation reported here is a defect in theplacenta; this is despite the fact that Cited1 is expressed in anumber of tissues during mouse embryo development (17). Itis possible that functional redundancy is masking other roles ofCited1; this can occur if there is redundancy at the geneticpathway level or if a single gene can substitute for Cited1function. It is possible that other Cited genes can functionallycompensate for the loss of Cited1 during embryonic develop-ment. Cited1, Cited2, and Cited4 are each expressed duringembryonic development in the mouse (17, 37, 75). In the em-bryo, both Cited1 and Cited2 are expressed in nascent meso-derm, myocardium, cranial neural crest cells, presomitic me-soderm, and somites. No overlap in expression of Cited1 andCited4 has been identified in the embryo; however, these genesare both expressed in adult mammary epithelial cells (74, 75).Therefore, it is possible that the Cited1 loss-of-function muta-tion results in embryonic defects but that compensatory effectsof Cited2 mask these. This would require that they play similarmolecular roles during embryonic development, a propositionsupported by the observations that both Cited1 and Cited2 bindCBP/p300, ER�/�, and TFAP2 (6, 11, 73, 74). Generation ofcompound mutant embryos that lack both Cited1 and Cited2could resolve this issue.

In summary, the X-linked gene Cited1 is required in themouse for normal placental development and for embryonicgrowth and viability. Cited1 mutants develop large maternalblood sinusoids, a phenotype consistent with its expression introphoblasts including those that line these sinusoids. The Cit-ed1 mutant mouse line that we have generated will allow fur-ther dissection of the function of this gene during embryonicand adult life. Importantly, it may prove to be a model forintrauterine growth restriction (IUGR) in humans. IUGR is asignificant cause of infant mortality and morbidity, and it hasbeen suggested that infants with IUGR exhibit higher rates ofcoronary heart disease, type 2 diabetes, hypertension, andstroke as adults (7–10, 13, 49). Therefore, fetal growth not onlyimpacts the outcome of the perinatal period but also may affectadult well-being. The etiologies of IUGR are numerous but are

FIG. 8. Resin casts reveal the arrangement of the fetal vasculature. Light micrographs of Cited1�/Y (A, C, E, and G) and Cited1neo/Y (B, D, F,and H) showing the base (A and B) and side (C and D) of casts filled with red resin from the venous side and blue resin from the arterial sideon day 16.5 p.c. Scanning electron micrographs of capillaries filled from the arterial side, at the top of the labyrinthine layer having recentlyemerged from the arterioles (E and F) and at the tips of the casts where the capillaries are projecting into the center of the labyrinthine layer (Gand H), are shown. Bar, 1.25 mm (A and B), 1.2 mm (C and D), and 40 �m (E to H).

TABLE 5. Quantification of the capillary diameter from fetalvascular casts on day 16.5 p.c.a

Side filled

Capillary diameter (�m) for Cited1genotype

PWild-type(�m/Y, �p/

�m)Null (neo/Y)

Arterial 9.66 � 0.11 10.677 � 0.12 0.0001Venous 10.43 � 0.17 10.82 � 0.13 0.0401

a The diameter of resin casts of fetal capillaries filled from the arterial andvenous sides on day 16.5 p.c. was determined (see Materials and Methods). Meandiameters are presented with standard error of the mean. Nested analysis ofvariance showed that the diameters of capillaries filled from the arterial sidewere significantly different between wild-type (Cited1�p/�m, Cited1�m/Y) and null(Cited1neo/Y) mice.


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often associated with abnormalities in placental structure andfunction. Since it is difficult to fully assess the role of genesduring the development of placental insufficiency in the hu-man, there is a need for the development and analysis ofanimal models. The Cited1 mutant mouse line reported heremay serve as a useful model for placental insufficiency and thuscontribute to our understanding of the etiology of this disor-der.

ACKNOWLEDGMENTS

We are extremely grateful to Austin Smith for the 129/Olac genomiclibrary and the E14.2 ES cells; Margaret Budanovic from the Univer-sity of New South Wales Electron Microscopy Unit; James Cross andMichelle Tallquist for methods; Janet Rossant, Reinald Fundele, andDaniel Linzer for reagents; Stuart Gilchrist for help with the statisticalanalyses; Christine Biben for critical assessment of the manuscript; andunknown reviewers for their very helpful comments. Animal workcomplied with all relevant governmental and institutional policies.

D.B.S. is a Westfield-Belconnen Postdoctoral Fellow. S.L.W. is aRoyal Society of London International Postdoctoral Fellow and aWellcome Trust International Travelling Research Fellow. T.E.T. is anNHMRC Peter Doherty Postdoctoral Fellow. S.L.D. is a PharmaciaFoundation of Australia Senior Research Fellow. T.S. is supported bythe U.S. National Cancer Institute (R01-CA82230) and the AVONProject on Breast Cancer Research.

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cited1 is required in trophoblasts for placental development and

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