The Mmachc gene is required for pre-implantation embryogenesis in the mouse

    The Mmachc gene is required for pre-implantation embryogenesis in the mouse Maira A. Moreno-Garcia, Mihaela Pupavac, David S. Rosenblatt, Michel L. Tremblay, Loydie A. Jerome-Majewska PII: DOI: Reference:

S1096-7192(14)00156-5 doi: 10.1016/j.ymgme.2014.05.002 YMGME 5750

To appear in:

Molecular Genetics and Metabolism

Received date: Revised date: Accepted date:

10 March 2014 6 May 2014 6 May 2014

Please cite this article as: Moreno-Garcia, M.A., Pupavac, M., Rosenblatt, D.S., Tremblay, M.L. & Jerome-Majewska, L.A., The Mmachc gene is required for preimplantation embryogenesis in the mouse, Molecular Genetics and Metabolism (2014), doi: 10.1016/j.ymgme.2014.05.002

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ACCEPTED MANUSCRIPT The Mmachc gene is required for pre-implantation embryogenesis in the mouse Maira A Moreno-Garcia1, Mihaela Pupavac1, David S. Rosenblatt1,2, Michel L.Tremblay3,

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Loydie A. Jerome-Majewska1,2,4*

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Department of Human Genetics, McGill University, 1205 Avenue Docteur Penfield, N5/13,Montreal, Quebec, Canada H3A 1B1 2 Department of Pediatrics, McGill University, Research Institute- at Place Toulon 4060 Ste. Catherine West PT 420, Montreal Children's Hospital, Montreal, Quebec, Canada H3Z 2Z3 3 Department of Biochemistry, McGill University, Goodman Cancer Research Center 1160 Avenue Pine, Montreal, Quebec, Canada 4 Department of Anatomy and Cell Biology McGill University Strathcona Anatomy and Dentistry Building 3640 University Street, Montreal, Quebec, Canada H3A2B2

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* Corresponding author: [email protected]

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Abstract

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Running title: Mmachc and pre-implantation

Patients with mutations in MMACHC have the autosomal recessive disease of cobalamin metabolism known as cblC. These patients are unable to convert cobalamin into the two active forms, methylcobalamin and adenosylcobalamin and consequently have elevated homocysteine and methylmalonic acid in blood and urine. In addition, some cblC patients have structural abnormalities, including congenital heart defects. Mmachc is conserved in the mouse and shows tissue and stage-specific expression pattern in midgestation stage embryos. To create a mouse model of cblC we generated a line of mice with a gene-trap insertion in intron 1 of the Mmachc gene, (MmachcGt(AZ0348)Wtsi). Heterozygous mice show a 50% reduction of MMACHC protein, and have significantly higher levels of homocysteine and methylmalonic acid in their blood. The MmachcGt allele was inherited with a transmission ratio distortion in matings with heterozygous 1

ACCEPTED MANUSCRIPT animals. Furthermore, homozygous MmachcGt embryos were not found after embryonic day 3.5 and these embryos were unable to generate giant cells in outgrowth assays. Our findings confirm

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that cblC is modeled in mice with reduced levels of Mmachc and suggest an early requirement

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for Mmachc in mouse development.

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Keywords: cobalamin / Mmachc / pre-implantation / ratio distortion / trophectoderm

Introduction

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CblC (OMIM 277400) is an autosomal recessive disorder and the most frequent inborn error of cobalamin metabolism (Watkins & Rosenblatt, 2011). The clinical and biochemical features

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of patients with this disorder include diverse neurological, hematological, ophthalmological, dermatological, cardiac, vascular, and renal defects as well as elevation of both homocysteine

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and methylmalonic acid in the blood and urine (Martinelli et al, 2011; Carrillo-Carrasco et al, 2012; Carrillo-Carrasco & Venditti, 2012). Mutations in MMACHC cause the cblC disorder. MMACHC encodes for the MMACHC protein, an immediate downstream acceptor of cobalamin (Cbl) (Hannibal et al, 2009). MMACHC is not a member of any previously identified gene family, its C-terminal end does not seem to be conserved in eukaryotes outside mammalia, and no homologous proteins are found in prokaryotes (Lerner-Ellis et al, 2006). MMACHC along with MMADHC interact and orchestrate the fate of processed cobalamin (Deme et al, 2012; Plesa et al, 2011; Gherasim et al, 2013). At E11.5, Mmachc is expressed in head mesenchyme, dorsal root ganglia, lung endoderm, mesonephric mesenchyme, notochord, ectoderm, endothelium of blood vessels and heart (Pupavac et al, 2011; Moreno-Garcia et al, 2013). To generate a mouse model of cblC and to gain insights into the requirement of Mmachc during

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ACCEPTED MANUSCRIPT mouse embryonic development, we used embryonic stem cells with a gene-trap in the first intron of Mmachc to generate mice with a loss-of-function allele. Here, we report that Mmachc

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heterozygous mice (Mmachc+/Gt) are fertile, viable and have a lifespan comparable to wild type

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(Mmachc+/+) mice. Furthermore, the MmachcGt allele was inherited with a transmission ratio distortion on two different mixed genetic backgrounds; C57Bl6/129Sv and CD1/C57B6/129Sv.

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Unexpectedly, although homozygous MmachcGt/Gt blastocysts were observed at embryonic day

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(E) 3.5, MmachcGt/Gt embryos were not found post-implantation. Furthermore, MmachcGt/Gt blastocysts were unable to generate trophectoderm in vitro outgrowth assays. Our data suggest a

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requirement for Mmachc in the pre-implantation period.

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Results

Generation of mice with a gene-trap insertion in the Mmachc gene

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To explore the requirement for the Mmachc gene during mouse development, we generated Mmachc deficient mice using embryonic stem cells (ES) containing a gene-trap in the Mmachc gene locus. The gene-trap was inserted into intron 1 of the Mmachc locus (Figure 1A). The genetrap insertion was verified by Southern blot analysis (Figure 1B), DNA sequencing and PCR, which allowed us to distinguish the wild type and gene-trap alleles (Figure 1B). The mutated allele is predicted to generate a non-functional truncated MMACHC protein containing the first 27 of 279 amino acids of MMACHC (Supplemental Figure 1). Western blot analysis on lysates from E11.5 wild type and heterozygous embryos shows a 50% decrease of MMACHC protein in heterozygous embryos compared to wild type embryos (Figure 1C), confirming that this mutation results in decreased MMACHC protein production.

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ACCEPTED MANUSCRIPT Disruption of Mmachc results in hyperhomocysteinemia and methylmalonic acidemia In man, mutations in Mmachc result in increased levels of homocysteine and methylmalonic acid

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in urine and blood. To determine if reduced levels of MMACHC in heterozygous mice perturbs

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levels of these metabolites, we measured homocysteine and methylmalonic acid levels in plasma from adult females (n= 5 per genotype). We found a significant increase in the concentrations of

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these metabolites in plasma of MmachcGt/+ (Figure 2) mice when compared to Mmachc+/+

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animals (p<0.05, t-test).

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Non-Mendelian segregation of the MmachcGt allele

To determine if Mmachc+/+, Mmachc+/Gt and MmachcGt/Gt embryos were born at the expected

on

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mixed

genetic

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Mendelian frequency, we genotyped 197 offspring from inter se crosses between heterozygotes backgrounds:

C57Bl/6

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129Sv/EvTac

(C57/129)

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CD1/C57Bl6/129Sv/EvTac (CD1/C57/129) (Table I and II). From these crosses, mice homozygous for the MmachcGt allele were not found at weaning (Table I and II), indicating that MmachcGt homozygous embryos are lost before weaning. In addition, we observed significantly more heterozygous mice than expected from heterozygous crosses on the C57/129 genetic background (Table I, χ2 = 10.23, p < 0.05). We observed approximately an equal number of males and females born from all matings. In order to determine if the observed segregation distortion was due to a maternal or paternal affect, we examined segregation of the MmachcGt allele from matings between wild type females to heterozygous males or vice versa on the mixed C57/129 genetic background (Table I). Intriguingly, the segregation distortion was only found in offspring from mating between wild-type females to heterozygous males (χ2 = 13.16, p < 0.05). To determine if the observed transmission ratio distortion was due to the genetic background we

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ACCEPTED MANUSCRIPT examined segregation of the MmachcGt allele on the CD1 genetic background. Mendelian segregation of the MmachcGt allele was initially found in these matings suggesting that the

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distortion was related to the genetic background (Table II). In addition, Mendelian segregation of the MmachcGt allele was found in embryos collected from the mixed CD1/C57/129 genetic

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background (Table III, χ2 = 0.00, p > 0.05).

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To determine when MmachcGt/Gt embryos arrested, we collected embryos between E3.5 and

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E16.5 from heterozygous crosses on CD1/C57/129 (Table IV) and C57/129 (Table V) backgrounds. At E3.5 MmachcGt/Gt embryos were found at the expected Mendelian frequency

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(14:40:12, χ2 = 3.09, p > 0.05). However, no MmachcGt/Gt embryos were ever found postimplantation on the CD1/C57/129 mixed genetic background (Table IV) or the mixed C57/129

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genetic background (Table V). This indicates that MmachcGt/Gt embryos arrest pre-implantation

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and that segregation distortion was present before birth on both genetic backgrounds.

Loss of Mmachc compromises the outgrowth potential of TE giant cells The absence of MmachcGt/Gt embryos post-implantation combined with no excess of empty deciduas suggests that these embryos arrest pre-implantation. To determine if MmachcGt/Gt embryos are able to form blastocyst and proceed through the early steps of implantation ex vivo, E3.5 conceptuses were collected from pregnant Mmachc+/Gt females after inter se mating (n = 26) and cultured for 5 days. Conceptuses were scored as blastocysts when the blastocoel cavity was evident and occupied more than half the volume of the conceptus, or as compact morulas when the blastocoel cavity was not distinguishable (Figure 2). E3.5 conceptuses with expanded blastocysts were either wild type (n = 6) or Mmachc+/Gt (n = 11), whereas conceptuses designated as compacted morulas were either MmachcGt/Gt (n = 7) or Mmachc+/Gt (n = 2) (Figure

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ACCEPTED MANUSCRIPT 2). Thus, MmachcGt/Gt conceptuses were significantly delayed in forming blastocysts when compared to their wild type or heterozygous littermates (Fisher’s exact test, p < 0.05). During the

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5 day culture period, a small but detectable blastocoel cavity was found in 1 MmachcGt/Gt embryo, suggesting that trophectoderm was able to differentiate. However, the majority of

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MmachcGt/Gt blastocysts were unable to hatch and attach to the plate (n = 6) compared to wild

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type and heterozygous blastocysts (n = 19) that were able to do so. In addition, few giant cells,

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the derivative of the trophectoderm, were found in outgrowth from the 1 MmachcGt/Gt embryo that successfully hatched (Figure 3). Thus, in total our data suggest that the trophectoderm

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lineage of MmachcGt/Gt embryos is compromised.

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Discussion

Mice containing a gene-trap in intron 1 of the Mmachc gene were generated to assess the role of

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Mmachc in mouse development and physiology. We measured homocysteine and MMA levels in adult females on a mixed genetic background and found significantly higher levels of these metabolites in heterozygous females, suggesting that normal levels of MMACHC is required for cobalamin metabolism in mice. Furthermore, embryos homozygous for the MmachcGt allele were only observed at E3.5. Thus, MmachcGt/Gt embryos died between E3.5 and E5.5, indicating that the mutation was lethal either before or around the time of implantation. Intriguingly although vitamin B12 deficiency is associated with reduced fertility in human, patients with cblC are born (Kuhne et al 1991; Pront et al 2008). Thus, our results suggest that loss of function of Mmachc in mice is much more severe than that usually seen in cblC patients. Interestingly, similar observations have been found for other genes in the cobalamin pathway. Homozygosity for protein loss of function mutations in Cubn, Amn, Mtr, and Mthfd1 also results

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ACCEPTED MANUSCRIPT in embryonic lethality with death occurring between E7.5 and E13.5 (Tomihara-Newberger et al, 1998; Smith et al, 2006; Swanson et al, 2001; Christensen et al, 2013). A hypomorphic mutation

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in Mtrr results in severe embryonic phenotypes including intrauterine growth restriction,

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developmental delay, and congenital malformations (Padmanabhan et al, 2013). These data combined suggest, that embryonic requirements for genes in the cobalamin pathway are different

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in mice and human.

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No morphological abnormalities were found in heterozygous adult (Mmachc+/Gt) mice, despite the 50% decrease of MMACHC protein observed in the embryos at E11.5, and increased

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homocysteine and MMA levels in adults. However, we observed significant transmission ratio distortion of the MmachcGt allele on the C57/129 genetic background, and this distortion was not

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apparent when the CD1 mixed genetic background was introduced. Mmachc maps to distal mouse chromosome 4 in a region that is not imprinted but was reported to show transmission

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ratio distortion in crosses between C57Bl/6J and M. Spretus, with the M. Spretus allele being selected over the maternal C57Bl/6J (Ceci et al, 1989). It is interesting to note that Shb, a nonrelated gene 23cM from Mmachc, was shown to preferentially ovulate Shb- oocytes, which results in increased numbers of Shb+/- mice, and reduced numbers of Shb+/+ mice (Kriz et al, 2007). It is possible that this gene, or other modifier genes on this chromosome, is responsible for the transmission ratio distortion that we observed. To explore why homozygous null embryos (MmachcGt/Gt) fail to develop past E3.5, we cultured conceptuses from intercrosses of heterozygous animals in vitro. All MmachcGt/Gt conceptuses were delayed when compared to wild type and heterozygous littermates, and were compromised in generation of trophectoderm-derived giant cells ex vivo. The trophectoderm is a polarized epithelium, which generates the blastocoel cavity and mediates attachment and implantation in

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ACCEPTED MANUSCRIPT the uterus. Thus, our data suggest that in the absence of Mmachc, the trophectoderm is compromised thus leading to pre-implantation lethality. We postulate that abnormal

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homocysteine recycling to methionine may result in abnormal expression of genes, such as

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Tead4, which is critical for trophectoderm differentiation and depends on normal levels of methionine for its regulation (Ikeda et al 2012). Future studies will examine the specific

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requirement for Mmachc in the trophectoderm.

Materials and methods

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Gene trap

Embryonic Stem cell line with MmachcGt allele (AZ0348) was purchased from the Sanger

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Institute (http://www.sanger.ac.uk/PostGenomics/genetrap/vectors/) and blastocyst injection and generation of the chimeric mice was done in transgenic facility of the McIntyre Medical Sciences

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Building, Montreal, Quebec. ES cells carrying the MmachcGt allele were injected into blastocysts from C57Bl/6J females pregnant for 3.5 days, using standard protocols. Embryos were then transferred to day 2.5 pseudopregnant females. After birth, chimeras were identified on the basis of coat colour. F1 mice carrying the MmachcGt allele were on a mixed C57/129 genetic background. MmachcGt mutation is maintained on a mixed C57/129 genetic background. To introduce the CD1 genetic mix, CD1 females (Jackson Laboratory) were mated to MmachcGt males and maintained by intercross matings. All experiments were performed in accordance with the Canadian Council on animal care, and mice were housed in the Montreal Children’s Hospital Animal Facility. Female mice were mated to males, and the day on which a vaginal plug was detected was considered E0.5. Mice were fed a normal breeding diet and were weaned at 3 weeks after birth.

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ACCEPTED MANUSCRIPT Genotyping A three-primer PCR reaction was used to identify the presence or absence of the MmachcGt

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allele. Primers F1 (5’-cttgccatcaatacgggact-3’) and R1 (5’-ctggaaagctcaatggccta-3’) detected the wild type allele (217bp) (Figure 1a). Primer F1 together with primer R2 (5’-

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aagggtctttgagcaccaga-3’) detected the MmachcGt allele (460bp) (Figure 1b). PCR protocol was

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the following: 94°C for 3 min. [94°C for 30 sec., 60°C for 50 sec., 72°C for 1 min.] for 34 cycles

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and 72°C for 5 min. DNA samples were obtained from blastocyst embryo, tail, ear, or yolk sac tissue. Tail, ear, or yolk sac samples were incubated in buffer containing 50mM KCl, 10mM

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Tris-HCl (pH 8.3), 2.5 mM MgCl 2, 0.1 mg/mL gelatin, 0.45% v/v Nonidet P40, 0.45% v/v Tween 20, and 100μg/mL proteinase K. The samples were incubated in this buffer overnight at

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55°C, then incubated for 30 minutes at 95°C and subjected to PCR analysis. For blastocyst genotyping, each blastocyst was washed in 1X PBS (GIBCO®PBS ready to use)

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and transferred into a PCR tube. Blastocysts were lysed in 10 µL of buffer containing 500 mM KCl, 100mM Tris-HCl (pH 8), gelatin (0.1 mg/mL), 0.45% NP-40, 0.45% Tween 20, and proteinase K (0.5 mg/mL); the samples were incubated at 60°C for 3 min, at 55°C for 4 hours, and at 100°C for 10 min, and subjected to PCR analysis.

Southern blot Genomic DNA from Mmachc+/+ and Mmachc+/Gt animals was extracted according to standard phenol/chloroform extraction methods (Invitrogen). Extracted genomic DNA was digested overnight with BamHI restriction enzyme (New England BioLabs). Digested DNA was run overnight on a 0.8% agarose gel in 1xTBE buffer at 60 Volts, and was then transferred to a nylon membrane using the salt transfer method as described in (Brown, 2001). To generate the probe,

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ACCEPTED MANUSCRIPT genomic DNA was amplified using forward primer 5’-ccttaatccgggccatattt-3’, and reverse primer 5’-acaacacgacctggtcatca-3’. The resulting 1363bp amplicon was run out on a 1% agarose gel,

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purified, cloned into a TOPOII vector (Invitrogen), and sequenced. The probe was then labelled

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and hybridized to blots according to AlkPhos Direct Labeling and Detection System with CDPStar (GE). Using this kit, the probe was labeled with thermostable alkaline phosphatase, which

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upon addition of CDP-Star substrate produced a chemiluminescent signal that was captured on

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film.

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Sample collection of blood and biochemical analyses

Intra-cardiac blood samples were collected and centrifuged for 10 min at 3000 rpm. Aliquots of

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plasma were stored frozen at−20C until analysis. Homocysteine and Methylmalonic acid

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concentrations were determined as described previously (Stabler et al.1988).

Blastocyst culture

Mouse uterus was dissected at E3.5. The uterus was placed in a glass dish containing Dulbecco’s modified Eagle’s medium plus 15% fetal bovine serum, antibiotics (1% streptomycin) and 2.5 mM HEPES. Using a Pasteur pipette, the blastocysts were flushed out of the uterus, and into the media. Each blastocyst was then transferred into a 96-well plate and cultured for 5 days (37˚C and 5% CO2). One day later, attachment of the blastocyst was confirmed. Cell lysis, and genotyping was performed on the 5th day of culture.

Western blotting analysis

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ACCEPTED MANUSCRIPT Embryos were harvested at 11.5 days post coitum and lysed in cell lysis buffer (0.05 M NaH2PO4 (pH 8), 0.15 M NaCl, 0.1 M imidazole, 0.5% Chaps, and Complete Protease Inhibitors (Roche)).

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Protein concentration of the supernatant was determined using Advanced Protein Assay Reagent

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(DC protein essay kit) and BSA as standard protein. Equal amounts of protein (30 μg) were subjected to SDS-PAGE electrophoresis on a 10% polyacrylamide gel and transferred to an

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Immun-Blot PVDF membrane (BIO-RAD Laboratories; Cat. #162-0177). The membrane was

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then blocked with 5% non-fat milk for 1 h at room temperature. The membrane was incubated with the primary antibodies: MMACHC monoclonal antibody (NeuroMab clone N230/21),

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(1:2500); and β-actin antibody (Cell Signaling), (1:5000); and with their respective secondary antibodies. Western blot analysis was repeated at least three times. The immune-reactive bands

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were detected by ECL plus Western Blotting Detection System, (GE Healthcare; Cat.

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#RPN2132).

Statistical Analysis

Relative protein expression was reported as mean ± SEM. To compare values across heterozygous and wild type groups we used one-way analysis of variance (ANOVA). The fit of observed genotype distribution with the expected Mendelian distribution was evaluated by Pearson's chi-squared test (χ2) test. The minimum level of statistical significance was set at p < 0.05. The chi-squared test was used to calculate whether the ratios of wild type, heterozygous and homozygous embryos or offspring obtained deviated from the expected Mendelian ratios with a statistical significance. Since we found that MmachcGt/Gt embryos arrest at E3.5, the statistical significance of excess of the heterozygotes was calculated at all stages after E3.5.

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Acknowledgements

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We would like to thank our colleague Junhui Liu who generated the initial animals used or this

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study. We would also like to thank Dr. Sally Stabler for metabolite measurements. This work was supported by a grant from the Canadian Institutes of Health Research (#15078) to D.S.R.

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and L.JM. L.J.M. and D.S.R. are members of the Research Institute of the McGill University

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Health Centre, which is supported in part by the FRSQ. MAMG was supported by Toronto

Montreal Children's hospital foundation.

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Author contribution

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Dominion Bank (TD) Postdoctoral fellowship in Child Health Research Excellence of the

DSR and LAJM conceived the study; MAMG, DSR and LAJM wrote the manuscript; MAMG

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and MP carried out experiments and analyzed data.

Conflict of interest

The authors declare no conflict of interest.

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Figure legends Figure 1. Genotyping and protein expression of Mmachc. (A) Schematic showing the β-geo gene-trapping vector containing a splice acceptor (SA) and polyadenylated (pA) tail. This construct was randomly inserted into intron 1 of the Mmachc gene to produce the MmachcGt 14

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allele. (B) Image of an agarose gel showing PCR products resulting from DNA amplification using primers in (A). F1 and R1 primers amplify the wild type allele resulting in a 217bp PCR band. F1 and R2 amplify the gene-trapped allele resulting in a 460bp PCR band. Lane 1 shows ladder (100 bp), lane 2 shows amplification of a heterozygous Mmachc+/Gt sample, lane 3 shows amplification of a wild-type Mmachc+/+ sample, lane 4 shows amplification of a homozygous MmachcGt/Gt sample, and lane 5 is a water control containing no DNA; (C) BamHI digested genomic DNA collected from Mmachc+/+ and Mmachc+/Gt mice and film exposure of nylon membrane after hybridization of probe shown in A, showing the expected single band at 4.7 kb in the wild type sample, and the expected 4.7 kb and 2.6 kb bands in the heterozygous sample. (D) Western blot of the MMACHC protein in wild type and heterozygous mouse samples. The protein bands were quantified densitometrically and normalized to the expression of β-actin in the same sample. Values are expressed as relative intensities; n = 3 in each group, mean±SEM.

D

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Figure 2. Increased levels of homocysteine and methylmelonic acid in wild type and heterozygous mice. The levels of (A) homocysteine (B) and methylmalonic acid (MMA) was measured in plasma of adult Mmachc and Mmachc+/Gt mice (n=5 per group). * indicates statistical significance (p>0.05, t – test) in the levels of homocysteine and MMA between wild type and heterozygous animals.

AC CE P

TE

Figure 3. MmachcGt/Gt blastocysts fail to yield trophectodermal outgrowth in vitro. A and C: wild type and heterozygous blastocysts with normal inner cell mass (ICM, black arrowheads), blastocoel cavity (black star), and trophectoderm (TE, red arrowheads). B and D: Normal outgrowths with morphologically distinguishable inner cell mass (ICM, black arrowheads), and giant cells (gc, ) E) MmachcGt/Gt blastocyst that is delayed with no cavity. MmachcGt/Gt blastocyst failed to hatch or attach in vitro. (F) Non-representative image of one MmachcGt/Gt blastocyst that attached but failed to show outgrowth or differentiation of trophectoderm after 72 hours in vitro. Scale bar = 50 µm. ZP = zona pellucida, gc=giant cells

Table I. Genotype distribution of offspring at weaning from parents on mixed C57/129 genetic background. Offspring Genotype Distribution

Parental Genotype ♀ Mmachc

♂ +/Gt

Mmachc Mmachc

+/+

+/Gt

Mmachc

+/Gt

Mmachc

+/Gt

Mmachc

Number of litters

χ

df

p

2

+/+

+/Gt

Gt/Gt

18

81

0

18

10.23

1

0.0014*

46

88

-

19

13.16

1

0.0003*

54

56

-

16

0.04

1

0.8488

+/+

*Statistically significant deviation from expected Mendelian genotype distribution (p < 0.05) 15

ACCEPTED MANUSCRIPT

Table II. Genotype distribution of offspring at weaning from parents on mixed CD1/C57/129 genetic background.

Mmachc

+/Gt

Mmachc

+/Gt

Mmachc

+/Gt

Gt/Gt

40

40

-

38

60

df

p

9

0.00

1

1.000

0

15

1.30

1

0.2534

NU

Mmachc

+/+

+/Gt

2

χ

RI

♂

+/+

Number of litters

SC

♀

PT

Offspring Genotype Distribution

Parental Genotype

Parental genotype ♀

MA

Table III. Distribution of genotyped embryos collected from matings on mixed CD1/C57/129 genetic background. Offspring Genotype Distribution

ES

♂

(+/Gt)

23

20

4

16

11

4

11.5

28

29

5

12.5

15

18

3

14.5

2

6

1

Total

84

84

17

Mmachc

+/Gt

AC CE P

+/+

TE

10.5

D

(+/+)

9.5

Mmachc

Number of litters

χ

2

0.00

df

p

1

1.000

ES = Embryonic stage.

Table IV. Distribution of genotyped embryos collected from heterozygous intercross matings on CD1/C57/129 genetic background. Parental genotype ♀

Mmachc

ES

♂

+/Gt

Mmachc

Offspring Genotype Distribution

Number of litters

χ

df

p

3.09

2

0.2132

5.85

1

0.0155*

+/+

+/Gt

Gt/Gt

3.5

14

40

12

11

6.5

5

17

0

4

7.5

10

23

0

4

9.5

3

25

0

3

13.5

2

8

0

1

2

+/Gt

16

ACCEPTED MANUSCRIPT Total

34

113

12

23

34.32

2

0.0001*

PT

*Statistically significant deviation from expected Mendelian genotype distribution (p < 0.05) ES = Embryonic stage

Mmachc

♂

+/Gt

Mmachc

+/Gt

+/Gt

9.5

8

68

10.5

3

18

11.5

3

6

0

2

13.5

1

8

0

2

16.5

3

5

0

1

Total

18

0

19

SC

Gt/Gt

Number of litters

+/+

0

11

0

3

NU

♀

ES

Offspring Genotype Distribution

MA

Parental genotype

RI

Table V. Distribution of genotyped embryos from heterozygous intercross matings on C57/129 genetic background.

105

2

χ

df

p

19.35

1

0.0001*

AC CE P

TE

D

*Statistically significant deviation from expected Mendelian genotype distribution (p < 0.05) ES = Embryonic stage

17

AC CE P

TE

D

MA

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

Figure 1

18

AC CE P

TE

D

MA

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

Figure 2

19

AC CE P

TE

D

MA

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

Figure 3

20

ACCEPTED MANUSCRIPT Highlights

PT

RI

SC NU MA D TE

 

We generated of a novel mouse model of cblC. We report abnormal levels of homocysteine and methylmalonic acid in serum of adult heterozygous mice. We report Pre-implantation lethality of Mmachc homozygous mutant embryos. We show a requirement for Mmachc in the trophectoderm lineage and suggest that mutations in this gene may be associated with infertility and pre-implantation loss.

AC CE P

 

21