Ddx1 knockout results in transgenerational wild-type lethality in mice

Abstract

DEAD box 1 (DDX1) is a member of the DEAD box family of RNA helicases which are involved in all aspects of RNA metabolism. DDX1 has been implicated in a variety of biological processes, including 3'-end processing of mRNA, DNA repair, microRNA processing, tRNA maturation and mRNA transport. To study the role of DDX1 during development, we have generated mice carrying a constitutive Ddx1 knock-out allele. Ddx1(+/-) mice have no obvious phenotype and express similar levels of DDX1 as wild-type mice indicating compensation from the intact Ddx1 allele. Heterozygote matings produce no viable Ddx1(-/-) progeny, with Ddx1(-/-) embryos dying prior to embryonic day (E) 3.5. Intriguingly, the number of wild-type progeny is significantly decreased in heterozygote crosses, with two different heterozygote populations identified based on parental genotype: (i) normal Ddx1(+/-) mice which generate the expected number of wild-type progeny and (ii) Ddx1*(/-) mice (with * signifying a non-genetically altered allele) which generate a significantly reduced number of wild-type mice. The transgenerational inheritance of wild-type lethality observed upon crossing Ddx1*(/-) mice is independent of parental sex and occurs in cis through a mechanism that is different from other types of previously reported transgenerational epigenetic inheritance.

Figure 1. Genomic map of the gene-trap insertion site.

ESCs containing a single gene-trap insertion in Ddx1 were purchased from BayGenomics. (a) The insertion containing a β-geo gene, splice acceptor (SA) and a polyadenylation signal (PA) is located between exons 14 and 15 of Ddx1. The insertion generates a truncated DDX1 protein fused to LacZ. Locations of primers (RGo) and Southern blot probes used for genotyping are also shown. (b) Southern blot analysis of RRT447 cell line using a ³²P-labeled cDNA probe specific to β-geo. (c) Southern blot analysis of wild-type and Ddx1^+/− mice using a ³²P-labeled cDNA probe specific to Ddx1. (d) PCR amplification of genomic DNA for routine genotyping using primers shown in (A). (e) Progeny from heterozygous intercrosses (Ddx1^+/− or Ddx1^+/−) were collected and genotyped at different developmental stages. No Ddx1^−/− progeny were observed out of a total of 758 postnatal offspring, 225 E6-10 embryos and 91 E9.5 blastocysts genotyped. A significant decrease in the percentage of wild-type mice was observed post E3.5 (P < 0.001). Fisher’s exact tests were performed to determine significant differences between the expected and observed ratios of Ddx1^+/+ to Ddx1^+/− mice.

Figure 2. Heterozygous mice generate a bimodal distribution of progeny genotypes.

(a) Litters from heterozygous intercrosses (Ddx1^+/− or Ddx1^+/−) that contained at least 5 pups were plotted as a percentage of wild-type mice generated (n = 178). A normal random distribution plotted around the expected value of 33% wild-type is included for comparison. (b) Ddx1^+/− and Ddx1*^/− intercrosses were separated (n = 32 and n = 146, respectively) and the percentage of wild-type mice generated was plotted. (c) The percentage of wild-type mice at ages E3.5, E6-10 and P0 from Ddx1^+/− (n = 22, 61, 229, respectively) or Ddx1*^/− (n = 69, 164, 529, respectively) intercrosses were plotted against the expected percentage. (d) Backcrosses (wild-type X heterozygote) from both FVB and C57BL/6 (combined) mice were separated by genotype and sex of the heterozygote. The percentage of wild-type genotyped was normalized to the Ddx1^+/− backcross. Fisher’s exact tests were performed to determine significant differences.

Figure 3. Ddx1 mRNA and protein expression levels are similar in wild-type and heterozygous animals.

(a) Western blot analysis of 50 μg of whole brain lysates from P0-3 mice of the indicated genotypes. Blots were immunostained with anti-DDX1 antibody (top) and anti-actin antibody (bottom). (b) Quantitative real-time PCR of P0-3 mouse brain RNA from the indicated genotypes. qPCR was carried out with 3’ Ddx1 primers and Gapdh primers as a control (n≥4 for each sample). Expression levels for Ddx1 are plotted relative to wild-type. Error bars show standard error of the mean. (c) Semi-quantitative RT-PCR analysis of cDNAs generated from P0-3 mouse brain RNA. cDNA samples were amplified with Ddx1 primers 3’ to the gene-trap (top panel), primers specific to β-geo (second panel), primers to the 3’ end of β-geo and the 3’ region of Ddx1 (third panel), and primers to Gapdh as a control (bottom panel).

Figure 4. Methylation analysis at the Ddx1 transcription start site.

(a) A CpG island consisting of 55 CpG sites was predicted flanking the transcription (txn) start site of Ddx1 from -156 to +487. MethF and MethR indicate binding sites of primers used to amplify the region following bisulfite conversion. (b) A lollipop diagram shows the methylation status of each of the 55 CpGs, where a white circle indicates no methylation and a black circle indicates methylation. At least 4 clones from each genotype (Ddx1^+/+, Ddx1^+/−, and Ddx1*^/−) were analyzed for their methylation patterns. A cross indicates indeterminate methylation.

Figure 5. Inheritance model of the Ddx1* allele.

(a) Depiction of the two types of wild-type alleles as determined by parental crosses. (b) Ddx1^+/− intercrosses produce the expected ratio of wild-type to heterozygote progeny, whereas Ddx1*^/− mice intercrosses produce an abnormal ratio of wild-type to heterozygote progeny. (c) Ddx1^+/− backcrosses produce the expected ratio of wild-type to heterozygote progeny, whereas partial wild-type lethality is observed in Ddx1*^/− backcrosses. (d) Proposed mechanism for wild-type lethality. Under normal conditions, each Ddx1 allele produces 1X Ddx1 RNA, resulting in a total of 2X DDX1 RNA and protein. Ddx1* alleles generate ~2X Ddx1 RNA to compensate for inactivation of the mutant Ddx1 allele. Ddx1*^/+ and Ddx1*^/* mice are predicted to produce ~3X and 4X Ddx1 RNA, respectively. This increase results in early embryonic lethality, with higher penetrance observed with increased levels of DDX1.