The paternally expressed imprinted Retrotransposon-like 1 (Rtl1/Peg11) is a retrotransposon-derived gene that has evolved a function in eutherian placentation. Seven miRNAs, including miR-127, are processed from a maternally expressed antisense Rtl1 transcript (Rtl1as) and regulate Rtl1 levels through RNAi-mediated post-transcriptional degradation. To determine the relative functional role of Rtl1as miRNAs in Rtl1 dosage, we generated a mouse specifically deleted for miR-127. The miR-127 knockout mice exhibit placentomegaly with specific defects within the labyrinthine zone involved in maternal-fetal nutrient transfer. Although fetal weight is unaltered, specific Rtl1 transcripts and protein levels are increased in both the fetus and placenta. Phenotypic analysis of single (ΔmiR-127/Rtl1 or miR-127/ΔRtl1) and double (ΔmiR-127/ΔRtl1) heterozygous miR-127 and Rtl1 deficient mice indicate that Rtl1 is the main target gene of miR-127 in placental development. Our results demonstrate that miR-127 is an essential regulator of Rtl1 mediated by a trans-homologue interaction between reciprocally imprinted genes on the maternally and paternally inherited chromosomes.

The CRISPR/Cas system efficiently introduces double strand breaks (DSBs) at a genomic locus specified by a single guide RNA (sgRNA). The DSBs are subsequently repaired through non-homologous end joining (NHEJ) or homologous recombination (HR). Here, we demonstrate that DSBs introduced into mouse zygotes by the CRISPR/Cas system are repaired by the capture of DNA sequences deriving from retrotransposons, genomic DNA, mRNA and sgRNA. Among 93 mice analysed, 57 carried mutant alleles and 22 of them had long de novo insertion(s) at DSB-introduced sites; two were spliced mRNAs of Pcnt and Inadl without introns, indicating the involvement of reverse transcription (RT). Fifteen alleles included retrotransposons, mRNAs, and other sequences without evidence of RT. Two others were sgRNAs with one containing T7 promoter-derived sequence suggestive of a PCR product as its origin. In conclusion, RT-product-mediated DSB repair (RMDR) and non-RMDR repair were identified in the mouse zygote. We also confirmed that both RMDR and non-RMDR take place in CRISPR/Cas transfected NIH-3T3 cells. Finally, as two de novo MuERV-L insertions in C57BL/6 mice were shown to have characteristic features of RMDR in natural conditions, we hypothesize that RMDR contributes to the emergence of novel DNA sequences in the course of evolution.

Paternally expressed 11/Retrotransposon-like 1 (Peg11/Rtl1) knockout (KO) mice show mid-to late fetal lethality or late fetal growth retardation associated with frequent neonatal lethality. The lethal phenotype is largely dependent on genetic background and becomes more severe with each succeeding generation in the course of backcross experiments to C57BL/6 (B6). We previously suggested that these lethal and growth phenotypes in the fetal stages were due to severe defects in placental fetal capillaries in the labyrinth layer. In this study, we re-examined KO fetuses and placentas and confirmed that the severe clogging of fetal capillaries was associated with KO fetuses showing mid-fetal lethality with internal bleeding. Importantly, the basal region of the fetal capillary network was specifically damaged, also leading to poor expansion of the labyrinth layer and placental size reduction in the later stage. An apparent down-regulation of transmembrane protein 100 (Tmem100), mesenchyme homeobox 2 (Meox2) and lymphatic vessel endothelial hyaluronan receptor 1 (Lyve1) expression were observed in earlier stage placentas even before apparent size reduction became, suggesting that these genes are involved in the maintenance of fetal capillaries associated with Peg11/Rtl1 during development.

RTL1 (also termed paternal expressed 11 (PEG11)) is considered the major imprinted gene responsible for the placental and fetal/neonatal muscle defects that occur in the Kagami–Ogata and Temple syndromes (KOS14 and TS14, respectively). However, it remains elusive whether RTL1 is also involved in their neurological symptoms, such as behavioral and developmental delay/intellectual disability, feeding difficulties, motor delay, and delayed speech. Here, we demonstrate that the mouse RTL1 protein is widely expressed in the central nervous system (CNS), including the limbic system. Importantly, two disease model mice with over‐ and under‐expression of Rtl1 exhibited reduced locomotor activity, increased anxiety, and impaired amygdala‐dependent cued fear, demonstrating that Rtl1 also plays an important role in the CNS. These results indicate that the KOS14 and TS14 are neuromuscular as well as neuropsychiatric diseases caused by irregular CNS RTL1 expression, presumably leading to impaired innervation of motor neurons to skeletal muscles as well as malfunction of the hippocampus‐amygdala complex. It is of considerable interest that eutherian‐specific RTL1 is expressed in mammalian‐ and eutherian‐specific brain structures, that is, the corticospinal tract and corpus callosum, respectively, suggesting that RTL1 might have contributed to the acquisition of both these structures themselves and fine motor skill in eutherian brain evolution.