The Research Of Early Embryonic Development And Disease Models In Nonhuman Primate

A mammal develops from a single zygote upon the oocyte fertilization by a sperm.The zygote divides and gives rise to the morula,which further matures to a blastocyst.The blastocyst reaches the uterus,implants in the endometrium,and then continues its development into a fetus.In this process,preimplantation embryo goes through a series of important biological events including maternal to zygotic transition,zygotic genome activation,and cell lineage differentiation.All these events are relying on a highly coordinated cascade of molecule regulations in the embryo and especially vulnerable to environment stress.High temperature and endocrine-disrupting compound,for example,can interfere with the normal development of gametes and embryos,and even produce epigenetic changes that can be inherited.Due to the close phylogenetic relationship between humans and non-human primates(NHPs),the study on the development of embryos in NHPs can provide a better understanding of human early embryonic development.Moreover,NHPs disease models is idea animal model for human disease study.In this thesis,we focus on the molecule mechanisms and biomedical applications of preimplantation embryos from rhesus monkeys and cynomolgus monkeys,using different in vitro culture systems.We used single-cell transcriptome sequencing technology to profile gene expressions of rhesus monkey preimplantation embryos at different developmental stages.These results provide a comprehensive transcriptome landscape of rhesus monkey embryo from intracytoplasmic sperm injection(ICSI).Our data show for the first time that there is a transcription retardation in NHPs preimplantation embryos,despite normal morphology.Meanwhile,compared with the normal ICSI blastocyst,the transcription profiles of blastocysts from somatic cell nuclear transfer(SCNT),androgenesis(AG)and parthenogenesis(PG)embryos showed more prevalent retardation,indicate the failure of zygote genome activation which might result in the abnormal development of AG and SCNT embryos.Using the CRISPR/Cas9 system to target DAX1 gene of preimplantation embryos and successfully obtained a gene-modified cynomolgus monkey.The male fetuses present similar aberrant phenotypes of adrenal hypoplasia congenita(AHC)and hypogonadotropic hypogonadism(HH)diagnosed in human patients.We further detected abnormal activation of Wnt/?-catenin signaling in the fetal DAX1-deficient testis,which could affect testis neovascularization and cause AHC-HH.This study showed that the adrenal and gonadal developmental defects caused by DAX1 deficiency in male cynomolgus begin at the gonadal decision-making stage in fetal stage,we also proved that NHP models are better than rodent models to represent human disease.Through generation of chimeric preimplantation embryos,we proved that cynomolgus monkey pluripotent stem cells,including embryonic stem cells(ESCs),induced pluripotent stem cells(i PSCs),and nuclear transfer-embryonic stem cells(NT-ESCs),can be incorporated into host embryos and develop into chimeras with all three germ layers and extraembryonic ectoderm.After analyzing single cell transcriptomes of chimeric embryos,we found that the chimeric efficiency is related to the pluripotency of injected stem cells and is also regulated by the microenvironments of chimeric embryos.In summary,we investigated the molecular mechanisms involved in the development of NHP preimplantation embryos.Further,the chimeras and NHP disease models generated from the preimplantation embryos would be powerful tools for stem cell and biomedical research.This study is the first complete transcription mapping of primate preimplantation embryos,and it is important supplement for the study of primate early development regulation.The establishment of monkey model via CRISPR/Cas9 technology which fully reflects the advantages in the study of human diseases.