Preimplantation Development In Mouse Research Articles

Cross-species comparison of mouse and human preimplantation development with an emphasis on lineage specification.

Human embryogenesis still remains largely unexplored. This review helps identify some of our current gaps in knowledge pertaining to preimplantation development, which may have implications for understanding fundamental aspects of human development, assisted reproductive technologies, and stem cell biology. Preimplantation development is arguably one of the most critical stages of embryogenesis. Beginning with the formation of the totipotent zygote post-fertilization, a series of cell divisions, and a complex coordination of physical cues, molecular signals and changes in gene expression lead to the formation of the blastocyst, a structure capable of implanting into the uterine wall. The blastocyst is composed of more specified cellular lineages, which will give rise to every tissue of the developing organism as well as the extra-embryonic lineages which support fetal growth. While the mouse has been used as a model to understand the events of preimplantation development for decades, in recent years, an expanding body of work has been conducted using the human embryo. These studies have identified some crucial species differences, particularly in the transcriptional and spatio-temporal expression of lineage markers and responses to cell signaling perturbations. This review compares recent findings on preimplantation development in mouse and human, with a focus on the specification of the first cellular lineages. Highlighting differences and noting mechanisms that require further examination in the human embryo is of critical importance for both the accurate translation of results from the mouse model and our overall understanding of mammalian development. We further highlight the latest advancement in reproductive research, the development of the 3D stem cell-based models known as 'blastoids'. The knowledge discussed in this review has major clinical implications for assisted reproductive technologies such as in vitro fertilization and for applications in stem cell biology.

O-099 TEAD4 regulates trophectoderm differentiation upstream of CDX2 in human preimplantation embryos

Abstract Study question What is the main pathway regulating trophectoderm (TE) differentiation during pre-implantation development in mouse versus human embryos? Summary answer TEAD4 is acting upstream of CDX2 and is involved in TE differentiation, as TEAD4-null human embryos exhibit compromised TE lineage differentiation. What is known already TEAD4 is the earliest transcription factor during early embryo development, required for the expression of TE-associated genes leading to successful TE differentiation and subsequent blastocoel formation in mouse. Functional knock-out studies in mouse, inactivating Tead4 by site-specific recombination have shown that Tead4-null embryos do not express TE specific genes, including Caudal-Type Homeobox Protein 2 (Cdx2) and GATA Binding Protein 3 (Gata3), but expression of inner cell mass (ICM)-specific genes, remains unaffected. Furthermore, ablation of Tead4 compromises embryonic development and subsequent blastocoel formation in mouse. The role of TEAD4, during human pre-implantation development has not been functionally characterized yet. Study design, size, duration CRISPR-Cas9 was introduced in mouse zygotes and editing efficiency was evaluated by next-generation sequencing (NGS) on 4.5dpc embryos (n = 55). Developmental kinetics were monitored in CRISPR-Cas9 targeted (n = 83), sham-injected (n = 26) and non-injected media-control (n = 51) mouse embryos. Immunofluorescence analysis was performed in Tead4 targeted (n = 57) and non-injected media-control embryos (n = 94). The same methodology was applied in human donated in vitro matured (IVM) metaphase-II (MII) oocytes, which were CRISPR-Cas9 targeted (n = 74) during ICSI or used as media-Control (n = 33). Participants/materials, setting, methods A gRNA-Cas9 mixture targeting exon 2 of Tead4/TEAD4 was microinjected in respectively mouse 2PN (pronuclear) stage zygotes, or human IVM MII oocytes along with the sperm. Generated embryos were cultured in vitro for 4 days in mouse or 6.5 days in human. Embryonic development and morphology were assessed daily, followed by a detailed scoring at the late blastocyst stage. Successful targeting following CRISPR-Cas9 introduction was assessed by immunostaining and NGS analysis of the targeted locus. Main results and the role of chance In mouse, we confirmed previous findings, as the developmental capacity of Tead4 targeted embryos was significantly reduced starting from the morula stage and blastocyst formation rates were 8.97% in the targeted group, compared to 87.23% in the control and 87.50% in the sham group, respectively. Immunofluorescence analysis of late morula and blastocyst stage embryos confirmed the absence of Tead4, Cdx2 and Gata3, resulting from the successful interruption of the Tead4 locus (n = 57). Exon 2 of TEAD4 was successfully targeted in human. In total, 21 embryos from various developmental stages were successfully NGS analyzed and 90,48% (19 out of 21) of the embryos carried genetic modifications as a result of CRISPR-Cas9 genome editing and seven blastocysts were identified carrying exclusively frameshift mutations. In contrast to mouse, the developmental capacity of human targeted embryos (25%) did not differ significantly from the control group (23%). However, the blastocyst morphology and quality were compromised in the targeted group showing mostly grade C TE scores, containing very few cells. Immunofluorescence analysis of targeted blastocysts (n = 6) confirmed successful editing by complete absence of TEAD4 and its downstream TE marker CDX2. Limitations, reasons for caution CRISPR-Cas9 germline genome editing results in multiple editing outcomes with variable phenotypic penetrance, the mosaic nature of which complicates the phenotypic analysis and developmental behaviour of the injected embryos. Wider implications of the findings Elucidation of the evolutionary conserved molecular mechanisms that regulate self-renewal of the trophoblast lineage can give us fundamental insights on early implantation failure. Trial registration number Not Applicable

Dynamic Alternative Splicing During Mouse Preimplantation Embryo Development.

The mechanism of alternative pre-mRNA splicing (AS) during preimplantation development is largely unknown. In order to capture the dynamic changes of AS occurring during embryogenesis, we carried out bioinformatics analysis based on scRNA-seq data over the time-course preimplantation development in mouse. We detected numerous previously-unreported differentially expressed genes at specific developmental stages and investigated the nature of AS at both minor and major zygotic genome activation (ZGA). The AS and differential AS atlas over preimplantation development were established. The differentially alternatively spliced genes (DASGs) are likely to be key splicing factors (SFs) during preimplantation development. We also demonstrated that there is a regulatory cascade of AS events in which some key SFs are regulated by differentially AS of their own gene transcripts. Moreover, 212 isoform switches (ISs) during preimplantation development were detected, which may be critical for decoding the mechanism of early embryogenesis. Importantly, we uncovered that zygotic AS activation (ZASA) is in conformity with ZGA and revealed that AS is coupled with transcription during preimplantation development. Our results may provide a deeper insight into the regulation of early embryogenesis.

Maternal Setdb1 Is Required for Meiotic Progression and Preimplantation Development in Mouse.

Oocyte meiotic progression and maternal-to-zygote transition are accompanied by dynamic epigenetic changes. The functional significance of these changes and the key epigenetic regulators involved are largely unknown. Here we show that Setdb1, a lysine methyltransferase, controls the global level of histone H3 lysine 9 di-methyl (H3K9me2) mark in growing oocytes. Conditional deletion of Setdb1 in developing oocytes leads to meiotic arrest at the germinal vesicle and meiosis I stages, resulting in substantially fewer mature eggs. Embryos derived from these eggs exhibit severe defects in cell cycle progression, progressive delays in preimplantation development, and degeneration before reaching the blastocyst stage. Rescue experiments by expressing wild-type or inactive Setdb1 in Setdb1-deficient oocytes suggest that the catalytic activity of Setdb1 is essential for meiotic progression and early embryogenesis. Mechanistically, up-regulation of Cdc14b, a dual-specificity phosphatase that inhibits meiotic progression, greatly contributes to the meiotic arrest phenotype. Setdb1 deficiency also leads to derepression of transposons and increased DNA damage in oocytes, which likely also contribute to meiotic defects. Thus, Setdb1 is a maternal-effect gene that controls meiotic progression and is essential for early embryogenesis. Our results uncover an important link between the epigenetic machinery and the major signaling pathway governing meiotic progression.

A non-canonical mode of microtubule organization operates throughout pre-implantation development in mouse

In dividing animal cells, the centrosome, comprising centrioles and surrounding pericentriolar-material (PCM), is the major interphase microtubule-organizing center (MTOC), arranging a polarized array of microtubules (MTs) that controls cellular architecture. The mouse embryo is a unique setting for investigating the role of centrosomes in MT organization, since the early embryo is acentrosomal, and centrosomes emerge de novo during early cleavages. Here we use embryos from a GFP::CETN2 transgenic mouse to observe the emergence of centrosomes and centrioles in embryos, and show that unfocused acentriolar centrosomes first form in morulae (~16–32-cell stage) and become focused at the blastocyst stage (~64–128 cells) concomitant with the emergence of centrioles. We then used high-resolution microscopy and dynamic tracking of MT growth events in live embryos to examine the impact of centrosome emergence upon interphase MT dynamics. We report that pre-implantation mouse embryos of all stages employ a non-canonical mode of MT organization that generates a complex array of randomly oriented MTs that are preferentially nucleated adjacent to nuclear and plasmalemmal membranes and cell-cell interfaces. Surprisingly, however, cells of the early embryo continue to employ this mode of interphase MT organization even after the emergence of centrosomes. Centrosomes are found at MT-sparse sites and have no detectable impact upon interphase MT dynamics. To our knowledge, the early embryo is unique among proliferating cells in adopting an acentrosomal mode of MT organization despite the presence of centrosomes, revealing that the transition to a canonical mode of interphase MT organization remains incomplete prior to implantation.

The CDC14B Phosphatase Is a Negative Regulator of Pre-Implantation Embryonic Development in Mouse.

Following fertilization, the transition from a highly differentiated oocyte to totipotent 2-cell blastomeres requires two unique mitotic cell cycles. The first cell cycle is characterized by a prolonged G1 phase and DNA replication (S phase) that occurs separately in the female and male pronuclei. During the second cell cycle, the G2 phase is prolonged and occurs concurrently with zygotic genome activation, which is essential for progression past the 2-cell embryonic stage. CDC14B, a dual specificity phosphatase that counteracts cyclin dependent kinase 1 (CDK1) action, regulates mitosis in somatic cells and prevents premature meiotic resumption in mouse oocytes. It is not known if CDC14B plays a role during the unique mitotic cell cycles during preimplantation development. We demonstrate that CDC14B is present in preimplantation mouse embryos and that it localizes to mitotic centrosomes and spindles. Over-expression of CDC14B in 1-cell embryos prevents development to the blastocyst stage as 40% and 60% of the embryos arrest at the 1 and 2-cell stages, respectively. The embryos that arrest at the 1-cell stage contain reduced CDK1 activity, suggesting that this block is due to a cell cycle defect. Notably, the embryos that arrest at the 2-cell stage fail to activate the zygotic genome despite having normal levels of CDK1 activity. This function is likely specific to embryos because over-expression of CDC14B in meiotically incompetent oocytes that are transcriptionally active does not repress global transcription. These data suggest that CDC14B is a negative regulator of the 1-to-2-cell transition and of zygotic genome activation in mouse embryogenesis. K.S. was supported by HD055822 and M.G.B. was supported by 5D43TW000671. This research was supported by a grant from the NIH (HD22681) to R.M.S. (poster)

A shift from kinesin 5-dependent metaphase spindle function during preimplantation development in mouse

Microtubules within meiotic and mitotic spindles continually move towards spindle poles in a process termed poleward flux, which is essential for spindle integrity and faithful chromosome segregation. Kinesin 5 is a longstanding candidate for a molecular motor that might drive poleward flux, and has been shown to drive flux and to be necessary for spindle bipolarity in Xenopus egg extracts. However, kinesin 5 is not necessary for poleward flux or for maintaining metaphase spindle bipolarity in intact mammalian cells, and the reason for the different results in these systems is unknown. The experiments presented here test the hypothesis that these results might reflect developmental differences in spindle function by examining the role of kinesin 5 in mouse eggs and preimplantation embryos. In contrast to cultured somatic cells, poleward flux in mouse eggs is critically dependent upon kinesin 5. Inhibition of poleward flux leads to spindle shortening as a result of continued microtubule depolymerisation at the pole, and eventual loss of spindle bipolarity. Spindle bipolarity is also dependent upon kinesin 5 during the first three embryonic cleavages, but becomes kinesin 5-independent in the majority of spindles by the blastocyst stage. This switch occurs asynchronously in different blastomeres but is independent of clonal cell heritage and of whether the blastomere is within the inner cell mass or the trophoectoderm. These experiments reveal a novel developmental switch in the requirements for spindle function and chromosome segregation during preimplantation development.

The Expression and Stage-Specific Localization of Protein Kinase C Isotypes during Mouse Preimplantation Development

Signaling events mediate many processes that act during embryogenesis to initiate the program of early development. Within the cell many of these changes are mediated through the activation or inactivation of kinases and phosphatases. Protein kinase C (PKC) is one kinase that has been shown to be involved in at least two developmental transitions during early development, fertilization and embryonic compaction. PKC is a family of kinases whose various isotypes have differing requirements for activation of the kinase that include the availability of calcium, diacylglycerol, and negatively charged phospholipids. The presence of more than one isotype in an egg or blastomere of the embryo would provide the possibility that different isotypes mediate distinct signaling pathways in the cells. To address this possibility the different isotypes of PKC were examined at the mRNA and protein levels during preimplantation development in the mouse. Our results demonstrate that seven isotypes of PKC are present during preimplantation development in mouse, some are of maternal origin and others appear after fertilization. Two isotypes have a stage-dependent nuclear localization. In addition, within each blastomere PKC isotypes occupy different subcellular locations in a stage-dependent fashion.

Preimplantation development in the streptozotocin-induced diabetic mouse

Streptozotocin (STZ) was used to develop a diabetic mouse model in which to study the development of the preimplantation embryo. STZ doses of 0, 160, 190, 210 and 240 mg kg-1 were given; 190 mg kg-1 was found to be the most suitable as the standard diabetogenic dose, providing about 60% mice with plasma glucose greater than 20 mM. The STZ-diabetic mice responded to superovulation with 10 i.u. of gonadotrophin in the same manner as control mice, producing similar embryo numbers at 48 h, 72 h and 96 h post-hCG. Furthermore, the proportion of 2-cell embryos collected from STZ-diabetic mice which developed to blastocysts in vitro was similar to that of 2-cell embryos from control mice. The STZ-diabetic mouse model after superovulation thus produced normal early preimplantation embryos whose development can be examined in detail in a diabetic environment.