Exposure of chimaeric embryos to exogenous FGF4 leads to the production of pure ESC-derived mice.

Producing chimaeras constitutes the most reliable method of verifying the pluripotency of newly established cells. Moreover, forming chimaeras by injecting genetically modified embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) into the embryo is part of the procedure for generating transgenic mice, which are used for understanding gene function. Conventional methods for generating transgenic mice, including the breeding of chimaeras and tetraploid complementation, are time-consuming and cost-inefficient, with significant limitations that hinder their effectiveness and widespread applications. In the present study, we modified the traditional method of chimaera generation to significantly speed up this process by generating mice exclusively derived from ESCs. This study aimed to assess whether fully ESC-derived mice could be obtained by modulating fibroblast growth factor 4 (FGF4) levels in the culture medium and changing the direction of cell differentiation in the chimaeric embryo. We found that exogenous FGF4 directs all host blastomeres to the primitive endoderm fate, but does not affect the localisation of ESCs in the epiblast of the chimaeric embryos. Consequently, all FGF4-treated chimaeric embryos contained an epiblast composed exclusively of ESCs, and following transfer into recipient mice, these embryos developed into fully ESC-derived newborns. Collectively, this simple approach could accelerate the generation of ESC-derived animals and thus optimise ESC-mediated transgenesis and the verification of cell pluripotency. Compared to traditional methods, it could speed up functional studies by several weeks and significantly reduce costs related to maintaining and breeding chimaeras. Moreover, since the effect of stimulating the FGF signalling pathway is universal across different animal species, our approach can be applied not only to rodents but also to other animals, offering its utility beyond laboratory settings.

The number of nuclei in compacted embryos, assessed by optical coherence microscopy, is a non-invasive and robust marker of mouse embryo quality.

Optical coherence microscopy (OCM) visualizes nuclei in live, unlabeled cells. As most cells are uninucleated, the number of nuclei in embryos may serve as a proxy of the cell number, providing important information on developmental status of the embryo. Importantly, no other non-invasive method currently allows for the cell number count in compacted embryos. We addressed the question of whether OCM, by providing the number of nuclei in compacted mouse embryos, may help evaluate embryo quality. We subjected compacted embryonic Day 3 (E3.0: 72 h after onset of insemination) mouse embryos to OCM scanning and correlated nuclei number and developmental potential. Implantation was assessed using an outgrowth assay (in vitro model meant to reflect embryonic ability to implant in vivo). Embryos with more cells at E3.0 (>18 cells) were more likely to reach the blastocyst stage by E4.0 and E5.0 (P ≪ 0.001) and initiate hatching by E5.0 (P < 0.05) than those with fewer cells (<12 cells). Moreover, the number of cells at E3.0 strongly correlated with the total number of cells in E4.0 and E5.0 embryos (ρ = 0.71, P ≪ 0.001 and ρ = 0.61, P ≪ 0.001, respectively), also when only E4.0 and E5.0 blastocysts were considered (ρ = 0.58, P ≪ 0.001 and ρ = 0.56, P ≪ 0.001, respectively). Additionally, we observed a strong correlation between the number of cells at E3.0 and the number of trophectoderm cells in E4.0 and E5.0 blastocysts (ρ = 0.59, P ≪ 0.001 and ρ = 0.57, P ≪ 0.001, respectively). Importantly, embryos that had more cells at E3.0 (>18 cells) were also more likely to implant in vitro than their counterparts with fewer cells (<12 cells; P ≪ 0.001). Finally, we tested the safety of OCM imaging, demonstrating that OCM scanning affected neither the amount of reactive oxygen species nor mitochondrial activity in the embryos. OCM also did not hinder their preimplantation development, ability to implant in vitro, or to develop to term after transfer to recipient females. Our data indicate that OCM imaging provides important information on embryo quality. As the method seems to be safe for embryos, it could be a valuable addition to the current repertoire of embryo evaluation methods. However, our study was conducted only on mouse embryos, so the proposed protocol would require optimization in order to be applied in other species.

Inhibition of stearoyl-CoA desaturase 1 in the mouse impairs pancreatic islet morphogenesis and promotes loss of ß-cell identity and α-cell expansion in the mature pancreas.

Abnormalities that characterize the pathophysiology of type 2 diabetes (T2D) include deficiencies of ß-cells and the expansion of α-cells in pancreatic islets, manifested by lower insulin release and glucagon oversecretion. The molecular mechanisms that determine intra-islet interactions between pancreatic α- and ß-cells are still not fully understood. The present study showed that stearoyl-coenzyme A (CoA) desaturase 1 (SCD1), an enzyme that is implicated in fatty acid metabolism, serves as a checkpoint in the control of endocrine cell equilibrium in pancreatic islets. Our data showed that SCD1 activity is essential for proper α-cell and ß-cell lineage determination during morphogenesis of the pancreas and the maintenance of mature ß-cell identity. The inhibition of SCD1 expression/activity led to both a decrease in the expression of ß-cell signature genes (e.g., Pdx1, Nkx6.1, MafA, and Neurod1, among others) and induction of the expression of the dedifferentiation marker Sox9 in mature pancreatic islets. The transcriptional repression of Pdx1 and MafA in SCD1-deficient ß-cells was related to the excessive methylation of promoter regions of these transcription factors. In contrast, SCD1 ablation favored the formation of α-cells over ß-cells throughout pancreas organogenesis and did not compromise α-cell identity in adult pancreatic islets. Such molecular changes that were caused by SCD1 downregulation resulted in the mislocalization of α-cells within the core of islets and increased the ratio of pancreatic α- to ß-cell mass. This was followed by islet dysfunction, including impairments in glucose-stimulated insulin release, simultaneously with elevations of basal glucagon secretion. Altogether, these findings provide additional mechanistic insights into the role of SCD1 in the pathogenesis of T2D.

Cytoplasmic polyadenylation by TENT5A is required for proper bone formation.

Osteoblasts orchestrate bone formation through the secretion of type I collagen and other constituents of the matrix on which hydroxyapatite crystals mineralize. Here, we show that TENT5A, whose mutations were found in congenital bone disease osteogenesis imperfecta patients, is a cytoplasmic poly(A) polymerase playing a crucial role in regulating bone mineralization. Direct RNA sequencing revealed that TENT5A is induced during osteoblast differentiation and polyadenylates mRNAs encoding Col1α1, Col1α2, and other secreted proteins involved in osteogenesis, increasing their expression. We postulate that TENT5A, possibly together with its paralog TENT5C, is responsible for the wave of cytoplasmic polyadenylation of mRNAs encoding secreted proteins occurring during bone mineralization. Importantly, the Tent5a knockout (KO) mouse line displays bone fragility and skeletal hypomineralization phenotype resulting from quantitative and qualitative collagen defects. Thus, we report a biologically relevant posttranscriptional regulator of collagen production and, more generally, bone formation.

Attempts to obtain fully xenogeneic fetuses in rat ↔ mouse model†,‡.

The full-term development of the xenogeneic embryo in the uterus of the mother of different species is very restricted and can occur only in certain groups of closely related mammals. In the case of mouse ↔ rat chimeras, the interspecific uterine barrier is less hostile to interspecific chimeric fetuses. In current work, we tested the development of mouse and rat fetuses in uteri of females of the opposite species. We created chimeric mouse ↔ rat blastocysts by injection of mouse embryonic stem cells (ESCs) into eight-cell rat embryos and rat ESCs into eight-cell mouse embryos. Chimeras were transferred to the foster mothers of the opposite species. Despite a huge number of transferred embryos (>1000 in total for both variants), only one live fetus derived solely from the mouse ESCs was isolated at E13.5 from the rat uterus. All other fetuses and newborns were chimeric or were built only from the cells of the recipient embryo. We examined the possible reason for such an outcome and found that the xenogeneic fetuses are eliminated at the perigastrulation stage of development. Thus, we conclude that in the rat ↔ mouse combination even when extraembryonic tissues of the chimeric embryo are composed solely of the cells of the same species as the female to which embryos are transferred, the full-term development of the pure xenogeneic fetus is very unlikely.

Postovulatory ageing modifies sperm-induced Ca2+ oscillations in mouse oocytes through a conditions-dependent, multi-pathway mechanism.

Postovulatory ageing of mammalian oocytes occurs between their ovulation and fertilization and has been shown to decrease their developmental capabilities. Aged oocytes display numerous abnormalities, including altered Ca2+ signalling. Fertilization-induced Ca2+ oscillations are essential for activation of the embryonic development, therefore maintaining proper Ca2+ homeostasis is crucial for the oocyte quality. In the present paper, we show that the mechanism underlying age-dependent alterations in the pattern of sperm-triggered Ca2+ oscillations is more complex and multifaceted than previously believed. Using time-lapse imaging accompanied by immunostaining and molecular analyses, we found that postovulatory ageing affects the amount of Ca2+ stored in the cell, expression of Ca2+ pump SERCA2, amount of available ATP and distribution of endoplasmic reticulum and mitochondria in a manner often strongly depending on ageing conditions (in vitro vs. in vivo). Importantly, those changes do not have to be caused by oxidative stress, usually linked with the ageing process, as they occur even if the amount of reactive oxygen species remains low. Instead, our results suggest that aberrations in Ca2+ signalling may be a synergistic result of ageing-related alterations of the cell cycle, cytoskeleton, and mitochondrial functionality.

Dynamics of cytoplasm and cleavage divisions correlates with preimplantation embryo development.

In vitro fertilization has become increasingly popular as an infertility treatment. In order to improve efficiency of this procedure, there is a strong need for a refinement of existing embryo assessment methods and development of novel, robust and non-invasive selection protocols. Studies conducted on animal models can be extremely helpful here, as they allow for more extensive research on the potential biomarkers of embryo quality. In the present paper, we subjected mouse embryos to non-invasive time-lapse imaging and combined the Particle Image Velocimetry analysis of cytoplasmic dynamics in freshly fertilized oocytes with the morphokinetic analysis of recordings covering 5 days of preimplantation development. Our results indicate that parameters describing cytoplasmic dynamics and cleavage divisions independently correspond to mouse embryo's capacity to form a high-quality blastocyst. We also showed for the first time that these parameters are associated with the percentage of abnormal embryonic cells with fragmented nuclei and with embryo's ability to form primitive endoderm, one of the cell lineages differentiated during preimplantation development. Finally, we present a model that links selected cytoplasmic and morphokinetic parameters reflecting frequency of fertilization-induced Ca2+-oscillations and timing of 4-cell stage and compaction with viability of the embryo assessed as the total number of cells at the end of its preimplantation development. Our results indicate that a combined analysis of cytoplasmic dynamics and morphokinetics may facilitate the assessment of embryo's ability to form high-quality blastocysts.

Mouse↔rat aggregation chimaeras can develop to adulthood.

In order to examine interactions between cells originating from different species during embryonic development we constructed interspecific mouse↔rat chimaeras by aggregation of 8-cell embryos. Embryos of both species expressed different fluorescent markers (eGFP and DsRed), which enabled us to follow the fate of both components from the moment of aggregation until adulthood. We revealed that in majority of embryos the blastocyst cavity appeared inside the group of rat cells, while the mouse component was allocated to the deeper layer of the inner cell mass and to the polar trophectoderm. However, due to rearrangement of all cells and selective elimination of rat cells, shortly before implantation all primary lineages became chimaeric. Moreover, despite the fact that rat cells were always present in the mural trophectoderm, majority of mouse↔rat chimaeric blastocysts implanted in mouse uterus, and out of those 46% developed into foetuses and pups, half of which were chimaeric. In contrast to mural trophectoderm, polar trophectoderm derivatives, i.e. the placentae of all chimaeras were exclusively of mouse origin. This strongly suggests that the successful postimplantation development of chimaeras is enabled by gradual elimination of xenogeneic cells from the nascent placenta. The size of chimaeric newborns was within the limits of control mouse neonates. The rat component located preferentially in the anterior part of the body, where it contributed mainly to the neural tube. Our observations indicate that although chimaeric animals were able to reach adulthood, high contribution of rat cells tended to diminish their viability.

Mouse blastomeres acquire ability to divide asymmetrically before compaction.

The mouse preimplantation embryo generates the precursors of trophectoderm (TE) and inner cell mass (ICM) during the 8- to 16-cell stage transition, when the apico-basal polarized blastomeres undergo divisions that give rise to cells with different fate. Asymmetric segregation of polar domain at 8-16 cell division generate two cell types, polar cells which adopt an outer position and develop in TE and apolar cells which are allocated to inner position as the precursors of ICM. It is still not know when the blastomeres of 8-cell stage start to be determined to undergo asymmetric division. Here, we analyze the frequency of symmetric and asymmetric divisions of blastomeres isolated from 8-cell stage embryo before and after compaction. Using p-Ezrin as the polarity marker we found that size of blastomeres in 2/16 pairs cannot be used as a criterion for distinguishing symmetric and asymmetric divisions. Our results showed that at early 8-cell stage, before any visible signs of cortical polarity, a subset of blastomeres had been already predestined to divide asymmetrically. We also showed that almost all of 8-cell stage blastomeres isolated from compacted embryo divide asymmetrically, whereas in intact embryos, the frequency of asymmetric divisions is significantly lower. Therefore we conclude that in intact embryo the frequency of symmetric and asymmetric division is regulated by cell-cell interactions.

In Memoriam - Prof. Andrzej Krzysztof Tarkowski (1933-2016).

Professor Andrzej Krzysztof Tarkowski passed away last September (2016) at the age of 83. His findings, have become indispensable tools for immunological, genetic, and oncological studies, as well as for generating transgenic animals which are instrumental for studying gene function in living animals. His work and discoveries provided a tremendous input to the contemporary developmental biology of mammals.