Noise control and utility: From regulatory network to spatial patterning

Stochasticity (or noise) at cellular and molecular levels has been observed extensively as a universal feature for living systems. However, how living systems deal with noise while performing desirable biological functions remains a major mystery. Regulatory network configurations, such as their topology and timescale, are shown to be critical in attenuating noise, and noise is also found to facilitate cell fate decision. Here we review major recent findings on noise attenuation through regulatory control, the benefit of noise via noise-induced cellular plasticity during developmental patterning, and summarize key principles underlying noise control

Embryology

Embryology is a branch of science concerned with the morphological aspects of organismal development. The genomic and molecular revolution of the second half of the 20th century, together with the classic descriptive aspects of this science have allowed greater integration in our understanding of many developmental events. Through such integration, modern embryology seeks to provide practical knowledge that can be applied to assisted reproduction, stem cell therapy, birth defects, fetal surgery and other fields. This book focuses on human embryology and aims to provide an up-to-date source of information on a variety of selected topics. The book consists of nine chapters organized into three sections, namely: 1) gametes and infertility, 2) implantation, placentation and early development, and 3) perspectives in embryology. The contents of this book should be of interest to biology and medical students, clinical embryologists, laboratory researchers, obstetricians and urologists, developmental biologists, molecular geneticists and anyone who wishes to know more about recent advances in human development

The development of a cell-based assay for quality control in human in vitro fertilisation

Quality control systems are critical to improving laboratory practices and contribute significantly to success in human in vitro fertilisation (IVF). The mouse embryo assay (MEA) is commonly used in production quality control, as well as in product research and development. Despite its popularity there are some well-known issues associated with it; these include, but are not limited to, insensitivity to sub-optimal conditions, ethical issues due to animal use and variation in end-point interpretation between operators. The aim of this thesis was to therefore develop a cell-based assay that could facilitate research and development whilst addressing the issues presented above. Firstly, using the pluripotent P19 embryonal carcinoma cell line, 2D and 3D cell-based assays were developed and optimised, and their response to culture perturbations such as varying osmolality and energy source levels were tested. Secondly, given that mammalian cells are generally resistant to changes in culture condition, characteristic features of the mammalian cold stress response, mTOR inhibitor rapamycin and AMPK inhibitor dorsomorphin were incorporated as potential tools for decreasing cellular resistance to altering culture conditions. The P19 assay demonstrated sensitivity to changes in energy substrate availability when treated with rapamycin and dorsomorphin, and was subsequently used to systematically screen different media formulations in order to identify the ranges of pyruvate, glutamine and calcium lactate concentrations required for optimum cell growth. Lastly, the developed 2D assay and the MEA were treated with the same products and their responses were directly compared to highlight similarities, and to identify differences, between the two assays. These assays/comparisons showed that the P19 assay identified differences in human IVF culture media that were not identified by the MEA. The P19 assay may therefore represent a sensitive alternative to the MEA that can detect discrete changes in culture conditions and indicate the extent to which different energy substrates affect cell growth and proliferation. It is important to note that the two assays differ in their robustness and the P19 assay responded differently to repeat experiments with the same treatment. Overall, this novel P19 assay may provide an effective means of validating and testing products used in IVF whilst reducing animal use in research and developmen

Comparative Analysis of Small Non-Coding RNA and Messenger RNA Expression in Somatic Cell Nuclear Transfer and In Vitro-Fertilized Bovine Embryos During Early Development Through the Maternal-to-Embryonic Transition

Cloning animals using somatic cell nuclear transfer (scNT) was first successfully demonstrated with the birth of Dolly the sheep, but the process of cloning remains highly inefficient. By improving our understanding of the errors that may occur during cloned cattle embryo development, we could obtain a greater understanding of how specific molecular events contribute to successful development. The central dogma of biology refers to the process of DNA being transcribed into messenger RNA (mRNA) and the translation of mRNA into proteins, which ultimately carry out the functions encoded by genes. The epigenetic code is defined as the array of chemical modifications, or “marks”, to DNA molecules that do not change the genome sequence but do allow for control of gene expression. During early development, genome reprogramming involves the removal of epigenetic marks from the sperm and egg and re-establishment of marks for the embryonic genome that code for proper gene expression to support embryo development. The point during this process at which the embryo’s genes are turned on is known as embryonic genome activation (EGA). Small non-coding RNAs (sncRNAs), including microRNAs (miRNAs), may also contribute to the this process. For example, miRNA molecules do not code for proteins themselves, but rather bind to mRNAs and effectively block their translation into protein. We hypothesized that aberrant expression of sncRNAs in cloned embryos may lead to anomalous abundance of mRNA molecules, thus explaining poor development of cloned embryos. First, we used RNA sequencing to examine the total population of sncRNAs in cattle embryos produced by in vitro fertilization (IVF) and found a dramatic shift in populations at the EGA. Next, we collected both sncRNA and mRNA from scNT cattle embryos, and again performed sequencing of both RNA fractions. We found that few sncRNAs were abnormally expressed in scNT embryos, with all differences appearing after EGA at the morula developmental stage. However, notable differences in the populations of sncRNAs were evident when comparing embryos by developmental stage. For populations of mRNA, we observed dramatic differences when comparing scNT and IVF cattle embryos, with the highest number of changes occurring at the EGA (8-cell stage) and after (morula stage). While changes in specific miRNA molecules (miR-34a and miR-345) were negatively correlated with some of their predicted target mRNAs, this pattern was not widespread as would be expected if these sncRNAs are functionally binding to all of the predicted mRNA targets. Collectively, our observations suggest that other mechanisms leading to altered expression of mRNA in cloned embryos may be responsible for their relatively poor development

Causes and consequences of chromosome segregation errors in the mouse preimplantation embryo

La division cellulaire est un processus biologique universel nécessaire à la reproduction, au développement, à la survie cellulaire ainsi qu’à la réparation des tissus. Une ségrégation chromosomique exacte pendant la mitose est essentielle pour une répartition égale des chromosomes répliqués entre les cellules filles. Des erreurs dans la ségrégation des chromosomes mènent à une condition appelée aneuploïdie, définie par un nombre inadéquat de chromosomes dans une cellule. L’aneuploïdie est associée à une altération de la santé cellulaire, la tumorigénèse, des malformations congénitales et l'infertilité. Contre toute attente, les embryons préimplantatoires de mammifères, dont les humains, consistent souvent en un mélange de cellules euploïdes et de cellules aneuploïdes. Ce mosaïcisme est inexorablement causé par des erreurs dans la ségrégation des chromosomes au cours des divisions mitotiques suivant la fécondation et est associé à un potentiel de développement réduit lors des traitements de fertilité. Malgré sa découverte il y a 25 ans, les mécanismes qui sous-tendent l’apparition de l'aneuploïdie mosaïque dans les embryons préimplantatoires sont toujours méconnus. Pour explorer les causes et les conséquences des erreurs de ségrégation chromosomique, des approches d'imagerie de fine pointe ont été utilisées sur des embryons préimplantatoires murins. L'analyse de la dynamique de la ségrégation des chromosomes via l’imagerie de cellules vivantes a permis d’identifier les chromosomes retardataires, lors de l’anaphase, comme la forme la plus répandue des erreurs de ségrégation. Ces chromosomes retardataires entraînent fréquemment une encapsulation de chromosome unique dans une structure appelée micronoyau. D'autres expériences d'imagerie par immunofluorescence sur des cellules vivantes ou fixées ont révélé que les chromosomes des micronoyaux subissent des dommages importants à l'ADN et sont mal répartis de manière récurrente lors des divisions cellulaires subséquentes dans la phase préimplantatoire. D’autres approches ont aussi permis d’examiner l'efficacité du mécanisme de contrôle de l’assemblage du fuseau mitotique, (SAC pour Spindle Assembly Checkpoint). Les résultats obtenus attestent que le SAC fonctionne, cependant la signalisation liée au SAC n’est pas efficace et ne permet pas de différer l'anaphase, malgré la présence de chromosomes retardataires et ce indépendamment de la taille des cellules. Les résultats présentés révèlent aussi qu’une inhibition partielle d’une cible du SAC, le complexe de promotion de l'anaphase (APC/C), cause une mitose prolongée et une réduction des erreurs de ségrégation. En outre, les études présentées démontrent que la fonction déficiente du SAC pendant le développement préimplantatoire est la cause principale d’une forte incidence de chromosomes retardataires qui entraînent une mauvaise ségrégation chromosomique répétée et qui causent une aneuploïdie mosaïque dans l’embryon. De plus, ce travail fournit la preuve que la modulation pharmacologique de la signalisation SAC-APC/C permet d’éviter les erreurs de ségrégation des chromosomes dans les embryons précoces. En conclusion, ces résultats apportent de nouvelles perspectives sur les causes et la nature des erreurs de ségrégation chromosomique dans les embryons. De plus, ce travail apporte de nouvelles explications mécanistiques sur l'apparition du mosaïcisme dans les embryons ce qui aura des implications importantes dans la détection et la prévention thérapeutique potentielle de l'aneuploïdie mosaïque dans les embryons préimplantatoires.Cell division is a universal biological process necessary for reproduction, development, cell survival and the maintenance and repair of tissues. Accurate chromosome segregation during mitosis is essential to ensure replicated chromosomes are partitioned equally into daughter cells. Errors in chromosome segregation often result in cells with abnormal numbers of chromosomes, a condition termed aneuploidy, which is associated with impaired cellular health, tumorigenesis, congenital defects and infertility. Counterintuitively, preimplantation embryos from many mammalian species, including humans, often consist of a mixture euploid and aneuploid cells. Such mosaic aneuploidy in embryos is inexorably caused by errors in chromosome segregation during mitotic divisions following fertilization and has been associated with reduced developmental potential in fertility treatments. However, ever since its discovery 25 years ago, how and why mosaic aneuploidy arises in the preimplantation embryo has remained elusive. To explore the causes and consequences of embryonic chromosome segregation errors, advanced imaging approaches were employed in the mouse preimplantation embryo. Live cell imaging analysis of chromosome segregation dynamics identified lagging anaphase chromosomes as the most prevalent form of chromosome mis-segregation in embryos. Lagging chromosomes frequently result in the encapsulation of single chromosomes into micronuclei, which occur in embryos in vitro and in vivo. Further live imaging and immunofluorescence experiments revealed chromosomes within micronuclei are subject to extensive DNA damage and centromeric identity loss, failing to assemble functional kinetochores and being recurrently mis-segregated during ensuing cell divisions in preimplantation development. To uncover the underlying causes for the increased propensity for chromosome mis-segregation in embryos, live imaging and loss-of-function approaches were used to examine the effectiveness of the mitotic safeguard mechanism, the Spindle Assembly Checkpoint (SAC). These studies demonstrated that the SAC normally functions to prevent segregation errors during preimplantation development but SAC signaling at misaligned chromosomes fails to delay anaphase. Moreover, SAC failure in embryos is most evident during mid-preimplantation development, independent of cell size. Partial inhibition of SAC target, the Anaphase Promoting Complex (APC/C), extended mitosis and reduced chromosome segregation errors in embryos. These studies have uncovered deficient SAC function during preimplantation development as a major cause for the high incidence of lagging chromosomes in embryos, which result in repeated mis-segregation of single chromosomes in a manner that necessarily causes mosaic aneuploidy. Additionally, this work provides proof-of-principle demonstration that pharmacological modulation of SAC-APC/C signalling can avert chromosome segregation errors in the early embryo. Altogether, these findings present new insights into the causes and nature of chromosome mis-segregation in embryos, providing novel mechanistic explanations for the occurrence of mosaicism that will have substantial implications for the detection and potential therapeutic prevention of aneuploidy in preimplantation embryos

Wnt/ß-catenin signalling and the dynamics of fate decisions in early mouse embryos and embryonic stem (ES) cells

Wnt/β-catenin signalling is a widespread cell signalling pathway with multiple roles during vertebrate development. In mouse embryonic stem (mES) cells, there is a dual role for β-catenin: it promotes differentiation when activated as part of the Wnt/β-catenin signalling pathway, and promotes stable pluripotency independently of signalling. Although mES cells resemble the preimplantation epiblast progenitors, the first requirement for Wnt/β-catenin signalling during mouse development has been reported at implantation [1,2]. The relationship between β-catenin and pluripotency and that of mES cells with epiblast progenitors suggests that β-catenin might have a functional role during preimplantation development. Here we summarize the expression and function of Wnt/β-catenin signalling elements during the early stages of mouse development and consider the reasons why the requirement in ES cells do not reflect the embryo

Paternal effects on pre-implantation embryo development in cattle

Currently, sire fertility is measured using sire conception rate (SCR), which is not always indicative of embryo development. Since the majority of pregnancy loss in dairy cattle occurs during the early embryonic period, it is important to determine the effect of sire during this time period. Therefore, the goal of this research is to identify sires with high and low capacities to produce embryos and elucidate the effect of sire on early embryo development. To investigate this, 65 Holstein sires with SCRs ranging from -14.2 to 5.3 were run through an in vitro embryo production system and embryo development was monitored. Based on their in vitro development performance, eight high performing (HP) and 9 low performing (LP) sires were identified. The average blastocyst rate (BL) was 48 percent for HP and 14 percent for LP sires, respectively. In this dataset, there was no correlation between SCR and BL. However, there was an increase in embryos arrested at the 5-6 cell stage in LP sires compared to HP sires. Next, embryos were produced from HP and LP to determine autophagy levels, and blastocyst cell number. LP sires had a higher rate of autophagy than high performing sires, with no effect of SCR. However, the ratio of trophectoderm to inner cell mass cells in blastocysts did not differ between sire performance groups. RNA-Seq on 4-cell embryos identified 687, and 1411 genes with increased expression in HP and LP sires, respectively. Genes with increased expression in HP sires were involved in mRNA and cell cycle regulation, chromosome segregation, and sperm mitochondria clearance. Genes with increased expression in embryos from LP sires were indicative of sperm mitochondria retention, and an increase in DNA damage and apoptosis. Lastly, embryos were produced in vivo from HP and LP sires. Interestingly, LP sires generated twice the number of degenerated embryos as HP sires. In conclusion, this research demonstrates a clear effect of sire on preimplantation embryonic development, where LP sires produced a higher proportion of embryos with developmental delays, increased autophagy and expression of DNA damage and pro-apoptotic genes, resulting in embryonic arrest at the 5-6 cell stage. Interestingly, SCR was not indicative of preimplantation development in this study. The in vitro model used in this study to identify sires with negative effects in embryo development represents a useful tool to build a robust predictor of sire fertility that accounts for the sire's influence on the early stages of pregnancy

Rethinking embryology in vitro: A synergy between engineering, data science and theory

Pluripotent stem cells, in the recent years, have been demonstrated to mimic different aspects of metazoan embryonic development in vitro. This has led to the establishment of synthetic embryology: a field that makes use of in vitro stem cell models to investigate developmental processes that would be otherwise inaccessible in vivo. Currently, a plethora of engineering-inspired techniques, including microfluidic devices and bioreactors, exist to generate and culture organoids at high throughput. Similarly, data analysis and deep learning-based techniques, that were established in in vivo models, are now being used to extract quantitative information from synthetic systems. Finally, theory and data-driven in silico modeling are starting to provide a system-level understanding of organoids and make predictions to be tested with further experiments. Here, we discuss our vision of how engineering, data science and theoretical modeling will synergize to offer an unprecedented view of embryonic development. For every one of these three scientific domains, we discuss examples from in vivo and in vitro systems that we think will pave the way to future developments of synthetic embryology.Peer ReviewedPostprint (published version