Periodic Reporting for period 4 - ELONGAN (Gene editing and in vitro approaches to understand conceptus elongation in ungulates)
Okres sprawozdawczy: 2022-04-01 do 2023-09-30
The project aimed to understand conceptus elongation, a largely unexplored developmental period exclusive from ungulates, which include the four most relevant mammalian livestock species in Europe: cows, pigs, sheep and goat. Key developmental processes occurring during this period include the formation of a flat embryonic disc resembling that of humans, and gastrulation, which entails the differentiation of epiblast cells into the three germ layers. The importance of studying these developmental processes is dual. On one side, failures during this period account for most reproductive losses in livestock species, exerting a negative impact on European Bioeconomy. On the other, given the similarities in embryonic disc formation between humans and ungulates, ungulate embryos serve as a model to understand human diseases and conditions that origin during early development, such as spina bifida. The overall objectives of this project have been 1) to uncover the role of specific genes on developmental processes by means of CRISPR-mediated ablation, 2) to determine the metabolic and proteomic composition of the uterine fluid to understand the embryonic requirements for conceptus elongation, and 3) to develop an in vitro system to achieve conceptus elongation without the need of experimental animals. The conclusions of the project organized by objectives have been 1) striking differences have been observed between the transcriptional regulation of mouse early development and that of ungulates and humans, the role of transcription factors essential for mouse development is not conserved between mammals, 2) a detailed analysis of the proteomics and metabolomics composition of the uterine fluid has provided clues to develop the in vitro system of the last objective and to design future nutritional and pharmacological strategies to prevent embryonic loss in farm animals, and 3) a fully in vitro system –not requiring from experimental animals- has been developed to achieve embryonic development to the beginning of gastrulation, an unprecedented advance in ungulates that provides a unique animal experimentation-free and real embryo model to study mammalian flat gastrulation.
The main results of the project organized by objectives have been the following:
Objective 1:
The project has developed techniques that boosted the efficiency of CRISPR-mediated gene ablation (KO) in ungulate embryos from <5 % to ~40 % by diminishing the incidence of a troublesome phenomenon called mosaicism. The project has also provided a technique that improves targeted insertion rates from ~25 to ~50 % by chemical activation of a DNA repair pathway (HDR).
Using CRISPR technology, the project has uncovered that the role of the most relevant transcription factors involved in first TE commitment in mice (TEAD4, CDX2 and GATA3) is not conserved in mammals, as these genes are not required for trophoectoderm commitment and development in ungulates. Uncovering that the text-book knowledge gathered from mice -the predominant mammalian model in Developmental Biology- is not applicable to early preimplantation development of other mammals, including ungulates and very likely humans, constitutes a paradigm shift impacting both Developmental Biology and stem cell studies.
Other relevant findings derived from employing CRISPR technology in this project have been:
1) Determine that direct progesterone signaling is not required for bovine embryo development up to maternal recognition of pregnancy, by both ablating its receptor PGR and performing removal/addition in vitro culture experiments.
2) Uncover the essential role of the protein ZP4 (absent in mice, but present in ungulate, rabbits and humans) during pre-hatching embryo development. ZP4 was found to confer mechanical properties to the zona pellucida which are essential to prevent blastomeres disaggregation and to ensure proper permeability.
3) Discover the role of the protein TMEM95 in mammalian fertilization. TMEM95 became the third known sperm protein essential for mammalian fertilization and the first proven to be essential in two mammalian species (cattle and mice).
4) Uncover that SOX2 role in epiblast commitment is conserved between mammals, as –in agreement with mice KO data- its ablation in ungulate embryos prevents epiblast formation.
5) Identify the developmental stage at which bovine embryos carrying naturally occurring deleterious mutations in the genes APAF1, SMC2 and TFBM1 show developmental defects, a relevant information to take decisions on the reproductive and genotyping management of dairy farms.
Objective 2
The project has provided a quantitative analysis of the proteins and biochemical compounds present in the bovine uterine fluid (UF) nourishing embryos before implantation. This analysis identified great differences between the UF nourishing Day 7 blastocysts and that nourishing Day 14 elongated conceptuses, suggesting that embryo metabolic requirements are stage-specific and providing relevant clues for the development of the in vitro system of objective 3 and nutritional and pharmacological strategies to prevent embryo loss.
Objective 3
The project has developed a pioneer in vitro system that supports ungulate embryo development to the embryonic disc stage up to the beginning of gastrulation. Such advanced developmental stage had not been reached before in vitro in any farm animal and allows the study of early developmental processes without the need of experimental animals. The system is currently employed in two joint projects with private companies to test the suitability of diverse artificial reproductive techniques routinely used in the cattle industry without the need of transferring embryos to recipient animals, and to test human assisted reproductive technologies employing bovine embryos as a model. The system have been also employed to reduce the number of experimental animals required for the aims of objective 1 and to test the role of different signaling pathways during ungulate early development (TFGβ, MEK and BMP4).
The knowledge generated has been disseminated so far through 15 publications in indexed journals; 60 communications presented in 30 international scientific conferences; 4 conferences, 1 workshop and a training course oriented towards cattle industry, 2 workshops oriented towards policy makers, and 13 press releases.
Objective 1:
The project has developed techniques that boosted the efficiency of CRISPR-mediated gene ablation (KO) in ungulate embryos from <5 % to ~40 % by diminishing the incidence of a troublesome phenomenon called mosaicism. The project has also provided a technique that improves targeted insertion rates from ~25 to ~50 % by chemical activation of a DNA repair pathway (HDR).
Using CRISPR technology, the project has uncovered that the role of the most relevant transcription factors involved in first TE commitment in mice (TEAD4, CDX2 and GATA3) is not conserved in mammals, as these genes are not required for trophoectoderm commitment and development in ungulates. Uncovering that the text-book knowledge gathered from mice -the predominant mammalian model in Developmental Biology- is not applicable to early preimplantation development of other mammals, including ungulates and very likely humans, constitutes a paradigm shift impacting both Developmental Biology and stem cell studies.
Other relevant findings derived from employing CRISPR technology in this project have been:
1) Determine that direct progesterone signaling is not required for bovine embryo development up to maternal recognition of pregnancy, by both ablating its receptor PGR and performing removal/addition in vitro culture experiments.
2) Uncover the essential role of the protein ZP4 (absent in mice, but present in ungulate, rabbits and humans) during pre-hatching embryo development. ZP4 was found to confer mechanical properties to the zona pellucida which are essential to prevent blastomeres disaggregation and to ensure proper permeability.
3) Discover the role of the protein TMEM95 in mammalian fertilization. TMEM95 became the third known sperm protein essential for mammalian fertilization and the first proven to be essential in two mammalian species (cattle and mice).
4) Uncover that SOX2 role in epiblast commitment is conserved between mammals, as –in agreement with mice KO data- its ablation in ungulate embryos prevents epiblast formation.
5) Identify the developmental stage at which bovine embryos carrying naturally occurring deleterious mutations in the genes APAF1, SMC2 and TFBM1 show developmental defects, a relevant information to take decisions on the reproductive and genotyping management of dairy farms.
Objective 2
The project has provided a quantitative analysis of the proteins and biochemical compounds present in the bovine uterine fluid (UF) nourishing embryos before implantation. This analysis identified great differences between the UF nourishing Day 7 blastocysts and that nourishing Day 14 elongated conceptuses, suggesting that embryo metabolic requirements are stage-specific and providing relevant clues for the development of the in vitro system of objective 3 and nutritional and pharmacological strategies to prevent embryo loss.
Objective 3
The project has developed a pioneer in vitro system that supports ungulate embryo development to the embryonic disc stage up to the beginning of gastrulation. Such advanced developmental stage had not been reached before in vitro in any farm animal and allows the study of early developmental processes without the need of experimental animals. The system is currently employed in two joint projects with private companies to test the suitability of diverse artificial reproductive techniques routinely used in the cattle industry without the need of transferring embryos to recipient animals, and to test human assisted reproductive technologies employing bovine embryos as a model. The system have been also employed to reduce the number of experimental animals required for the aims of objective 1 and to test the role of different signaling pathways during ungulate early development (TFGβ, MEK and BMP4).
The knowledge generated has been disseminated so far through 15 publications in indexed journals; 60 communications presented in 30 international scientific conferences; 4 conferences, 1 workshop and a training course oriented towards cattle industry, 2 workshops oriented towards policy makers, and 13 press releases.
As detailed above, the project has generated knowledge that goes beyond the state of the art in different fields. The three major advances are summarized below:
1) Uncover the divergent or conserved roles between mice and farm animals of 10 genes on reproductive processes including fertilization, pre-implantation embryo development and first lineage commitment. The knowledge generated has broken long established dogmas in mammalian Developmental Biology established by mouse KO models.
2) Provide a pioneer fully in vitro biosystem able to generate and develop ungulate embryos up to the beginning of gastrulation. This system currently constitutes the only real embryo and animal experimentation-free biosystem to study embryo development beyond blastocyst hatching available in mammals.
3) Develop techniques for genome edition in farm animal embryos that boosted (2- to 10-fold) direct KO and KI generation efficiencies compared to the state of the art.
1) Uncover the divergent or conserved roles between mice and farm animals of 10 genes on reproductive processes including fertilization, pre-implantation embryo development and first lineage commitment. The knowledge generated has broken long established dogmas in mammalian Developmental Biology established by mouse KO models.
2) Provide a pioneer fully in vitro biosystem able to generate and develop ungulate embryos up to the beginning of gastrulation. This system currently constitutes the only real embryo and animal experimentation-free biosystem to study embryo development beyond blastocyst hatching available in mammals.
3) Develop techniques for genome edition in farm animal embryos that boosted (2- to 10-fold) direct KO and KI generation efficiencies compared to the state of the art.