Performing Department
Animal and Avian Sciences
Non Technical Summary
Poultry is the third-largest agricultural commodity. Eggs and poultry meat constitute the chief protein component of the American diet and represent the principal source of protein worldwide. With the rapid growth of the world population, increased global demand for high-quality poultry products warrants innovative ways to improve chicken reproductive efficiency. Besides common reproductive diseases in the ovary and oviduct, flock fertility and reduced sperm production are common concerns in poultry production. Chicken primordial germ cells (PGCs) are the unipotent precursors of sperm cells and ova, essential for flock fertility. PGCs pass genetic material from one generation to the next. Therefore, a deeper understanding of the formation and differentiation of PGCs is critical to advance approaches to improve the reproduction efficiency of poultry. Specifically, the differentiation mechanisms of chicken embryonic stem cells (ESCs) into primordial germ cells (PGCs) and PGCs into spermatogonial stem cells (SSCs) are critical for sperm production in chickens. The primary breeding companies maintain pedigreed lines under constant selection for production traits within the layer, broiler, and turkey industries.The main goal of broiler chicken reproduction is to produce high-quality fertilized eggs. Flock fertility is the most significant reproductive issue the broiler industry currently faces. The percentage of fertile eggs produced depends on ovulation and semen production levels. The trait is of considerable interest in the broiler industry and is an issue for males more than females primarily because of the effect of semen quality on chick output. There is a negative correlation between growth and fertility; thus, the broiler and turkey industries mainly perform labor-intensive artificial insemination. Therefore, opportunities exist to improve fertility in broiler stocks. Beingunipotent precursors of sperm cells and ova, chicken ESCs, and PGCs are critical for determining fertilized egg rate and flock fertility and, importantly, transmit genetic information to future generations. Although a dynamic selection of the early germ cell populations is an essential biological feature linked to fertility, their heterogeneity and hallmarks in avian species have not been fully characterized. We still do not know the regulatory mechanisms of formation, development, and differentiation and are faced with a limited number of PGCs per embryo. Thus, we hypothesize that regulatory elements epigenetically control the fates of germ cell lineages in chicken embryonic stem cells. Ascertaining these regulatory elements is essential to modulate the transition from quiescence to proliferation and differentiation in chicken ESCs and PGCs. Specifically, characterizing the regulatory mechanisms will provide deep insights into chicken stem cell biology, uncovering biomarkers of PGCs that are more likely or able to differentiate, particularly in the broiler breeder industry. It is possible to develop an epigenetic strategy for the sex control or identification of early embryos, i.e., not like genomic modification (GM) methods, increasing the economic benefits of poultry production as the broiler industry prefers more male chicks, and vice versa for layers. Besides providing new knowledge of chicken germ cell developmental transitions and plasticity, the project's results will significantly help us understand chicken embryonic stem cell biology, improving the efficiency of inducing somatic cells to become PGCs and resulting in significant interest among poultry science and producers.
Animal Health Component
0%
Research Effort Categories
Basic
60%
Applied
20%
Developmental
20%
Goals / Objectives
The project hypothesis is that regulatory elements epigenetically control the fates of germ cell lineages in chicken embryonic stem cells.Aim 1: Identify transcriptomic signatures and regulatory elements of chicken germ cell lineages. Our working hypothesis is that integrated transcriptome and chromatin accessibility analyses at the single-cell level will identify critical regulatory elements involved in germ cell lineage differentiation.Aim 2: Validate critical regulatory elements in vitro that direct PGC formation in stem cell lineage differentiation. Our working hypothesis is that some essential regulatory elements direct and coordinate the differentiation of cell lineages in PGC formation. We will unravel essential regulatory elements in stem cell differentiation into PGC, including epigenetic factors.Aim 3: Validate the roles of important regulatory elements in vivo in broiler reproduction. Our working hypothesis is that mechanisms regulating oogenesis and spermatogenesis from PGCs can be defined from the genes and regulatory elements. We will select the most significant genes, and regulatory elements, evaluating their effects on the quantity of sperm and ova in our PGC-mediated model.
Project Methods
In our proposed research, we will implement multiple novel methodologies with fertilized eggs, including single-cell analysis and the time-of-flight (CyTOF) methods, followed by computational biological analysis.The research includes:1) Identifying transcriptomic signatures and regulatory elements of chicken germ cell lineages.The working hypothesis is that integrated transcriptome and chromatin accessibility analyses at the single-cell level will identify critical regulatory factors involved in cell lineage fates. We will perform scRNA-seq and scATAC-seq studies on embryonic stem cells, classify cell lineages, and ascertain the intermediate stages of stem cell differentiation. Meanwhile, we will verify the biomarkers or signatures of subtype germ cells, cell lineage trajectory, and pseudotemporal ordering of germ cells. The intermediate lineages provide information on differentiation checkpoints or branching points for channeling cells into more than one lineage. We will identify overlapping regulatory regions or nearby genes expressed in cell populations and establish a catalog of regulatory areas. Cluster analysis will determine the subpopulations and place markers or signatures of cell lineages. Then, the pseudotemporal order, cell cycle, and regulon analyses will be inferred. Finally, the germ cell status will be validated. This Aim will help us understand the molecular basis of cell specificity and potentially the origins of cell subpopulations.2) Validating critical regulatory elements in vitro that direct PGC formation in stem cell lineage differentiation.Our working hypothesis is that regulatory elements direct cell lineages in PGC formation. We will unravel vital regulatory elements and histone modifications in the stem cell lineage's differentiation and proliferation. Using comparative analysis among species, we will discover universal regulatory elements based on identified markers above. Cytometry by time of flight (CyTOF) is used to quantify labeled targets on the surface and interior of single cells with mass cytometry. It allows the quantification of multiple cellular components simultaneously. We will also validate the CyTOF lineages using the antibody cocktail labeled with heavy metal ion tags.The readout is by time-of-flight mass spectrometry, combining many antibody specificities in a single sample without significant spillover between channels. Thus, we can corroborate the CyTOF results and validate essential marker genes in the subpopulations. We believe the analysis will decipher the roles of regulatory elements and epigenetic factors in stem cell differentiation.3) Validating the roles of critical regulatory elements in vivo in broiler reproduction.Our working hypothesis is that the regulatory aspects discovered in Aims 1 and 2 directly enhance the formation of PGCs and the differentiation trajectory through oogenesis or spermatogenesis, thus serving essential roles in poultry reproduction, a crucial trait within the broiler industry. Several genes and regulatory elements will be involved in this process, but we will select the two or three most significant ones based on stringent Bonferroni criteria. By comparing the quality and quantity of sperm and ova, we will evaluate the roles of the most substantial genes or regulatory elements discovered in improving the quality and quantity of PGCs. The findings will provide novel approaches to enhance reproductive efficiency in poultry and demonstrate a unique use of genome editing to enhance traits by modulating regulatory elements.