Source: WASHINGTON STATE UNIVERSITY submitted to NRP
A SINGLE CELL CHROMATIN ACCESSIBILITY ATLAS OF THE DEVELOPING BOVINE PLACENTA
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1032039
Grant No.
2023-67012-42232
Cumulative Award Amt.
$162,824.37
Proposal No.
2024-03723
Multistate No.
(N/A)
Project Start Date
Nov 1, 2023
Project End Date
Feb 28, 2025
Grant Year
2024
Program Code
[A1201]- Animal Health and Production and Animal Products: Animal Breeding, Genetics, and Genomics
Recipient Organization
WASHINGTON STATE UNIVERSITY
240 FRENCH ADMINISTRATION BLDG
PULLMAN,WA 99164-0001
Performing Department
(N/A)
Non Technical Summary
Reproductive efficiency and success are critical aspects contributing to the profitability and sustainability of cattle operations in the United States. Only 30% of animals conceive and carry pregnancy to term, amounting to a cost of almost $1 billion per year from pregnancy losses on beef and dairy farms. Placental development, among other aspects of pregnancy, is essential for successful reproduction. Therefore, the aims of this project are to leverage cutting-edge genomic technologies to understand genetic regulation of placental development at single cell resolution, and utilize this knowledge to further understand the influence of genetic variation on fertility traits in cattle. This work will contribute to our understanding of the biological processes necessary for reproductive success. Further, it will allow for more precise identification of reproductively superior animals with the use of genomics, which will contribute to more profitable and sustainable cattle operations.
Animal Health Component
25%
Research Effort Categories
Basic
75%
Applied
25%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3043499108050%
3043499105050%
Knowledge Area
304 - Animal Genome;

Subject Of Investigation
3499 - Dairy cattle, general/other;

Field Of Science
1080 - Genetics; 1050 - Developmental biology;
Goals / Objectives
Reproductive efficiency and success are critical aspects contributing to the profitability and sustainability of cattle operations in the United States. Only 30% of animals conceive and carry pregnancy to term, amounting to a cost of almost $1 billion per year from pregnancy losses on beef and dairy farms. Placental development, among other aspects of pregnancy, is essential for successful reproduction. Therefore, the aims of this project are to leverage cutting-edge genomic technologies to understand genetic regulation of placental development at single cell resolution, and utilize this knowledge to further understand the influence of genetic variation on fertility traits in cattle.This project has two specific objectives:Objective 1: Define open chromatin regions, transcription factor binding sites, and predicted cis-regulatory elements across cell populations of the bovine placenta during early gestation (day 40 when cotyledons arise) and mid-gestation (day 170, when placentomes are fully developed and functional) by implementing single nuclei Assay for Transposase-Accessible Chromatin with sequencing (ATAC-seq) paired with existing single nuclei RNA-seq.Objective 2: Examine enrichment of quantitative trait loci (QTL) in regulatory regions defined in this study in cell populations across development of the bovine placenta and computationally predict the biological consequences of these variants in specific cell types.This work will contribute to our understanding of the biological processes necessary for reproductive success. Further, it will allow for more precise identification of reproductively superior animals with the use of genomics, which will contribute to more profitable and sustainable cattle operations.
Project Methods
Methods:Objective 1: Nuclei will be isolated from snap frozen tissues using the Chromium Nuclei Isolation Kit following the Nuclei Isolation Protocol for Single Cell ATAC (PN-1000493) approved by the 10X Genomics platform. Final nuclei concentration will be determined with a Countess II FL (Invitrogen), and a minimum of 2,000 nuclei per sample (target 5,000 nuclei as described in the human single-nuclei ATAC-seq atlas) will be submitted for library preparation and sequencing. Library preparation from nuclei and sequencing will be performed at the University of Missouri Genomics Technology Core by scientists skilled in preparing these libraries from a variety of tissue types and that have extensive experience with troubleshooting. Sequencing will be performed on an Illumina NovaSeq 6000 for 100 cycles (50 bp paired end reads), to achieve 25,000 reads per nucleus. Data analysis: All computational analyses will be performed on the Washington State University High Performance Computing Cluster. Raw BCL files output from the 10X Genomics platform will be demultiplexed and converted to fastq files, mapped to the cattle reference genome ARS-UCD1.2, and processed into a feature matrix using ENSEMBL annotation in the Cell Ranger software (cellranger-2.0.0, 10X Genomics). Count matrices will be processed as previously described by removing doublets with the python package Scrublet, and dimension reduction with SnapATAC. Integration with single-nuclei RNA-seq and differences in open chromatin across cell populations and developmental time points will be investigated with Seurat. Enriched transcription factor binding sites in open chromatin will be identified with JASPAR and candidate driver TFs will be identified as previously described.61,89 Cell-cell communication by ligand-receptor interactions will be predicted between cell populations at each developmental time point using CellPhoneDB. Cis-regulatory elements will be profiled between expressed genes and open chromatin regions using linkage disequilibrium score regression with a Benjamini-Hochberg correction for multiple testing as previously described. Cell trajectory will be predicted with Monocle3 to identify regulatory regions related to binucleate cell formation. All methodologies described are within the skillsets of the PD and collaborating mentors.Objective 2: Data mining: Genetic variants and QTLs associated with fertility traits will be obtained from Animal QTLdb and FAANGMine databases. Literature searches for additional variants not curated within these databases will be performed. Data analysis: Enrichment of variants in open chromatin regions will be performed as previously described across different cell types and developmental time points using a hypergeometric test to evaluate significant QTL enrichment in regions with linkage disequilibrium (r2 > 0.1), as many QTLs have not been fine-mapped. The effects of genetic variation on transcription factor binding sites and regulatory regions will be predicted using deltaSVM models that calculate binding scores as previously described. Biological significance will be investigated with pathway analyses with GO and KEGG enrichment and literature searches. The methods described are within the skillsets of the PD and collaborating mentors.Outcome measures:Objective 1: Identification of open chromatin regions consistent with previously analyzed single-nuclei RNA-seq as described in the preliminary data, as these two datasets have been found to directly relate.Objective 2: The locations of QTLs related to fertility are correctly overlayed from curated databases and located in regions of open chromatin as others have described.

Progress 11/01/23 to 10/31/24

Outputs
Target Audience:This project primarily engaged reproductive and developmental biologists, geneticists, and livestock industry professionals, reaching both graduate students and established researchers. The research yielded exciting findings that have been effectively disseminated through multiple channels, ensuring broad impact. The project's contributions extended beyond academia to include livestock producers and industry stakeholders, particularly those interested in improving reproductive efficiency and reducing pregnancy loss in cattle. Additionally, the findings were shared with interdisciplinary research groups, such as the Interdisciplinary Reproduction and Health Group at the University of Missouri, fostering valuable discussions and collaborations. The results of this work have been published in two peer-reviewed manuscripts in Biology of Reproduction, with a third manuscript currently in preparation. Findings were also presented at several major scientific conferences, including a poster at the International Plant & Animal Genome (PAG) Conference in January 2023 and another at the International Ruminant Reproduction Symposium in May 2023. Preliminary research findings were shared in March 2023 with the Interdisciplinary Reproduction and Health Group through a Works in Progress Seminar at the University of Missouri, further extending the project's reach. The project's significance was also recognized through multiple invited presentations, including a platform talk by the PD's Primary Mentor at the International Ruminant Reproduction Symposium in May 2023, an invited platform talk by the PD in the Cattle/Sheep/Goat Section at the PAG Conference in January 2024, and an invited talk at the Society for the Study of Reproduction Annual Meeting in July 2024. These presentations reached a diverse audience of scientists, students, and industry professionals, enhancing awareness and engagement with the research. Beyond publications and presentations, the project played a crucial role in mentorship and education. Three graduate students and three undergraduate students gained hands-on experience in data analysis and bioinformatics, using this research and data as a framework for developing their skills. The findings were also presented as an example of the influence of gene regulation on a specific phenotype in a 300-level undergraduate animal genetics course at Washington State University, helping students build a deeper understanding of reproduction and genomics in cattle. The research has direct implications for the livestock industry, particularly in understanding genetic factors that contribute to pregnancy loss. By identifying key genetic and epigenetic mechanisms affecting placental development and reproductive success, this work supports more efficient and profitable beef and dairy operations. The project's contributions to both scientific knowledge and industry practices ensure a lasting impact, with results laying the foundation for future research and funding opportunities. Through its combination of high-impact research, education, and outreach, this project has advanced the understanding of cattle reproduction while also fostering the next generation of scientists in the field. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The PD has actively participated in numerous academic events and professional development opportunities. These experiences deepened the PD's expertise in reproductive biology, advanced bioinformatics, and mentorship, all of which contributed to professional advancement. Training activities: Expanded knowledge of reproductive biology and bioinformatics through one-on-one mentorship with the Primary Mentor and guidance from the Mentoring Committee. Provided hands-on training to three graduate students and three undergraduate students in data analysis techniques for single-cell RNA and ATAC sequencing, using the project's datasets and pipelines as instructional tools. Recruited and trained a graduate student in reproductive biology and bioinformatics within the PD's newly established laboratory. In addition to research training, the PD engaged in several professional development activities, presenting findings at academic conferences and seminars. Professional development activities: Delivered a Works-in-Progress seminar for the Interdisciplinary Reproduction and Health Group (IRHG) at the University of Missouri, presenting "Single nuclei RNA sequencing uncovers trophoblast cell types and lineages in the bovine placenta" (March 2023). Presented a poster at the International Ruminant Reproduction Symposium (IRRS) titled "A single-cell transcriptomic atlas of the developing bovine placenta" (July 2023). Delivered an invited platform talk in the Cattle/Sheep/Goat Workshop at the International PAG Conference, titled "Gene expression and chromatin accessibility dynamics of the developing bovine placenta at single-cell resolution" (January 2024). Presented an invited talk at the Society for the Study of Reproduction (SSR) Annual Meeting, titled "Single-cell multiome analysis of the bovine placenta identifies gene regulatory networks in trophoblast differentiation" (July 2024). How have the results been disseminated to communities of interest?The research findings from this fellowship have been actively disseminated through peer-reviewed publications, oral presentations, and poster sessions, reaching scientific communities in reproductive biology and genetics. To date, one manuscript has been published in Biology of Reproduction, with another in preparation. Additionally, the PD delivered three oral presentations, one lecture for an undergraduate course, and one poster presentation during the fellowship, sharing discoveries with both academic and industry audiences. Manuscripts: Davenport, Kimberly M., O'Neil, Eleanore V., Ortega, M. Sofia, Patterson, Amanda, Kelleher, Andrew M., Warren, Wesley C., and Thomas E. Spencer. Single cell insights into development of the bovine placenta. Biology of Reproduction. 2024 January 13. doi:10.1093/biolre/ioad123. Oral Presentations: Reproductive biology audience - Works-in-Progress seminar for the Interdisciplinary Reproduction and Health Group (IRHG) at the University of Missouri, presenting "Single nuclei RNA sequencing uncovers trophoblast cell types and lineages in the bovine placenta" (March 2023). Genetics audience - invited platform talk in the Cattle/Sheep/Goat Workshop at the International PAG Conference, titled "Gene expression and chromatin accessibility dynamics of the developing bovine placenta at single-cell resolution" (January 2024). Reproductive biology audience - invited talk at the Society for the Study of Reproduction (SSR) Annual Meeting, titled "Single-cell multiome analysis of the bovine placenta identifies gene regulatory networks in trophoblast differentiation" (July 2024). Undergraduate audience - general results and findings were presented to a 300-level undergraduate animal genetics course as an example of how gene regulation can influence different biological processes such as development of the placenta (September 2024). Poster Presentations: Reproductive biology audience - poster presentation at the International Ruminant Reproduction Symposium (IRRS) titled "A single-cell transcriptomic atlas of the developing bovine placenta" (July 2023). What do you plan to do during the next reporting period to accomplish the goals?The next reporting period will focus on finishing data analyses for Objective 2 and preparing an additional scientific manuscript for submission. The new graduate student and PD will work collaboratively on accomplishing these final goals.

Impacts
What was accomplished under these goals? Objective 1 has been completed, and Objective 2 is in progress. Objective 1 was accomplished during the first year of the fellowship and involved tissue collection from newly acquired animals at 40 and 170 days of gestation, nuclei isolation from cotyledon tissue, single-nuclei multiome sequencing, and comprehensive data analysis. Notably, the University of Missouri Genomics Core successfully performed library preparation for single-nuclei multiome sequencing for the first time as part of this project. The results were disseminated through oral and poster presentations, as well as peer-reviewed publications in genetics and reproductive biology. Additionally, RNA in-situ hybridization was incorporated to validate the expression of key genes in histological sections of cotyledon tissue, expanding the original scope of the objective and enhancing the study's scientific merit. This work identified essential cell types and transcription factor regulatory networks hypothesized to drive trophoblast differentiation in the bovine placenta, and therefore ensuring proper development and maintenance of pregnancy. The outputs from Objective 1 included: Two peer-reviewed journal articles in Biology of Reproduction (one published, one accepted). Three invited oral presentations at major conferences: the International Plant & Animal Genome (PAG) Conference, the Society for the Study of Reproduction (SSR) Annual Meeting, and the International Ruminant Reproduction Symposium (IRRS), with two presented by the PD (PAG and SSR) and one by the PD's Primary Mentor (IRRS). One poster presentation by the PD at the IRRS. Objective 2 is currently in progress and contributing to the PD's transition to an independent faculty position. Data analyses are being performed by a graduate student recruited by the PD to help finish the research, and train in reproductive biology, genetics, and bioinformatics. Data were mined from the Animal QTL Database and overlayed with open chromatin regions of trophoblast cells, which were identified in Objective 1. Further data analyses are ongoing to further dissect the relationship between genetic variation and mechanisms critical for pregnancy establishment and maintenance.

Publications

  • Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: 9. Davenport, Kimberly M., ONeil, Eleanore V., Ortega, M. Sofia, Patterson, Amanda, Kelleher, Andrew M., Warren, Wesley C., and Thomas E. Spencer. Single cell insights into development of the bovine placenta. Biology of Reproduction. 2024 January 13. doi:10.1093/biolre/ioad123.