Recipient Organization
UNIV OF MARYLAND
(N/A)
COLLEGE PARK,MD 20742
Performing Department
Animal and Avian Sciences
Non Technical Summary
With each new pregnancy, the mammary gland cycles through a lactation phase of tissue growth and differentiation followed by an involution phase of programmed cell death and tissue remodeling. These dramatic transformations are achievable because lineage-restricted progenitor cells lacking true stem cell potential still exhibit sufficient cellular plasticity to regenerate new tissue and restore lactation capacity. Adult stem cells were widely thought to be drivers of mammary transformation; however, their existence, numbers, and identities remain controversial. Early fetal mammary cells are the only mammary cells known to exhibit true stemness. We hypothesize that gene expression profiling of bovine fetal mammary stem cells will greatly facilitate new strategies using mammary stem cells to advance dairy animal health and performance. Therefore, we will use cell and molecular biology approaches to 1) fully characterize the gene expression signatures associated with stem cell potential within fetal bovine mammary stem cells; 2) identify distinct stem cell types based on those signatures; and 3) determine when and where these cells spatially reside within the fetal mammary rudiment. Collectively, this information will considerably advance the field of bovine mammary stem cell biology and facilitate new research into improving mammary performance, udder and human breast health, and tissue regeneration.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
The overall goals of this research are to: 1) fully characterize the gene expression signatures associated with stem cell potential within fetal bovine mammary stem cells (MaSCs); 2) identify distinct stem cell types based on those signatures; and 3) determine when and where these cells spatially reside within the fetal mammary rudiment. Completion of these aims will serve as the foundation for a high impact and innovative research program focused on the advancement of dairy ruminant mammary stem cell biology. To our knowledge, no other lab has performed these experiments with the methods proposed below.Objective 1: Identify unique cell types and their individual gene expression signatures within the bovine fetal mammary rudiment and surrounding mesenchyme. Single cell RNA-Sequencing (scRNA-Seq) provides individual cell transcriptome information that will reveal potential transcriptional regulators and signaling pathways controlling developmental potential in emerging bovine fetal stem cells. Completion of this aim will provide transcriptomic signatures for bovine fetal MaSCs. Furthermore, our findings may visualize the transcriptional heterogeneity within the fetal MaSC population and mammary mesenchyme lineage. Lastly, gene expression signatures will collectively indicate which signaling pathways are activated between fetal MaSCs and surrounding mammary mesenchyme.Objective 2: Characterize the spatiotemporal gene expression profiles of fetal MaSC genes in the bovine fetal mammary rudiment. Single cell multiplex-spatial-transcriptomics (MST, formerly called Spatial Genomic Analysis) visualizes and quantitates gene expression within all single cells of tissue sections via multiplexed, single molecule fluorescence in situ hybridization. The resulting data produces an overlapping, quantifiable, and spatial gene expression atlas for a minimum of 10-12 genes. Cumulative gene expression data for each cell can then be used to perform a clustering analysis based on similar transcriptomic signatures. Cells types clustered together can then be mapped back to their positions in the original tissue, thereby creating a fetal MaSC atlas of the mammary rudiment.
Project Methods
Our research strategy it to is to utilize two high-resolution transcriptomic approaches to acquire detailed spatial and temporal gene expression information and reveal the molecular identities of fetal mammary stem cells (MaSCs).Objective 1: Identify unique cell types and their individual gene expression signatures within the early bovine fetal mammary epithelium and surrounding mesenchyme.Methods:While the exact stage of fetal MaSC formation has not been determined in cattle, mammary organogenesis commences with stereotyped fetal structures conserved across species. Therefore, our experiments will assess the mammary bud stage (~43 days gestation, ~25 mm crown-rump length) and sprout stage (80 days, ~120 mm crown-rump length), which correspond to "naïve" and "lineage-primed" fetal MaSC multipotent states in mice. Crown-rump length and fetal age are highly correlated (r2= .979) in Holstein cattle. Healthy bovine fetuses will be harvested, selected by crown-rump length, and cleaned by Applied Reproductive Technology LLC (Madison, WI, USA). Fetuses will be transferred into HypoThermosol FRS preservation solution (Sigma Aldrich) and shipped cold-overnight (within 24 hrs). This shipping method adequately preserves fresh organ tissue that intended to produce high quality single cell transcriptomes. Upon delivery, fetal mammary rudiments located near the female genital tubercle will be dissected, minced, dissociated, and filtered through a 40 μm nylon filter (BD Biosciences) according to published protocols. Depending on cell counts, cells may be pooled from other fetuses at very similar crown-rump lengths. Single cell suspensions will be tested for viability and FAC-sorted to remove dead cells if necessary. Cell preps (2500 cells per sample (min 800-1000), 4 fetal replicates per stage) will be submitted to the University of Maryland Brain and Behavior Institute Advanced Genomic Technologies Core for cDNA library construction and single cell RNA-sequencing (Drop-Seq Platform: 10x Genomics Chromium Single Cell 3', / (25K reads per cell x 1000-2500 cells) = 2 libraries/run). Analyses will be conducted using Cell Ranger (UMD Deepthought2 HPC cluster) or publicly available software such as the NCBI SRA and the Galaxy platform. We expect to identify cell clusters of at least two fetal lineages: fetal mammary epithelia (stem cells) and mesenchyme. To characterize any heterogeneity within the fetal MaSCs, we will perform subcluster analysis on the mammary epithelia cluster. Gene ontology (GO) enrichment analyses will reveal potential pathways that are involved in supporting mammary epithelial-mesenchyme interactions, such as Parathyroid Hormone-Like Hormone, Ectodysplasin A, FGF, and Wnt signaling.Expected outcomes and pitfalls: We expect to obtain sufficient cell numbers and viability to perform single cell RNA-Seq. If this is not feasible, we will resort to FAC-sorting fetal mammary epithelial cells from mammary mesenchyme using Lgr5 receptor and PthrP Receptor antibodies, respectively. We will then perform bulk-RNA-Seq on sorted cell fractions. If students are unable to receive sufficient training, we will seek collaborators to help us to complete data analyses. We will use this data set to choose new candidate genes for single cell multiplex spatial transcriptomics (MST) described in Objective 2. We will also use this information to help derive or generate bovine fetal MaSC cell culture lines.Objective 2:Characterize the spatiotemporal gene expression profiles of fetal MaSC genes in the bovine fetal mammary rudiment.Methods:Tissue preparation:Bovine fetuses will be acquired as described in Objective 1. Fetal mammary tissue will be dissected, fixed in RNase-free 4% paraformaldehyde, and cryosectioned at 10 µm for single-cell MST.Cellular Segmentation Immunohistochemistry:Preliminary immunohistochemistry will be performed on sections using P-cadherin, E-cadherin, or β-catenin antibodies to validate an antibody for segmentation purposes. Segmentation defines cell boundaries and allows for single mRNAs of each gene to be quantified within individual cells using published MATLAB code.Quantitative PCR for initial detection of geneexpression:Excised fetal mammary tissue will be allocated for cDNA synthesis. QPCR will be performed on fetal mammary cDNA to ensure that candidate genes intended for evaluation by single cell MST are expressed.Multiplex-Spatial-Transcriptomics:Single cell MST produces quantitative single-cell transcriptional profiling through imaging of individual mRNAs across multiple genes. This technique visualizes and quantitates gene expression via multiplexed, single molecule fluorescencein situhybridization. The data produces an overlapping, quantifiable, and spatial gene expression atlas for multiple genes covering all of the cells in a single mammary gland section. mRNA detection is based on fluorescent-based signal amplification of hybridization chain reaction RNA hairpins that bind to DNA oligonucleotide "probes" hybridized to specific mRNAs. Each probe set is designed to label a specific gene's transcripts within a tissue section. Quantified gene transcripts will be used to construct a cell signature. Cells with similar signatures will then be mapped back to their cell position in the original section. As a result, subpopulations of cells can be spatially identified within tissue sections. Our first set of twelve candidate genes were selected based on published spatiotemporal expression and function in fetal MaSCs (mouse, E13-E16) or putative adult bovine MaSCs. Individual probe sets consisting of at least 15 reverse compliment DNA oligonucleotide 20-mers will be designed using Stellaris Probe Designer software (Biosearch Technologies) and validated via NCBI BLAST. Probe sets will be manufactured by Integrate DNA Technologies. Fluorophore-conjugated hybridization-chain-reaction amplifiers will be purchased from Molecular Instruments (488 nm, 594 nm, 647 nm, Cy7). Multiplexed hybridizations and imaging will be performed with 4 probe sets per round (3 serial rounds total per section). Following the last round of hybridization, sections will be immunolabeled for segmentation analysis. Imaging will be done on a 6-channel PerkinElmer confocal spinning disk (University of Maryland CMNS Imaging Incubator). Mammary mesenchyme tissue surrounding the mammary epithelia will serve as negative controls for MaSC gene expression analysis. Biological replicates will consist of 3 sections from at least 3 fetuses per bud and sprout stage (n=3).Expected outcomes and pitfalls:Our success will allow us to expand the number of genes assayed, as well as expand this technique for use in other stages of mammary development. Although not feasible during this timeline, we will immediately repeat this technique with new candidate genes discovered from our own data sets generated from completion of Objective 1.Efforts:Our findings will be disseminated to our primary target audiences (the mammary gland/dairy science research communities) via oral and poster presentations given at scientific conferences and symposia. We will also deliver our findings by submitting at least two peer-reviewed manuscripts to reputable scientific journals. Any novel techniques, reagents, or models we generate or derive will be published or made availableto the research community.Evaluation:The success of our project will be determined by evaluating the number and quality of journal publications we produce. We will also gauge success by evaluating if our findings could be used as pilot data to successfully pursue additional grant funding. Other milestones will include successful graduate and undergraduate Honorsthesis defenses.