Using mass cytometry (CyTOF), this project will identify unique mammalian hypothalamic cell types important for energy homeostasis.
The hypothalamus of the mammalian brain controls many homeostatic processes including energy balance, fertility, body temperature regulation and growth via molecularly distinct cell types. However, the identity and the role of these cells largely remain unknown. We will delineate specific cell types using a proteomics approach and later probe their function. To accomplish this we will use mass cytometry (CyTOF), which is a flow cytometry variant that dramatically increases the number of measurement parameters by using isotopically pure rare earth metals and atomic mass spectrometry rather than fluorophores and fluorescence detection. By making over 40 simultaneous molecular measurements per cell, mass cytometry dramatically increases the intersectional power of fluorescence flow cytometry to identify known and unknown cell types, and profile their molecular status. As a high-dimensional single-cell measurement technique, it is more similar to single-cell RNA sequencing (scRNA-seq). While mass cytometry cannot measure thousands of parameters/cell, it does have several advantages over scRNA-seq: 1. it is higher throughput and lower cost than scRNA-seq, able to measure over 1,000,000 cells per hour at the cost of 0.03¢ per cell, 2. it is not susceptible to “drop-out” or PCR amplification bias, 3. most importantly, mass cytometry provides direct measurement of bioactive molecules within the cell, such as cell surface receptors, intracellular signaling molecules, transcription factors, proliferation and viability markers. Until now, mass cytometry has not been used to examine cells of the nervous system, because it is a newly developed technology, and also because neural tissue is difficult to dissociate into a single-cell suspension. We will optimize protocols to overcome this barrier, facilitating our construction of a directed proteomic single cell atlas for the hypothalamus. Following this optimization step, we will generate baseline single cell proteomic atlas for the hypothalamus. This will be used as a platform to identify unique cell types and determine the changes that occur during energy balance challenges (e.g. starvation versus obesity). This information will allow us to generate novel transgenic animal models to determine the function of unique hypothalamic cell types critical in energy homeostasis.
Establish a comprehensive single-cell molecular atlas of the hypothalamus:
1) We will optimized and validated our cell dissociation protocol and antibody staining panels capable of discerning hypothalamic cell types.
2) We will generated a cell atlas using a force-directed dimensionality reduction approach called FLOW-MAP.
3) We will generate similar data from animals that have been food restricted or fed high-fat diet to delineate cell-types critical for energy balance.
4) We will identify unique cell types, generate animal models and determine their function.
50% of the funds will be used for salary support for one MSTP student (Brandon Podyma) and one biology graduate student (Qijun Tang). The rest of the funds will be used to acquire the necessary reagents and the use of core facilities for this project.