Illuminating the genetics of obesity by single-cell sequencing of human neurons

Illuminating the genetics of obesity by single-cell sequencing of human neurons

EBPOD 2017: Project 7

This is one of 11 joint postdoctoral fellowships offered by EMBL-EBI, the NIHR Cambridge Biomedical Research Centre and the University of Cambridge’s School of the Biological Sciences in 2017.

Principal Investigators

Abstract

Single-cell RNA sequencing (scRNAseq) allows the diversity of cell types in the human body to be distinguished with unprecedented detail1, and can be coupled with perturbations such as gene knockout or exposure to exogenous factors to measure the resulting transcriptional changes (perturb-seq)2,3. We will use these methods to define disease-relevant human neuronal populations by their functional responsiveness to hormones and therapeutic drugs, and will examine the effect of target gene knockout on these responses. Our multidisciplinary team is seeking a motivated postdoc interested working with several cutting-edge technologies to address these fundamental questions. 

Motivation

The hypothalamus of the human brain contains distinct neuron types that regulate appetite and energy expenditure4-7. The activity of some of these neuron types is modulated by hormones and other factors, but the identity of other factor-responsive cells and the relevant signaling pathways are still poorly understood. It has been difficult to address these questions in animal models given the heterogeneity of responsive cell populations, the rarity of relevant cell types, and the difficulty in isolating them from the adult brain. 

We have recently developed robust methods to differentiate human pluripotent stem cells (hPSCs) into hypothalamic neurons8. ScRNAseq can be used to characterize the genes and pathways that are involved in response to a genetic or environmental perturbation in cell types of interest2,3. Identifying the cell types that respond to environmental factors and therapeutic compounds and the role that obesity-associated genes play in regulating these responses will provide biological insights and have strong translational potential. 

Data and resources

We are able to robustly generate generate hypothalamic neurons from multiple hPSC lines and have shown by scRNAseq that they contain neuron types essential for the regulation of appetite and energy homeostasis, sleep, temperature, stress, fertility, or growth. These cells spontaneously fire action potentials and some of them respond to physiologically relevant factors including the neurotransmitter serotonin, the amino acid leucine, and the “satiety hormone” leptin. We have also assembled a unique collection of hundreds of hPSCs whose genomes have been deeply sequenced. 

Objectives

Aim 1. Identify hypothalamic cell types that respond to physiologically relevant factors.
Studies in animal models have mapped many of the cell types that respond to serotonin and leptin, but these studies have not been extended to humans, in which distinct cell populations might be involved. Furthermore, the identify of cells that participate in a feeling of satiety by responding to the amino acid lysine or the gut hormone GLP1 are largely unknown. This question is of immediate therapeutic relevance since the main small molecule therapy for obesity (liraglutide) targets the receptor for GLP1, but its central mechanism of action is poorly understood. We will differentiate hPSCs into hypothalamic neurons, expose them to vehicle control or the compound of interest, dissociate cultures, and compare their transcriptomes by scRNAseq. This analysis will reveal the molecular identify of cell types that respond to these cues by significantly altering their gene expression, and reveal which pathways are affected. 

Aim 2. Test the effect of candidate gene knockout on functional responsiveness.
Numerous genes associated obesity have been identified by targeted exome sequencing and genome wide-association studies (GWAS)6. However, with few exceptions, the mechanisms by which these genes contribute to obesity are poorly understood. We will therefore use CRISPR/Cas9-based gene editing in hPSCs9 to knock out candidate genes and study their effect on gene expression in different hypothalamic neuron populations by scRNAseq under steady-state conditions, as well as and in response to stimulation with efficacious exogenous factors identified in Aim 1. 

Aim 3. Test the contribution of common genetic variants on hypothalamic neuron phenotypes.
Together with collaborators at Harvard University and the WT-Sanger Institute, we deeply sequenced of hundreds of hPSCs. We identified cell lines carrying a particularly heavy or light burden of genetic variants associated with obesity (BMI) that affect genes predominately expressed in the brain10. We will select multiple cell lines with a high or low burdens of BMI-associated variants, differentiate them into hypothalamic neurons, and profile them by scRNAseq in the presence and absence of relevant exogenous factors. 

Future directions

The proposed studies can be extended to include candidate therapeutic agents or combinations of factors to test for potential synergistic action.
Since appetite regulation is highly conserved, experiments on human cell in vitro can be translated into mouse models in vivo to test for effects on affect feeding behavior and body weight. Together, these studies have the potential to lead to new human therapies for obesity, one of the biggest public health challenges facing us today. 

Position description and training opportunities

The successful postdoc candidate will have strong training in statistics and informatics, and ideally have experience working with genomic or transcriptional data sets and an interest in biological problems. The position will enable the candidate to work as part of a multidisciplinary team. The Stegle lab will train the candidate in cutting-edge bioinformatics tools to analyse scRNAseq data sets. The Merkle lab will train the candidate in stem cell biology, directed neuronal differentiation, and in vitro cellular phenotyping. The Yeo laboratory will provide expertise on the biology of obesity-associated genes, and will enable promising leads to be followed up in animal models in vivo.  

References

1. Buettner, F. et al. Computational analysis of cell-to-cell heterogeneity in single-cell RNA-sequencing data reveals hidden subpopulations of cells. Nature Biotechnology 33, 155–160 (2015). 

2. Dixit, A. et al. Perturb-Seq: Dissecting Molecular Circuits with Scalable Single-Cell RNA Profiling of Pooled Genetic Screens. Cell 167, 1853–1866.e17 (2016). 

3. Campbell, J. N. et al. A molecular census of arcuate hypothalamus and median eminence cell types. Nat. Neurosci. 20, 484–496 (2017). 

4. Schwartz, M. W., Woods, S. C., Porte, D., Seeley, R. J. & Baskin, D. G. Central nervous system control of food intake. Nature 404, 661–671 (2000). 

5. Coll, A. P., Farooqi, I. S. & O'Rahilly, S. The hormonal control of food intake. Cell 129, 251–262 (2007). 

6. O'Rahilly, S. & Farooqi, I. S. Genetics of obesity. Philos. Trans. R. Soc. Lond., B, Biol. Sci. 361, 1095–1105 (2006). 

7. Yeo, G. S. H. & Heisler, L. K. Unraveling the brain regulation of appetite: lessons from genetics. Nat. Neurosci. 15, 1343–1349 (2012). 

8. Merkle, F. T. et al. Generation of neuropeptidergic hypothalamic neurons from human pluripotent stem cells. Development 142, 633–643 (2015). 

9. Merkle, F. T. et al. Efficient CRISPR-Cas9-mediated generation of knockin human pluripotent stem cells lacking undesired mutations at the targeted locus. Cell Rep 11, 875–883 (2015). 

10. Locke, A. E. et al. Genetic studies of body mass index yield new insights for obesity biology. Nature 518, 197–206 (2015).