Developmental Immunology Laboratory – Georg Holländer

Our single cell genomics related research focuses on three separate aspects of thymus biology: 

1.  The Homunculus in our Thymus: A Cellular Genomics Approach

Thymic epithelial cells (TEC) are unique because they express almost the complete set of proteincoding genes. TEC thus reconstitute a pansomatic expression profile, almost as a molecular homunculus. However, important questions central to a molecular understanding of this promiscuous gene expression (PGE) in TEC have remained unexplored because the necessary experimental systems and analytical methods have until recently been unavailable. The planned research programme will use recently established methods and will develop new approaches to characterise in detail the molecular features of PGE in TEC. We will use unbiased genome-wide transcriptome and epigenome, and selected protein expression analyses at single cell resolution to map intracellular and population-wide expression landscapes. Improved cellular proteomics will record protein expression from as few as several thousand cells. Explicitly, our outlined research programme will address three open questions:

I. What are the diversity, complexity and dynamics of gene expression in distinct TEC populations at single cell resolution?
II. By what mechanisms do TEC escape temporal and cell lineage-specific epigenetic gene silencing to fully accomplish PGE?
III. How plastic is PGE over the life course and in response to perturbations, and what perturbations affect the functional effectiveness of negative thymic selection?

2. Mapping the transcriptome of initial embryonic TEC differentiation for the identification of Foxn1 target genes essential for early thymus organogenesis.

The Foxn1 protein is expressed in the thymus exclusively in TEC where it serves as a cell-autonomous master regulator of differentiation and growth. The goal of these experiments is to identify Foxn1-controlled target genes essential in early TEC development. Our identification of Foxn1 target genes was so far restricted to postnatal TEC using steady-state conditions. We now extend this analysis to time points early in thymus organogenesis when Foxn1 has just been expressed to be able to identify its candidate genes in a thymus primordium that still only comprises a few hundred TEC. At this stage methodological biases and limited cell number preclude population-based RNASeq analyses. However, with the advent of reliable and robust transcriptome detection methods for single cells (e.g. SMARTseqII), these limitations have successfully been overcome. We will therefore take advantage of our proven expertise in single cell TEC isolation and transcriptome analysis to identify Foxn1 candidate genes during early thymus organogenesis.

3. Coordination of transcriptomic and spatial information during development

Two major limitations of single cell genomics is (i) the loss of information about the original location within the sample of the sequenced cell and (ii) low throughput at high cost with only hundreds of cells analysed per day. We will therefore develop in a Oxford –Cambridge-WTSI collaboration genetic technologies to i) record within the genome the lineage history of each cell, for readout at any stage of interest and ii) to provide a unique fluorescent signature to cells to enable us to provide spatial and temporal context to their transcriptional profiles.

Sansom SN, Shikama N, Zhanybekova S, Nusspaumer G, Macaulay IC, Deadman ME, Heger A, Ponting CP, Holländer GA. Population and single cell genomics reveal the Aire-dependency, relief from Polycomb silencing and distribution of self-antigen expression in thymic epithelia. Genome Res. 2014 24:1918-31.