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Thymic epithelial cells (TEC) are essential for thymocyte differentiation and repertoire selection. Despite their indispensable role in generating functional T cells, the molecular mechanisms that orchestrate TEC development from endodermal progenitors in the third pharyngeal pouch (3rd PP) are not fully understood. We recently reported that the T-box transcription factor TBX1 negatively regulates TEC development. Although initially expressed throughout the 3rd PP, Tbx1 becomes downregulated in thymus-fated progenitors and when ectopically expressed impairs TEC progenitor proliferation and differentiation. Here we show that ectopic Tbx1 expression in thymus fated endoderm increases expression of Polycomb repressive complex 2 (PRC2) target genes in TEC. PRC2 is an epigenetic modifier that represses gene expression by catalyzing trimethylation of lysine 27 on histone H3. The increased expression of PRC2 target genes suggests that ectopic Tbx1 interferes with PRC2 activity and implicates PRC2 as an important regulator of TEC development. To test this hypothesis, we used Foxn1Cre to delete Eed, a PRC2 component required for complex stability and function in thymus fated 3rd PP endoderm. Proliferation and differentiation of fetal and newborn TEC were disrupted in the conditional knockout (EedCKO) mutants leading to severely dysplastic adult thymi. Consistent with PRC2-mediated transcriptional silencing, the majority of differentially expressed genes (DEG) were upregulated in EedCKO TEC. Moreover, a high frequency of EedCKO DEG overlapped with DEG in TEC that ectopically expressed Tbx1. These findings demonstrate that PRC2 plays a critical role in TEC development and suggest that Tbx1 expression must be downregulated in thymus fated 3rd PP endoderm to ensure optimal PRC2 function.

We present tetramer-associated T-cell receptor sequencing (TetTCR-Seq), a method to link T cell receptor (TCR) sequences to their cognate antigens in single cells at high throughput. Binding is determined using a library of DNA-barcoded antigen tetramers that is rapidly generated by in vitro transcription and translation. We applied TetTCR-Seq to identify patterns in TCR cross-reactivity with cancer neo-antigens and to rapidly isolate neo-antigen-specific TCRs with no cross-reactivity to the wild-type antigen.

Our understanding of nerve regeneration can be enhanced by delineating its underlying molecular activities at single neuron resolution in small model organisms such as Caenorhabditis elegans. Existing cell isolation techniques cannot isolate regenerating neurons from the nematode. We present femtosecond laser microdissection (fs-LM), a new single cell isolation method that dissects intact cells directly from living tissue by leveraging the micron-scale precision of fs-laser ablation. We show that fs-LM facilitated sensitive and specific gene expression profiling by single cell RNA-sequencing, while mitigating the stress related transcriptional artifacts induced by tissue dissociation. Single cell RNA-sequencing of fs-LM isolated regenerating C. elegans neurons revealed transcriptional program leading to successful regeneration in wild-type animals or regeneration failure in animals lacking DLK-1/p38 kinase. The ability of fs-LM to isolate specific neurons based on phenotype of interest allowed us to study the molecular basis of regeneration heterogeneity displayed by neurons of the same type. We identified gene modules whose expression patterns were correlated with axon regrowth rate at a single neuron level. Our results establish fs-LM as a highly specific single cell isolation method ideal for precision and phenotype-driven studies. MainSpinal cord injuries result in permanent functional deficit as axons in the adult central nervous system fail to regenerate after trauma 1 . State-of-the-art interventions promote neuron survival and axon regrowth by engaging neuron-intrinsic and pro-regenerative pathways 2 . However, clinical outcome of such interventions are still poor: regrowth can only be stimulated in a small number of neurons, while a seemingly homogeneous neuron population can exhibit diverse or even opposite responses to the same intervention 3,4 . A deeper understanding of nerve regeneration can be attained by dissecting the process at single neuron resolution in a well-defined nervous system. For such studies, Caenorhabditis elegans has been established as a valuable model organism in conjunction with femtosecond laser axotomy 5,6 , which can reproducibly induce a variety of relevant regeneration phenotypes in vivo. The transparent body of C. elegans further allows analysis of such phenotypes, including axon regrowth rate, guidance, and fusion at a single neuron resolution in vivo with fluorescence microscopy. Past nerve regeneration studies in C. elegans have led to the discovery of many conserved nerve regeneration pathways [7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26] , notably the dual leucine zipper kinase (DLK-1/p38) signaling cascade, whose role in neural development, regeneration, and degeneration has been confirmed in numerous vertebrate and invertebrate species 7,[27][28][29][30][31] .Nerve regeneration research in C. elegans has relied on costly and time-consuming mutant screens that test individual genes for function in axon regeneration (Supplementary Note 1...

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