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Down the Rabbit Hole – Easter Egg Hunt with Single Cell Sequencing


[↓] The Origins of Easter Bunny

[↓] Is Easter Bunny male or female?

[↓] Can Easter Bunny get sick?

[↓] What came first, rabbit or the egg?

[↓] SUGAR-seq: Can we sequence candy?

The Origins of the Easter Bunny [↑]

Easter celebration is a strong religious and cultural tradition in the west. The most prominent secular symbol of the Christian holiday, the Easter Bunny, was reportedly introduced to America by the German immigrants who brought over their stories of an egg-laying hare, while the decoration of eggs and gifting sweets is believed to date back to at least the 13th century. The exact origins of this mythical mammal are unclear and if it existed, it would probably belong to the egg-laying mammalian order Monotremata, like the famous platypus or its less famous cousin echidna. However, Australians would disagree as they have their own Easter Bunny representative belonging to the Marsupialia order, the Easter Bilby.

Regardless, rabbits are known to be prolific procreators, making them and the egg ancient symbols of fertility and new life. So, what else do eggs, bunnies and candy have in common? Single cell sequencing has been used to study all three!

We would have loved to get our hands on a monotreme single cell sequencing data to compare to today’s rabbits and birds, but no one has done single cell sequencing on any of the species belonging to this order. Marsupial and monotreme mammals fill an important gap in vertebrate phylogeny between reptile-mammal divergence 310 million years ago and the eutherian (placental) mammal radiation. [1] Instead, we show here how single cell sequencing can be used to study rabbit biology and implement this model organism in study of human diseases, chicken development in the egg, and… yes, you read it right… sweets!

Is Easter Bunny male or female? [↑]

Although there is no data on monotremes, some of our readers might be interested if the Easter Bunny was female or male, with former implying it indeed laid eggs, while the latter could be possible if it just stole them from chickens. If we assume the Easter Bunny was born from an egg, single cell sequencing data would be useful to delineate steroidogenic cell lineage specification in mammals and birds [2]. Therian mammals have an XX:XY sex chromosome system and the Y chromosome-linked SRY gene acts as the master sex determinant, directing testis formation. However, SRY is absent in non-mammals, and birds have ZZ:ZW sex chromosomes, rendering gonadal sex determination to be governed by the Z-linked gene, DMRT1, which operates via a dosage mechanism, making temperature a master epigenetic regulator for this gene.

Estermann et al. used single cell sequencing to characterize cell subpopulations responsible for supporting gonadal cell lineage. [3] They identified a new mesenchyme-derived cell type with PAX2 as a novel marker specific for chicken steroidogenic cell progenitors. So, if anyone obtains a tissue sample from the Easter Bunny, make sure to let us know, as our technology and expertise are well known for being able to process almost any tissue type, and the data from these studies will help us discern its sex and origin.

Can Easter Bunny get sick? [↑]

When you travel a lot between the continents, make sure to get your vaccinations, especially when it comes to cross-species contact. However, the Easter Bunny probably doesn’t have this luxury (unless it steals vaccines too) and has to rely on its own immune system to protect it, which we don’t know anything about. Luckily for us, Li et al. performed single cell sequencing on peripheral blood mononuclear cells (PBMCs) to compare the transcriptomes of immune cells from 12 different species, including rabbits and birds. [4] Distinct molecular profiles were characterized for different immune cell types, including T cells, B cells, natural killer cells, monocytes, and dendritic cells. Notably, the ligand and receptor pair VIM-CD44 was highly conserved among the immune cells across the species. Such studies not only identify immune cell types, their interactions, and regulatory molecular mechanisms, but also identify potential targets for immune-related disease therapy. In all, regardless of its probably well-built immune system, if the Easter Bunny ever gets sick and needs help from us humans, single cell immune profiling technology will allow us to quickly connect its immunity to our current known species single cell data. We are ready!

What came first, rabbit or the egg? [↑]

As we are still waiting for that Easter Bunny tissue sample to verify whether it lays eggs or steals them, we can prepare the reference database by looking at the differences in mammalian vs avian development. We have abundant single cell data on mouse development due to extensive studies of different tissues, as presented by a mouse single cell atlas published by Tabula Muris Consortium et al. [5] and a few specific studies of rabbit retina and heart development. [6,7]

On the egg side of this tale, Feregrino et al. constructed a single cell transcriptomics atlas to study chicken limb development, however, a complete chicken version of Tabula Muris has not been finalized yet. Recently, Yamagata wrote a comprehensive review on studies contributing to Tabula Gallus, a nascent single cell avian development atlas project including different -omics modalities such as Epigenome, Proteome, Glycome, and Connectome. [8] We eagerly expect the results of such a multinational, cross-field project that will eventually also be beneficial for a better understanding of human health and diseases, as well as our Easter Bunny origin. In conclusion, we must admit that the egg came first, since the rabbit single cell research and transcriptomics atlas still needs to be constructed.

SUGAR-seq: Can we sequence candy? [↑]

We are not sure if the gifts of sweets were originally meant for the Easter Bunny to leave the eggs next to or was it leaving the candy along with the eggs for us. The historians will have to tackle that one. In the meantime, we would like to introduce you to SUGAR-seq (SUrface-protein Glycan And RNA-seq), a single cell multiomics technique that profiles cells based on several modalities, including the levels and types of N-glycans on the cell surface.

Kearney et al. used integrated SUGAR-seq and glycoproteome analysis to identify tumor-infiltrating T cells (TILs) with unique surface glycan properties that report their epigenetic and functional state. [9] They showed that Tregs and activated/exhausted CD8+ T cells displayed the most extensive N-glycosylation in contrast to naïve CD4+ cells and memory CD4/CD8+, which displayed the least. Moreover, integration of SUGAR-seq with single cell TCR sequencing revealed that highly abundant clones were enriched in activated/exhausted CD8+ T cells compared to memory cells. Furthermore, as glycosylation is conserved in multicellular organisms, SUGAR-seq can be used to study glycan biology in diverse species without the need for specialized reagents. This is also another way to confirm the origins of the Easter Bunny and see if the centuries of nibbling on candy is what made it different than the rest of known rabbits, marsupials and monotremes.

Conclusion? [↑]

Single cell multiomics is a powerful new technology which allows for integration of multi-modal single cell profiling, giving us a more holistic approach to characterize cell heterogeneity within a tissue, link individual cell phenotype to genotype, study immune cell clonality and its effect on disease or development, but also provide insights into mythical creatures such as the Easter Bunny.

The origin of the Easter Bunny and the debate whether it is male or female persists, but we are closer to unveiling these mysteries than ever. Until then, enjoy your holidays and make sure to enjoy your Easter candy (but not too much since we can tell!).

We Wish You a Happy Easter Everyone

References [↑]

[1] Deakin et al. The evolution of marsupial and monotreme chromosomes. Cytogenet. Genome Res., 2012. [PubMed]

[2] Estermann et al. Gonadal Sex Differentiation: Supporting Versus Steroidogenic Cell Lineage Specification in Mammals and Birds. Front. Cell Dev. Biol., 2020. [PubMed]

[3] Estermann et al. Insights into gonadal sex differentiation provided by single-cell transcriptomics in the chicken embryo. Cell Rep., 2020. [PubMed]

[4] Li et al. Molecular mechanisms governing circulating immune cell heterogeneity across different species revealed by single-cell sequencing. Clin. Transl. Med., 2022. [PubMed]

[5] The Tabula Muris Consortium et al. Single-cell transcriptomics of 20 mouse organs creates a Tabula Muris. Nature, 2018. [PubMed]

[6] Bargagna-Mohan et al. Corneal nonmyelinating Schwann cells illuminated by single-cell transcriptomics and visualized by protein biomarkers J. Neurosci. Res., 2021. [PubMed]

[7] Liang et al. Cellular and molecular landscape of mammalian sinoatrial node revealed by single-cell RNA sequencing Nat. Commun., 2021. [PubMed]

[8] Yamagata, Masahito. Towards Tabula Gallus. Int. J. Mol. Sci. Nature, 2018. [PubMed]

[9] Kearney et al. SUGAR-seq enables simultaneous detection of glycans, epitopes, and the transcriptome in single cells Science Advances, 2021.[PubMed]