Transcriptomic analysis of the gray short-tailed opossum (Monodelphis domestica) B-cell genes

Abstract

Marsupials and eutherians are mammals that differ in their physiological traits, predominately their reproductive and developmental strategies; eutherians give birth to well-developed young, while marsupials are born highly altricial after a much shorter gestation. These developmental traits result in differences in the development of the immune system of eutherian and marsupial species. B-cells are key to humoral immunity, are found in multiple lymphoid organs, and have the unique ability to mediate the production of antigen-specific antibodies in the presence of pathogens. Marsupial B-cell investigations have become increasingly important in understanding an adaptive immune system that develops primarily ex utero. In comparison to eutherians and monotremes, marsupial B-cells have four Immunoglobulin (Ig) heavy (H) chain isotypes (IgA, IgG, IgM and IgE) and two light (L) chain isotypes; lambda (Ig?) and kappa (Ig?). The gray short-tailed opossum (Monodelphis domestica) is a well-established model marsupial species, with a well annotated genome. The B-cell transcriptome of an individual opossum was investigated by Next Generation RNA-Seq techniques at the single-cell level. A total of 273 single-cells and 575,721 contigs were generated, annotation of the transcriptome identified 14,654 unique genes. The first study of this thesis analysed the IgH and IgL usage in the opossum B-cell repertoire. Not surprisingly, IgM had the highest expression in the repertoire, followed by IgA, IgG, and very few cells expressing IgE. Despite Ig? being the most complex IgL isotype, the ratio of Ig? to Ig? was 35:65. IgL isotypes have been identified to have a greater contribution to antibody diversification than IgH isotypes, due to the complexity and abundance of IgL variable (V) gene segments. The second study of this thesis examined the whole opossum B-cell transcriptome and analysed the most highly expressed genes. The most abundant gene transcripts were Sydnecan-4, making up 0.66% of the entire transcriptome. IgM and IgG cells produced significantly more transcripts of the golgi glycoprotein 1 and ELMO domain-containing protein genes in comparison to IgA. Since IgE expressing cells were very low in number, a definitive comparison could not be made between all IgH cells. Highly expressed genes associated with the marsupial immune system included MHC class II DRa chain and MHC class II DAB chain. The diverse array of genes identified in the opossum single-cell transcriptome reveals the importance of marsupial B-cells in producing endogenous antibody responses, and has allowed for a comparative analysis with other mammalian lineages.

Date of Award	2019
Original language	English