Sex chromosomes and germline transcriptomics explored by single-cell sequencing and RNA-tomography

Abstract

In our study of germ cell differentiation, we applied two recently developed technologies on the germline of various model organisms: single-cell mRNA sequencing and RNA-tomography. For the first time we could look at gene expression with such a high resolution, and this led us to discover the function of unknown genes, and describe the localization and dynamics of gene expression. In Chapter 1 I introduce the relevant biology and technology for the following chapters. I briefly explain the soma vs. germline difference, sexual reproduction and the differences between the male and female germline. Next, I introduce meiosis, a cell division intimately linked to germ cell maturation. Subsequently I address the special role of sex chromosomes during germ cell differentiation. Finally, I introduce the experimental and computational tools utilized in this work. In Chapter 2 we studied the female human germline during fetal development using single-cell mRNA sequencing. All humans have two copies of their genome in every cell: one inherited from the mother and one from the father. These are called alleles. Cells use many genes from one specific allele only, but these differences are erased in mature germ cells. If genes are only used from one of the two genomes, it is called allele specific gene expression. There are two big classes of such genes in females: imprinted genes, and genes on the X chromosome. We found surprising differences between these two groups in how they lose their allele specific expression. While imprinted genes are activated in a strict order, the X-chromosome seems to activate without a defined order. In Chapter 3 we applied single-cell mRNA sequencing and pseudotemporal reconstruction to study male germ cells in mice. We found that one of the earliest steps in differentiation is regulated by a reverse logic: the silencing of “undifferentiated genes” seems to be a larger change than the activation of “differentiation genes”. Next, we looked at later steps of germ cell differentiation and found that genes on sex chromosomes (the X and Y chromosome) are silenced in a sequential order, right before a massive activation of sperm specific (“differentiated”) genes. In Chapter 4 we created a gene expression atlas of C elegans from its head to its tail, and we compared organ specific gene expression across sexes. C elegans have two sexes: male and hermaphrodite. Both sexes are producing sperm capable of fertilization, but if both are present, male sperm has an advantage over hermaphrodite sperm. We described and validated several male specific genes in the germline, which could help understanding this competition and advantage. In Chapter 5, I discuss of the work presented in this thesis.