Grutzner, FrankShearwin, LindaPajpach, Filip2023-05-042023-05-042022https://hdl.handle.net/2440/138213The monotreme lineage, including the platypus and echidna, can be described as the oldest surviving mammals. Their evolutionary history and extraordinary biology make them excellent organisms to study key aspects of mammalian evolution. These egg-laying mammals have fascinated scientists and the general public for centuries. One of the most interesting aspects of monotreme biology is their complex sex chromosomes system. With ten sex chromosomes in platypus and nine in echidna males, monotremes feature the highest number of sex chromosomes reported in mammals and one of the highest among vertebrates. Investigation of this sex chromosome system in the past has been limited due to the lack of molecular tools and material available for monotremes. Although considerable progress has been made, there are still open questions, such as understanding the processes like chromosome segregation, interphase organization, mitotic and meiotic behavior, and sex chromosome evolution. To provide a better understanding of the evolution of genes involved in chromosome segregation in mammals, the investigation of some of the key components of chromosome segregation in monotremes was conducted. In this regard, the chromosome passenger complex (CPC) is a protein complex essential for chromosome segregation and consists of borealin, survivin, inner centromere protein and Aurora kinase B or C. This research revealed the absence of Aurora kinase C in monotremes and hence completed the picture of the evolution of this protein family in mammals. It also showed the conservation of other genes associated with chromosome segregation in monotremes, suggesting their functions have been preserved in mammals. Lastly, this research identified differences in meiotic behavior of the cohesin between platypus and echidna, suggesting the evolution of lineage specific cohesin functions in platypus but not echidna. Evolution of mammalian sex chromosomes is another important aspect and monotremes are a key lineage to investigate this due to its basal evolutionary position. There are two independent evolutionary origins of mammalian sex chromosomes – one that led to the XY sex chromosome system in therian mammals (marsupials and placentals), while monotremes evolved multiple sex chromosomes (5X and 5Y in platypus males, 5X and 4Y in echidna males) independently. This means that monotreme and therian sex chromosome systems are unrelated, and the therian X shares extensive homology with platypus chromosome 6. This provides an opportunity to investigate the monotreme autosome that leads to the therian sex chromosome. The meiotic behaviour and segregation pattern of platypus chromosome 6 was analyzed, revealing striking sex chromosome-like behavior of this chromosome, such as non-random segregation, association with the sex chromosome complex and presence of an unpaired region. Based on these results, we propose novel evolutionary scenarios to explain the evolution of mammalian sex chromosomes. Monotreme sex chromosomes are also unique for their interphase organization and recent work revealed interphase association of the sex chromosomes which had not been observed in other species. Given that monotreme sex chromosomes contain X and Y specific copies of the CCCTC-binding factor (CTCF) which facilitates chromosomal interactions, this work investigated the interphase organization of sex chromosomes and a potential role of the sex linked CTCF protein in chromatin interactome of males and females. The results suggest the CTCFX and CTCFY proteins bind to individual DNA binding sites, with Y chromosomes containing only Y copy specific binding site, which potentially contributes to male specific functions. Furthermore, a report of novel inter-chromosomal interactions in platypus males that may be mediated by CTCF is reported in this work. In conclusion, this research has revealed conserved and unique features of regulation of chromosome segregation in monotremes. Furthermore, this research has shown sex chromosome-like features of a monotreme autosome, advancing the understanding of how sex chromosomes can evolve from autosomes. Lastly, this work identified conserved and unique features of monotreme interphase organization of sex chromosomes and CTCF protein, which suggests sex specific functions of CTCF at interphase, further expanding the knowledge of regulation of mammalian sex chromosome interactomes.enMonotreme, sex chromosomes, evolution, meiosis, chromosome segregation, mammalThesis