Function and Regulation of the Y-Linked Axonemal Dynein Genes During Drosophila Spermatogenesis

Abstract

The germline is considered to be immortal, meaning an organism’s germ cells have the potential to give rise to all subsequent generations. With so much at stake, the germline goes to great lengths to protect itself while also maintaining reproductive potential, resulting in fascinating and innovative biology. This thesis focuses on two aspects of germ cell development in Drosophila males that appear disadvantageous yet are prevalent across drosophilids. The first is the expression of the Y chromosome gigantic genes – these genes are essential for fertility yet they are riddled with megabases of repetitive DNA. The second is the assembly of the long cilia found within the sperm’s tail – Drosophila have some of the longest sperm in the animal kingdom, yet little is known about how these long cilia are assembled. This thesis will describe the innovations that have allowed germ cells to overcome these challenges and will go on to discuss how these burdens may benefit the fly. In Drosophila, the Y chromosome is largely heterochromatic, encoding only a handful of genes, which are essential for male fertility. Intriguingly, some of these genes are amongst the largest genes identified to date, spanning several megabases. For example, the gene kl-3, which encodes an axonemal dynein motor protein required for sperm motility, spans 4.3Mb with only 14kb of coding sequence. The introns of these genes contain megabases of simple satellite DNA repeats (e.g. (AATAT)n) that comprise over 99% of the locus. Although this “intron gigantism” has been observed in several genes across species, including the mammalian Dystrophin gene, its regulation and functional relevance remains elusive. The transcription/processing of such gigantic genes/RNA transcripts poses a significant challenge. I identified that the Y-linked gigantic genes require a unique gene expression program in order to overcome these challenges. By monitoring Y-linked gene expression over developmental time, I found that transcription of these loci takes 80-90 hours. I further identified two RNA-binding proteins that specifically bind to Y-linked gene transcripts. Loss of either RNA binding protein resulted in sterility due to the loss of Y-linked gene products. I found that this unique gene expression program functions on two fronts: it increases the ability of RNA polymerase to transcribe the repetitive introns, and it aids in processing the large transcripts. I speculate that this program may be utilized to modulate gene expression patterns during development. During Drosophila spermatogenesis, germ cells undergo drastic morphological changes to yield a 1.9mm sperm. The cilia found within the sperm tail are cytoplasmic cilia – a specialized type of cilia where the axoneme (the microtubule structural component) resides within the cytoplasm instead of within a specialized ciliary compartment. Cytoplasmic cilia likely allow for efficient assembly of longer cilia, however, the mechanism for their assembly remains unknown. I found that mRNAs encoding four axonemal dynein heavy chain genes (three of which are Y-linked gigantic genes) colocalize in a novel ribonucleoprotein (RNP) granule, which localizes near the site of axoneme assembly during sperm elongation. Precise localization of this RNP granule mediates incorporation of the axonemal dynein motor proteins into the axoneme. This work is the first to uncover how cytoplasmic cilia are efficiently assembled to allow for the production of 1.9mm sperm, and highlights that there are other cilia assembly mechanisms besides the ancient and conserved mechanism by which traditional cilia assemble.