The role of ASCIZ and Dynein light chain-1 in embryonic development

© 2018 Dr. Ashleigh KingASCIZ (ATM-substrate Chk2-interacting Zn2+ finger protein) is a conserved and highly specialised transcription factor for Dynein light chain 1 (Dynll1) expression. ASCIZ is able to sense DYNLL1 protein levels and thereby mediates auto-regulation of Dynll1 gene expression. It is known that both proteins are essential for normal development. The aim of this thesis was to determine to what extent and how this important interaction regulates embryogenesis and normal mouse development, and whether loss of Dynll1 phenocopies or exceeds the Asciz phenotype. By using genetically modified mice with a targeted null mutation I have found that Dynll1 is essential for early embryonic development. Dynll1 null mice exhibit incomplete embryonic turning, neural tube closure failure and severe cardiac oedema. These defects are consistent with a strong ciliopathy. DYNLL1 is a subunit of the cilia-specific Cytoplasmic Dynein-2 motor complex and the Dynll1 null phenotypes are strikingly reminiscent of phenotypes seen in mice lacking the Cytoplasmic Dynein-2 light-intermediate chain, Dync2li1, the only other previously targeted null mutation of a Dynein-2 subunit. In addition, I have generated the first conditional mouse mutant for a Cytoplasmic Dynein-2 subunit to better understand the role of retrograde intraflagellar transport (IFT) in development. This limb bud mesoderm-specific Dynll1 null mutant highlights an important role for the Dynein-2 complex in endochondral bone development and digit patterning via the cilia- dependent Hedgehog (Hh) signalling pathway. Importantly, this mouse model replicates characteristic clinical features of the bone defects of human Jeune syndrome patients with mutations in Dynein-2 subunits. Analysis of these Dynll1 mouse mutants has provided mechanistic insight into how the protein regulates cilia functions. Loss of Dynll1 led to impaired Dynein-2 complex stability, evident by reduced protein levels of other subunits, and resulted in severely impaired Hh signalling and IFT but did not interfere with cilia formation. Interestingly, both for the germline and limb-specific conditional null mice, phenotypes for Asciz mutants were considerably milder than those of Dynll1 mutants. Because Asciz and Dynll1 have recently been shown to be important for the development of conventional B- lymphocytes (B-2 cells), I hypothesized that they would also be critical for the development of the embryonic-origin B-1a cell lineage. Consistently, conditional deletion of Asciz or Dynll1 from embryonic innate B-lymphocyte progenitors resulted in a profound decrease in B-1a cell numbers. Together, the results presented in this thesis demonstrate that Dynll1 is essential for embryogenesis, limb development and patterning via its role in Cytoplasmic Dynein-2 dependent Hh signal transduction. Additionally, Dynll1 is required for the early establishment of foetal B-1a cells. Interestingly, Asciz and Dynll1 largely contribute to these early developmental processes in a linear pathway