Primary cilia are microtubule-based signaling organelles, emanating from the cell body on most cells in growth arrest. They comprise a highly conserved membrane structure, and coordinate a variety of cellular processes in development and homeostasis, including cell differentiation, migration, cell cycle regulation, and cell division. Defects in ciliary function is associated with a plethora of multi-systemic disorders affecting a variety of functions, including hearing, vision, kidneys, and liver. These are collectively termed ciliopathies.
Maintenance and formation of the primary cilium critically relies on the trafficking to and from the organelle, as well as along the axoneme in a process mediated by the concerted actions of intraflagellar transport particles and BBSome complexes. At the base of the cilium, endo- and exocytotic vesicles accumulate, collectively supplying the cilium with signaling proteins, receptors, axonemal building blocks, and membrane. Collectively, these processes ensure the carefully regulated exit and entry of signaling mediators to/from the cilium, enabling the multitude of sensory functions of the organelle, including signaling through the Transforming Growth Factor b receptors (TGFb) and the hedgehog signaling (HH) network.
Centrioles play a central role in cilium formation. During every cell cycle, they transition between basal bodies associated with primary cilium formation, and spindle poles responsible for organizing the mitotic spindle. These processes are of critical importance to brain development, in which the rapid expansion of the neuronal stem cells forms the basis of the mammalian brain. Primary microcephaly (MCPH) is a neuro developmental disorder in which patients are born with significantly smaller brains than average. Retarded cell cycle progression as a result of compromised centrosome structure and/or function constitutes the main underlying mechanism in disease etiology.
This dissertation includes one review on ciliary coordination of cell migration (ARTICLE III), one methods chapter addressing a line of protocols for evaluating coordination of signaling at the ciliary pocket (ARTICLE II), and two research articles (ARTICLES I and IV). In research ARTICLE I we address the expression of primary cilia in human testicular development. We find, that peritubular myoid cells lining the seminiferous tubules of mammalian testes express primary cilia in adult tissue, whereas Sertoli cells within the tubules do not. Immature interstitial Leydig cells form primary cilia, however, mature Leydig cells do not. Further, in vitro analysis in Leydig cells reveal that these cells do form primary cilia upon serum depletion, and that receptors and mediators of the HH signaling network accumulate along the cilium upon pathway activation. Our results suggest a potential role for HH signaling through the primary cilium during human testicular development. In ARTICLE IV we identify ribosomal rRNA processing protein homolog 7A (RRP7A) as a novel disease gene in MCPH. We find that RRP7A localizes to cilia and radial glia cells lining the developing human ventricular zone. P19CL6 cells with a targeted mutation to Rrp7a and zebrafish with a loss of function mutation in rrp7a show, that depletion of RRP7A results in reduced brain size and dysfunctional neurogenesis. In addition, analysis of patient dermal fibroblasts show, that RRP7A localizes to nucleoli, centrosomes, and cilia, and that patient cells carrying a single amino acid substitution in RRP7A display reduced nucleoli localization. Moreover, patient-derived fibroblasts display defects in ribosomal RNA processing. Ultimately, we show that mutation in RRP7A lead to cell cycle retardation which is partly characterized by defects in timely resorption of primary cilia. In summary, we present the identification of defective ribosome biogenesis as a novel cause of human MCPH.
Collectively, the results presented in this PhD dissertation provide novel insight into the mechanisms governed by the primary cilium, and clearly reflect the critical level of complexity in signaling and function of this organelle. Further, the identification of a novel mechanism in the etiology of MCPH greatly contributes to the expansion of the current understanding of genetic mechanisms underlying human disease and development.