Our lab discovered that the primary cilium is required for Hedgehog (Hh) signaling in mice. Primary cilia use the centriole, the core of the centrosome, as a template. The centrosome has historically been regarded as the microtubule organizing center of animal cells especially due to its localization at the spindle poles during mitosis. Experiments in Drosophila have shown that the centrosome is dispensable for mitosis, but suggest that the most important role of the centrosome may be in nucleation of the primary cilium. No genetic studies have been carried out to define the roles of the centrosome in mammals. Because the centrosome also houses many signaling molecules like kinases (protein kinase A, Aurora kinase), cyclins (E and A) and proteasomes, we are taking a genetic approach in mice to define the function of mammalian centrosomes and, in particular to test whether they act as signaling centers. To define the role of the mammalian centrosome, we are studying mutations in Sas4 (also called Cenpj or Cpap in mammals), which encodes a protein essential for centriole duplication. So far, our work supports a role for centrosomes in Hh signaling, just like primary cilia.
In Aim 1 of this proposal, we will investigate any disruptions in the different signaling pathways in detail in Sas4 mutants. We will also examine the roles of Sas4 in the cell cycle (especially mitosis), cell polarity and cell survival in the embryo. Polycystic kidney diseae (PKD) is associated with disruption of cilia and/or centrosomes, and the proteins responsible for PKD localize to cilia/centrosomes. Because centrosomes template cilia and may act as signaling centers in kidneys, we are using conditional genetic ablation of centrosomes caused by loss of Sas4 to test whether this leads to cystic kidney disease. Our preliminary results indicate that the disruption of Sas4/centrosomes indeed leads to PKD in young mice.
In Aim2, we will use conditional alleles of genes required for cilia formation to assess the differences between the absence of cilia and centrosomes during kidney development. This will define whether the functions of centrosomes extend beyond cilia formation or if the only essential function of centrosomes in the mammalian kidney is to template cilia. Our data thus far indicate that cilia and centrosomes are mainly required for Hh signaling and disruption of cilia or centrosomes in kidneys leads to cyst formation. Therefore, in Aim 3 we will test whether cilia/centrosome-dependent Hh signaling plays a role in cystic kidney disease. We will test whether the cyst formation is associated with Hh signaling using lineage tracing experiments in established PKD mouse models. If Hh signaling is activated during cyst formation, we will employ genetic approaches to test the importance of Hh signaling during the formation of kidney cysts.
We propose to study centrosomes, cilia, and Hedgehog signaling which are associated with many human malignancies. Centrosomes and their derivatives the cilia are also tightly connected with human syndromes collectively termed ciliopathies, many of which exhibit polycystic kidney disease.
Our aim i s to use genetic approaches in the mouse to better understand the connections between these organelles, signaling and human disease.
