Cilia and flagella are cell surface organelles with microtubule-based axonemal cores. Although these organelles have been known to biologists for centuries, only in the last five years has it been recognized that cilia are crucial for mammalian embryonic development as well as for the function of multiple adult organs (Pan et al., 2005
). Many potential ciliary proteins have been identified in various species in recent years using biochemical, comparative genomic and proteomic methods. Nevertheless, the spectrum of factors required for the formation and/or function of cilia, as well as the molecular mechanisms underlying the regulation of cilia biogenesis, have yet to be fully revealed.
Two multiprotein complexes, the intraflagellar transport (IFT, complex A and B) complexes, are present in the green alga Chlamydomonas reinhardtii
(Rosenbaum and Witman, 2002
). The IFT complexes move within the flagella, suggesting that they are likely to be involved in the transportation of molecules inside the flagella. Mutations in protein components of the IFT complexes (the IFT proteins), as well as in the microtubule motor proteins kinesin II and cytoplasmic dynein, result in the degeneration of flagella, indicating that IFT is required for flagella formation (Pan et al., 2005
Cilia have been implicated in the pathogenesis of many human genetic diseases, such as polycystic kidney disease (PKD), Bardet-Biedl syndrome (BBS), Meckel-Gruber syndrome (MKS) and Joubert’s syndrome (JBTS) (Fliegauf et al., 2007
). Most of the proteins known to be connected with these diseases are localized to the cilia or to the basal bodies, centrosome-like structures from which cilia originate. The identities of additional genes, such as those mutated in MKS2 (Roume et al., 1998
) and JBTS2 (Valente et al., 2005
) patients, are yet to be discovered.
The discovery that cilia play essential roles in signal transduction in multiple pathways, especially the Hedgehog (Hh) pathway, greatly advanced our understanding of both the function of cilia and the mechanism of intracellular signaling (Bisgrove and Yost, 2006
). The Hh proteins, a family of secreted proteins, regulate the development of multiple organ systems in both vertebrates and invertebrates (Hooper and Scott, 2005
). Loss of Hh signaling in mammals results in disruption of left-right asymmetry, loss of ventral cell fate in the central nervous system (CNS), loss of digits and many other defects (Chiang et al., 1996
, Hh regulates the activities of the transcription factor Cubitus interruptus (Ci) (Methot and Basler, 2001
). Ci is a dual-function protein that acts as both a transcriptional activator and repressor. In the absence of Hh, Ci is proteolytically processed into a transcriptional repressor that maintains repression of Hh target genes. When Hh is present, proteolytic processing of Ci is inhibited and Ci acts as a transcriptional activator that turns on the transcription of Hh target genes. The signal from Hh is transmitted to Ci through a signaling cascade that starts with the binding of Hh ligand to its cell surface receptor, Patched (Ptc). As a result, the G-protein-coupled receptor-like protein Smoothened (Smo) is activated, leading to the inhibition of Ci processing and activation of Ci activator function.
Many components of the mammalian Hh pathway serve similar functions to their Drosophila
counterparts (Hooper and Scott, 2005
). However, significant differences do exist. One difference is the duplication of most Hh pathway genes and their subsequent functional divergence in vertebrates. For example, there are three mammalian homologs of Ci, which constitute the Gli family (Gli1, Gli2 and Gli3). Gli1 does not appear to be subject to proteolytic processing. Therefore, Gli1 functions as a transcriptional activator only. Both Gli2 and Gli3 undergo proteolytic processing in vivo, but Gli3 is much more efficiently processed than Gli2, making it the major repressor (Pan et al., 2006
; Wang et al., 2000
). Hh pathway regulation between Drosophila
and vertebrates is also divergent in that some vertebrate-specific Hh pathway components, such as Hip (Hhip – Mouse Genome Informatics) and Rab23, have been identified (Chuang and McMahon, 1999
; Eggenschwiler et al., 2001
In recent years, we and others have found that mouse and zebrafish mutants with cilia defects exhibit compromised Hh signaling (reviewed by Bisgrove and Yost, 2006
; Tobin et al., 2008
). Our detailed analysis indicates that IFT-related proteins are crucial for both Gli activator and repressor functions (Liu et al., 2005
). Recent protein localization studies suggest that multiple components of the mouse Hh signaling pathway are localized in the primary cilia (Corbit et al., 2005
; Haycraft et al., 2005
; Rohatgi et al., 2007
). By contrast, Drosophila
cilia mutants do not exhibit defects in Hh signaling (Han et al., 2003
; Sarpal et al., 2003
). Therefore, roles for cilia in Hh signal transduction are likely to be restricted to vertebrates.
Calcium signaling was first associated with cilia function with the discovery that the intracellular calcium level rises upon the bending of cilia on canine MDCK cells (Praetorius and Spring, 2001
). It was later shown that polycystin 2 (PC2; Pkd2), a calcium-channel protein localized to the cilia, and its binding partner polycystin 1 (PC1; Pkd1), are essential for initiating the calcium influx that triggers the calcium level change in renal epithelia (Nauli et al., 2003
). A PC2-dependent intracellular calcium surge was also observed on the left side of the embryonic node at E8, after node cilia-mediated nodal flow is initiated (McGrath et al., 2003
). However, PC1 is not expressed in the node and is not required for establishing left-right asymmetry in the mouse, suggesting that the mechanism involved in opening the PC2 channel in the embryo is different from that in the kidney (Karcher et al., 2005
might play a role in PC2 activation because overexpression of Shh
leads to an increase in the intracellular calcium level on the left side of the node (Tanaka et al., 2005
Additional studies suggest further roles for calcium during gastrulation and left-right axis determination. In chicken, a high level of extracellular calcium is observed on the left side of Hensen’s node, and this asymmetric distribution of calcium is translated into asymmetry of the embryo through Notch signaling (Raya et al., 2004
). Calcium waves are also observed in the zebrafish and frog organizers during gastrulation (reviewed by Webb and Miller, 2006
). It has been suggested that calcium waves might be important for convergent extension movements (Wallingford et al., 2001
). However, the roles for calcium in cilia formation have not been investigated, despite the fact that several calcium-binding proteins (e.g. calmodulin, calcineurin, centrin) are localized to the cilia or basal body.
In the current study, we have identified C2cd3, a novel vertebrate-specific C2 domain-containing protein, as an essential regulator of ciliogenesis in the mouse. Through the study of mouse mutants carrying two different loss-of-function alleles of this gene, we show that C2cd3 is essential for mouse embryonic development through regulating the intracellular transduction of Hh signals and proteolytic processing of Gli3. Cilia biogenesis is severely disrupted in the absence of C2cd3. We speculate that C2cd3, a putative calcium-dependent lipid-binding protein that is localized at the basal body of cilia, mediates calcium-dependent vesicular transport and/or recruitment of proteins, including Hh pathway components, during cilia biogenesis. Therefore, the discovery of C2cd3 might lead to a better understanding of the connection between calcium signaling, cilia formation and cilia-dependent signal transduction.