Ciliopathies in humans include the phenotypes seen in syndromes such as MKS, NPHP, JBTS, and BBS where most of the genes identified encode proteins that localize to cilia or basal bodies. One of the common phenotypes associated with defects in cilia signaling is the formation of renal cysts. This is seen for the cilia-localized proteins polycystin 1 and polycystin 2, which are affected in PKD. Mutations in the PKD genes do not disrupt cilia structure but do result in the loss of a flow-regulated mechano-sensitive calcium signal, and in the case of polycystin-1 altered AP-1 pathway activation and increased mTOR activity (Low et al., 2006
; Shillingford et al., 2006
). Proteins involved in the cystic renal pathology in NPHP patients localize to the base of cilia; however, the roles of many of the NPHP proteins are largely unknown. Mutations in nph-1
in C. elegans
cause defects in morphology of a subset of cilia on a few amphid neurons in the hermaphrodite and in sensory neurons that are specific to the male (Jauregui et al., 2008
) and result in several phenotypes typically associated with disruption of cilia signaling pathways (Jauregui and Barr, 2005
; Winkelbauer et al., 2005
). It is uncertain whether these phenotypes are due to loss of a specific signaling pathway mediated by the NPH proteins or due to ultrastructural ciliary defects that may disrupt multiple pathways. In contrast to PKD- and NPHP-associated proteins, evidence suggests that MKS1 and mecklin, which are involved in renal cyst formation in MKS, affect cell polarization and centriole migration and severely disrupt cilia assembly, at least based on siRNA knockdown data (Dawe et al., 2007b
). An intriguing finding that is emerging with regards to MKS, NPHP, and JBTS is that genes associated with one of these syndromes are being identified as responsible for the phenotypes in one or both of the other disorders. These data argue that the syndromes represent a spectrum of the same underlying defect and that the resulting phenotypes will be dictated by the nature of the mutation occurring in these genes. However, other than their colocalization to cilia or basal bodies, there is currently very little known about how proteins associated with MKS, NPHP, or JBTS are functionally related or how mutations in one of the genes may affect the localization and/or activity of another.
To begin addressing these questions, we are using C. elegans
as a model system to elucidate the functional relationships between cilia and proteins associated with human cystic kidney disease syndromes. Here we analyzed the connection between the MKS1 homolog XBX-7 and its related B9 protein family members and the NPHP1 and NPHP4 homologues, NPH-1 and NPH-4. Intriguingly, we demonstrate that all three B9 genes in C. elegans
are coregulated by the same transcription factor in ciliated sensory neurons and that the B9 proteins localize to the transition zones at the base of the sensory cilia. Similar localization to basal bodies has been reported for the XBX-7 homolog in mammalian cells, suggesting conserved function (Dawe et al., 2007b
). However, siRNA-mediated knockdown of MKS1 expression in mammalian cells or RNAi knockdown of the TZA-1 homolog in P. tetraurelia
suggest these proteins are required for cilia assembly or formation (Dawe et al., 2007b
; Ponsard et al., 2007
). Additionally, during review of this manuscript a conditional mutant in the tza-1
mouse homolog was described. These mutant mice had absent or truncated cilia and systemic phenotypes typically associated with disruption of cilia in mammals (Town et al., 2008
). In contrast, our analysis of single, double, or triple mutations in the B9 genes in C. elegans
did not reveal cilia morphology defects unless these mutations were combined with mutations in either nph-1
. It is plausible that the B9 gene
single mutants exhibit ultrastructural abnormalities in cilia or subsets of cilia that would not be detectable by the methods used in this analysis. Alternatively, the discrepancy could be due in part to the nature of the alleles we analyzed. xbx-7
both result in internal deletions that do not cause frame shifts. Thus, the proteins encoded by xbx-7(tm2705)
may retain partial function with respect to their potential roles in ciliogenesis. In the case of the xbx-7(tm2705)
mutants, this could explain the consistently milder phenotype relative to that seen in the other B9 gene
mutants when crossed with an nph gene
mutant. Although the tza-1(tm2452)
mutation is an internal in-frame deletion, we show that this mutation results in loss of both XBX-7 and TZA-2 from the transition zones, which would thus be expected to acerbate the phenotype as was observed. In contrast, the tza-2(ok2092)
allele results in deletion of most of the protein including the B9 domain and is thought to represent a null mutation.
Our genetic analyses in C. elegans indicate that the B9 proteins form part of a complex at the transition zone. This is supported by data showing that XBX-7 localization was dependent on the presence of both TZA-1 and TZA-2, and TZA-2 required only TZA-1 for proper localization. In contrast, disruption of TZA-2 or XBX-7 did not alter localization of TZA-1. We interpret these data to indicate that TZA-1 functions early in the assembly of a complex containing the B9 proteins followed by TZA-2 and subsequently by XBX-7. It is unknown whether the loss of fluorescent signal from the transition zones was due to the unincorporated proteins being diffused throughout the neurons or alternatively due to destabilization and degradation of these proteins. However, using a yeast two-hybrid assay, we observed a positive interaction between TZA-1 and TZA-2, suggesting at least in the case of TZA-2 that the protein was mislocalized due to failure in assembly into the complex at the transition zone. In contrast, we did not observe a physical interaction between XBX-7 and the other B9 proteins, suggesting that there is an unknown factor that is responsible for anchoring XBX-7 in the complex. Whether there is a similar hierarchy of complex assembly with regards to the mammalian proteins remains to be determined.
Because the NPHP protein homologues NPH-1 and NPH-4 are part of a complex that localizes similarly to the B9 proteins at the transition zone (Winkelbauer et al., 2005
), and mutations in the NPHP proteins result in cystic renal disease in humans and mice, we explored the possibility that the C. elegans
B9 and NPH proteins were functionally related or perhaps components of the same complex. However, our analysis of the localization of NPH-1 and NPH-4 in the B9 gene
mutants revealed there were no effects on their localization to the transition zones nor were the B9 proteins affected by mutations in nph-1
. Thus, there does not appear to be a direct link between the B9 proteins and the NPH proteins with regards to complex formation at the base of cilia.
Cilia mutants in C. elegans exhibit numerous behavioral and sensory defects including abnormalities in chemotaxis, osmotic avoidance deficiencies, altered dauer formation, and a marked reduction in foraging activity in the presence of food. In the B9 gene mutants, we did not observe defects in osmotic avoidance, chemotaxis, or dauer formation, in agreement with the presence of overtly normal cilia morphology. However, both the tza-1(tm2452) and tza-2(ok2092) mutants did exhibit subtle yet significant reductions in foraging behavior, which was not evident in the xbx-7(tm2705) mutants. Interestingly, we also found that nph-4(tm925) mutants had defects in foraging activity that was significantly more severe than either tza-1(tm2452) or tza-2(ok2092) mutants. These observations led us to evaluate whether the foraging behavior defects in the B9 gene mutants were due to disruption of the same pathway affected in the nph-4(tm925) mutants. We analyzed this in B9 gene;nph-4 double mutants and found that mutations affecting both the B9 proteins and NPH-4 resulted in a markedly more severe dwelling phenotype than either mutation alone. This phenotype was similar to that seen in worms completely lacking cilia, suggesting redundant functions of the B9 proteins and NPH-4 at the base of cilia.
The B9 gene;nph-4
double mutants were also unable to normally dye-fill. This was due to abnormalities in bundling, orientation, or length of the cilia along with gross cilia positioning defects caused by dendrite malformation. Additionally, we observed the same altered morphology in B9 gene;nph-1
double mutants, suggesting that because NPH-4 is critical for the proper localization of NPH-1 to the transition zones, the defects observed in the B9 gene;nph-4
double mutants can be attributed to the loss of NPH-1. The complex phenotype observed in these worms is unique among mutants defective in dye-filling. daf-19
mutant worms exhibit phasmid neuron dendrites of varying lengths similar to those seen in the B9 gene;nph gene
double mutants, but daf-19
mutants lack all ciliary and transition zone structures (Swoboda et al., 2000
). Notably, centrioles positioned at the distal tips of the dendrites are detected by electron microscopy in daf-19
mutants (Perkins et al., 1986
). Because each of the B9 and nephrocystin genes are strongly regulated by the DAF-19 transcription factor and are therefore not expressed in daf-19
mutant worms, it seems likely that the B9 proteins and nephrocystins will not be critical for positioning the centrioles at the distal tips of the dendrites. Disruption of the forkhead domain transcription factor gene fkh-2
results in shortened dendrites and cilia morphology defects, but this phenotype is specific to AWB neurons alone (Mukhopadhyay et al., 2007
). B9 gene;nph gene
double mutants may most closely resemble mec-8
mutants in which amphid cilia fail to fully penetrate the sheath glia, are sometimes misoriented laterally, and do not fasciculate at the sheath/socket channel (Perkins et al., 1986
). However, gross dendrite morphology defects have not been reported in mec-8
mutants. Although we observed grossly normal sheath cells in B9 gene;nph gene
double mutants, we have not directly determined whether these cells cooperate with the socket cells to properly form the channels through which the cilia normally project. It is likely, however, that the channels do properly form since the B9 gene;nph gene
mutants are still able to uptake DiI to some extent, indicating that on occasion some cilia are correctly formed and exposed to the external environment.
The additive effects seen in the B9 gene;nph gene double mutants indicate that the B9 proteins and NPH proteins likely function redundantly to coordinate how cilia and dendrites will form. It is possible that the phenotypes we see in these double mutants are related to those observed when the B9 proteins are singularly disrupted in other biological systems. Together, our findings provide insights into the allelic nature of the genes that can be involved in multiple syndromes and more importantly indicate that the mammalian homologues of the B9 domain proteins TZA-1 and TZA-2 are strong candidates as loci involved in the pathology of MKS, NPHP, or JBTS patients for which the underlying genetic defects have not yet been identified.