In this study, we demonstrate that Wnt5a plays a role in mice in two cellular processes regulated by the PCP pathway, establishment of epithelial PCP and CE.
We found a reciprocal expression pattern of Wnt5a and Frzb along the axis for planar polarization in the cochlea at the time that PCP is being established (). We showed that Wnt5a suppresses the effect of Frzb on stereociliary bundle orientation in vitro (). We further observed imperfect stereociliary bundle alignments in Wnt5a−/− animals () and a genetic interaction between Wnt5a and Ltap/Vangl2 in regulating stereociliary bundle orientation (). Together, these observations indicate that Wnt5a contribute to the establishment of uniform bundle orientation.
Although Frzb addition has a strong effect on PCP in vitro, only minor imperfection in the alignment of stereocilia in Wnt5a−/− mice was observed. This observation suggests that additional pathways parallel or redundant to Wnt5a are involved in PCP regulation in the cochlea. The presence of redundant pathways made it difficult to determine the molecular mechanism underlying the role of Wnt5a in PCP signaling in mice. It is not clear whether the reciprocal expression of Wnt5a and Frzb along the axis for planar polarization is involved in generating a graded Wnt signal to direct the establishment of PCP in the cochlea. The lack of demonstrated effectiveness of Wnt5a-CM on stereociliary orientation further prevented us from reversing the source of Wnt5a in vitro to test a possible instructive role for Wnt5a. However, the data from our grafted organ cultures () indicates an intrinsic difference between the medial and lateral regions of the cochlear epithelium for PCP and supports the presence of instructive cues along the mediolateral axis of the cochlear epithelium.
The composition of potential mediolateral instructive cues, however, remains unknown. The putative compensatory pathways for Wnt5a may include additional Wnts (Colosimo and Tolwinski, 2006
; Dabdoub et al., 2003
; Price et al., 2006
) and Hh (Colosimo and Tolwinski, 2006
). BMPs might also contribute to PCP regulation in the cochlea, as BMP signaling can be modulated by a secreted frizzled molecule (Lee et al., 2006
; Muraoka et al., 2006
) and BMPs are expressed asymmetrically along the mediolateral axis of the cochlear epithelium (Morsli et al., 1998
; Takemura et al., 1996
). Future studies towards the understanding of additional pathways and the generation of genetic tools to interrupt or reverse the presence of Wnts and Wnt antagonists in vivo will be critical to determine the molecular role of Wnts in PCP regulation.
CE consists of cellular intercalations along the mediolateral axis and extension along the perpendicular longitudinal axis. In Xenopus and zebrafish, Wnt5 and Wnt11 are required for CE. However, their expression pattern is not consistent with an instructive but rather a permissive role (Heisenberg et al., 2000
; Kilian et al., 2003
; Smith et al., 2000
; Tada and Smith, 2000
; Ulrich et al., 2003
). In the cochlea, the expression of Wnt5a is asymmetric along the mediolateral and the longitudinal axes () and is appropriate to serve as a cue for CE along both axes. The effect of Frzb on cochlear extension in vitro and the suppression of this effect by Wnt5a provided the first hint that Wnts may be involved in CE in mammals (). Our observation of craniorachischisis and shortened and widened cochleae in some Wnt5a−/−
animals (), and the genetic interaction between Wnt5a and Ltap/Vangl2 in enhancing the penetrance of cochlear CE and neural tube closure (Figs. ,) further supported a role for Wnt5a in the PCP pathway for CE regulation.
An intriguing question is how the mammalian PCP pathway concurrently regulates the establishment of PCP and CE in the cochlea during terminal differentiation. In Wnt5a mutants, phenotypes from the two processes show different penetrance. The cochlear CE defect has a higher penetrance than the stereociliary defect in Wnt5a−/− animals (), while Wnt5a+/−; LtapLp/+ double heterozygous animals have a 100% penetrant stereociliary orientation defect in the third row of OHCs but no apparent cochlear CE defect (). The dissociation of cochlear CE and stereocilia orientation defects in some Wnt5a mutants suggests that the molecular mechanisms underlying CE and stereociliary bundle orientation are not identical.
Wnts can trigger both canonical (β-catenin-dependent) and non-canonical (β-catenin-independent) downstream pathways. No canonical Wnt activity was detected in the cochlea during terminal differentiation (supplemental Fig. 1
), making it unlikely that Wnt5a acts via a β-catenin-dependent pathway. This said, it remains possible that canonical activity occurs at a level below the detection threshold with the BAT-gal reporter. However, our data is consistent with the current view on the modulation of the specificity of downstream Wnt pathways by the context of intracellular factors and receptor. Candidate vertebrate cytoplasmic PCP proteins have been shown to promote the PCP pathway while inhibiting the canonical Wnt pathway (Moeller et al., 2006
; Schwarz-Romond et al., 2002
; Simons et al., 2005
). For example, a candidate PCP gene, Ankrd6/Diversin, inhibits canonical Wnt activity while promoting PCP signaling (Moeller et al., 2006
; Schwarz-Romond et al., 2002
). The presence of specific Fz receptors in the cochlea may further direct Wnts in the cochlea to act via a noncanonical pathway. Fz3 and Fz6 are expressed in the developing organ of Corti and are redundantly required for uniform stereociliary orientation (Wang et al., 2006b
). However, Fz3 and Fz6 fail to mediate the activation of the canonical Wnt pathway in vitro when Wnt5a is juxtaposed with a domain enabling its binding to LRP5/6, the coreceptor required for the canonical Wnt pathway (Liu et al., 2005
). Our study now provides the first complementary genetic evidence to demonstrate functional involvement of Wnt5a in the PCP signaling, a noncanonical Wnt pathway, in mammals. While additional studies are clearly needed to further dissect the exact molecular role and the signaling cascade downstream of Wnt5a in PCP regulation, it is tempting to speculate that an intricate modulation or suppression of canonical Wnt signaling by multiple components of the PCP pathway may have evolved in the vertebrates to divert Wnts to PCP signaling.