In this study, we demonstrate that, in addition to its initial role in renal vesicle formation, Wnt9b plays a later role in renal tubule morphogenesis. Mice carrying a hypomorphic mutation of Wnt9b or mice that have had a floxed allele of Wnt9b deleted with either KspCre or the tamoxifen inducible CaggCreErTm;Wnt9b−/flox develop cystic kidneys. Cystogenesis does not appear to be caused by increased cell numbers as we have not detected differences in the rates of cell proliferation or apoptosis in mutant epithelia either prior to or concurrent with cyst formation. Instead, we hypothesize that cyst formation is the result of defects in planar cell polarity. We show that cells within the epithelial tubule are elongated perpendicular to the proximal/distal axis of the tubule and that this process is dependent on Wnt9b. We hypothesize that proper cell orientation is required for convergent extension movements and oriented cell divisions. Although cells within the normal collecting ducts and proximal tubules of embryonic kidneys divide in a random orientation, the number of cells composing the wall of the tubule decreases during the embryonic period. We hypothesize that convergent extension movements drive the number of cells within the circumference (or wall) of a tubule to decrease as the tubule elongates. This process, at least in part, establishes the tubule diameter and contributes to tubule length. Once the tubule diameter is established, cell division becomes oriented parallel to the proximal/distal axis to ensure that the kidney tubules continue to elongate while they maintain their diameter. Our data suggest that Wnt9b plays essential roles in both of these processes, perhaps by mediating cell orientation.
In stark contrast to its β–catenin dependent/canonical role during tubule induction17,18
, we have shown that the role of Wnt9b in establishing and maintaining tubule diameter is β–catenin independent. Instead, Wnt9b appears to signal through the non-canonical Rho/Jnk pathway during tubule morphogenesis. Interestingly, recent studies showed that attenuation of Rho kinase led to shorter, wider tubules in cultured kidneys51,52
, a phenotype that may reflect attenuation of Wnt9b signaling. Our data support a hypothesis where Wnt pathway usage is not determined by the individual ligand but instead by the cellular environment in which the signal is received. Depending on the cell type, Wnt9b can signal through both pathways within the same organ system.
Several factors involved in cystic kidney diseases are localized to, and/or are necessary for the function of, the apical mono-cilia 32,53–64
. In addition, recent studies suggest that the primary cilium may play a role in inhibiting canonical Wnt signaling (perhaps promoting non-canonical Wnt signaling) in early mouse and zebrafish embryos 65,66
. A simple model for Wnt pathway usage in the kidney is that the cilia and/or ciliary factors block canonical signaling by Wnt9b and promote non-canonical signaling. Indeed, we see no defects in the expression or localization of several ciliary factors such as Pc-1 and Pc-2 (factors involved in the progression of human autosomal dominant PKD) 61,67
and inversin (Inv) mRNA (the ortholog of the gene mutated in Nephronophthisis type II)60
in Wnt9b mutants nor is Wnt9b mRNA expression affected in Inv−/−
mice (data not shown).
Although this study has revealed a great deal about the mechanisms that regulate tubule diameter, several questions remain unanswered. One such question is why Wnt9b cysts are primarily restricted to the cortex of the kidney? There are several possible answers. The simplest is that another molecule compensates for Wnt9b in the medullary region. Several Wnts, including Wnt5a, Wnt7b, Wnt4 and Wnt11, are expressed in the medullary region of both wild type and Wnt9b mutant kidneys (69
and Karner and Carroll, unpublished observations) and any one of these factors may compensate for loss of Wnt9b. Alternatively, there may be a parallel, Wnt independent, signaling pathway that regulates PCP in the medulla. A recent study showed that mice lacking the PCP determinant Fat4 developed kidney cysts primarily within the medullary region 34
. Compensation by either another Wnt or Fat4 would explain the paucity of medullary cysts in Wnt9b mutants. However, it is important to note that we do observe increased numbers of cells within the circumference of the Wnt9b mutant collecting ducts as well as defects in cell orientation during embryonic stages but do not observe cysts in this nephron segment until post-natal stages. Similar findings were observed by Yu et al. 69
suggesting that other processes, such as defects in fluid secretion or cellular growth (hypertrophy) most likely contribute to cyst formation.
Another question raised by these findings is how Wnt9b, produced by the collecting ducts, affects planar cell polarity in the relatively distant proximal tubules? The simplest hypothesis is that Wnt9b, secreted from the collecting ducts, travels vertically through the stroma to polarize the epithelia. However, it is formally possible that Wnt9b travels through the lumen or through the plane of the epithelium (transcytosis) to regulate morphogenesis. This question is complicated by the fact that we do not know the cell type that is the target of Wnt9b nor do we know at precisely what step during tubule morphogenesis Wnt9b acts, as evidenced by the disparate onset of cystogenesis in KspCre;Wnt9b−/flox and CaggCreErTm;Wnt9b−/flox mutants. Our model is that Wnt9b signals relatively late or continuously during tubule morphogenesis. However, it is possible that it Wnt9b establishes polarity early on in the process of tubule formation, acting on the metanephric mesenchyme or renal vesicles. Finally, it is possible that Wnt9b does not signal directly to the epithelial cells, instead directly signaling to the intervening stroma, which secondarily affects morphogenesis. Determining which of these mechanisms is utilized will be facilitated by the identification of molecular targets of Wnt9b.
A final question that remains is whether Wnt9b contributes to human forms of PKD. Wnt9b continues to be expressed in the adult kidney suggesting that it may play a role in kidney maintenance and/or repair and that improper regulation of this molecule in adults leads to cystogenesis. For instance, improper activation of canonical Wnt9b activity (or failure to divert Wnt9b signaling through the non-canonical branch) in adult kidneys due to loss of ciliary signaling may play a causal role in cystogenesis. Determining if this is the case will require simultaneous ablation of Wnt9b in kidneys that lack intact ciliary signaling or in injured kidneys.
In sum, our findings show that Wnt9b, produced by the kidney collecting ducts, non-autonomously regulates morphogenesis of the developing kidney tubules. We suggest that Wnt9b is required for PCP and the PCP dependent cellular processes convergent extension and oriented cell division. These processes are in turn required to establish and maintain the tubular diameter and length during the embryonic period but are dispensible in healthy, differentiated tubules. A better grasp of the regulation and downstream targets of Wnt9b will significantly impact our understanding of epithelial tubule morphogenesis and the treatment of polycystic kidney disease.