We have identified in vivo biologic activities for Wisp3 using zebrafish. These insights are important because the role of Wisp3 in mammals has been difficult to determine. Although mutations in WISP3
have been found in patients with PPD, a skeletal disease associated with precocious cartilage failure, affected tissue has only become available after it is histologically indistinguishable from common, end-stage osteoarthritis (2
). Importantly, we showed that Wisp3 missense mutants that are disease-causing in humans had lost or reduced biologic activities in zebrafish. In mice, neither Wisp3 deficiency nor Wisp3 overexpression caused a discernable phenotype (ref. 27
and Y. Nakamura, unpublished observations). The lack of a mouse model of PPD and our inability to detect endogenous Wisp3
mRNA or Wisp3 protein expression in mammalian tissues has limited our ability to interrogate its biologic function.
zwisp3 mRNA was detectable by whole-mount in situ hybridization and zWisp3 protein was detectable by Western blot during embryonic development (Figure ). Overexpression of zWisp3 during early zebrafish development revealed an ability to inhibit BMP and Wnt signaling (Figures and ). We did not observe the opposite phenotype when zWisp3 was depleted using morpholinos, which is not surprising because there was no detectable endogenous zWisp3 protein expression during gastrulation.
The ability of hWISP3 and zWisp3 to inhibit BMP and Wnt signaling was also observed in mammalian cells (Figures and ). We found that hWISP3 and mBMP4 physically interacted, suggesting an inhibitory mechanism involving direct ligand binding. Consistent with this hypothesis is the finding that the C78R missense mutant retained its ability to bind mBMP4 and its ability to inhibit BMP signaling, whereas the C145Y and Q338L mutants lost both abilities. Another CCN family member, Ctgf, has also previously been shown to physically interact with BMP and to inhibit BMP signaling (42
), indicating that this biologic activity may be conserved within the CCN family. Rather than binding Wnt ligand to inhibit signaling, zWisp3 instead bound the coreceptors LRP6 and FzD8, binding most strongly to LRP6 (Figure ). Two other CCN family members, Cyr61 and Ctgf, have also been shown to modulate canonical Wnt signaling in Xenopus laevis
), again suggesting conserved biologic activity within the CCN family.
Importantly, we demonstrated that missense mutations in zWisp3, which are associated with the human disease PPD, affected the BMP and Wnt inhibitory functions in zebrafish. Missense mutations in the IGFBP, VWC, and CT domains each had comparable effects on Wnt signaling, implying that proper folding of multiple domains is necessary for the protein’s Wnt-inhibitory function. In contrast, the C78R mutation had a milder effect on BMP signaling, indicating that the IGFBP domain may be dispensable for this biologic activity. However, it is premature to speculate whether this difference points to Wisp3 having a more important role in regulating the Wnt pathway than the BMP pathway in humans.
Depletion of zWisp3 using either translation blocking or a splice-site morpholino caused mandibular cartilage undergrowth and affected the shape and strength of other pharyngeal cartilages (Figure ). These effects were unexpected because whole-mount in situ hybridization did not identify zwisp3
expression in chondrocytes. Chondrocytes could be expressing zwisp3
at levels detectable only by RT-PCR, similar to what has been observed in humans and mice, or the in situ hybridization protocol may not have had sufficient sensitivity to detect expression in cartilage. BMP signaling is important during zebrafish pharyngeal cartilage morphogenesis, as demonstrated by the malformations observed in zebrafish with mutations in smad5, a cytoplasmic effector of the BMP pathway (43
). In mammals, BMP signaling regulates cartilage development (44
) and homeostasis (45
), the latter being demonstrated by the occurrence of precocious osteoarthritis in mice with postnatal disruption of a BMP receptor (45
). In humans, genetic evidence implicates subtle perturbations in the regulation of BMP and related signaling pathways as risk factors for common osteoarthritis (46
). Wnt signaling has also been implicated in cartilage differentiation (48
) and skeletal homeostasis (50
). Mouse models of precocious osteoarthritis in the context of altered Wnt signaling have not yet been described, although disturbed skeletal homeostasis has been reported in mice lacking the Wnt coreceptor Lrp5 (51
). In humans, coding variants in secreted Wnt inhibitors have been associated with common osteoarthritis (53
), and studies of human articular cartilage explants detect Wnt and BMP signaling responses to mechanical stress (55
). Future research is needed to determine whether the pharyngeal cartilage fragility in the zWisp3 morphants relates to the precocious osteoarthritis observed in humans with PPD and whether both are consequences of perturbed BMP and/or Wnt signaling.
Biologic activities for Wisp3 have previously been sought ex vivo using mammalian systems (19
). Some studies suggest that Wisp3 deficiency might cause PPD by reducing the normal expression of several cartilage matrix proteins or the ability of chondrocytes to handle reactive oxygen species (19
). The fragility of the pharyngeal cartilage elements in the morphants could reflect altered abundance of structural matrix components. Formal testing of this hypothesis in zebrafish morphants must await the development of proteomic and/or immunohistochemical methods that can detect differences in tissue structure and composition. Surprisingly, no apparent defects in skeletal growth or matrix production were observed in the Wisp3 knockout mice (27
), although there is precedent for mutations that cause recognizable skeletal phenotypes in humans being nonpenetrant or exceedingly subtle in mice (56
). Other studies suggested that Wisp3 controls tumor cell growth and invasiveness (20
). The specificity of these effects is difficult to determine given the absence of increased rates of cancer in humans with PPD and in mice with targeted disruption of mWisp3
, although studies using mice with increased predisposition to cancer may be illuminating.
Multiple signaling pathways are involved in complex developmental processes. For example, during gastrulation, Wnt and BMP signaling are required for specification of ventral fates (28
). Specification of dorsal cell fates depends on the action of secreted inhibitors of these pathways. In addition, some inhibitors affect multiple pathways, such as Cerberus, which inhibits BMP and Wnt signaling during the formation of the vertebrate head (16
). BMP and Wnt signaling are both required during skeletal differentiation, growth, and homeostasis, and we have shown that zWisp3 has the ability to modulate both pathways. Given the ability of zWisp3 to inhibit BMP and Wnt signaling, when overexpressed in zebrafish or when added to mammalian cell cultures, it is also surprising that Wisp3 overexpression in mice causes no phenotype. There are several potential explanations for the interspecies difference in the biologic activity of overexpressed Wisp3 in vivo. These include differences in the protein’s posttranslational modification, abundance, stability, or interacting partners. One inter-species difference we observed in this study is that zWisp3 existed as a full-length protein when expressed in HEK293T cells, whereas hWISP3 or mWisp3 protein was often cleaved, leading to the production of smaller carboxyterminal fragments (Figure C and Figure , D and E). These smaller fragments, while abundant in CM, did not coprecipitate with either mBMP4 or hLRP6, which suggests that they are not biologically active inhibitors. Similar cleavage of overexpressed protein in transgenic mice may render the protein nonfunctional. Whereas studies in mice have not been revealing, the zebrafish assay will be useful in determining whether other missense mutations in patients suspected of having PPD are disease-causing or rare, benign polymorphisms.