After more than 30 years of intense study, the major intercellular signalling systems that orchestrate embryonic development and tissue maintenance are reasonably well understood. Although there are many crucial details yet to be worked out, there is a tendency among researchers in the field to think that the major players in these signalling pathways — and the ways in which they interact — are known. In a paper in Nature, Szenker-Ravi et al.1 remind us that this is not necessarily the case.
WNT proteins are signalling molecules whose activity controls many processes, from tissue organization and body-axis formation during embryonic development to maintenance and regulation of stem cells in adult tissue. WNTs signal through several distinct intracellular pathways, but these pathways share an initial step: WNT molecules outside the cell bind to and activate receptors of the Frizzled family that span the cell membrane. Frizzled receptors must be present in the membrane for intracellular WNT-pathway activation.
In vertebrates, an auxiliary regulatory process is involved in controlling the accumulation of Frizzled receptors, and hence in determining WNT signalling levels. The system involves three groups of proteins2,3: an LGR (LGR4, 5 or 6), an extracellular R-Spondin (RSPO1, 2, 3 or 4), and an E3 ubiquitin ligase enzyme (ZNRF3 or RNF43). In the absence of RSPOs, the ubiquitin ligase tags Frizzled receptors with ubiquitin molecules, which mark the receptors for degradation. This results in low membrane concentrations of Frizzled, so WNT signalling is attenuated4–6 (Fig. 1a). Conversely, when RSPOs are present, they bind to LGRs2,3, and the resulting complex binds the ubiquitin ligase and prevents it from tagging Frizzled. The receptors accumulate, and WNT signalling can occur4–6 (Fig. 1b).
This mechanism has a crucial role in regulating WNT signalling in stem-cell compartments characterized by LGR expression, for instance in the intestine7 and hair follicles8. Because WNT signalling has been extensively studied in these contexts, the LGR–RSPO–ligase complex has become part of the standard picture of how WNT activity is regulated.
Szenker-Ravi and colleagues’ study began with a genetic analysis of five families affected by either tetra-amelia syndrome, which is characterized by the lack of all four limbs and by lung abnormalities, or by a previously undescribed syndrome involving severe limb malformations. The authors found that these two syndromes are caused by five mutations in the RSPO2 gene that disrupt different protein domains. Using in vitro assays, the researchers demonstrated that the mutations prevent RSPO2 from binding to LGR or RNF43, and so inhibit WNT signalling.
So far, Szenker-Ravi and co-workers’ data fit nicely with the known roles of RSPOs, LGRs and ubiquitin ligases. And, like people carrying RSPO2 mutations, mice lacking Rspo2 have limb abnormalities9. The authors expected that the loss of LGR activity would have the same effect. But they got a surprise when they analysed mice lacking the Lgr4, 5 and 6 genes — the triple-mutant embryos did not have limb or lung abnormalities. This suggests that, in some tissues, RSPO2 (and perhaps other RSPOs) can act independently of LGRs, potentiating WNT signalling in the absence of its usual binding partner.
To test this idea directly, the group next investigated whether cells isolated from LGR triple-mutant embryos are capable of RSPO-mediated WNT signalling. They found no evidence of WNT signalling when these cells were exposed to Rspo1 or Rspo4, but WNT activity was detected in the presence of Rspo2 or Rspo3. Thus, RSPO2 and RSPO3 seem to be able to induce WNT signalling independently of LGRs. However, these RSPOs still seem to act through their normal ubiquitin ligase targets, because Szenker-Ravi et al. found that modulation of ZNRF3 alters WNT signalling in triple-mutant cells. Consistent with this picture, the authors showed that deletion of rspo2 in the frog Xenopus laevis led to missing limbs, whereas deletion of the znrf3 and rnf43 genes led to extra limbs.
This study demonstrates that the accepted model of WNT-receptor modulation does not hold in the case of limb and lung development. Szenker-Ravi et al. hypothesize that a separate, unidentified receptor is necessary for this LGR-independent WNT signalling (Fig. 1c). Notably, a study published earlier this year10 identified one potential candidate. That work showed that RSPO2 and RSPO3 can bind to ZNRF3 or RNF43 in conjunction with heparin sulfate proteoglycan (HSPG) molecules in lieu of LGRs, to enable WNT signalling in vitro. Future work will be required to test whether HSPGs play this part in the context of lung and limb development. In addition, it remains to be determined whether the HSPG–RSPO–ZNRF3 complex promotes WNT signalling by preventing ZNRF3 activity, or whether another mechanism is at work. Either way, it will be important to determine the extent of any functional similarities between LGR- and HSPG-based complexes, and to uncover whether there is any pattern to the use of LGR or HSPG as a cofactor in a particular tissue.
Szenker-Ravi and colleagues’ work also points to ways to broaden our understanding of processes that require WNT signalling, such as limb development. For example, analysis of the early stages of limb development in frog embryos lacking znrf3 and rnf43 could reveal why these mutations lead to extra limbs. Do ZNRF3 and RNF43 act as ‘master regulators’ of limb numbers, as the authors propose? Consistent with this idea, WNT activity has a role in initiating the formation of the limb bud11 (which eventually gives rise to the limb). Alternatively, rather than being master regulators, these proteins might mediate limb numbers indirectly. For example, extra limbs might arise as a secondary consequence of expansion of the pool of limb progenitor cells, or they might arise because of changes in the formation of a signalling centre at the tip of the limb bud that directs limb outgrowth — both WNT-dependent processes12,13.
Finally, it will be interesting to evaluate LGR-independent, RSPO-mediated WNT signalling in cancer. Chromosomal abnormalities that lead to activation of RSPO2 or RSPO3 have been shown to drive WNT-dependent colon tumours14. Szenker-Ravi and colleagues’ demonstration that these two RSPOs can modulate WNT activity independent of LGR adds a twist to these findings, and should prompt scientists to look for cancer-causing mutations in RSPO2 or RSPO3 in cells outside LGR-expressing cell compartments.
Nature 557, 495-496 (2018)