Novel mutations in Lrp6 orthologs in mouse and human neural tube defects affect a highly dosage-sensitive Wnt non-canonical planar cell polarity pathway

Abstract

Wnt signaling has been classified as canonical Wnt/b-catenin-dependent or non-canonical planar cell polarity (PCP) pathway. Misregulation of either pathway is linked mainly to cancer or neural tube defects (NTDs), respectively. Both pathways seem to antagonize each other, and recent studies have implicated a number of molecular switches that activate one pathway while simultaneously inhibiting the other thereby partially mediating this antagonism. The lipoprotein receptor -related protein Lrp6 is crucial for the activation of the Wnt/b-catenin pathway, but its function in Wnt/PCP signaling remains largely unknown. In this study, we investigate the role of Lrp6 as a molecular switch between both Wnt pathways in a novel ENU mouse mutant of Lrp6 (Skax26 m1Jus ) and in human NTDs. We demonstrate that Skax26 m1Jus represents a hypermorphic allele of Lrp6 with increased Wnt canonical and abolished PCP-induced JNK activities. We also show that Lrp6Skax26-Jus genetically interacts with a PCP mutant (Vangl2 Lp ) where double heterozygotes showed an increased frequency of NTDs and defects in cochlear hair cells' polarity. Importantly, our study also demonstrates the association of rare and novel missense mutations in LRP6 that is an inhibitor rather than an activator of the PCP pathway with human NTDs. We show that three LRP6 mutations in NTDs led to a reduced Wnt canonical activity and enhanced PCP signaling. Our data confirm an inhibitory role of Lrp6 in PCP signaling in neurulation and indicate the importance of a tightly regulated and highly dosage-sensitive antagonism between both Wnt pathways in this process.

Neurodevelopmental Perspectives on Wnt Signaling in Psychiatry

“…Furthermore, there may be a biochemical connection between the Wnt/β-catenin and Wnt/PCP pathways in facilitating neural tube closure. While the LRP6 receptor is generally thought to be dedicated to Wnt/β-catenin signaling, recent data show that NTD-associated LRP6 mutations decrease Wnt/β-catenin activity while simultaneously increasing Wnt/PCP activity [58]. This supports the notion that these seemingly separable Wnt pathways engage in reciprocal inhibition and other forms of cross talk to form integrated signaling networks involved in developmental decision-making [13,61].…”

“…A role for the Wnt/β-catenin pathway is indicated in this process as well. For example, mice with LOF mutations in either Axin1 or Tcf3 exhibit incomplete neural tube closure [53,54], and various mutations in LRP6 cause NTDs in mice [55,56,57], and are present in human NTD patients [58,59]. Recent evidence suggests defects in this case may be due in part to inappropriate fate specification of the laterally placed neural plate border cells governed by the Wnt/β-catenin pathway [60].…”

Planar Cell Polarity Pathway in Kidney Development and Function

“…Another possibility is that the activities of the canonical Wnt and the PCP pathways are interdependent. For example, a hypermorphic allele of the canonical Wnt coreceptor lipoprotein receptor-related protein, Lrp6, increased Wnt canonical and, simultaneously, abolished PCP-induced JNK activities [117]. Several proteins have been shown to act as molecular switches between the Wnt/ -catenin and PCP signaling pathways [116,118] lending credibility to this mechanism.…”

“…Indeed, several recent publications presented convincing data documenting antagonistic relationship between Wnt/ -catenin activity and PCP signaling [117,118]. The inv/inv mouse has cystic kidneys and laterality defects (situs inversus) [141], and knockdown of inv in zebrafish leads to a shortened anterior-posterior axis and widened somites, a classical manifestation of defective PCP signaling.…”

Genetic studies of ANKRD6 as a molecular switch between Wnt signaling pathways in human neural tube defects

“…The wild‐type DIVERSIN open reading frame was cloned in pCDNA3.1 Myc/His A and the human mutations were introduced in this backbone by in site‐directed mutagenesis. The effect of the DIVERSIN variants on PCP signaling and on canonical Wnt/β catenin signaling was tested using the AP1 responsive JNK reporter assay (PathDetect Kit, Stratagene) and the TCF/LEF‐1 responsive Wnt/β‐catenin assay using the TOPFLASH /pGL3‐OT in mammalian HEK cells respectively as previously described (Allache et al, ). Briefly, HEK cells were transfected with (100 ng pAP1‐Luc, 300 ng pSV‐β‐Galactosidase, and 100 ng wild‐type or mutant DIVERSIN or empty pCDNA3.1 vector alone as control) DNA/well in 24‐well plates.…”