Activation of WNT signaling restores the facial deficits in a zebrafish with defects in cholesterol metabolism

Abstract

Inborn errors of cholesterol metabolism occur as a result of mutations in the cholesterol synthesis pathway (CSP). Although mutations in the CSP cause a multiple congenital anomaly syndrome, craniofacial abnormalities are a hallmark phenotype associated with these disorders. Previous studies have established that mutation of the zebrafish hmgcs1 gene (Vu57 allele), which encodes the first enzyme in the CSP, causes defects in craniofacial development and abnormal neural crest cell (NCC) differentiation. However, the molecular mechanisms by which the products of the CSP disrupt NCC differentiation are not completely known. Cholesterol is known to regulate the activity of WNT signaling, an established regulator of NCC differentiation. We hypothesized that defects in cholesterol synthesis are associated with reduced WNT signaling, consequently resulting in abnormal craniofacial development. To test our hypothesis we performed a combination of pharmaceutical inhibition, gene expression assays, and targeted rescue experiments to understand the function of the CSP and WNT signaling during craniofacial development. We demonstrate reduced expression of four canonical WNT downstream target genes in homozygous carriers of the Vu57 allele and reduced axin2 expression, a known WNT target gene, in larvae treated with Ro-48-8071, an inhibitor of cholesterol synthesis. Moreover, activation of WNT signaling via treatment with WNT agonist I completely restored the craniofacial defects present in a subset of animals carrying the Vu57 allele. Collectively, these data suggest interplay between the CSP and WNT signaling during craniofacial development.

Regulation of Wnt Signaling Pathways at the Plasma Membrane and Their Misregulation in Cancer

“…Presence of canonical Wnt ligand stimulates enrichment of cholesterol, an essential component of the ordered membrane domains, in the inner membrane leaflet and receptor clustering that is likely aided by other membrane components such as heparin sulfate and phosphatidylinositol-4,5-bisphosphate (PIP2) ( Mii et al, 2017 ; Erazo-Oliveras et al, 2018 ). Defects in synthesis of cholesterol thus reduces Wnt signaling and results in abnormalities in craniofacial development and neural crest cell differentiation ( Sezgin et al, 2017a ; Castro et al, 2020 ). The glycosphingolipid mannosyl glucosylceramide, which assembles with sterols into ordered membrane domains, likewise enhances presynaptic Wnt1/Wingless (Wg) signaling in the lipid rafts and promotes synaptic bouton formation at the Drosophila neuromuscular junction ( Huang et al, 2018 ).…”

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“…Adequate cholesterol levels are also needed for proper Wnt signaling to occur. Recent studies have shown that defects in cholesterol synthesis reduce Wnt signaling, causing abnormalities in craniofacial development and neural crest cell differentiation (Sezgin et al, 2017;Castro et al, 2020). Similarly, inadequate cholesterol modulation of presynaptic nAChR may produce alterations in the timing for KCC2 expression in prenatal brain and delay or even impede synaptic pruning, thus having a role in the observed increased dendritic arborization, in addition to the synaptic density modifications due to reduced APC expression.…”

Isogenic hiPSC models of Turner syndrome development reveal shared roles of inactive X and Y in the human cranial neural crest network

“…3, Table S1), at the expense of oxidative phosphorylation, which has also been linked to NCC specification 42 . In addition, cholesterol biosynthesis genes are significantly upregulated in 45,X NCCs, which while critical for modulating NCC signaling 43 may also alter their patterning 44 , and is independently consistent with hypercholesteremia seen in TS patients 5,24 .…”