Probes for WNT5A

Wnt5a is a non-canonical Wnt ligand that is essential for normal embryonic development in mammals. The role of Wnt5a in early intestinal development has been examined in gene ablation models, where _Wnt5a__−/−_ mice exhibit strikingly shortened intestines. However, the exact cellular source of Wnt5a has remained elusive, until a recent study found that FoxL1-expressing mesenchymal cells (FoxL1+ cells), which are localized directly beneath the intestinal epithelium, express Wnt5a. To determine whether FoxL1+ cells are a required source of Wnt5a during intestinal development, we derived _FoxL1-Cre; Wnt5a__f/f_ mice, which is the first mouse model to ablate Wnt5a in a cell type-specific manner in the intestine _in vivo_. Our results show that Wnt5a deletion in FoxL1+ cells during fetal life causes a shortened gut phenotype in neonatal mice, and that our model is sufficient to increase rate of apoptosis in the elongating epithelium, thus explaining the shortened gut phenotype. However, in contrast to previous studies using Wnt5a null mice, we did not observe dysregulation of epithelial structure or apical-basal protein localization. Altogether, our findings establish a developmental role for FoxL1+ mesenchymal cells in controlling non-canonical Wnt signaling during midgut elongation.

Mutations in the FOXF1 gene, encoding the mesenchymal Forkhead Box (FOX) transcription factor, are linked to Alveolar Capillary Dysplasia with Misalignment of Pulmonary Veins (ACDMPV), a severe congenital disorder associated with the loss of alveolar capillaries and lung hypoplasia. While proangiogenic functions of FOXF1 have been extensively studied, the role of FOXF1 in mesenchymal-epithelial signaling during lung development remains uncharacterized. Herein, we utilized murine lung organoids to demonstrate that the S52F FOXF1 mutation (found in ACDMPV patients) stimulates canonical WNT/β-catenin signaling in type 2 alveolar epithelial cells (AEC2s), leading to increased proliferation of AEC2s and decreased differentiation of AEC2s into AEC1s. Alveolar organoids containing Foxf1WT/S52F lung fibroblasts and wild-type epithelial cells grew faster on Matrigel and exhibited AEC2 hyperplasia. AEC2 hyperplasia and loss of AEC1s were found in the lungs of Foxf1WT/S52F embryos, a mouse model of ACDMPV. Activation of canonical WNT/β-catenin signaling in AEC2s of lung organoids and Foxf1WT/S52F mice was associated with decreased expression of non-canonical WNT5A ligand in lung fibroblasts. Mechanistically, FOXF1 directly activates the Wnt5a gene transcription through an evolutionarily conserved +6320/+6326 region located in the first intron of the Wnt5a gene. Site-directed mutagenesis of the +6320/+6326 region prevented the transcriptional activation of the Wnt5a enhancer by FOXF1. Treatment with exogenous WNT5A ligand inhibited the effects of the S52F FOXF1 mutation on canonical WNT/β-catenin signaling in alveolar organoids, preventing aberrant AEC2 cell expansion and restoring differentiation of AEC1s. Activation of either FOXF1 or WNT5A may provide an attractive strategy to improve lung function in ACDMPV patients.

While prior work has established that articular cartilage arises from Prg4-expressing perichondrial cells, it is not clear how this process is specifically restricted to the perichondrium of synovial joints. We document that the transcription factor Creb5 is necessary to initiate the expression of signaling molecules that both direct the formation of synovial joints and guide perichondrial tissue to form articular cartilage instead of bone. Creb5 promotes the generation of articular chondrocytes from perichondrial precursors in part by inducing expression of signaling molecules that block a Wnt5a autoregulatory loop in the perichondrium. Postnatal deletion of Creb5 in the articular cartilage leads to loss of both flat superficial zone articular chondrocytes coupled with a loss of both Prg4 and Wif1 expression in the articular cartilage; and a non-cell autonomous up-regulation of Ctgf. Our findings indicate that Creb5 promotes joint formation and the subsequent development of articular chondrocytes by driving the expression of signaling molecules that both specify the joint interzone and simultaneously inhibit a Wnt5a positive-feedback loop in the perichondrium.

Vesicoureteral reflux (VUR) is a common, familial genitourinary disorder, and a major cause of pediatric urinary tract infection (UTI) and kidney failure. The genetic basis of VUR is not well understood. A diagnostic analysis sought rare, pathogenic copy number variant (CNV) disorders among 1737 patients with VUR. A GWAS was performed in 1395 patients and 5366 controls, of European ancestry. Altogether, 3% of VUR patients harbored an undiagnosed rare CNV disorder, such as the 1q21.1, 16p11.2, 22q11.21, and triple X syndromes ((OR, 3.12; 95% CI, 2.10 to 4.54; P=6.35×10-8) The GWAS identified three study-wide significant and five suggestive loci with large effects (ORs, 1.41-6.9), containing canonical developmental genes expressed in the developing urinary tract (WDPCP, OTX1, BMP5, VANGL1, and WNT5A). In particular, 3.3% of VUR patients were homozygous for an intronic variant in WDPCP (rs13013890; OR, 3.65; 95% CI, 2.39 to 5.56; P=1.86×10-9). This locus was associated with multiple genitourinary phenotypes in the UK Biobank and eMERGE studies. Analysis of Wnt5a mutant mice confirmed the role of Wnt5a signaling in bladder and ureteric morphogenesis. These data demonstrate the genetic heterogeneity of VUR. Altogether, 6% of patients with VUR harbored a rare CNV or a common variant genotype conferring an OR >3. Identification of these genetic risk factors has multiple implications for clinical care and for analysis of outcomes in VUR.

Diseases of the airway such as Tracheobronchomalacia (TBM) and Complete tracheal rings (CTR) are prevalent conditions associated with abnormal patterning of the trachealis muscle and cartilage. However, the underlying mechanisms of tracheal patterning are poorly understood. We have demonstrated that Wnt signaling via Wls plays an essential role in determining the mesenchymal patterning of the trachea. Notum, a direct target of Wnt signaling, encodes an enzyme that inactivates Wnt ligands, thus attenuating Wnt signaling's strength in developing trachea. In Notum deficient mice, chondroblasts and smooth muscle cells of the trachea were specified properly. Meanwhile, deletion of Notum impaired and delayed mesenchymal condensations of chondroblasts causing abnormal cartilage and stenosis. Further, tracheal muscle organization was disrupted. We hypothesize that chondrocyte condensation influences the trachealis muscle organization during tracheal tubulogenesis. Methods: We utilized genetically modified mice wherein Notum, Wnt5a, and Ror2 were deleted in the germline or conditionally ablated in tracheal mesenchyme. Tracheal smooth muscle cells were genetically labeled using γSMAeGFP mice. RNA scope, whole-mount stain, immunofluorescence, and fluorescent microscopy were utilized to visualize changes in trachealis muscle cell and cartilage organization induced by in vivo gene deletion and ex vivo treatments. Results: Analysis of muscle cells of control tracheas at E12, E14, and E16 showed progressive trachealis muscle organization with myocytes oriented perpendicular to the elongation axis of the trachea. In contrast, Notum deficient tracheas showed disordered orientation of the muscle cells with changes in the cytoskeleton. The anomalous muscle cell arrangement was increasingly observed after E14 after mesenchymal condensations were completed, and cartilage formed. Ex vivo studies with ABC99, a pharmacological inhibitor of Notum affected trachealis muscle organization, recapitulating the in vivo data. The Planar Cell Polarity (PCP) branch of the non-canonical Wnt signaling pathway mediates organization and functioning of the trachealis muscle. Mesenchymal deletion of the non-canonical Wnt5a and its receptor Ror2 impaired trachealis muscle cell orientation, causing changes in myocytes' cytoskeletal organization similar to changes observed in Notum deficient trachea. Different from Notum deletion, changes in myocyte organization preceded cartilaginous mesenchymal condensation. Despite the abnormal muscle organization in Wnt5a, cartilaginous mesenchymal condensations occurred, although reduced in number. We conclude that tracheal chondrogenesis affects trachealis muscle formation by constraining the expansion and altering the cytoskeletal organization of tracheal myocytes after mesenchymal condensation takes place. Thus, delayed tracheal chondrogenesis may partially underlie the pathology of tracheal stenosis. These studies were partially supported by NIH-NHLBI R01HL144774-01A1 to DS.