Probes for ROR2

The choroid plexus (ChP) produces cerebrospinal fluid and forms an essential brain barrier. ChP tissues form in each brain ventricle, each one adopting a distinct shape, but remarkably little is known about the mechanisms underlying ChP development. Here, we show that epithelial WNT5A is crucial for determining fourth ventricle (4V) ChP morphogenesis and size in mouse. Systemic Wnt5a knockout, or forced Wnt5a overexpression beginning at embryonic day 10.5, profoundly reduced ChP size and development. However, Wnt5a expression was enriched in Foxj1-positive epithelial cells of 4V ChP plexus, and its conditional deletion in these cells affected the branched, villous morphology of the 4V ChP. We found that WNT5A was enriched in epithelial cells localized to the distal tips of 4V ChP villi, where WNT5A acted locally to activate non-canonical WNT signaling via ROR1 and ROR2 receptors. During 4V ChP development, MEIS1 bound to the proximal Wnt5a promoter, and gain- and loss-of-function approaches demonstrated that MEIS1 regulated Wnt5a expression. Collectively, our findings demonstrate a dual function of WNT5A in ChP development and identify MEIS transcription factors as upstream regulators of Wnt5a in the 4V ChP epithelium.

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.