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Fig. S2 Cloning and verification of a Cadherin-2 variant as in vivo adhesion reporter protein. (A) Schematic representation of different Cadherin-2 variants (black diamond in EC1 represents cis-dimerizing activity). (B, C) Full-length Cadherin-2 (B) and dominant-negative Cdh2ΔN (C) mRNA-injected embryos analyzed by ISH for atoh1a expression at 24 hpf show severe morphological defects in the hindbrain (dorsal view, anterior is left). (D–F) In contrast, embryos injected with Cdh2ΔEC2-4ΔC:mCherry (D, lateral view of head, overlay with mCherry expression) do not reveal defects, neither by morphology nor by expression of wnt1 (E) or atoh1a (F). Furthermore, mRNA-injection of this variant was unable to rescue pac-/-R embryos (unpublished data). (G–I) Single optical section (1 μm) of a cell in the URL at 65 hpf, co-electroporated with full-length Cdh2:GFP and Cdh2ΔEC2-4ΔC:mCherry plasmid DNA. (G) Full-length Cadherin-2 preferentially clusters in the anterior cell and along the lateral plasma membrane (white arrowheads). The Cdh2ΔEC2-4ΔC:mCherry reporter variant (I) colocalizes with full-length Cadherin-2:GFP in the same regions (see GFP and mCherry overlayed in H). (J) Co-immunoprecipitation (IP) using the Cadherin-2:GFP fusion protein as bait (Input: lane 2) reveals direct interaction between Cdh2ΔEC2-4ΔC:mCherry and Cadherin-2:GFP (IP: lane 2). Full-length Cadherin-2:mCherry used as positive control shows similar interactions (IP: lane 4). (K–L) Bodipy Ceramide membrane staining (K) overlayed with Cdh2ΔEC2-4ΔC:mCherry fluorescence (L) expressed from injected mRNA shows membrane localization of this variant (L, M) and intact cellular morphologies in the cerebellum at 48 hpf. LRL, lower rhombic lip; MHB, mid-hindbrain boundary; URL, upper rhombic lip.