Fig. 1

Expression and interaction of zebrafish Mdka, Mdkb and Ptn with Ptprz1b. (A) Hybridization chain reaction RNA in situ hybridization (HCR) of mdka, mdkb and ptn expression in wildtype embryos at 10 hpf (neural plate stage) and 12 hpf (early neural keel stage). Upper panels are representative maximum-intensity-projection (MIP) dorsal views with dlx3b (magenta) demarcating the neural plate/keel boundary. Arrow heads indicate mdka and mdkb expression (cyan) at 10 hpf. The lower panels are reconstructed orthogonal views from the position indicated by yellow dashed lines in the upper panels. The outlines of neural plate/keel are marked by white dashed lines, and otic vesicles flanking r5 are labelled with white asterisks. Scale bars = 50 μm. (B) Fluorescent RNA in situ hybridization (FISH) of mdka, mdkb and ptn expression in wildtype embryos at 14 hpf. Left: Schematic diagram of hindbrain organization, dorsal view with dashed box indicating rhombomere region (r1 to r6) analyzed by FISH. NC = negative control (mdka sense probe). Representative MIP images showing dorsal views and pseudo-colored in fire LUT with an intensity range from 0 to 255 by ImageJ. Dashed lines delineate lateral edges of rhombomeres, white asterisks indicate position of otic vesicles at r5. Scale bars = 50 μm. (C) Mean fluorescent intensity of FISH signals in (B) along anteroposterior axis of r1 to r6. (D) Representative MIP images after proximity ligation assay (PLA) of controls and different pairs of ligands (Mdka, Mdkb, Ptn) and receptor (Ptprz1b). Embryos injected with HA-ptprz1b mRNA alone served as the negative control (NC). Positive controls (PC) are mdka-MYC mRNA-injected embryos using two pairing secondary antibodies that recognize the same primary antibody. Embryos co-injected with mdka-MYC and secreted EGFP (secEGFP) mRNA served as random collision controls. PLA signals are represented in cyan, and DAPI in magenta. Scale bars = 20 μm. (E) Statistical analysis of PLA levels normalized to DAPI signals after thresholding (PLA/DAPI area ratios). Data are presented as scattered dots with mean ± SD. Statistical analysis was performed on Estimation Stats (https://www.estimationstats.com) to compare each dataset with the random collision control. P values are calculated from a two-sided permutation t-test under a CI of 95% and listed in Fig. S1G. Asterisks indicate statistical significance (P < 0.05). (F) Representative super resolution single plane images of PLA signals from Mdka-MYC/Mdkb-MYC/Ptn-MYC and mEGFP-Ptprz1b, respectively. mEGFP-Ptprz1b is colored in cyan, PLA in magenta, and DAPI in gray. PLA signals on plasma membrane are indicated by yellow arrowheads. Scale bars = 20 μm. (G-J) FCCS measurements were performed on MZ ptprz1b embryos at 4 hpf that had been injected with the indicated mRNAs at 1-cell stage. Green and red curves are auto-correlation functions plotted from green and red fluorophore channels, respectively. Blue curves represent the cross-correlation function. All curves were fitted by a 3D-2 particles model as represented by black curves, respectively. (G) MZ ptprz1b mutant embryos expressing Ptn-mEGFP and PMT-mApple were used as negative control. (H) MZ ptprz1b mutant embryos expressing membrane bound PMT-mEGFP-mApple were used as positive control. (I) Data obtained from MZ ptprz1b embryos that were co-injected with ptn-mEGFP and ptprz1b-mApple mRNA. The relative cross-correlation values of FCCS measurements are shown in (J) in the form of mean ± SD. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)