Ultrastructure and 3D reconstruction of olfactory HR-like/NaR-like ionocyte cell pairs and multicellular complexes in the wild-type zebrafish larva. (A–G) Representative example of an ionocyte cell pair in the 7 dpf zebrafish larval olfactory pit. The HR-like ionocyte is shown in red, with the NaR-like ionocyte in cyan. (A) Volume-EM 3D reconstruction of the cell pair; see also Supplementary Movie 6. (B) Location of the cell pair (red arrowhead) in the left olfactory pit (just within the OSN zone). Coronal section; anterior to the top. (C,D) Sections at the approximate levels shown in A. Black arrowhead in D marks an electron-dense structure in the extracellular space between the cell pair (see also S). (E–E’’’) 3D reconstructions of the apical part of the cell pair, showing the microvillous apical knob of the HR-like ionocyte (red), which projects above the surrounding olfactory supporting cells. Side views (top panels) and top-down views (lower panels). The neck of the HR-like ionocyte is wrapped by a thin layer of cytoplasm of the NaR-like cell (cyan). (F,G) Tight junctions (zonulae occludentes) of the ionocyte pair. (F) Enlargement of the box in C, showing color-coded labeling of the junctions. Green, shallow tight junction between the two ionocytes; yellow, deep tight junction between NaR-like ionocyte and olfactory supporting cell; orange, deep tight junction between NaR-like ionocyte and multiciliated cell; blue, deep tight junction between HR-like ionocyte and olfactory supporting cell. Abbreviations: ci-OSN, ciliated olfactory sensory neuron; sc, olfactory supporting cell; sg, secretory granule. (G) 3D reconstruction of the tight junctions (top-down view); see also Supplementary Movie 7. (H–K) Examples of NaR-like olfactory ionocytes in a live 5 dpf embryo, labeled by EGFP (cyan) in the Notch reporter line Tg(tp1bglob:EGFP). (H) Co-expression of EGFP (Notch reporter; cyan) with HCR RNA-FISH for trpv6 (magenta), confirming the cell as an NaR-like ionocyte (see also Fig. 3B–B’’). (H’,I–K) EGFP (cyan) channel only. (I,J) Additional examples in longitudinal view. (K) The apices of EGFP+ cells (NaR-like ionocytes) appear as crescents in a top-down view. (L–S) Examples of three- and four-cell ionocyte complexes in the wild-type zebrafish olfactory epithelium at 7 dpf. (L–P) Example of a 3-cell complex, consisting of an HR-like ionocyte (red), NaR-like ionocyte (cyan), and possible second NaR-like ionocyte (dark blue). A nearby ciliated OSN (yellow) is included for context. (L) 3D reconstruction of the ionocyte complex and ciliated OSN; see also Supplementary Movie 8. (M) Location of the cells in the OSN zone of the right-hand olfactory pit (red arrowhead). Coronal section; anterior to the top. (N) Longitudinal section through the complex, showing close association between the HR-like ionocyte (red) and the ciliated OSN (yellow) at the base, and location relative to the basal lamina (pink). (O) Enlargement of the box in N, showing color-coded labeling of the tight junctions of the HR-like (red) and NaR-like (cyan) cells (color code as in F). (P) 3D reconstruction of the tight junctions of the HR-like (red) and NaR-like (cyan) cells (top-down view; compare to G; see also Supplementary Movie 9). (Q) Example section through a three-cell complex consisting of an HR-like cell (red), an NaR-like cell (cyan), and possible second HR-like cell (pink). (R,S) Example sections through a four-cell complex consisting of two HR-like/NaR-like pairs. The cell pairs are separate at their apices (R), but are closely associated beneath the epithelial surface (S). Black arrowhead in S marks electron-dense structures between the ionocytes. Scale bars: B, 20 µm; C; 1 µm (applies to D–E’’’, G); F, 0.5 µm; M, 20 µm; N, 3 µm; O, 1 µm (applies to P); Q, 2 µm; R, 2 µm (applies to S).
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