The supraorbital superficial line of neuromast is formed by a novel hybrid‐origin mechanism. (A) Low magnification (10x objective) confocal image of nerves (Hgn39dGFP; yellow) and mature neuromasts (TMRE; magenta) at 5 mm SL with the area of interest behind the eye indicated (red box). (B–B′) Comparison of (B) Tg(CldnB:GFP) (yellow) and (B′) Sox2 (magenta) expression at the connection between neuromast O2 and the nAVsos nerve (red arrowheads) in a 6 mm SL specimen. Note that the nerve is also Sox2‐positive. CldnB:GFP‐positive cells extending from existing neuromasts are interneuromast cells with Sox2‐positive nuclei (white arrowheads). Scale = 20 µm. (C–F) Live time series images of a single Tg(CldnB:GFP/Hgn39d:GFP) (yellow) specimen labeled with TMRE (magenta). In all images, neuromasts HM1, IO4, and O2 are labeled. The intersection between the nerve originating from HM1 (white arrowhead) and strands of interneuromast cell‐derived primordial tissue originating from IO4 and O2 (white brackets) is noted with a white box. Scale = 50 µm. (C) Initial intersection of the nerve and primordia at 5 mm SL. The nAVsos nerve extending from HM1 (white arrowhead) has no lateral line tissue along it and intersects (white box) with the CldnB:GFP‐labeled primordium extensions (yellow) from O2 and IO4 (white brackets). (D) Adherence of primordium tissue to the nAVsos nerve at 6 mm SL. The nAVsos nerve (white arrowhead) now has lateral line tissue running parallel to it on the portions of the nerve that extend past the intersection point (white box) with the O2 and IO4 primordia (white brackets). (E) Proliferation of primordium tissue in the SO superficial line at 7 mm SL. The primordium extending from O2 (white bracket) still intersects (white box) with the SO superficial line, while the primordium from IO4 is beginning to condense into canal neuromast IO4. The first neuromast of the SO Superficial line (SO Sup 1) has begun to condense beyond the intersection point. (F) Differentiation of a supraorbital superficial neuromast by 8 mm SL. Both SO Sup 1 and canal neuromast IO4* have differentiated into mature neuromasts with functional hair cells (magenta), and neuromast O2 remains permanently connected to the SO superficial line (white bracket; white box). (F′) Same view as (F) showing the same specimen immunolabeled for neuromast marker Sox2 (magenta) and nerve marker acetylated alpha‐tubulin (cyan). The nAVsos nerve is labeled. (G–H) Representative images of SO supraorbital development in specimens labeled for Sox2 (magenta) and acetylated alpha‐tubulin (cyan) at (G) 5 mm, (H) 6 mm, (I) 7 mm, and (J) 8 mm SL. Annotations as in Panels C–F. Scale = 50 µm. (K) Schematic of the hybrid‐origin mechanism of SO superficial line formation at the imaged stages 5–8 mm SL with placodal origin indicated.
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