Formation of the first mesonephric nephron. A: A cross section of a 5 mm larva stained with methylene blue and basic fuchsin shows a basophilic cluster sitting on top of the PT (arrow) at the caudal end of the swim bladder (white asterisk). The red line indicates the region of the cross sections for A-B. B: A cross section of a 5.5 mm larva stained similarly to A shows a nascent basophilic tubule making contact with the PT (arrow), but its lumen has not yet fused with the PT lumen. C: Injection of 40 kDa dextran-FITC into a 5.5 mm larva indicates that the nascent tubule is not yet functional and did not accumulate the fluorescent tracer (arrow). D: The tubule of a 6 mm larva did accumulate the fluorescent tracer (arrow), indicating that the first mesonephric nephron is functional at the 6 mm stage. PT: pronephric tubule; SB: swim bladder; dashed lines demarcate the swim bladder from the pronephric tubule.

The developing and regenerating mesonephros contain proliferating basophilic clusters that express wt1b. A–D: Larvae (A,B) and adult fish with kidney damage (C,D) were injected with BrdU and serial sections were alternately stained with H+E and anti-BrdU by immunohistochemistry. This shows that the developing mesonephros (A,B) and the regenerating adult mesonephros (C,D) both have basophilic clusters with BrdU⁺ cells (arrows). E,F: Tg(wt1b:GFP) transgenic fish with kidney damage were also injected with BrdU and stained similarly to C,D. This shows that basophilic clusters in the regenerating kidney also express wt1b-GFP (arrows). PT: pronephric tubule; MT: mesonephric tubule; H+E: hematoxylin and eosin.

Invasion of the mesonephric nephron into the pronephric tubule. A: A live time-course of Tg(lhx1a:eGFP)/Tg(cdh17:mCherry) double transgenic larvae shows that the nascent nephron infiltrates the underlying PT (middle panel), followed by downregulation of lhx1a:eGFP expression. B: Downregulation of endogenous lhx1a expression was also observed in nascent nephrons. PT: pronephric tubule; t: time; h: hours.

Mesonephric expression of the pan-tubule cdh17 marker. A–G: Whole mount in situ hybridizations of larvae show that the first mesonephric nephron expresses cdh17 at the 5.2–5.5 mm stage (B white arrow and inset). Additional nephrons appear later in both caudal and rostral positions relative to the first nephron (C,D: white arrows, arrowheads, and inset). The inset in C is a magnified image of the arrowhead. At the 9 mm stage (<30 dpf), the developing mesonephros has three distinct regions (head, trunk, and tail; F,G), resembling the adult mesonephros in H. H: The mesonephros of a sexually mature adult fish (90 dpf) has the head, trunk, and tail regions. SB: swim bladder; G: glomerulus.

Mesonephric expression of early and mature nephron markers. A,B: Whole mount in situ hybridizations of larvae show that the early acting transcription factor wt1b is expressed in clusters of nephron progenitor cells (arrows and arrowheads). C: The mature glomerular marker nphs1 is expressed in mesonephric glomeruli (white arrows and arrowheads) in addition to being maintained in the PG (black arrow). D–F: Magnified images of nephron progenitor clusters expressing the early acting markers wt1b, pax2a, and lhx1a. G–H: Magnified images showing a mature glomerulus (G) and an immature presumptive glomerulus (H) expressing nphs1 (arrows). PG: pronephric glomerulus.

Expression of slc20a1a in the PCT. A–C: Whole mount in situ hybridizations of larvae show that the first mesonephric PCT expresses slc20a1a at the 6 mm stage (B white arrow), with several more PCTs appearing at the 8 mm stage (C arrows). PCT: proximal convoluted tubule.

Expression of trpm7 in the PST. A–C: Whole mount in situ hybridizations of larvae show that the first mesonephric PST expresses trmp7 at the 6 mm stage (B white arrow), with several more PSTs appearing at the 8 mm stage (C arrows). PST: proximal straight tubule.

Expression of slc12a1 in the DE. A–C: Whole mount in situ hybridizations of larvae show that the first mesonephric DE forms on top of the pronephric DE and expresses slc12a1 at the 6 mm stage (B arrowhead). At the 8 mm stage, several more DEs appear on top of the pronephric DE (C white arrows) and pronephric DL (C white arrowheads), and in the rostral region of the pronephric DE (C red arrowhead and inset). The inset is a magnified image of the red arrowhead at a different angle. D: A magnified image of a branch point between the mesonephric DE (dashed line) and the pronephric DE. E: At the 8 mm stage, slc12a1 is downregulated at the junction point with the pronephric DE. DE: distal early segment; DL: distal late segment.

Expression of slc12a3 in the DL. A,B: Whole mount in situ hybridizations of larvae for the slc12a3 marker show that the first mesonephric nephron does not form a DL the 6 mm stage (B dashed box). C–E: At the 8 mm stage, both rostral and caudal nephrons express slc12a3 and form on top of the pronephric DE (red arrows) and pronephric DL (blue arrows). D and E are magnified representations of rostral and caudal nephrons at the 8 mm stage. DE: distal early segment; DL: distal late segment.

Expression of gata3 in the pronephric CD. A–C: Whole mount in situ hybridizations of larvae show that gata3 is not expressed in mesonephric nephrons at the 6 mm or 8 mm stage, but is expressed in a terminal segment of the pronephros. CD: presumptive collecting duct or ureter segment.