Characterization of mesonephric development and regeneration using transgenic zebrafish

Abstract

The zebrafish is a valuable vertebrate model for kidney research. The majority of previous studies focused on the zebrafish pronephros, which comprises only two nephrons and is structurally simpler than the mesonephros of adult fish and the metanephros of mammals. To evaluate the zebrafish system for more complex studies of kidney development and regeneration, we investigated the development and postinjury regeneration of the mesonephros in adult zebrafish. Utilizing two transgenic zebrafish lines (wt1b::GFP and pod::NTR-mCherry), we characterized the developmental stages of individual mesonephric nephrons and the temporal-spatial pattern of mesonephrogenesis. We found that mesonephrogenesis continues throughout the life of zebrafish, with a rapid growth phase during the juvenile period and a slower phase in adulthood such that the total nephron number of juvenile and adult fish linearly correlates with body mass. Following gentamicin-induced renal injury, the zebrafish mesonephros can undergo de novo regeneration of mesonephric nephrons, a process known as neonephrogenesis. We found that wt1b expression was induced in individually dispersed cells in the mesonephric interstitium as early as 48 h following injury. These wt1b-expressing cells formed aggregates by 72-96 h following injury which proceeded to form nephrons. This suggests that wt1b may serve as an early marker of fated renal progenitor cells. The synchronous nature of regenerative neonephrogenesis suggests that this process may be useful for studies of nephron development.

Fig. 1.

Mesonephrogenesis in cdh17::GFP/pod::mCherry double-transgenic zebrafish. A: schematic graph illustrating the transgene structures of pod::mCherry and cdh17::GFP. Tol2-L and Tol2-R are Tol2 transposon elements to facillite the transgenesis. B: lateral view of a 9 days postfertilization (dpf) larva. Podocytes in pronephric glomeruli (arrow) are marked with mCherry fluorescence (red) and pronephric tubules and ducts are labeled with GFP fluorescence (green). cdh17::GFP expression is also present in the intestinal epithelia, where the endogenous cdh17 is expressed (12). C: lateral view of a 14 dpf larva that developed the first pair of mesonephric glomeruli (arrowheads) with matured podocytes-expressing mCherry (red). D: ventral view of a 28 dpf juvenile. Multiple glomeruli (red) are visible at the anterior and medial portions of the mesonephric kidney with convoluted mesonephric tubules. E: ventral view of a 36 dpf juvenile. More mesonephric glomeruli and more convolutions of tubules developed in the anterior and medial regions. A few nephrons are present in between the rostral and medial regions as well as in the posterior region. F: ventral view of the whole mesonephric kidney in a 6-mo-old adult. ANDR, anterior nephron-dense region; MNSR, medial nephron-sparse region; MNDR, medial nephron-dense region; PNSR, posterior nephron-sparse region. Note symmetric groups of nephrons (arrowheads) in PNSR are segmentally distributed. Anterior to the left in all the panels.

Fig. 2.

Staging the development of a mesonephric nephron in wt1b::GFP/pod::mCherry double-transgenic zebrafish. A: dispersed wt1b::GFP-expressing cells in the nephrogenic region before nephron starts to develop. B: early aggregate (stage I): aggregate of wt1b::GFP⁺ cells with no defined lumen. C: tubular body (stage II): cluster of wt1b::GFP⁺ cells with a defined lumen (*) but no glomerulus. D, E: protoglomerular nephron (stage III): wt1b::GFP⁺ tubule with a defined glomerulus (arrow) but no pod::mCherry expression within the glomerulus. F: immature nephron (stage IV): mesonephric glomerulus (arrow) forms at the distal end of the tubule and wt1b::GFP and pod::mCherry (red) are both expressed in the podocytes within the glomerulus. G: mature nephron (stage V): wt1b::GFP expression is restricted to a cluster of cells at the urinary pole of the nephron (arrowhead) and pod::mCherry (red) is expressed in all the podocytes.

Fig. 3.

Temporal and spatial patterns of the initial mesonephrogenesis. A: lateral view of a 12-dpf larva of wt1b::GFP/pod::mCherry double-transgenic fish. wt1b::GFP expression (arrowhead) is visible at the location where the first mesonephric nephron is developing. B: lateral view of a 14-dpf larva at higher magnification showing the first wt1b::GFP-expressing aggregates in the mesonephric kidney located ventral to the 6th myotome. C: lateral view of two 20-dpf larvae showing that the locations of the first 2 mesonephric nephrons are consistent. D: histogram of the segmental distribution of the initial mesonephric nephrons indicates that the positioning of the initial mesonephric nephrons is conserved.

Fig. 4.

Mesonephrogenesis continues throughout the life of zebrafish. A: representative image of mesonephric tissue from an adult fish (age <6 mo) showing that nephrons at different developmental stages are present in adult fish. Although the majority of the nephrons are fully matured at stage V (arrowheads), 2 developing nephrons (arrows) are at stage III and stage IV with significant expression of wt1b::GFP. B: ratio of medial nephron number to total nephron number is 48 ± 5%. C: medial nephron number is logarithmically correlated to age, indicating a continuous increase of nephron number in the life of zebrafish. D: medial nephron number is positively correlated to the body mass of zebrafish. Red crosses in (C and D) represent the outliers not included in the regression calculations as described in materials and methods.

Fig. 5.

Gentamicin-induced renal injury triggers de novo regeneration of mesonephric nephrons. A, A': injection of PBS does not increase mesonephrogenesis at 5 days postinjection (dpi). The green puncta are wt1b::GFP-expressing cells at the neck of matured nephrons. B, B': by 5 dpi of gentamicin, there is an increased number of developing nephrons. C, C': at 9 dpi of gentamicin, wt1b::GFP expression indicates the progression of these newly made nephrons. D, D': level of nephrogenesis is still elevated at 14 dpi of gentamicin. E: quantification of wt1b-GFP⁺ nephron numbers postgentamicin-induced renal injury. *P < 0.05, **P < 0.01.

Fig. 6.

Renal injury induces wt1b::GFP expression in dispersed cells in the mesonephric kidney. A, A': 2 dpi of PBS does not induce wt1b:GFP expression. Most wt1b::GFP⁺ cells are in cluster at the urinary neck of intact mature nephrons in PBS-injected fish. B, B': at 2 dpi of gentamicin, wt1b:GFP expression (green) is induced in dispersed cells in the mesonephric kidney. A subset of these cells (arrowheads) are proliferative as shown with PCNA-staining (red). Inset: higher-magnification image showing a pair of wt1b:GFP-expressing cells are PCNA⁺. C, C': at 4 dpi of gentamicin, wt1b::GFP⁺ cells have formed aggregates that are PCNA⁺ (red).