The zebrafish is an important model for understanding kidney development. Here, an in situ hybridization protocol is optimized to detect gene expression in zebrafish larvae and juveniles during mesonephros development.
The zebrafish forms two kidney structures in its lifetime. The pronephros (embryonic kidney) forms during embryonic development and begins to function at 2 days post fertilization. Consisting of only two nephrons, the pronephros serves as the sole kidney during larval life until more renal function is required due to the increasing body mass. To cope with this higher demand, the mesonephros (adult kidney) begins to form during metamorphosis. The new primary nephrons fuse to the pronephros and form connected lumens. Then, secondary nephrons fuse to primary ones (and so on) to create a branching network in the mesonephros. The vast majority of research is focused on the pronephros due to the ease of using embryos. Thus, there is a need to develop techniques to study older and larger larvae and juvenile fish to better understand mesonephros development. Here, an in situ hybridization protocol for gene expression analysis is optimized for probe penetration, washing of probes and antibodies, and bleaching of pigments to better visualize the mesonephros. The Tg(lhx1a-EGFP) transgenic line is used to label progenitor cells and the distal tubules of nascent nephrons. This protocol fills a gap in mesonephros research. It is a crucial model for understanding how new kidney tissues form and integrate with existing nephrons and provide insights into regenerative therapies.
The zebrafish embryo is an important model for studying tissue development due to its small size, transparency, available tools, and survival without feeding for up to five days1,2. It has greatly contributed to the understanding of kidney development and the conservation between zebrafish and mammals3,4,5. The kidney plays an essential role in maintaining fluid homeostasis, filtering the blood, and excreting waste6. The nephron, the functional unit of the kidney, comprises a blood filter connected to a long tubule. In zebrafish, two kidney structures form throughout its life. The pronephros (the temporary embryonic kidney) forms first during early development. It consists of two nephrons running along the anterior-posterior axis and becomes functional at around 2 days post fertilization (dpf). The utility of the pronephros lies in its simplicity, having just two nephrons that are mostly linear and easy to visualize (although the proximal convoluted tubule begins to coil at three dpf)3. This has facilitated not only studies of its early development from the intermediate mesoderm, but also the segmentation pattern and tubule repair7,8.
The usefulness of zebrafish becomes limited after five dpf, when the yolk is diminished and the larvae rely on feeding in the aquatic system9. At around 2 weeks old, the larvae undergo metamorphosis into juveniles, where new tissues form and old tissues are lost and/or reorganized9. This is also when the mesonephros (the permanent adult kidney) forms10,11,12. The first adult nephron forms near the sixth somite, and fuses with the distal early tubule of the pronephros. Additional nephrons are added posterior to this position initially, but also toward the anterior later on. The primary nephrons in this first wave fuse to the tubules of the pronephros and share a common lumen to deposit their waste. Secondary nephrons form in the next wave and fuse to primary nephrons. This reiterative process creates a mesonephros that is branched, somewhat akin to the mammalian kidney. Presumably, the pronephros eventually loses its tubule identity and becomes two major collecting ducts where all nephrons drain their waste13.
Prior to the formation of the first adult nephron, single progenitor cells begin to appear in ~4 mm (total length) larvae (~10 dpf). These cells, which are marked in the Tg(lhx1a-EGFP) transgenic line, adhere to the pronephros and seem to migrate to future sites of nephrogenesis. The single cells coalesce into clusters, which differentiate into new nephrons12. It is unclear where these cells reside or what genes they express before the onset of mesonephrogenesis.
Understanding mesonephros development provides insights into the mammalian kidney in ways that the pronephros cannot. These include the formation of nephrogenic aggregates from single progenitor cells, functional integration of new nephrons with existing ones, and branching morphogenesis. However, there are limitations to studying postembryonic development. The larvae are less transparent due to their larger size and having pigmentation. The developmental timeline is not synchronous among individual animals and is highly dependent on environmental factors, such as feeding and crowding9,14. Although knockdown reagents exist, they are less effective in larvae compared to embryos15. In this protocol, an in situ hybridization method to determine gene expression during zebrafish mesonephros development is described. Several steps are optimized to increase visualization of the mesonephros and penetration and washing of the probe and antibody. The Tg(lhx1a-EGFP) transgenic line is used along with a probe for EGFP to label single progenitor cells, nephrogenic aggregates, and the distal tubules of nascent nephrons. This method will provide a deeper understanding of mesonephros development and insight into regenerative therapies.
The use of zebrafish larvae and juveniles is approved by the IUP IACUC (protocol #02-1920, #08-1920). Details of the solution content are listed in the Table of Materials.
1. Raising larvae
NOTE: It will take up to 21 days or more to raise larvae and juveniles to the stage of interest.
2. Day 1 – 2: Fixing larvae
3. Day 3: Measuring larvae
4. Day 3 – 4: Dehydration
5. Day 5: Rehydration
6. Day 5: Proteinase K digest
7. Day 5: Bleaching
8. Day 5: Prehybridization
NOTE: For steps done at 70 °C, it is important to work quickly to minimize the vial cooling down.
9. Day 6: Probe hybridization
10. Day 7: Probe washing
11. Day 7: Blocking
12. Day 8 – 9: Antibody incubation
13. Day 10 – 11: Antibody washing
14. Day 12: Staining
15. Day 12: Imaging
Using the Tg(lhx1a-EGFP) transgenic line, it was demonstrated that this in situ hybridization protocol is effective in labeling kidney progenitor cells and various nephron structures during mesonephros development. As expected, the central nervous system is also labeled in this transgenic line (not shown). The initial mesonephric nephron forms at approximately 5.2 mm, dorsal to the pronephros (Figure 2A), and the distal tubule of this nephron is labeled by EGFP10,12. Progenitor clusters are present at this stage and later on (Figure 2A–B, arrowheads), and single progenitor cells are also labeled (Figure 2C). This method provides an additional tool in studying kidney development and helps shed light on understanding the human kidney.
Figure 1: Measuring larvae. Fixed larvae in a Petri plate are placed on top of a flat ruler. Under a dissecting microscope, the larvae are measured and separated by groups of similar sizes. Please click here to view a larger version of this figure.
Figure 2: Mesonephros development. At around 5.2 mm in the Tg(lhx1a-EGFP) transgenic line, the first mesonephric nephron is formed dorsal to pronephros and swim bladder (SB) (A). Clusters of progenitor cells are present during mesonephros development (A–B, arrowheads) in addition to single progenitor cells (C, bracket). In larger juveniles, background staining can occur in the somites (C, arrows). Please click here to view a larger version of this figure.
The in situ hybridization method described here is aimed toward studying mesonephros development. However, it can be applied to study the development of other tissues and organs during metamorphosis, such as the gut, nervous system, scales, and pigmentation14. Probes can be generated for endogenous genes or fluorescent markers in transgenic lines.
It is critical for the larvae to remain intact in order to observe the organs and tissues in their native context. To retain tissue integrity, it is important to minimize the time of proteinase K treatment. It is important to determine the best treatment time for each new batch of enzyme. Alternatively, acetone can be used instead of proteinase K for improved tissue integrity16. Excessive bleaching also reduces tissue integrity. Minimal bleaching and allowing the eyes to retain some pigmentation help in visualizing the larvae during washes. It is common for the eyes to fall off during the hybridization step, which is an indicator of tissue fragility. The use of DMSO with the fixing and proteinase K solution is crucial for tissue penetration17.
A limitation of this method is the poor penetration of reagents in larger animals. To improve penetration, the head and tail can be removed with a razor blade before fixation17. The gut can be removed with fine tweezers and tungsten needles after fixation to allow direct access of the reagents to the mesonephros. Long probes (greater than 1 KB) can have poor penetration of the tissue, but they can be hydrolyzed into short fragments (around 0.3 KB) to improve penetration18. For probes with weak signals, control probes with the sense sequence can be used to differentiate between the background and the probe signal. Larger animals will have a higher background staining of the somites (Figure 2C, arrows). However, this can be minimized with longer washes of the probe and antibodies.
The protocol here can also be applied to dissected and isolated tissues and organs, such as the adult zebrafish kidney12,19. Although there are published descriptions of similar protocols9,16, none of them describe the entire process from rearing larvae to in situ hybridization with this level of detail. Therefore, this method provides an additional tool in deciphering the development of the vertebrate kidney.
The authors have nothing to disclose.
Funding was provided by the Pennsylvania Academy of Science, and the Commonwealth of Pennsylvania Biologists, and the Indiana University of Pennsylvania (School of Graduate Studies and Research, Department of Biology, and the Cynthia Sushak Undergraduate Biology Fund for Excellence). The Tg(lhx1a-EGFP) transgenic line was provided by Dr. Neil Hukriede (University of Pittsburgh).
Anti-fluorescein antibody | Roche/Sigma-Aldrich | 11426338910 | |
Bleaching solution | 0.8% KOH, 0.9% H2O2 in PBST | ||
Blocking reagent | Roche/Sigma-Aldrich | 11096176001 | Use for blocking solution, prepare according to manufacture's instruction |
Cell strainer | Fisher Scientific | 22-363-549 | 100 μm |
E3 medium | 5 mM NaCl, 0.33 mM CaCl2, 0.33 mM MgSO4, 0.17 mM KCl, 0.0001% methylene blue | ||
Eyelash manipulator | Fisher Scientific | NC1083208 | Use to manipulate larvae |
Fixing solution | 4% paraformaldehyde, 1% DMSO in PBS; heat at 65°C while shaking until the powder dissolves, then add DMSO after it cools down | ||
Fluorescein probe synthesis | Roche/Sigma-Aldrich | 11685619910 | |
Glass vial | Fisher Scientific | 03-338B | |
Hatchfry encapsulation | Argent | ||
Hyb- solution | 50% formamide, 5X SSC, 0.1% Tween-20 | ||
Hyb+ solution | HYB-, 5 mg/mL torula RNA, 50 ug/mL heparin | ||
MAB (10X) | 1 M maleic acid, 1.5 M NaCl, pH 7.5 | ||
MABT | 1X MAB, 0.1% Tween-20 | ||
Maleic acid | Sigma-Aldrich | M0375 | |
Paraformaldehyde | Sigma Aldrich | 158127 | |
PBS (10X) | 8% NaCl, 0.2% KCl, 1.44% Na2HPO4, 0.24% KH2PO4 | ||
PBST | 1X PBS, 0.1% Tween-20 | ||
PBST2 | 1X PBS, 0.2% Tween-20 | ||
Powder food | Mix 2 g of each of spirulina and hatchfry encapsulon in 50 mL of fish system water and shake well | ||
Proteinase K | Sigma-Aldrich | P5568 | Use to permeabilize larvae |
Proteinase K solution | 20 μg/mL, 1% DMSO final concentration in PBST | ||
Spirulina microfine powder | Argent | ||
SSC (20X) | 3 M NaCl, 0.3 M sodium acetate anhydrous, pH 7, autoclave | ||
SSCT (0.2X) | Dilute from 20X SSC, 0.1% Tween-20 | ||
SSCT (2X) | Dilute from 20X SSC, 0.1% Tween-20 | ||
Staining buffer | 100 mM Tris pH 9.5, 50 mM MgCl2, 100 mM NaCl, 0.1% Tween-20 | ||
Staining solution | 200 μg/mL iodonitrotetrazolium chloride, 200 μg/mL 5-Bromo-4-chloro-3-indolyl phosphate disodium salt, in staining buffer | ||
Stopping solution | 1 mM EDTA, pH 5.5, in PBST | ||
Torula (yeast) RNA | Sigma-Aldrich | R6625 | |
Tricaine | Sigma Aldrich | E10521 | 2%, pH 7 |
Wash buffer | 50% formamide, 2X SSC, 0.1% Tween-20 |