In Situ Hybridization in Zebrafish Larvae and Juveniles during Mesonephros Development

Summary

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.

Abstract

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.

Introduction

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 days^1,2. It has greatly contributed to the understanding of kidney development and the conservation between zebrafish and mammals^3,4,5. 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 conne....

Protocol

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.

1. Set up adult zebrafish to mate by adding 1 male and 1 female fish in a mating tank in the late afternoon after their last meal.
   ​NOTE: Not al.......

Discussion

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. .......

Materials

Name	Company	Catalog Number	Comments
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