Mating and Egg Staging: A Method to Generate Embryos and Sort Them by Developmental Stage

Overview

The article describes a protocol to generate zebrafish embryos through natural mating and sorting their developmental stages. The protocol is used further for analysis of pancreatic β-cells by using transgenic fishes.

Protocol

1. Generating embryos through natural mating

1. Culture embryos to 3 months of age, reproductive maturity.
2. Segregate adult male and female fish from the desired strain into divided mating tanks on the evening before embryo collection, and add 2 males and 3 females to each tank.
   NOTE: Use of the transgenic insulin2a:mCherry fluorescent reporter strain allowed the analysis of pancreatic β-cells.
3. Transfer fish to the mating tank with fresh system water and remove the divider immediately after the lights come on the following morning.
4. Allow the fish to mate naturally until embryos are observed in bottom tank. Collect embryos in 30 min intervals until the desired amount is collected. Store each collected time point in separate Petri dishes in embryo media at 28.5 °C.
5. Perform microinjection of genetic material or placement in experimental culture media, if desired, and culture the embryos in fresh Hank's embryo medium in 10-cm Petri dishes at 28.5 °C.
   NOTE: For gene expression analysis in morpholino (MO) injected or mutant animals, note that each manipulation may have incidental impacts on gene expression. Mutants can exhibit genetic compensation at the level of transcription that is not observed by MO-based gene targeting.

2. Stage embryos

1. Culture the embryos in groups of 50–75 embryos per 10-cm Petri dish to promote consistent developmental timing of all embryos.
2. Monitor the developmental morphology using dissecting microscope at blastomere, epiboly, and somite stages to ensure developmental progression.
   NOTE: Remove any dying or malformed embryos to prevent developmental delay in the dish.
3. Separate the embryos based on the developmental age. Stage the embryos and larvae using total body length after 24 hpf.
   NOTE: The somites are the chevron-shaped mesodermal tissue present on the dorsal part of the embryo.

Materials

Name	Company	Catalog Number	Comments
ins2a:mCherry	ZIRC	ZL1483
Mating tanks 1.0L Crossing Tank Set	Aquaneering	ZHCT100
Dissecting Microscope	Zeiss