Wholemount In Situ Hybridization for Astyanax Embryos

Summary

This protocol enables visualization of gene expression in embryonic Astyanax cavefish. This approach has been developed with the goal of maximizing gene expression signal, while minimizing non-specific background staining.

Abstract

In recent years, a draft genome for the blind Mexican cavefish (Astyanax mexicanus) has been released, revealing the sequence identities for thousands of genes. Prior research into this emerging model system capitalized on comprehensive genome-wide investigations that have identified numerous quantitative trait loci (QTL) associated with various cave-associated phenotypes. However, the ability to connect genes of interest to the heritable basis for phenotypic change remains a significant challenge. One technique that can facilitate deeper understanding of the role of development in troglomorphic evolution is whole-mount in situ hybridization. This technique can be implemented to directly compare gene expression between cave- and surface-dwelling forms, nominate candidate genes underlying established QTL, identify genes of interest from next-generation sequencing studies, or develop other discovery-based approaches. In this report, we present a simple protocol, supported by a flexible checklist, that can be widely adapted for use well beyond the presented study system. It is hoped that this protocol can serve as a broad resource for the Astyanax community and beyond.

Introduction

In situ hybridization is a common method for staining fixed tissues to visualize gene expression patterns¹. This technique has been performed for years in other traditional² and non-traditional³ model systems, for a variety of biological studies. However, several steps and reagents are necessary to successfully perform this procedure. For investigators who have never performed this technique, initiating the process can be intimidating owing to the many steps involved. Further, the lengthy nature of this procedure lends itself to technical errors, which can be challenging to troubleshoot.

The overall goal of this article is to present a simple and straightforward method that will render this hybridization technique accessible to a wide audience. To reduce the introduction of errors, we present a straightforward approach that yields high quality gene expression staining and minimizes non-specific background signal. This procedure is similar to other approaches developed in traditional model systems, such as Danio rerio⁴. Here, we aim to facilitate careful implementation of each step using a downloadable checklist (Supplemental File 1), to promote careful implementation of the protocol. The rationale for doing this is to facilitate organization through the many steps involved in this procedure. This article is appropriate for researchers interested in performing whole-mount in situ hybridization in developing embryos, but have not yet performed the procedure. The advantage of the chosen approach for Astyanax researchers is that it has been tested and proven in both cavefish and surface fish morphs, thereby facilitating comparative expression analyses. The presented method can be used by researchers in studies on Astyanax and other systems.

Protocol

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Cincinnati (Protocol #10-01-21-01). 1. Fixation Isolate desired number of Astyanax mexicanus embryos from a breeding tank and fix ~50 embryos at a time. If embryos are large and old, it may be necessary to fix 25 at a time to ensure even fixation. Depending on the age of the embryo, utilize the IACUC-approved method of anesthesia. For olde…

Representative Results

In this report, we provide a simple and straightforward approach to perform labeling of embryonic Astyanax specimens for high-quality gene expression analysis. This technique can be carried out in either four or five days, and each principal step in the procedure is represented in a color-coded flowchart (Figure 1). Once completed, stained embryos should harbor a dark purple chromatic label in tissues expressing the particular gene of interest. We ha…

Discussion

Owing to the vulnerability of RNA to degradation, one of the most critical steps in the protocol concerns the sterile synthesis of the RNA probe. However, if a probe is carefully generated, and provides good results, it can be reused in subsequent staining reactions. A second crucial step is the careful production of all reagents used throughout the protocol. Since this protocol involves several days and many small steps, it is essential that all reagents are accurately produced, and stored in a sterile manner. Further, …

Materials

10 mL Serological Pipette	VWR	89130-888
1000 mL Filtration Unit	VWR	89220-698
15 mL Conical	VWR-Greiner	82050-278
25 mL Serological Pipette	VWR	89130-890
250 mL Filtration Unit	VWR	89220-694
5 mL Serological Pipette	VWR	89130-886
50 mL Conical	VWR-Falcon	21008-940
500 mL Filtration Unit	VWR	89220-696
Anti-Digoxigenin-AP, Fab fragments	Sigma-Roche	11093274910
BCIP	Sigma-Aldrich	B8503-1G	1 g
Blocking Solution	Sigma-Roche	11 096 176 001	50 g
Citric Acid	Fisher Scientific	A104-500	500 g
DIG RNA Labeling Kit (SP6/T7)	Sigma-Roche	11175025910
Eppendorf Tubes	VWR	20170-577
Ethanol	Fisher-Decon	04-355-223	1 Gal
Formamide	Thermo Fisher Scientific	17899	100 mL
Glass dram vials	VWR	66011-041	1 Dr
Glass Pipettes	Fisher Scientific	13-678-8A
HCl	Thermal-ScientificPharmco-AAPER	284000ACS	500 mL
Heparin	Sigma	H3393-25KU
Magnesium Chloride-crystalline	Fisher Scientific	M33-500	500 g
Maleic Acid	Sigma	M0375-100g	100g
Methanol	Fisher Scientific	A452-4	4L
Molecular-grade Water (RNase-free)	VWR	7732-18-5	500 mL
NaCl	Fisher Scientific	S271-3	3 kg
NaOH pellets	Fisher Scientific	S318-500	500 g
NBT Substrate powder	ThermoFisher Scientific	34035	1 g
Normal Goat Serum	Fisher-Invitrogen	31873
Nutating Mixer	VWR	82007-202
Paraformaldehyde	Sigma	158127-500g	500 g
PBS 10x	Fisher Scientific	BP399-20	20L
Proteinase K (200mg/10ml)	Qiagen	19133	10 mL
Plastic Pipettes	VWR-Samco	14670-147
RNAse	Sigma	R2020-250mL	250 mL
Shaking Water Bath 12 L	VWR	10128-126	12 L
Standard Analog Shaker	VWR	89032-092
Tris	Sigma Millipore-OmniPur	9210-500GM	500 g
tRNA Yeast	Fisher-Invitrogen	15401011	25 mg
Tween 20	Sigma	P9416-50mL	50 mL
Vortex-Genie 2	Fisher Scientific-Scientific Industries, Inc	50-728-002
Lithium Chloride (LiCl)	Sigma-Aldrich	203637-10G	10 g