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.
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.
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.
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, …
The authors have nothing to disclose.
The authors wish to thank members of the Gross lab for helpful comments on this manuscript. We wish to acknowledge four high school students who utilized this protocol during summer internships in 2017 and 2018, including Christine Cao, Michael Warden, Aki Li, and David Nwankwo. HL was supported by a UC Biology STEM Fellowship during the summer of 2017. This work was supported by grants from the National Science Foundation (DEB-1457630 to JBG), and the National Institutes of Dental and Craniofacial Research (NIH; DE025033 to JBG).
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 |