A 3-D Tail Explant Culture to Study Vertebrate Segmentation in Zebrafish

Summary

Here, we present the protocol for 3-D tissue culture of the zebrafish posterior body axis, enabling live study of vertebrate segmentation. This explant model provides control over axis elongation, alteration of morphogen sources, and subcellular resolution tissue-level live imaging.

Abstract

Vertebrate embryos pattern their major body axis as repetitive somites, the precursors of vertebrae, muscle, and skin. Somites progressively segment from the presomitic mesoderm (PSM) as the tail end of the embryo elongates posteriorly. Somites form with regular periodicity and scale in size. Zebrafish is a popular model organism as it is genetically tractable and has transparent embryos that allow for live imaging. Nevertheless, during somitogenesis, fish embryos are wrapped around a large, rounding yolk. This geometry limits live imaging of PSM tissue in zebrafish embryos, particularly at higher resolutions that require a close objective working distance. Here, we present a flattened 3-D tissue culture method for live imaging of zebrafish tail explants. Tail explants mimic intact embryos by displaying a proportional slowdown of axis elongation and shortening of rostrocaudal somite lengths. We are further able to stall axis elongation speed through explant culture. This, for the first time, enables us to untangle the chemical input of signaling gradients from the mechanistic input of axial elongation. In future studies, this method can be combined with a microfluidic setup to allow time-controlled pharmaceutical perturbations or screening of vertebrate segmentation without any drug penetration concerns.

Introduction

Metameric segmentation of organisms is widely used in nature. Repeated structures are essential for functionality of lateral organs such as vertebrae, muscles, nerves, vessels, limbs, or leaves in a body plan¹. As a result of such physiological and geometric constraints of the axial symmetry, most phyla of Bilateria-such as annelids, arthropods, and chordates-exhibit segmentation of their embryonic tissues (e.g., ectoderm, mesoderm) antero-posteriorly.

Vertebrate embryos sequentially segment their paraxial mesoderm along the major body axis into somites with species-specific intervals, counts, and size distributions.....

Protocol

This protocol involves use of live vertebrate embryos younger than 1 day post-fertilization. All the animal experiments were performed under the ethical guidelines of Cincinnati Children's Hospital Medical Center; animal protocols were reviewed and approved by the Institutional Animal Care and Use Committee (Protocol # 2017-0048).

1. Embryo collection

1. Set up pairs of zebrafish in crossing tanks the night before the embryo collection day. For precise staging control of embryo development, use barriers between mating pairs.
2. Raise barriers before preferred spawning time and collect eggs within 15 minutes in a 100 mm P....

Results

This protocol enables flat geometric culturing of live zebrafish tail explants. Tissue culture presents three major advantages over whole embryos: 1) control of axis elongation speed, 2) control over various signaling (morphogen) sources by simple dissection, and 3) near-objective, high magnification and high NA live imaging.

Chemically untreated slide chambers allow the tail explant to elongate its major axis (Figure 2A) by the skin ectoderm wrapping around the t.......

Discussion

This article presents a detailed protocol of a tissue culture explant technique we developed and used recently⁵ for zebrafish embryos. Our technique builds on the previous explant methods in chick⁸ and zebrafish^9,10,11 model organisms. Tail explants prepared with this protocol can survive as long as >12 h in a simple slide chamber, continuing to elongate its major body axis and .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1 mL Sub-Q Syringe with PrecisionGlide Needle	Becton, Dickinson and Co.	REF 309597	for dechorionating embryos and manipulations
200 Proof Ethanol, Anhydrous	Decon Labs	2701	for immunostaining
Antibiotic Antimycotic Solution (100×)	Sigma-Aldrich	A5955	for tissue dissection media
Calcium Chloride Anhydrous, Powder	Sigma-Aldrich	499609	for tissue dissection media
Dimethylsulfoxide	Sigma-Aldrich	D5879	for immunostaining
Disposable Scalpel, #10 Stainless Steel	Integra-Miltex	MIL4-411	for preparing tape slide wells
Ethyl 3-aminobenzoate methanesulfonate salt (Tricaine)	Sigma-Aldrich	886-86-2	(optional) for anesthesizing tissues older than 20 somites stage
Fetal Bovine Serum (FBS)	ThermoFisher	A3160601	additional for tissue culture media
Goat anti-Mouse IgG2b, Alexa Fluor 594	Invitrogen	Cat#A-21145; RRID: AB_2535781	secondary antibody for immunostaining
L-15 Medium with L-Glutamine w/o Phenol Red	GIBCO	21083-027	for tissue dissection media
Methanol	Sigma-Aldrich	179337	for immunostaining
Microsurgical Corneal Knife 2.85 mm Angled Tip Double Bevel Blade	Surgical Specialties	72-2863	for tissue dissection
Mouse monoclonal anti-ppERK	Sigma-Aldrich	Cat#M8159; RRID:AB_477245	for ppERK immunostaining
NucRed Live 647 ReadyProbes Reagent	Invitrogen	R37106	(optional) for live staining of cell nuclei
Paraformaldehyde Powder, 95%	Sigma-Aldrich	158127	for fixation of samples for immunostaining
Rat Tail Collagen Coating Solution	Sigma-Aldrich	122-20	(optional) for chemically activating slide chambers
Stage Top Incubator	Tokai Hit	tokai-hit-stxg	(optional) for temperature control during live imaging
Transparent Tape 3/4''	Scotch	S-9782	for preparing tape slide wells
Triton X-100	Sigma-Aldrich	X100	for immunostaining
Tween 20	Sigma-Aldrich	P1379	for immunostaining
Zebrafish: Tg(actb2:2xMCP-NLS-EGFP)	Campbell et al., 2015	ZFIN: ZDB-TGCONSTRCT-150624-4	transgenic fish with nuclear localized EGFP
Zebrafish: Tg(Ola.Actb:Hsa.HRAS-EGFP)	Cooper et al., 2005	ZFIN: ZDB-TGCONSTRCT-070117-75	transgenic fish with cell membrane localized EGFP