The Power of Simplicity: Sea Urchin Embryos as in Vivo Developmental Models for Studying Complex Cell-to-cell Signaling Network Interactions

Podsumowanie

This video article details a straightforward in vivo methodology that can be used to systematically and efficiently characterize components of complex signaling pathways and regulatory networks in many invertebrate embryos.

Streszczenie

Remarkably few cell-to-cell signal transduction pathways are necessary during embryonic development to generate the large variety of cell types and tissues in the adult body form. Yet, each year more components of individual signaling pathways are discovered, and studies indicate that depending on the context there is significant cross-talk among most of these pathways. This complexity makes studying cell-to-cell signaling in any in vivo developmental model system a difficult task. In addition, efficient functional analyses are required to characterize molecules associated with signaling pathways identified from the large data sets generated by next generation differential screens. Here, we illustrate a straightforward method to efficiently identify components of signal transduction pathways governing cell fate and axis specification in sea urchin embryos. The genomic and morphological simplicity of embryos similar to those of the sea urchin make them powerful in vivo developmental models for understanding complex signaling interactions. The methodology described here can be used as a template for identifying novel signal transduction molecules in individual pathways as well as the interactions among the molecules in the various pathways in many other organisms.

Wprowadzenie

Gene regulatory networks (GRNs) and signal transduction pathways establish the spatial and temporal expression of genes during embryonic development that are used to build the adult animal body plan. Cell-to-cell signal transduction pathways are essential components of these regulatory networks, providing the means by which cells communicate. These cellular interactions establish and refine the expression of regulatory and differentiation genes in and among the various territories during embryogenesis^1,2. Interactions among secreted extracellular modulators (ligands, antagonists), receptors, and co-receptors control the activities of signal transduction pathways. An assortment of intracellular molecules transduces these inputs resulting in altered gene expression, division, and/or shape of a cell. While many of the key molecules used at the extracellular and intracellular levels in the major pathways are known, it is an incomplete knowledge due in large part to the complexity of individual signaling pathways. In addition, different signaling pathways often interact with one another either positively or negatively at the extracellular, intracellular, and transcriptional levels^3,4,5,6. Importantly, the core components of signal transduction pathways are highly conserved in all metazoan species, and, remarkably, most of the major signaling pathways often perform similar developmental functions in many species when comparing organisms from closely related phyla in particular ^7,8,9,10,11.

The study of signaling during development is a daunting task in any organism, and there are several significant challenges to studying signaling pathways in most deuterostome models (vertebrates, invertebrate chordates, hemichordates, and echinoderms): 1) In vertebrates there are large numbers of possible ligand and receptor/co-modulator interactions, intracellular transduction molecules, as well as potential interactions among different signaling pathways due to the complexity of the genome^12,13,14; 2) The complex morphology and morphogenetic movements in vertebrates often make it more difficult to interpret functional interactions in and among signal transduction pathways; 3) Analyses in most non-echinoderm invertebrate deuterostome model species are limited by short windows of gravidity with the exception of some tunicate species^15,16.

The sea urchin embryo has few of the above-mentioned limitations and offers many unique qualities for performing a detailed analysis of signal transduction pathways in vivo. These include the following: 1) The relative simplicity of the sea urchin genome significantly reduces the number of possible ligand, receptor/co-receptor and intracellular transduction molecule interactions¹⁷; 2) The GRNs controlling the specification and patterning of the germ layers and major embryonic axes are well established in sea urchin embryos, aiding in the understanding of the regulatory context of the cell/territory receiving the signals^18,19; 3) Many signal transduction pathways can be studied between early cleavage and gastrula stages when embryos consist of a single layered epithelium whose morphology is easier to analyze; 4) The molecules involved in signaling pathways in the sea urchins are easily manipulated; 5) Many sea urchins are gravid for 10 to 11 months a year (e.g. Strongylocentrotus purpuratus and Lytechinus variegatus).

Here, we present a method to systematically and efficiently characterize components of the signaling pathways that specify and pattern territories in sea urchin embryos to illustrate the advantages that several invertebrate model systems offer in the study of complex molecular mechanisms.

Protokół

1. High Throughput Morpholino Design Strategy

1. Identify a gene(s) of interest (e.g. candidate gene approach, cis-regulatory analysis, RNAseq and/or proteomic differential screens).
2. Use genomic, transcriptomic, and gene expression data available on frequently updated websites (e.g. SpBase http://www.echinobase.org^​20 and S. purpuratus Genome Search http:///urchin.nidcr.nih.gov/blast/index.html) to determine that the spatiotemporal expression profile overlaps with the developmental mechanism in question. If no expression data is available, then generate qPCR primers and/or an antisense in situ probe to assess the gene expression pattern.
3. After determining the spatial and temporal expression pattern, obtain the DNA sequence from the genomic web sites.
   1. For generating translation-blocking morpholino oligonucleotides, obtain sequence of the 5' un-translated region (5' UTR) directly upstream from the initiation codon from expressed sequence tag (EST) databases available on SpBase or S. purpuratus Genome Search websites. If this information is unavailable for the gene of interest, then perform 5' RACE.
   2. To design a splice-blocking morpholino, search for the genomic sequence including the exons and introns using the scaffold blastn tool in SpBase or S. purpuratus Genome Search tool.
4. Use the oligonucleotide designing website http://oligodesign.gene-tools.com in order to design the desired 25 base pair morpholino sequence.
   1. For a translational-blocking morpholino, use the mRNA sequence from 5' to 3' as the source of translational target sequence. Include the 5' UTR sequence (approximately 70 nucleotides upstream of the transcription start site) plus 25 bases of coding region (downstream of the start site) with the start codon marked with parenthesis (ATG).
   2. For a splice-blocking morpholino, select intron-exon or exon-intron boundary sequence and include 50 bases (25 bases of exon sequence and 25 bases of intron sequence) around the boundary regions. Scan the genome with the morpholino sequence to verify that the sequence is unique.

2. Microinjection of Morpholino Oligonucleotides

1. Prepare 3 mM stock solutions of the morpholino oligonucleotides by adding 100 µL of nuclease-free water into the 300 nmol morpholino vial.
   NOTE: Do not use DEPC treated water for the re-suspension because diethyl pyrocarbonate can damage the morpholino.
2. For the first reconstitution spin down the vial containing the stock oligonucleotide solution for 30 s at full speed (14,000 - 16,000 x g), briefly vortex, heat at 65 °C for 5 to 10 min, briefly vortex, and keep the morpholino stock at room temperature for at least 1 h. Morpholino stock solutions are stored from -20 °C to +4 °C.
3. Prepare injection solution containing morpholino oligonucleotides at the desired concentration. This solution usually contains 20% glycerol or 125 mM KCl as a carrier and 15% FITC-Dextran (fluorescein isothiocyanate dextran 10,000 MW 2.5 mg/μL stock solution). FITC-Dextran and other fluorescent dextran conjugates are routinely used to identify injected embryos by epifluorescence microscopy. Store injection solutions at -20 °C.
4. Heat the morpholino solution at 65 °C in a heat block or water bath for at least 2 - 5 min.
5. Briefly spin the morpholino solution for 30 s at full speed (14,000 - 16,000 x g), vortex for 1 min and centrifuge at full speed (14,000 - 16,000 x g) for at least 10 min.
6. Load the injection needles with the morpholino solution. For a detailed microinjection protocol please see Stepicheva and Song, 2014²¹ and Cheers and Ettensohn, 2004²².

3. Fixation and In Situ Protocol at 24 h Post-fertilization (hpf) in S. purpuratus Embryos

NOTE: This protocol is modified from Arenas-Mena et al., 2000²³ and Sethi et al., 2014²⁴.

1. Fixation
   1. Add several drops of fixative (see below) to wells containing embryos, mix gently by pipetting, and allow them to settle. Remove the fixative solution, and then mix embryos with two additional 180 µL washes of fixative.
      NOTE: It is important to thoroughly mix the embryos during the washes by gentle pipetting up and down several times. Failure to do so can lower the signal to noise ratio.
   2. Leave the embryos to fix in the second fixative wash for 50 min to 1 h at room temperature (RT) in 4% electron microscopy grade paraformaldehyde consisting of 10 mM MOPS pH 7.0, 0.1% Tween-20, and artificial seawater (ASW). Make this solution fresh each time for best results. In addition, for ease and practicality embryos are fixed in 96-well plates.
      1. For 20 mL fixative use 5 mL 16% paraformaldehyde, 15 mL ASW, 200 µL 1 M MOPS pH 7.0, and 20 µL Tween-20.
   3. Wash 5 times at RT with 180 µL of MOPS wash buffer consisting of 0.1 M MOPS pH 7.0, 0.5 M NaCl and 0.1% Tween-20 for at least 5 min or until embryos drop to the bottom of the well. Again, It is important to thoroughly mix the embryos into the wash buffer by gently pipetting up and down several times. MOPS wash buffer can be used for 2 days if stored at 4 °C.
      1. For 40 mL MOPS wash buffer use 4 mL 1 M MOPS pH 7.0, 4 mL 5 M NaCl, 32 mL dH₂O, and 40 µL Tween-20.
      2. Fixed embryos can be stored at 4 ^oC for 2 days.
         NOTE: If storing fixed embryos longer, then add 0.2% sodium azide in MOPS wash buffer to prevent bacterial growth.
2. Pre-hybridization
   1. Aspirate the MOPS wash buffer and add 180 μL of a 2:1 ratio of MOPS wash buffer to hybridization buffer, mix embryos into the solution by gentle pipetting several times, and incubate for at least 20 min at RT. Hybridization buffer consists of 70% formamide, 0.1 M MOPS pH 7.0, 0.5 M NaCl, 1 mg/mL BSA and 0.1% Tween-20.
      1. For 40 mL hybridization buffer use 4 mL 1 M MOPS pH 7.0, 4 mL 5 M NaCl, 4 mL dH₂O, 0.04 g BSA, and 40 µL Tween-20. Mix well by vortexing, add 28 mL formamide, and vortex again.
   2. Remove the 2:1 ratio of MOPS wash and hybridization buffers and add 180 µL of a 1:2 ratio of MOPS wash buffer to hybridization buffer, mix embryos into the solution, and incubate for at least 20 min at RT.
   3. Before probe hybridization gently mix embryos into 100 - 150 µL of hybridization buffer. Incubate embryos at 50 °C for at least 1 h.
      NOTE: Incubating embryos overnight is acceptable. Before incubating, seal the wells with an adhesive sheet in order to prevent evaporation.
3. Hybridization
   1. In a separate tube add 0.1 - 0.3 ng/µL of probe and 500 µg/mL yeast tRNA into hybridization buffer, then vortex gently to create probe solution. Yeast tRNA is added to the probe solution to decrease non-specific binding of the anti-sense probe. Preheat this solution to 50 °C, and aspirate hybridization buffer.
   2. Mix pre-hybridized embryos into 100 µL of the probe solution, seal with an adhesive sheet, and hybridize at 50 °C for 2 to 7 days depending on the probe (the foxq2 probe can be incubated for 2-days).
      NOTE: Incubation times will vary based off the probe. Some genes expressed at low levels require up to a 7-day incubation. 96-well plates can be placed in a humidity box as insurance against evaporation if the adhesive sheet fails to seal properly.
   3. Wash 5 times at 50 °C with 180 μL of freshly made MOPS wash buffer for a total of 3 h. Then, wash 3 times (15 min) with 180 µL of MOPS wash buffer at RT. Remember to mix embryos into the wash buffer each time by gently pipetting several times.
4. Antibody Incubation
   1. Aspirate the MOPS wash buffer and mix embryos in 180 µL of blocking buffer consisting of 10% Normal Sheep Serum and 5 mg/mL BSA in MOPS wash buffer and incubate for at least 45 min at RT or 4 °C overnight.
   2. Remove blocking buffer and then mix embryos into blocking buffer containing a 1:1,500 dilution of alkaline phosphatase-conjugated anti-digoxigenin antibody diluted in blocking buffer. Incubate overnight at RT in a sealed plate to avoid evaporation. NOTE: Do not leave antibody on longer than overnight.
   3. Wash embryos 6 times (5 min or until embryos drop) in MOPS wash buffer at RT. Embryos can be stored overnight at 4 °C.
5. In situ development
   1. Wash embryos 3 times (10 min) at RT with freshly made pH 9.5 buffer consisting of 0.1 M Tris pH 9.5, 100 mM NaCl, 50 mM MgCl₂, 1 mM Levamisole, and 0.1% Tween-20.
      1. For 20 mL pH 9.5 buffer use 2 mL 1 M Tris pH 9.5, 400 µL 5M NaCl, 1 mL 1 M MgCl₂, 20 µL Tween-20, 16.6 mL dH₂O, and 0.0048 g Levamisole.
   2. Incubate embryos at 37 °C in staining buffer protected from light. Staining buffer consists of 10% Dimethyl Formamide, 4.5 µL/mL 4-Nitro blue tetrazolium chloride (NBT), and 3.5 µL/mL 5-Bromo-4-chloro-3-indolyl-phosphate (BCIP) in freshly made pH 9.5 buffer.
      1. For 1 mL of staining buffer use 100 µL Dimethyl Formamide, 4.5 µL NBT, and 3.5 µL BCIP.
   3. Stop the alkaline phosphatase reaction by washing 3 to 5 times in MOPS wash buffer. The reaction with the foxq2 probe typically takes 30 min to 1 h. Some probes may need overnight incubation at either RT or 4 °C.
   4. Mix embryos into a solution of 70% MOPS wash and 30% glycerol. The glycerol provides a refractive index necessary for microscopy. Embryos can be stored in this solution for several weeks. Seal the plates with plastic paraffin to prevent evaporation.
6. Slide preparation and image capturing
   1. Prepare a slide by arranging three small strips of double-sided tape into a triangle with small gaps between strips onto a slide.
   2. Transfer the embryos in the 70% MOPS wash and 30% glycerol solution to the center of the triangle and cover with a coverslip.
   3. Seal the coverslip by applying a layer of nail polish around the edges of the coverslip.
   4. Capture images using a compound light microscope with a 20X objective and an attached camera.

Wyniki

In the sea urchin embryo we have shown that 3 different Wnt signaling branches (Wnt/β-catenin, Wnt/JNK, and Wnt/PKC)^4,25 interact to form a Wnt signaling network that governs anterior-posterior (AP) patterning. One of the most important consequences of these signaling events is that the initial broadly expressed anterior neuroectoderm (ANE) GRN becomes restricted to a small territory around the anterior pole by the beginning ...

Dyskusje

The methodology presented here is an example that illustrates the power of using embryos with less genomic and morphological complexity than vertebrates to understand the signaling transduction pathways and GRNs governing fundamental developmental mechanisms.. Many labs are using similar assays during early sea urchin development to dissect the signaling pathways involved in other cell fate specification events (e.g. Notch, Hedgehog, TGF- β, and FGF signaling)^27,

Materiały

Name	Company	Catalog Number	Comments
Translational-blocking morpholino and/or splice-blocking morpholino	Gene Tools LLC	Customized	More information at www.gene-tools.com
Glycerol	Invitrogen	15514-011
FITC (dextran fluorescein isothiocyanate)	Invitrogen, Life Technologies	D1821	Make 25 mg/mL stock solution
Paraformaldehyde 16% solution EM Grade	Electron Microscopy Sciences	15710
MOPS	Sigma Aldrich	M1254-250G
Tween-20	Sigma Aldrich	23336-0010
Formamide	Sigma Aldrich	47671-1L-F
Yeast tRNA	Invitrogen	15401-029
Normal Goat Serum	Sigma Aldrich	G9023-10mL
Alkaline Phosphatase-conjugated anti-digoxigenin antibody	Roche	11 093 274 910
Tetramisole hydrochloride (levamisole)	Sigma Aldrich	L9756-5G
Tris Base UltraPure	Research Products Internationall Corp	56-40-6
Sodium Chloride	Fisher Scientific	BP358-10
Magnesium chloride	Sigma Aldrich	7786-30-3
BCIP (5-Bromo-4-Chloro-3-indolyl-phosphate	Roche	11 383 221 001
4 Nitro blue tetrazolium chloride (NBT)	Roche	11 383 213 001
Dimethyl Formamide	Sigma Aldrich	D4551-500mL
Potassium Chloride	Sigma Aldrich	P9541-5KG
Sodium Bicarbonate	Sigma Aldrich	S5761-500G
Magnesium Sulfate	Sigma Aldrich	M7506-2KG
Calcium Chloride	Sigma Aldrich	C1016-500G