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Visualizing egg and embryonic polarity
CHAPTER
Visualizing egg and embryonic polarity
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Lauren T. Smith, Athula H. Wikramanayake1 Department of Biology, University of Miami, Coral Gables, FL, United States 1 Corresponding author: e-mail address: [email protected]
CHAPTER OUTLINE 1 Introduction......................................................................................................252 2 Methods of Visualizing Oocyte and Egg Polarity...................................................254 2.1 Observation of the Germinal Vesicle and Polar Bodies............................254 2.2 Staining the Jelly Canal......................................................................255 2.3 Using the Pigment Band to Identify the Vegetal Pole..............................256 2.4 Visualizing the Microtubule Organizing Center Using Immunocytochemistry.........................................................................257 2.4.1 Overview........................................................................................258 2.4.2 Materials and solutions....................................................................258 2.4.3 Gamete collection...........................................................................259 2.4.4 Prefertilization treatments................................................................259 2.4.5 Fixation and permeabilization..........................................................260 2.4.6 Postfixation washes and blocking.....................................................260 2.4.7 Primary antibody preparation...........................................................261 2.4.8 Secondary antibody preparation.......................................................261 2.4.9 Mounting and observing specimen...................................................261 2.5 Visualizing the Vegetal Cortical Domain................................................261 2.6 Visualizing the Vegetal Cortex by Immunocytochemistry..........................262 2.6.1 Overview........................................................................................262 2.6.2 Materials and solutions....................................................................262 2.6.3 Gamete collection...........................................................................263 2.6.4 Prefertilization treatments................................................................263 2.6.5 Fixation and permeabilization..........................................................263 2.6.6 Postfixation washes and blocking.....................................................263 2.6.7 Primary antibody preparation...........................................................263 2.6.8 Secondary antibody preparation.......................................................264 2.6.9 Mounting and observing specimen...................................................264 2.7 Visualizing the Vegetal Cortical Domain Using Fluorescently Tagged Dsh...264 2.7.1 Overview........................................................................................264 2.7.2 Protamine sulfate-coated dish preparation........................................264 Methods in Cell Biology, Volume 150, ISSN 0091-679X, https://doi.org/10.1016/bs.mcb.2019.01.001 © 2019 Elsevier Inc. All rights reserved.
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2.7.3 Mouth pipette preparation...............................................................265 2.7.4 Injection solutions and micropipette preparation...............................265 2.7.5 Gamete collection...........................................................................266 2.7.6 Removing jelly coat.........................................................................266 2.7.7 Microinjection of sea urchin zygotes.................................................266 2.7.8 Observation under microscope........................................................266 Acknowledgments..................................................................................................266 References............................................................................................................267 Glossary................................................................................................................268
Abstract During development metazoan embryos have to establish the molecular coordinates for elaboration of the embryonic body plan. Typically, bilaterian (bilaterally symmetric animals) embryos establish anterior-posterior (AP) and dorsal-ventral (DV) axes, and in most cases the AP axis is established first. For over a century it has been known that formation of the AP axis is strongly influenced by the primary axis of the egg, the animal-vegetal (AV) axis. The molecular basis for how the AV axis influences AP polarity remains poorly understood, but sea urchins have proven to be important for elucidating the molecular basis for this process. In fact, it is the first model system where a critical role for Wnt signaling in specification and patterning the AV and AP axis was first established. One current area of research is focused on identifying the maternal factors that regulate localized activation of Wnt/βcatenin signaling at the vegetal pole during development. An essential tool for this work is the means to identify the AV polarity in oocytes and eggs. This permits investigation into how polarity is established and allows development of experimental strategies to identify maternal factors that contribute to and control axial polarity. This chapter provides protocols to accomplish this in sea urchin eggs and early embryos. We describe simple methods to visualize polarity including direct observation of eggs and oocytes, using a microscope for overt morphological signs of polarity, and more extensive methods involving localization of known factors indicative of inherent embryonic polarity, such as the upstream regulators of the Wnt/β-catenin pathway.
1 INTRODUCTION Most animal eggs have a primordial polarity referred to as the Animal-Vegetal (AV) axis that strongly influences early pattern formation during embryogenesis. The underlying mechanism for this process involves maternal determinants that are distributed asymmetrically along the AV axis. During cleavage stages these determinants are differentially inherited by blastomeres that arise at the different poles of the embryo and they impart cells with early specification cues. Typically, in bilaterian (bilaterally symmetrical) animals these specification cues instruct vegetal polederived cells to assume endomesodermal cell fates and instruct animal pole-derived
1 Introduction
blastomeres to assume ectodermal cell fates (Martindale, 2005; Martindale & Hejnol, 2009). In most animals, however, the molecular identity of the maternal determinants that specify cell fates along the AV axis remains largely uncharacterized. In contrast, work done in sea urchins has indicated that maternal factors active at the vegetal pole locally induces the highly conserved Wnt/β-catenin signaling pathway to initiate early pattern formation (Emily-Fenouil, Ghiglione, Lhomond, Lepage, & Gache, 1998; Logan, Miller, Ferkowicz, & McClay, 1999; Wikramanayake, Huang, & Klein, 1998). This pathway directly specifies endomesoderm at the vegetal pole, and then both directly and indirectly restricts neural fates to the anterior of the embryo, thus polarizing the embryo along an anterior-posterior (AP) axis (Range, 2014; Wikramanayake et al., 1998). This mechanism appears conserved, and it is now known that activation of Wnt/β-catenin at the future posterior pole is an early critical event for development in embryos of many bilaterian species (Petersen & Reddien, 2009). However, how the Wnt/β-catenin pathway is locally activated in early embryos is still poorly understood in most species. Researchers have begun to exploit embryological features of sea urchin eggs and early embryos to begin to identify the upstream maternal regulators of Wnt/β-catenin signaling in early embryos (Croce et al., 2011; Peng & Wikramanayake, 2013; Weitzel et al., 2004). The sea urchin embryo has provided critical insight into the specification and patterning of the AV axis starting with the classical work of Theodor Boveri who showed that the primary egg axis predicted where the germ layers are specified during embryogenesis (Boveri, 1901). Boveri’s experiments, which exploited a natural pigment band present toward the vegetal end of eggs of the Mediterranean Sea urchin Paracentrotus lividus to distinguish the animal pole from the vegetal pole, demonstrated that the pigment band was consistently inherited by the cells that formed the endomesoderm (Boveri, 1901). Sven Horstadius followed up on these observations with a set of experiments to ascertain if determinants for endomesoderm were also localized in the unfertilized egg. Horstadius used the pigment band as a marker for the vegetal end to bisect the egg equatorially. He then fertilized each half (Refer to Fig. 1B) and showed that while the vegetal half developed into an almost normal larva with all three germ layers, the animal halves developed into arrested embryos that were referred to as “dauerblastulae” (Horstadius, 1973). These results clearly demonstrated that determinants for endomesoderm were localized to the vegetal half of the unfertilized sea urchin egg (Horstadius, 1973). From the work done in several labs we now know that these determinants are most likely factors that locally activate the Wnt/β-catenin signaling pathway to specify endomesoderm in vegetal pole blastomeres (Croce et al., 2011; Emily-Fenouil et al., 1998; Logan et al., 1999; Peng & Wikramanayake, 2013; Weitzel et al., 2004; Wikramanayake et al., 1998). These maternal determinants have still not been identified, but with the advent of increasingly sensitive methods for identifying differentially expressed RNAs and proteins using methods such as RNA sequencing and mass spectrometry the time is now right for exploiting the beneficial embryological features of sea urchin eggs and embryos to identify the elusive localized determinants.
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FIG. 1 Visualization of the jelly canal in the sea urchin egg. (A) An egg from H. pulcherrimus stained with India ink shows the jelly canal at the animal pole. (B) An equatorial dissection of a H. pulcherrimus egg using the jelly canal as a landmark for egg polarity. Images taken from Maruyama, Y. K., Nakaseko, Y., & Yagi, S. (1985). Localization of cytoplasmic determinants responsible for primary mesenchyme formation and gastrulation in the unfertilized egg of the sea urchin Hemicentrotus pulcherrimus. Journal of Experimental Zoology, 236, 155–163.
Here, we provide protocols that can be used for visualization of the AV axis of the oocyte/egg and early embryos of sea urchins. These protocols allow investigators to identify the molecules underlying AV axis polarity in sea urchins using experimental strategies that exploit the advantages of experimental embryology and contemporary molecular methods in urchins.
2 METHODS OF VISUALIZING OOCYTE AND EGG POLARITY 2.1 OBSERVATION OF THE GERMINAL VESICLE AND POLAR BODIES Since the site of polar body release during meiosis marks the animal pole of metazoan oocytes (Martindale, 2005; Martindale & Hejnol, 2009), the position of the germinal vesicle or locating the polar bodies on the oocyte/egg surface immediately prior to meiosis are reliable ways to identify the animal pole of sea urchin oocytes. It is not known when the AV axis is initially polarized during oogenesis, but experimental observations indicate that this polarity is already specified prior to the first meiosis. Early in oogenesis the germinal vesicle of small primary oocytes is initially located centrally in the cell (Peng & Wikramanayake, 2013; Wessel, Voronina, & Brooks, 2004). Staining for the centrosome at this stage has shown that it is already present at the animal pole and staining for the Disheveled protein—a central
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upstream regulator of the Wnt signaling pathway—has shown that it has already begun to accumulate in a domain at the vegetal cortex termed the vegetal cortical domain (VCD) (Peng & Wikramanayake, 2013). Prior to the first meiosis the germinal vesicle moves to the animal pole of the oocyte, and in some echinoderms this has been shown to be a microtubule-dependent process (Miyazaki, Kato, & Nemoto, 2005; Schroeder, 1980a). Once the germinal vesicle has moved to the animal pole it is relatively easy to identify the AV polarity of the oocytes using light microscopy. This polarity is particularly striking in echinoderms such as asteroids and holothuroids, where one can collect large primary oocytes for experimentation and induce their maturation using 1-methyladenine (Foltz, Adams, & Runft, 2004; Smiley, 1990). Echinoid oocytes are more challenging to work with, and no consistent method is available to carry out in vitro maturation of these oocytes (Wessel et al., 2004). Echinoids spawn mature eggs that have completed meiosis, so other methods are needed to visualize the primary egg axis in these echinoderms. In P. lividus, the female pronucleus remains at the animal pole of the mature egg following meiosis and hence can be used as a reliable marker for the AV axis (Di Carlo, Romancino, Ortolani, Montana, & Giudice, 1996). However, this trait does not appear to be shared with other echinoids since in several other species the location of the pronucleus in mature eggs is random and does not correlate with the AV axis (Goda, Inoue, & Mabuchi, 2009; Maruyama, Nakaseko, & Yagi, 1985; Peng & Wikramanayake, 2013). However, Schroeder (1980b) showed that by careful collection of spawned eggs from Strongylocentrotus droebachiensis and observing them using Nomarski Differential Interference Contrast microscopy it is possible to identify the animal pole by the position of the polar bodies released during meiosis. However, the polar bodies are small and are easily displaced during spawning. Hence, this method is not particularly useful for manipulative experiments where one has to have absolute certainty about the polarity of the egg. In species where the AV axis can be reliably identified using the position of the germinal vesicle or the female pronucleus, it is possible to use a drawn out glass needle and bisect the egg equatorially, or dissect one of the poles to determine the importance of factors localized to the animal or vegetal cortex for normal development (Croce et al., 2011; Di Carlo et al., 1996; Horstadius, 1973; Maruyama et al., 1985). Additionally, drawn out glass needles can be used to separate the egg poles and collect them for identifying localized factors using sensitive methods such as RNA sequencing.
2.2 STAINING THE JELLY CANAL In addition to the position of polar body release, the jelly canal, a structure present in the jelly coat surrounding sea urchin eggs, has also been implicated as a definitive marker of the animal pole (Boveri, 1901; Schroeder, 1980b). Not much is known about how the jelly canal is formed, but it is observed to always align with the site
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of the polar body release (Schroeder, 1980b). The jelly canal is present in the jelly coat as a slender, conical channel that can be readily visualized by directly spawning females into a dense suspension of Sumi ink and seawater (Goda et al., 2009; Maruyama et al., 1985; Peng & Wikramanayake, 2013; Schroeder, 1980b). The Sumi ink suspension can be made by placing a few drops of seawater on the frosted end of a glass microscopy slide and rubbing a dry cake of Sumi Japanese ink against the frosted glass. For this method to work, it is critical to have a dense solution of Sumi ink in seawater and the particles have to be small enough to pass into the jelly canal. The eggs also have to be directly spawned into the Sumi ink solution, or they have to be collected as they are being released from the genital pores and quickly transferred to the Sumi ink solution. After a 5–10 min incubation, gently wash the eggs twice with seawater to dilute the ink suspension and observe the eggs under a microscope (Fig. 1; Goda et al., 2009; Peng & Wikramanayake, 2013; Schroeder, 1980b). Note that if eggs are spawned directly into seawater prior to adding them to the Sumi ink suspension you will not be able to visualize the jelly canal since the expanding jelly layer will squeeze this canal shut. In an alternate protocol, Goda et al. (2009) used black ink (Bokuju; Kuretake Co, Japan) that was dialyzed for 6 h against seawater. Lytechinus variegatus and Lytechinus pictus eggs were directly placed in a 1:4 ratio of the black ink and seawater for 1 min, rinsed with seawater and observed using light microscopy. By identifying the animal pole using the jelly canal as a marker, one can use drawn out glass needles to cut animal and vegetal halves and use them for experiments as needed (Fig. 1; Maruyama et al., 1985). One potential use of this method could be to carry out RNA sequencing of isolated animal and vegetal pole fragments to identify molecules enriched at the two egg poles.
2.3 USING THE PIGMENT BAND TO IDENTIFY THE VEGETAL POLE Classical embryologists have exploited the asymmetric distribution in the egg of organelles such as pigment granules to identify AV polarity in sea urchins. Several species of sea urchins produce eggs with pigment granules that are usually evenly distributed in the cortical region of the mature egg. In some species the cortical band of pigment granules is retracted from the vegetal pole before the metaphase of the fourth mitotic cycle leaving behind a clear cytoplasm that is inherited by the 16-cell stage micromeres (Schroeder, 1980b). Interestingly, Boveri (1901) showed that there is a pigment granule-free region toward the vegetal pole of P. lividus eggs, and hence, this pole could be identified by the “pigment band” located toward the vegetal end. Schroeder (1980b) reevaluated Boveri’s work and concluded that only 20% of P. lividus eggs displayed the pigment band prior to fertilization. A selection of Boveri’s original figures is shown in Fig. 2. The pigment band in P. lividus has been used by researchers to orient the AV axis of unfertilized eggs and carry out various embryological manipulations to ask questions about the distributions of determinants along the primary egg axis (Croce et al., 2011; Di Carlo et al., 1996; Horstadius, 1973). In one of these studies Croce et al. (2011) showed that fusing a vegetal cortex fragment to the animal pole membrane was sufficient to induce
2 Methods of visualizing oocyte and egg polarity
FIG. 2 Visualization of the pigment band and its fate during embryogenesis in Paracentrotus lividus. During oogenesis the pigment appears uniformly distributed but accumulates as a band toward the vegetal pole after oocyte maturation. This pigment band consistently gets incorporated into the vegetal cells during development. € von ovocyte, ei und larve des Strongylocentrotus lividus. Images taken from Boveri, T. (1901). Die polaritat €cher, 14, 630–653. Zoologische Jahrbu
ectopic endoderm in animal pole-derived blastomeres demonstrating the functional importance of the VCD for regulating polarity and patterning in the sea urchin embryo.
2.4 VISUALIZING THE MICROTUBULE ORGANIZING CENTER USING IMMUNOCYTOCHEMISTRY During oogenesis in echinoderms, the germinal vesicle moves from the center of the primary oocyte to the animal pole where meiosis takes place (Smiley, 1990). In some echinoderms the movement of the germinal vesicle to the animal pole prior to the
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FIG. 3 The Disheveled protein accumulates in a vegetal cortical domain early in oogenesis and is tightly embedded in the vegetal cortex. (A) Primary oocyte stained with an anti-Dsh antibody and phalloidin. The nascent accumulation of Dsh can be seen at the vegetal pole (arrow). At this time the centrosome is present at the animal pole (asterisk). (B) A mature egg stained with an anti-Dsh antibody. The striking accumulation of Dsh can be seen at the vegetal pole (arrow). Dsh protein is also found distributed throughout the egg cytoplasm. (C) Isolated egg cortices stained with the anti-Dsh antibody. The arrows point to the Dsh embedded in the vegetal cortical domain. Adapted from Peng, C. J. & Wikramanayake, A. H. (2013). Differential regulation of disheveled in a novel vegetal cortical domain in sea urchin eggs and embryos: Implications for the localized activation of canonical Wnt signaling. PLos One, 8(11), e80693.
first meiosis depends on microtubules (Miyazaki et al., 2005). The microtubules for this process are assembled by the microtubule organizing center (MTOC) which appears to assemble early during oogenesis (Peng & Wikramanayake, 2013; Smiley, 1990). In sea urchins, the maternal MTOC disappears following the second meiotic reduction. The sperm centrosome brought in at fertilization undergoes a duplication event and provides the MTOCs for mitosis (Schatten, Schatten, Mazia, Balczon, & Simerly, 1986). Therefore, using immunocytochemistry to localize MTOC proteins such as gamma-tubulin can be an effective way to detect AV polarity in oocytes. The MTOC in sea urchin oocytes can also be detected using phalloidin (Fig. 3A; Peng & Wikramanayake, 2013). For additional information regarding cytoskeleton labeling, reference Methods in Cell Biology, Volume 74, Chapter 16, Section IV.
2.4.1 Overview For protocols regarding the collection of gametes from sea urchins refer to chapter “Procuring animals and culturing of eggs and embryos” by Adams et al. Here, we provide the protocol for preparing oocytes, eggs and embryos for immunofluorescence staining.
2.4.2 Materials and solutions a. Buffers 10 Phosphate-Buffered Saline (PBS, Bio-Rad) Dilute to 1 before use
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b. Fixative 4% Paraformaldehyde (PFA, EMS) 1 PBS Add 10 mL 16% PFA in 30 mL of 1 PBS. PFA is a potential carcinogen and should be handled with gloves at all times, and fixation should be performed under a ventilated hood. In our lab we never bring fixatives into the room where we do embryo culturing and embryology. PFA should always be prepared fresh. c. Postfixation Solutions 100% Acetone (at 4 °C) d. Blocking Buffer PBST (BSA, Tween 20) 0.01% BSA, powder, stored at 4 °C 0.1% Tween 20 1 PBS For 100 mL PBST, use 0.0100 g BSA, 100 μL of Tween 20, and 100 mL 1 PBS. e. Staining Dilutions Primary antibodies stored at 4 °C Mouse anti-γ-tubulin antibody (1:50, Abcam). Secondary antibodies stored at 4 °C Use an antimouse secondary conjugated with an appropriate fluorophore. (e.g., Alexa Fluor 532 goat antimouse IgG (Invitrogen) or Alexa Fluor 568 donkey antimouse IgG (Invitrogen), or Alexa Fluor 635 goat antimouse IgG (Invitrogen)). Cytoskeleton labeling (F-actin) Fluorescein phalloidin (Invitrogen), stored at 20 °C, working dilution 3:100. e.g., 3 μL in 100 μL of Blocking Buffer. Note: Fluorescein phalloidin alone is effective in staining the centrosome in sea urchin oocytes (Peng & Wikramanayake, 2013). Therefore, if desired, one may forego using antibodies, and simply use phalloidin. Nuclear labeling 40 ,6-Diamidino-2-phenylindole, dilactate (DAPI, Invitrogen), blue, stored at 20 °C, working dilution 1:1000 to a final concentration of 300 nM. f. Mounting Solutions 50% Glycerol in 1 PBS
2.4.3 Gamete collection Refer to Chapter 1.
2.4.4 Prefertilization treatments The sea urchin egg has a thin egg envelope called the vitelline envelope (VE) that is attached to the plasma membrane via proteinaceous structures called vitelline posts (Kidd, 1978). In our experience, antibodies will freely pass through the VE of fixed,
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unfertilized eggs. At fertilization, however, the VE is converted into a fertilization envelope (FE) that is cross-linked by cortical granule enzymes and it becomes impenetrable to large proteins such as antibodies. The embryo will emerge from the FE at the late blastula stage by secreting a hatching enzyme. Hence, for successful antibody staining of any stage after fertilization up to the hatching blastula stage FEs need to be removed. To do this, cross-linking of the FE proteins is prevented by fertilizing eggs in ASW with 0.5 mM 3-amino-1,2,4-triazole (ATA, Sigma). Some labs prefer to use 10 mM para-amino benzoic acid (PABA, Sigma) to prevent crosslinking of the FE following fertilization (Logan et al., 1999). When embryos are collected for fixation at a particular stage, the egg envelopes can be removed by passing the embryos through a mesh strainer (BD Falcon). We typically use a 70 μm strainer for S. purpuratus and a 100 μm strainer for L. variegatus and L. pictus. Observe the eggs under a stereomicroscope to determine efficient envelope removal. If envelopes are still present, the eggs/embryos may be passed through the strainer again and usually twice is sufficient to strip the envelopes off embryos.
2.4.5 Fixation and permeabilization Once the desired stage is reached, embryos can be fixed and processed in a 1.5-mL microcentrifuge tube, or for larger numbers of embryos, 15 mL conical tubes. These can be gently centrifuged between washing and incubation steps. Be aware that embryos tend to stick to the side of plastic tubes when wash buffers are void of detergents and when tubes are not spun down properly. Alternately, fixed embryos can be left to settle by gravity because they readily sink once exposed to fixative. Compact embryo sample volume should not exceed 50 μL in a 1.5 mL tube, or 500 μL in a 15 mL tube. Fix embryos by adding 4% paraformaldehyde (PFA) solution in phosphate-buffered saline (PBS) pH 7.4. The amount of fixative added should be at least 10 the amount of sample. Samples can be fixed for 20–60 min at room temperature. The embryos should settle to the bottom during the fixation period. Remove and discard the fixative into a contained waste receptacle. Add enough ice-cold acetone to cover the sample (1 mL in a 1.5 mL tube) and incubate on ice for 10 min. This step permeabilizes the membranes of the embryo to increase efficiency of antibody penetration into cells. Following the 10 min acetone treatment, gently centrifuge the embryos using a hand centrifuge or in a centrifuge with a swing bucket rotor and then decant the acetone solution. Wash the embryos three times using 1 PBS solution (5 min incubations between washes should suffice). Note: Going forward, the protocol will be specific to using a 1.5 mL tube during this procedure.
2.4.6 Postfixation washes and blocking Transfer the embryos to a new 1.5 mL tube and allow them to settle on ice. Remove and discard the 1 PBS solution. Add Blocking Buffer up to 500 μL and incubate for 10 min. Remove and discard the Blocking Buffer solution. Repeat the addition and removal of 500 μL of Blocking Buffer three times.
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2.4.7 Primary antibody preparation Remove the Blocking Buffer from the sample until 100 μL is left. Add 100 μL of diluted 1° antibody solution to the sample. Incubate at room temperature for 1 h. Remove 1° antibody solution. Wash embryos with Blocking Buffer for 15 min on ice. Repeat the wash step two more times for a total of 3 washes. You can use a pipette to aspirate and expel the embryos gently in and out of the pipette tip to help remove excess primary antibody from the embryos. For smaller numbers of embryos one can reduce the amount of antibody solution used. Additionally, when working with very small numbers of embryos one can use Terasaki plates (Greiner Bio-One) to avoid losing embryos during the fixing and staining procedure.
2.4.8 Secondary antibody preparation Remove the blocking buffer following the third wash. PBST can be used to dilute the 2° antibody. Add diluted 2° antibody solution to sample. Incubate at room temperature for 45 min. To stain nuclei and the cell cortex, add DAPI (1:1000) and 6 μL of fluorescein phalloidin (3:100) for approximately 20 min. Longer incubation periods do not increase signal intensity, but instead increase background. There is an excellent discussion on some of the challenges associated with using phalloidin to stain F-actin in Strickland et al. (2004). Remove 2° antibody solution. Wash embryos with 1 PBS for 15 min on ice. Repeat the wash step two more times, for a total of 3 washes.
2.4.9 Mounting and observing specimen Each lab has their own methods for mounting fixed specimens for observation. Fixed samples can be mounted on slides, cover slipped and sealed with clear nail polish. The eggs and embryos should be protected from being distorted by using either clay “legs” or strips of coverslip glass (cut using a diamond knife) placed between the slide and the coverslip. The Dsh staining can be seen as a crescent at the vegetal pole (Fig. 3B). The pool of Dsh protein that accumulates at the vegetal pole is tightly embedded in the vegetal cortex and will remain attached even after isolation of the vegetal cortex (Fig. 3C).
2.5 VISUALIZING THE VEGETAL CORTICAL DOMAIN One exciting recent discovery in sea urchins is that oocytes and eggs have a distinct cytoarchitectural domain, termed the vegetal cortical domain (VCD), that is located at the vegetal pole cortex (Fig. 3; Peng & Wikramanayake, 2013). Microsurgical excision of the VCD blocks activation of the Wnt/β-catenin pathway at the vegetal pole of early embryos and leads to embryos that do not form endomesodermal tissues (Croce et al., 2011). Hence, the VCD appears to specify AV polarity of the egg and plays a critical role in activating Wnt/β-catenin signal transduction in vegetal pole blastomeres (Peng, Wang, & Wikramanayake, 2017; Peng & Wikramanayake, 2013). Moreover, the VCD has been shown to accumulate Disheveled (Dsh), a central regulatory protein in the Wnt signaling pathway (Fig. 3; Peng & Wikramanayake, 2013). In the sea
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urchin embryo, the Wnt/β-catenin pathway plays a critical role in specifying endomesoderm in cleavage stage embryos and the Dsh protein is required for the asymmetric activation of the pathway at the vegetal pole (Logan et al., 1999; Weitzel et al., 2004; Wikramanayake et al., 1998). The Dsh protein is expressed throughout the cytoplasm of the egg and in all cells of the early embryo. However, it is a differentially modified form of Dsh that accumulates at the VCD of unfertilized eggs and produces the domain inherited by the vegetal cells that nuclearize β-catenin in the early embryo (Peng & Wikramanayake, 2013). Staining of isolated egg cortices has shown that Dsh is embedded in the VCD confirming that the sea urchin egg cortex has a cytoarchitectural asymmetry at the vegetal pole (Fig. 3C), that appears to function as a scaffold for the localized activation of Wnt signaling (Peng & Wikramanayake, 2013). The ability to isolate large numbers of egg cortices and the availability of excellent anti-sea urchin Dsh antibodies facilitate the identification of the molecular basis in the VCD of sea urchins and the mechanism for the asymmetric activation of Wnt/β-catenin signaling at the vegetal pole of early embryos.
2.6 VISUALIZING THE VEGETAL CORTEX BY IMMUNOCYTOCHEMISTRY 2.6.1 Overview Visualization of the VCD can be achieved through immunofluorescent staining of Dsh using anti-Dsh antibodies or by microinjection of an mRNA coding for Dsh fused to a fluorescent protein (Peng & Wikramanayake, 2013; Weitzel et al., 2004). Three affinity-purified anti-Dsh polyclonal antibodies have been generated against three distinct epitopes on the L. variegatus Dsh protein (Peng & Wikramanayake, 2013). A His-tagged fusion protein of amino acids 1–101 of LvDsh protein including the DIX domain was used as one antigen to immunize rabbits. In addition, polyclonal antibodies were also produced using synthetic peptides corresponding to epitopes at the N-terminal (NH2-CASVTTDTRGDSQLPPERTG-COOH) and C-terminal (NH2CMVPMMPRQLGSVPEDLSGS-COOH) regions of Dsh as antigens. These polyclonal antibodies were affinity-purified using the immunizing antigens by Bethyl Labs (Montgomery, TX). The antibodies generated against the DIX domain, the N-terminal epitope, and the C-terminal epitope are referred to as anti-SUDshDIX, anti-SUDsh-N, and anti-SUDsh-C, respectively. All three antibodies have been successfully used in all sea urchin species tested thus far including S. purpuratus, L. variegatus, L. pictus, Paracentrotus lividus, Hemicentrotus pulcherrimus, and others. The anti-SUDshDIX antibody also cross reacts with Dsh proteins from other species including the sea anemone Nematostella vectensis (Lee, Kumburegama, Marlow, Martindale, & Wikramanayake, 2007). The anti-Dsh antibodies are available on request from the Wikramanayake lab. These antibodies will be available for use until they are depleted.
2.6.2 Materials and solutions i. Buffers Use same buffers as detailed in Section 2.4.
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ii. Fixative Use paraformaldehyde as a fixative as detailed in Section 2.4. iii. Postfixation solutions 100% Acetone. iv. Blocking buffer Use blocking buffer as detailed in Section 2.4. v. Antibody dilutions Primary Dsh antibodies stored at 4 °C Anti-SUDsh-C: S. purpuratus 1:400; L. pictus 1:200; L. variegatus 1:200 Anti-SUDsh-N: S. purpuratus 1:200; L. pictus 1:100; L. variegatus 1:100 Anti-SUDshDIX: S. purpuratus 1:400; L. pictus 1:400; L. variegatus 1:400 Secondary antibodies stored at 4 °C Use an antirabbit secondary antibody conjugated with an appropriate fluorophore as mentioned in Section 2.4.2, step e. Specifications regarding cytoskeleton and nuclear labeling can also be referenced in Section 2.4.2, step e. vi. Mounting solutions 50% Glycerol in 1 PBS. 0.1% Sodium azide.
2.6.3 Gamete collection Use same procedures as detailed in Section 2.4.
2.6.4 Prefertilization treatments Use same procedures as detailed in Section 2.4.
2.6.5 Fixation and permeabilization Use same procedures as detailed in Section 2.4.
2.6.6 Postfixation washes and blocking Use same procedures as detailed in Section 2.4.
2.6.7 Primary antibody preparation Remove the blocking buffer from the sample until 100 μL is left. Add 100 μL of diluted 1° antibody solution to the sample. Incubate at room temperature for 1 h. Remove 1° antibody solution. Wash embryos with blocking buffer for 15min on ice. Repeat the wash step two more times for a total of 3 washes. You can use a pipette to aspirate and expel the embryos gently in and out of the pipette tip to help remove excess primary antibody from the embryos. For smaller numbers of embryos one can reduce the amount of antibody solution used. Additionally, when working with very small numbers of embryos one can use Terasaki plates to avoid losing embryos during the fixing and staining procedure.
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2.6.8 Secondary antibody preparation Remove the blocking buffer following the third wash. Add diluted 2° antibody solution to sample. Incubate at room temperature for 45 min. To stain nuclei and the cell cortex add DAPI (1:1000) and 6 μL of fluorescein phalloidin (3:100) for approximately 20 min. Longer incubation periods do not increase signal intensity, but instead increase background. Remove 2° antibody solution. Wash embryos with 1 PBS for 15 min on ice. Repeat the wash step two more times, for a total of 3 washes.
2.6.9 Mounting and observing specimen See Section 2.4.9.
2.7 VISUALIZING THE VEGETAL CORTICAL DOMAIN USING FLUORESCENTLY TAGGED Dsh In marine invertebrate embryos expression of synthetic messenger RNAs has become a useful approach for studying gene function. In sea urchins, methods have been developed to carry out rapid microinjection to express RNAs coding for proteins of interest fused to a fluorescent protein to determine subcellular localization of the protein, and wild-type and various mutant proteins to test for protein function. For further information, reference chapter “Microinjection of oocytes and embryos with synthetic mRNA encoding molecular probes” by von Dassow et al.
2.7.1 Overview Upon fertilization in sea urchin eggs, activation leads to a rapid increase in the translation of proteins. Therefore, mRNAs injected into eggs or early blastomeres are readily translated into their protein products. A cDNA coding for a protein of interest fused to a fluorescent protein can be subcloned into an expression vector, such as pCS2 +, and synthetic mRNA can be transcribed using a specific RNA polymerase. It is important to note that RNases are fairly ubiquitous and if there is any contamination of reagents this will lead to degradation of the RNA. Therefore, all materials, benchtops, and solutions must be RNase-free while working with RNA.
2.7.2 Protamine sulfate-coated dish preparation Materials: 60-mm plastic cell culture dish lids (Fisher Scientific) 1% (w/v) protamine sulfate solution: 0.5 g protamine sulfate salt, ddH2O to 50 mL Gather several 60 mm plastic cell culture dish lids in rows. The lids are used because they are shallower and this makes it easier to position the needle for microinjections. Add enough 1% (w/v) protamine sulfate solution to cover the bottom of the lid. Let them sit for 1 min and then transfer the solution to an additional set of lids, or save the solution in an appropriate container for future use. Wash the coated lids by placing them in a ddH2O filled beaker. When all the dishes have been coated and washed, rinse them one additional time in ddH2O and allow them to dry at room temperature.
2 Methods of visualizing oocyte and egg polarity
2.7.3 Mouth pipette preparation Materials: 1 mL Tuberculin Slip Tip Syringe (BD Biosciences) Mouthpiece (can be purchased from Drummond Scientific as an aspirator tube assembly, model # 2-000-000) (See text for alternate mouthpieces) Rubber tubing, 1/800 ID, 3/1600 OD (Cole Parmer Instrument Company) Rubber pipette bulbs Mouth pipettes are used to move eggs and embryos during the injection process. These mouth pipettes can be made by connecting a mouthpiece to a glass micropipette using rubber tubing. In order for the glass micropipette to securely fit into the tubing, a pipette adapter must be made. Start at the tip of a 1 mL Tuberculin Slip Tip syringe and cut a length of 3 cm. Cut a rubber pipette bulb approximately 2.5 cm from the open end. Insert the wide end of the syringe tip into the cut end of the rubber pipette bulb. Use lab labeling tape to secure and seal the end of the rubber bulb to the end of the syringe. Next, insert the syringe tip of the pipette adapter into one end of a piece of 1/800 rubber tubing cut to 2–3 ft long. Insert the mouthpiece into the opposite end of the tubing. An alternative mouth pipette can be created by taking the rubber tubing of the dimensions mentioned above and inserting the pointed end of a p200 pipette tip into one end and the pointed end of a p1000 pipette tip into the other open end of the tubing. The glass pipette (see Section 2.7.4) can be inserted into the open end of the p1000 pipette and the other end can be used as the mouth piece.
2.7.4 Injection solutions and micropipette preparation Materials: Micropipette puller (similar to Sutter Instrument P-97 Flaming Brown) 1.0/0.75 mm (OD/ID) thin-wall single-barrel standard borosilicate glass tubing (World Precision Instruments) P2 pipetter 0.2 μm, 4 mm syringe driven filter (Millipore, Billerica, MA) 900 Glass Pasteur pipettes Bunsen burner Diamond knife The mRNA injection solutions are always prepared using nuclease-free water and 100% sterile glycerol. The final concentration of glycerol in injection solution is 40% (v/v) if you are using a continuous flow pressure injection system. It is good practice to filter the RNA solution to remove any particulate material that could clog the injection needles. We typically filter the injection solution using a 0.2 μm, 4 mm syringe driven filter (Millipore, Billerica, MA). Filtered mRNA solutions can be stable for several months at 70 °C. To fill injection needles with RNA solution, use a P2 pipetter and load needles by adding 0.5 μL of injection solution to the open end of the needle. Store filled needles at 4 °C for at least 15 min prior to use to allow the RNA solution to move to the needle tip by capillary action. Needles not in use should
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be stored at 20 °C while injecting. If filled needles need to be stored for a longer period of time (2–3 days) store them at 70 °C. Dejellied eggs are prepared for microinjection by lining them up at the bottom of a protamine sulfate-coated dishes. The eggs can be “rowed” using a mouth pipette. Start with a 900 glass Pasteur pipette. Heat the pipette over a Bunsen burner at the point where the wider bore of the pipette narrows to the smaller bore until it softens. Remove from the flame, and quickly pull the narrow end of the glass pipette horizontally to elongate the glass. The size of the bore of the pipette tip can be controlled by varying the time between removing the hot glass from the flame and beginning the pulling motion to lengthen the glass. If the pipette is pulled too soon, it will break or will form very narrow, unusable pipettes. If the glass is allowed to cool for too long, it will have a large bore size, which will make it difficult to control egg dispensing. Typically, you will want the pipette tip to be just large enough for one egg at a time to fit through the opening, making it easier to control the flow of eggs during mouth pipetting. Cut excess glass off using a diamond knife, leaving a 3–4 cm tip. To smoothen the tip before use, heat very briefly at the base of the flame from a Bunsen burner. Fasten the glass micropipette to the open end of the pipette adapter on the mouth pipette tubing.
2.7.5 Gamete collection Refer to Section 2.4.3.
2.7.6 Removing jelly coat Refer to Chapter 1.
2.7.7 Microinjection of sea urchin zygotes Refer to Chapter 9.
2.7.8 Observation under microscope For live imaging one can use glass bottom dishes similar to those available from MatTek Co. Use of the open dish allows a researcher to easily transfer the imaged sample to another dish for continued culture for later observations. These imaging dishes can be easily crafted by cutting a hole at the bottom of a small Petri dish and attaching a cover slip to the bottom using an adhesive or using vacuum grease. There is an excellent discussion on different ways to mount and image live embryos in Strickland et al. (2004) that readers should consult if they are interested in extended live imaging of embryos.
ACKNOWLEDGMENTS The work in AHW’s lab is supported by grant IOS1257967 from the National Science Foundation.
References
REFERENCES Boveri, T. (1901). Die polarit€at von ovocyte, ei und larve des Strongylocentrotus lividus. € Zoologische Jahrbucher, 14, 630–653. Croce, J., Range, R., Wu, S. Y., Miranda, E., Lhomond, G., Peng, J. C., et al. (2011). Wnt6 activates endoderm in the sea urchin gene regulatory network. Development, 138, 3297–3306. Di Carlo, M., Romancino, D. P., Ortolani, G., Montana, G., & Giudice, G. (1996). “BEP” RNAs and proteins are situated in the animal side of sea urchin unfertilized egg, which can be recognized by female pronuclear localization. Biochemistry and Biophysics Research Communications, 229, 511–517. Emily-Fenouil, F., Ghiglione, C., Lhomond, G., Lepage, T., & Gache, C. (1998). GSK3beta/ shaggy mediates patterning along the animal-vegetal axis of the sea urchin embryo. Development, 125, 2489–2498. Foltz, K., Adams, N., & Runft, L. (2004). Echinoderm eggs and embryos: Procurement and culture. In C. Ettensohn, G. Wray, & G. Wessel (Eds.), Development of sea urchins, ascidians, and other invertebrate deuterostomes: Experimental approaches: Vol. 74. Methods in cell biology (pp. 39–74). San Diego: Elsevier. Goda, M., Inoue, S., & Mabuchi, I. (2009). Calyculin-A induces cleavage in a random plane in unfertilized sea urchin eggs. Biological Bulletin, 216, 40–44. Horstadius, S. (1973). Experimental embryology of echinoderms. Oxford: Clarendon Press. Kidd, P. (1978). The jelly and vitelline coats of the sea urchin egg: New ultrastructural features. Journal of Ultrastructure Research, 64, 204–215. Lee, P. N., Kumburegama, S., Marlow, H. Q., Martindale, M. Q., & Wikramanayake, A. H. (2007). Asymmetric developmental potential along the animal-vegetal axis in the anthozoan cnidarian, Nematostella vectensis, is mediated by Dishevelled. Developmental Biology, 310, 169–186. Logan, C. Y., Miller, J. R., Ferkowicz, M. J., & McClay, D. R. (1999). Nuclear beta-catenin is required to specify vegetal cell fates in the sea urchin embryo. Development, 126, 345–357. Martindale, M. Q. (2005). The evolution of metazoan axial properties. Nature Genetics, 6, 917–927. Martindale, M. Q., & Hejnol, A. (2009). A developmental perspective: Changes in the position of the blastopore during bilaterian evolution. Developmental Cell, 17, 162–174. Maruyama, Y. K., Nakaseko, Y., & Yagi, S. (1985). Localization of cytoplasmic determinants responsible for primary mesenchyme formation and gastrulation in the unfertilized egg of the sea urchin Hemicentrotus pulcherrimus. Journal of Experimental Zoology, 236, 155–163. Miyazaki, A., Kato, K. H., & Nemoto, S. (2005). Role of microtubules and centrosomes in the eccentric relocation of the germinal vesicle upon meiosis reinitiation in sea-cucumber oocytes. Developmental Biology, 280, 237–247. Peng, J. C., Wang, L., & Wikramanayake, A. H. (2017). Origins of anterior-posterior polarity by localized activation of disheveled. Molecular Reproduction and Development, 84, 443. Peng, C. J., & Wikramanayake, A. H. (2013). Differential regulation of disheveled in a novel vegetal cortical domain in sea urchin eggs and embryos: Implications for the localized activation of canonical Wnt signaling. PLos One, 8(11), e80693. Petersen, C. P., & Reddien, P. W. (2009). Wnt signaling and the polarity of the primary body axis. Cell, 139, 1056–1068.
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Range, R. (2014). Specification and positioning of the anterior neuroectoderm in deuterostome embryos. Genesis, 52, 222–234. Schatten, H., Schatten, G., Mazia, D., Balczon, R., & Simerly, C. (1986). Behavior of centrosomes during fertilization and cell division in mouse oocytes and in sea urchin eggs. Proceedings of the National Academy of Sciences of the United States of America, 83, 105–109. Schroeder, T. E. (1980a). Expressions of the prefertilization polar axis in sea urchin eggs. Developmental Biology, 79(2), 428–443. Schroeder, T. E. (1980b). The jelly canal marker of polarity for sea urchin oocytes, eggs, and embryos. Experimental Cell Research, 128(2), 490–494. Smiley, S. (1990). A review of echinoderm oogenesis. Journal of Electron Microscopy Technique, 16, 93–114. Strickland, L., von Dassow, G., Ellenberg, J., Foe, V., Lenart, P., & Burgess, D. (2004). Light microscopy of echinoderm embryos. In C. Ettensohn, G. Wray, & G. Wessel (Eds.), Development of sea urchins, ascidians, and other invertebrate deuterostomes: Experimental approaches: Vol. 74. Methods in cell biology (pp. 39–74). San Diego: Elsevier. Weitzel, H. E., Illies, M. R., Byrum, C. A., Xu, R., Wikramanayake, A. H., & Ettensohn, C. A. (2004). Differential stability of B-catenin along the animal-vegetal axis of the sea urchin embryo mediated by disheveled. Development, 131, 2947–2956. Wessel, G. M., Voronina, E., & Brooks, J. M. (2004). Obtaining and handling echinoderm oocytes. In C. Ettensohn, G. Wray, & G. Wessel (Eds.), Development of sea urchins, ascidians and other invertebrate deuterostomes: experimental approaches: Vol. 74. Methods in cell biology (pp. 87–114). San Diego: Elsevier. Wikramanayake, A. H., Huang, L., & Klein, W. H. (1998). Beta-catenin is required for pattern formation along the animal-vegetal axis in the sea urchin embryo. Proceedings of the National Academy of Sciences of the United States of America, 95, 9343–9348.
GLOSSARY Animal-vegetal (AV) axis A primordial polarity found in oocytes and egg of most metazoans established during oogenesis that influences early pattern formation along the anteriorposterior vegetal axis in bilaterians. Bilaterian An animal displaying bilateral symmetry in their embryonic and adult body plan. Germinal vesicle The nucleus of an oocyte that has been arrested in prophase of meiosis 1 of oogenesis. Microtubule organizing center (MTOC) Structure in eukaryotic cells that nucleates microtubules responsible for cilia and flagella function and for nucleating mitotic and meiotic spindles. Vegetal cortical domain (VCD) A domain located at the vegetal pole cortex of the egg/ embryo where there is an accumulation of a differentially modified form of the Disheveled protein and is required for activation of Wnt signaling. Wnt signaling pathway An evolutionarily conserved signal transduction pathway found in all animals that strongly influences early pattern formation in early embryos.
Visualizing egg and embryonic polarity
11
Lauren T. Smith, Athula H. Wikramanayake1 Department of Biology, University of Miami, Coral Gables, FL, United States 1 Corresponding author: e-mail address: [email protected]
CHAPTER OUTLINE 1 Introduction......................................................................................................252 2 Methods of Visualizing Oocyte and Egg Polarity...................................................254 2.1 Observation of the Germinal Vesicle and Polar Bodies............................254 2.2 Staining the Jelly Canal......................................................................255 2.3 Using the Pigment Band to Identify the Vegetal Pole..............................256 2.4 Visualizing the Microtubule Organizing Center Using Immunocytochemistry.........................................................................257 2.4.1 Overview........................................................................................258 2.4.2 Materials and solutions....................................................................258 2.4.3 Gamete collection...........................................................................259 2.4.4 Prefertilization treatments................................................................259 2.4.5 Fixation and permeabilization..........................................................260 2.4.6 Postfixation washes and blocking.....................................................260 2.4.7 Primary antibody preparation...........................................................261 2.4.8 Secondary antibody preparation.......................................................261 2.4.9 Mounting and observing specimen...................................................261 2.5 Visualizing the Vegetal Cortical Domain................................................261 2.6 Visualizing the Vegetal Cortex by Immunocytochemistry..........................262 2.6.1 Overview........................................................................................262 2.6.2 Materials and solutions....................................................................262 2.6.3 Gamete collection...........................................................................263 2.6.4 Prefertilization treatments................................................................263 2.6.5 Fixation and permeabilization..........................................................263 2.6.6 Postfixation washes and blocking.....................................................263 2.6.7 Primary antibody preparation...........................................................263 2.6.8 Secondary antibody preparation.......................................................264 2.6.9 Mounting and observing specimen...................................................264 2.7 Visualizing the Vegetal Cortical Domain Using Fluorescently Tagged Dsh...264 2.7.1 Overview........................................................................................264 2.7.2 Protamine sulfate-coated dish preparation........................................264 Methods in Cell Biology, Volume 150, ISSN 0091-679X, https://doi.org/10.1016/bs.mcb.2019.01.001 © 2019 Elsevier Inc. All rights reserved.
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2.7.3 Mouth pipette preparation...............................................................265 2.7.4 Injection solutions and micropipette preparation...............................265 2.7.5 Gamete collection...........................................................................266 2.7.6 Removing jelly coat.........................................................................266 2.7.7 Microinjection of sea urchin zygotes.................................................266 2.7.8 Observation under microscope........................................................266 Acknowledgments..................................................................................................266 References............................................................................................................267 Glossary................................................................................................................268
Abstract During development metazoan embryos have to establish the molecular coordinates for elaboration of the embryonic body plan. Typically, bilaterian (bilaterally symmetric animals) embryos establish anterior-posterior (AP) and dorsal-ventral (DV) axes, and in most cases the AP axis is established first. For over a century it has been known that formation of the AP axis is strongly influenced by the primary axis of the egg, the animal-vegetal (AV) axis. The molecular basis for how the AV axis influences AP polarity remains poorly understood, but sea urchins have proven to be important for elucidating the molecular basis for this process. In fact, it is the first model system where a critical role for Wnt signaling in specification and patterning the AV and AP axis was first established. One current area of research is focused on identifying the maternal factors that regulate localized activation of Wnt/βcatenin signaling at the vegetal pole during development. An essential tool for this work is the means to identify the AV polarity in oocytes and eggs. This permits investigation into how polarity is established and allows development of experimental strategies to identify maternal factors that contribute to and control axial polarity. This chapter provides protocols to accomplish this in sea urchin eggs and early embryos. We describe simple methods to visualize polarity including direct observation of eggs and oocytes, using a microscope for overt morphological signs of polarity, and more extensive methods involving localization of known factors indicative of inherent embryonic polarity, such as the upstream regulators of the Wnt/β-catenin pathway.
1 INTRODUCTION Most animal eggs have a primordial polarity referred to as the Animal-Vegetal (AV) axis that strongly influences early pattern formation during embryogenesis. The underlying mechanism for this process involves maternal determinants that are distributed asymmetrically along the AV axis. During cleavage stages these determinants are differentially inherited by blastomeres that arise at the different poles of the embryo and they impart cells with early specification cues. Typically, in bilaterian (bilaterally symmetrical) animals these specification cues instruct vegetal polederived cells to assume endomesodermal cell fates and instruct animal pole-derived
1 Introduction
blastomeres to assume ectodermal cell fates (Martindale, 2005; Martindale & Hejnol, 2009). In most animals, however, the molecular identity of the maternal determinants that specify cell fates along the AV axis remains largely uncharacterized. In contrast, work done in sea urchins has indicated that maternal factors active at the vegetal pole locally induces the highly conserved Wnt/β-catenin signaling pathway to initiate early pattern formation (Emily-Fenouil, Ghiglione, Lhomond, Lepage, & Gache, 1998; Logan, Miller, Ferkowicz, & McClay, 1999; Wikramanayake, Huang, & Klein, 1998). This pathway directly specifies endomesoderm at the vegetal pole, and then both directly and indirectly restricts neural fates to the anterior of the embryo, thus polarizing the embryo along an anterior-posterior (AP) axis (Range, 2014; Wikramanayake et al., 1998). This mechanism appears conserved, and it is now known that activation of Wnt/β-catenin at the future posterior pole is an early critical event for development in embryos of many bilaterian species (Petersen & Reddien, 2009). However, how the Wnt/β-catenin pathway is locally activated in early embryos is still poorly understood in most species. Researchers have begun to exploit embryological features of sea urchin eggs and early embryos to begin to identify the upstream maternal regulators of Wnt/β-catenin signaling in early embryos (Croce et al., 2011; Peng & Wikramanayake, 2013; Weitzel et al., 2004). The sea urchin embryo has provided critical insight into the specification and patterning of the AV axis starting with the classical work of Theodor Boveri who showed that the primary egg axis predicted where the germ layers are specified during embryogenesis (Boveri, 1901). Boveri’s experiments, which exploited a natural pigment band present toward the vegetal end of eggs of the Mediterranean Sea urchin Paracentrotus lividus to distinguish the animal pole from the vegetal pole, demonstrated that the pigment band was consistently inherited by the cells that formed the endomesoderm (Boveri, 1901). Sven Horstadius followed up on these observations with a set of experiments to ascertain if determinants for endomesoderm were also localized in the unfertilized egg. Horstadius used the pigment band as a marker for the vegetal end to bisect the egg equatorially. He then fertilized each half (Refer to Fig. 1B) and showed that while the vegetal half developed into an almost normal larva with all three germ layers, the animal halves developed into arrested embryos that were referred to as “dauerblastulae” (Horstadius, 1973). These results clearly demonstrated that determinants for endomesoderm were localized to the vegetal half of the unfertilized sea urchin egg (Horstadius, 1973). From the work done in several labs we now know that these determinants are most likely factors that locally activate the Wnt/β-catenin signaling pathway to specify endomesoderm in vegetal pole blastomeres (Croce et al., 2011; Emily-Fenouil et al., 1998; Logan et al., 1999; Peng & Wikramanayake, 2013; Weitzel et al., 2004; Wikramanayake et al., 1998). These maternal determinants have still not been identified, but with the advent of increasingly sensitive methods for identifying differentially expressed RNAs and proteins using methods such as RNA sequencing and mass spectrometry the time is now right for exploiting the beneficial embryological features of sea urchin eggs and embryos to identify the elusive localized determinants.
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FIG. 1 Visualization of the jelly canal in the sea urchin egg. (A) An egg from H. pulcherrimus stained with India ink shows the jelly canal at the animal pole. (B) An equatorial dissection of a H. pulcherrimus egg using the jelly canal as a landmark for egg polarity. Images taken from Maruyama, Y. K., Nakaseko, Y., & Yagi, S. (1985). Localization of cytoplasmic determinants responsible for primary mesenchyme formation and gastrulation in the unfertilized egg of the sea urchin Hemicentrotus pulcherrimus. Journal of Experimental Zoology, 236, 155–163.
Here, we provide protocols that can be used for visualization of the AV axis of the oocyte/egg and early embryos of sea urchins. These protocols allow investigators to identify the molecules underlying AV axis polarity in sea urchins using experimental strategies that exploit the advantages of experimental embryology and contemporary molecular methods in urchins.
2 METHODS OF VISUALIZING OOCYTE AND EGG POLARITY 2.1 OBSERVATION OF THE GERMINAL VESICLE AND POLAR BODIES Since the site of polar body release during meiosis marks the animal pole of metazoan oocytes (Martindale, 2005; Martindale & Hejnol, 2009), the position of the germinal vesicle or locating the polar bodies on the oocyte/egg surface immediately prior to meiosis are reliable ways to identify the animal pole of sea urchin oocytes. It is not known when the AV axis is initially polarized during oogenesis, but experimental observations indicate that this polarity is already specified prior to the first meiosis. Early in oogenesis the germinal vesicle of small primary oocytes is initially located centrally in the cell (Peng & Wikramanayake, 2013; Wessel, Voronina, & Brooks, 2004). Staining for the centrosome at this stage has shown that it is already present at the animal pole and staining for the Disheveled protein—a central
2 Methods of visualizing oocyte and egg polarity
upstream regulator of the Wnt signaling pathway—has shown that it has already begun to accumulate in a domain at the vegetal cortex termed the vegetal cortical domain (VCD) (Peng & Wikramanayake, 2013). Prior to the first meiosis the germinal vesicle moves to the animal pole of the oocyte, and in some echinoderms this has been shown to be a microtubule-dependent process (Miyazaki, Kato, & Nemoto, 2005; Schroeder, 1980a). Once the germinal vesicle has moved to the animal pole it is relatively easy to identify the AV polarity of the oocytes using light microscopy. This polarity is particularly striking in echinoderms such as asteroids and holothuroids, where one can collect large primary oocytes for experimentation and induce their maturation using 1-methyladenine (Foltz, Adams, & Runft, 2004; Smiley, 1990). Echinoid oocytes are more challenging to work with, and no consistent method is available to carry out in vitro maturation of these oocytes (Wessel et al., 2004). Echinoids spawn mature eggs that have completed meiosis, so other methods are needed to visualize the primary egg axis in these echinoderms. In P. lividus, the female pronucleus remains at the animal pole of the mature egg following meiosis and hence can be used as a reliable marker for the AV axis (Di Carlo, Romancino, Ortolani, Montana, & Giudice, 1996). However, this trait does not appear to be shared with other echinoids since in several other species the location of the pronucleus in mature eggs is random and does not correlate with the AV axis (Goda, Inoue, & Mabuchi, 2009; Maruyama, Nakaseko, & Yagi, 1985; Peng & Wikramanayake, 2013). However, Schroeder (1980b) showed that by careful collection of spawned eggs from Strongylocentrotus droebachiensis and observing them using Nomarski Differential Interference Contrast microscopy it is possible to identify the animal pole by the position of the polar bodies released during meiosis. However, the polar bodies are small and are easily displaced during spawning. Hence, this method is not particularly useful for manipulative experiments where one has to have absolute certainty about the polarity of the egg. In species where the AV axis can be reliably identified using the position of the germinal vesicle or the female pronucleus, it is possible to use a drawn out glass needle and bisect the egg equatorially, or dissect one of the poles to determine the importance of factors localized to the animal or vegetal cortex for normal development (Croce et al., 2011; Di Carlo et al., 1996; Horstadius, 1973; Maruyama et al., 1985). Additionally, drawn out glass needles can be used to separate the egg poles and collect them for identifying localized factors using sensitive methods such as RNA sequencing.
2.2 STAINING THE JELLY CANAL In addition to the position of polar body release, the jelly canal, a structure present in the jelly coat surrounding sea urchin eggs, has also been implicated as a definitive marker of the animal pole (Boveri, 1901; Schroeder, 1980b). Not much is known about how the jelly canal is formed, but it is observed to always align with the site
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of the polar body release (Schroeder, 1980b). The jelly canal is present in the jelly coat as a slender, conical channel that can be readily visualized by directly spawning females into a dense suspension of Sumi ink and seawater (Goda et al., 2009; Maruyama et al., 1985; Peng & Wikramanayake, 2013; Schroeder, 1980b). The Sumi ink suspension can be made by placing a few drops of seawater on the frosted end of a glass microscopy slide and rubbing a dry cake of Sumi Japanese ink against the frosted glass. For this method to work, it is critical to have a dense solution of Sumi ink in seawater and the particles have to be small enough to pass into the jelly canal. The eggs also have to be directly spawned into the Sumi ink solution, or they have to be collected as they are being released from the genital pores and quickly transferred to the Sumi ink solution. After a 5–10 min incubation, gently wash the eggs twice with seawater to dilute the ink suspension and observe the eggs under a microscope (Fig. 1; Goda et al., 2009; Peng & Wikramanayake, 2013; Schroeder, 1980b). Note that if eggs are spawned directly into seawater prior to adding them to the Sumi ink suspension you will not be able to visualize the jelly canal since the expanding jelly layer will squeeze this canal shut. In an alternate protocol, Goda et al. (2009) used black ink (Bokuju; Kuretake Co, Japan) that was dialyzed for 6 h against seawater. Lytechinus variegatus and Lytechinus pictus eggs were directly placed in a 1:4 ratio of the black ink and seawater for 1 min, rinsed with seawater and observed using light microscopy. By identifying the animal pole using the jelly canal as a marker, one can use drawn out glass needles to cut animal and vegetal halves and use them for experiments as needed (Fig. 1; Maruyama et al., 1985). One potential use of this method could be to carry out RNA sequencing of isolated animal and vegetal pole fragments to identify molecules enriched at the two egg poles.
2.3 USING THE PIGMENT BAND TO IDENTIFY THE VEGETAL POLE Classical embryologists have exploited the asymmetric distribution in the egg of organelles such as pigment granules to identify AV polarity in sea urchins. Several species of sea urchins produce eggs with pigment granules that are usually evenly distributed in the cortical region of the mature egg. In some species the cortical band of pigment granules is retracted from the vegetal pole before the metaphase of the fourth mitotic cycle leaving behind a clear cytoplasm that is inherited by the 16-cell stage micromeres (Schroeder, 1980b). Interestingly, Boveri (1901) showed that there is a pigment granule-free region toward the vegetal pole of P. lividus eggs, and hence, this pole could be identified by the “pigment band” located toward the vegetal end. Schroeder (1980b) reevaluated Boveri’s work and concluded that only 20% of P. lividus eggs displayed the pigment band prior to fertilization. A selection of Boveri’s original figures is shown in Fig. 2. The pigment band in P. lividus has been used by researchers to orient the AV axis of unfertilized eggs and carry out various embryological manipulations to ask questions about the distributions of determinants along the primary egg axis (Croce et al., 2011; Di Carlo et al., 1996; Horstadius, 1973). In one of these studies Croce et al. (2011) showed that fusing a vegetal cortex fragment to the animal pole membrane was sufficient to induce
2 Methods of visualizing oocyte and egg polarity
FIG. 2 Visualization of the pigment band and its fate during embryogenesis in Paracentrotus lividus. During oogenesis the pigment appears uniformly distributed but accumulates as a band toward the vegetal pole after oocyte maturation. This pigment band consistently gets incorporated into the vegetal cells during development. € von ovocyte, ei und larve des Strongylocentrotus lividus. Images taken from Boveri, T. (1901). Die polaritat €cher, 14, 630–653. Zoologische Jahrbu
ectopic endoderm in animal pole-derived blastomeres demonstrating the functional importance of the VCD for regulating polarity and patterning in the sea urchin embryo.
2.4 VISUALIZING THE MICROTUBULE ORGANIZING CENTER USING IMMUNOCYTOCHEMISTRY During oogenesis in echinoderms, the germinal vesicle moves from the center of the primary oocyte to the animal pole where meiosis takes place (Smiley, 1990). In some echinoderms the movement of the germinal vesicle to the animal pole prior to the
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FIG. 3 The Disheveled protein accumulates in a vegetal cortical domain early in oogenesis and is tightly embedded in the vegetal cortex. (A) Primary oocyte stained with an anti-Dsh antibody and phalloidin. The nascent accumulation of Dsh can be seen at the vegetal pole (arrow). At this time the centrosome is present at the animal pole (asterisk). (B) A mature egg stained with an anti-Dsh antibody. The striking accumulation of Dsh can be seen at the vegetal pole (arrow). Dsh protein is also found distributed throughout the egg cytoplasm. (C) Isolated egg cortices stained with the anti-Dsh antibody. The arrows point to the Dsh embedded in the vegetal cortical domain. Adapted from Peng, C. J. & Wikramanayake, A. H. (2013). Differential regulation of disheveled in a novel vegetal cortical domain in sea urchin eggs and embryos: Implications for the localized activation of canonical Wnt signaling. PLos One, 8(11), e80693.
first meiosis depends on microtubules (Miyazaki et al., 2005). The microtubules for this process are assembled by the microtubule organizing center (MTOC) which appears to assemble early during oogenesis (Peng & Wikramanayake, 2013; Smiley, 1990). In sea urchins, the maternal MTOC disappears following the second meiotic reduction. The sperm centrosome brought in at fertilization undergoes a duplication event and provides the MTOCs for mitosis (Schatten, Schatten, Mazia, Balczon, & Simerly, 1986). Therefore, using immunocytochemistry to localize MTOC proteins such as gamma-tubulin can be an effective way to detect AV polarity in oocytes. The MTOC in sea urchin oocytes can also be detected using phalloidin (Fig. 3A; Peng & Wikramanayake, 2013). For additional information regarding cytoskeleton labeling, reference Methods in Cell Biology, Volume 74, Chapter 16, Section IV.
2.4.1 Overview For protocols regarding the collection of gametes from sea urchins refer to chapter “Procuring animals and culturing of eggs and embryos” by Adams et al. Here, we provide the protocol for preparing oocytes, eggs and embryos for immunofluorescence staining.
2.4.2 Materials and solutions a. Buffers 10 Phosphate-Buffered Saline (PBS, Bio-Rad) Dilute to 1 before use
2 Methods of visualizing oocyte and egg polarity
b. Fixative 4% Paraformaldehyde (PFA, EMS) 1 PBS Add 10 mL 16% PFA in 30 mL of 1 PBS. PFA is a potential carcinogen and should be handled with gloves at all times, and fixation should be performed under a ventilated hood. In our lab we never bring fixatives into the room where we do embryo culturing and embryology. PFA should always be prepared fresh. c. Postfixation Solutions 100% Acetone (at 4 °C) d. Blocking Buffer PBST (BSA, Tween 20) 0.01% BSA, powder, stored at 4 °C 0.1% Tween 20 1 PBS For 100 mL PBST, use 0.0100 g BSA, 100 μL of Tween 20, and 100 mL 1 PBS. e. Staining Dilutions Primary antibodies stored at 4 °C Mouse anti-γ-tubulin antibody (1:50, Abcam). Secondary antibodies stored at 4 °C Use an antimouse secondary conjugated with an appropriate fluorophore. (e.g., Alexa Fluor 532 goat antimouse IgG (Invitrogen) or Alexa Fluor 568 donkey antimouse IgG (Invitrogen), or Alexa Fluor 635 goat antimouse IgG (Invitrogen)). Cytoskeleton labeling (F-actin) Fluorescein phalloidin (Invitrogen), stored at 20 °C, working dilution 3:100. e.g., 3 μL in 100 μL of Blocking Buffer. Note: Fluorescein phalloidin alone is effective in staining the centrosome in sea urchin oocytes (Peng & Wikramanayake, 2013). Therefore, if desired, one may forego using antibodies, and simply use phalloidin. Nuclear labeling 40 ,6-Diamidino-2-phenylindole, dilactate (DAPI, Invitrogen), blue, stored at 20 °C, working dilution 1:1000 to a final concentration of 300 nM. f. Mounting Solutions 50% Glycerol in 1 PBS
2.4.3 Gamete collection Refer to Chapter 1.
2.4.4 Prefertilization treatments The sea urchin egg has a thin egg envelope called the vitelline envelope (VE) that is attached to the plasma membrane via proteinaceous structures called vitelline posts (Kidd, 1978). In our experience, antibodies will freely pass through the VE of fixed,
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unfertilized eggs. At fertilization, however, the VE is converted into a fertilization envelope (FE) that is cross-linked by cortical granule enzymes and it becomes impenetrable to large proteins such as antibodies. The embryo will emerge from the FE at the late blastula stage by secreting a hatching enzyme. Hence, for successful antibody staining of any stage after fertilization up to the hatching blastula stage FEs need to be removed. To do this, cross-linking of the FE proteins is prevented by fertilizing eggs in ASW with 0.5 mM 3-amino-1,2,4-triazole (ATA, Sigma). Some labs prefer to use 10 mM para-amino benzoic acid (PABA, Sigma) to prevent crosslinking of the FE following fertilization (Logan et al., 1999). When embryos are collected for fixation at a particular stage, the egg envelopes can be removed by passing the embryos through a mesh strainer (BD Falcon). We typically use a 70 μm strainer for S. purpuratus and a 100 μm strainer for L. variegatus and L. pictus. Observe the eggs under a stereomicroscope to determine efficient envelope removal. If envelopes are still present, the eggs/embryos may be passed through the strainer again and usually twice is sufficient to strip the envelopes off embryos.
2.4.5 Fixation and permeabilization Once the desired stage is reached, embryos can be fixed and processed in a 1.5-mL microcentrifuge tube, or for larger numbers of embryos, 15 mL conical tubes. These can be gently centrifuged between washing and incubation steps. Be aware that embryos tend to stick to the side of plastic tubes when wash buffers are void of detergents and when tubes are not spun down properly. Alternately, fixed embryos can be left to settle by gravity because they readily sink once exposed to fixative. Compact embryo sample volume should not exceed 50 μL in a 1.5 mL tube, or 500 μL in a 15 mL tube. Fix embryos by adding 4% paraformaldehyde (PFA) solution in phosphate-buffered saline (PBS) pH 7.4. The amount of fixative added should be at least 10 the amount of sample. Samples can be fixed for 20–60 min at room temperature. The embryos should settle to the bottom during the fixation period. Remove and discard the fixative into a contained waste receptacle. Add enough ice-cold acetone to cover the sample (1 mL in a 1.5 mL tube) and incubate on ice for 10 min. This step permeabilizes the membranes of the embryo to increase efficiency of antibody penetration into cells. Following the 10 min acetone treatment, gently centrifuge the embryos using a hand centrifuge or in a centrifuge with a swing bucket rotor and then decant the acetone solution. Wash the embryos three times using 1 PBS solution (5 min incubations between washes should suffice). Note: Going forward, the protocol will be specific to using a 1.5 mL tube during this procedure.
2.4.6 Postfixation washes and blocking Transfer the embryos to a new 1.5 mL tube and allow them to settle on ice. Remove and discard the 1 PBS solution. Add Blocking Buffer up to 500 μL and incubate for 10 min. Remove and discard the Blocking Buffer solution. Repeat the addition and removal of 500 μL of Blocking Buffer three times.
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2.4.7 Primary antibody preparation Remove the Blocking Buffer from the sample until 100 μL is left. Add 100 μL of diluted 1° antibody solution to the sample. Incubate at room temperature for 1 h. Remove 1° antibody solution. Wash embryos with Blocking Buffer for 15 min on ice. Repeat the wash step two more times for a total of 3 washes. You can use a pipette to aspirate and expel the embryos gently in and out of the pipette tip to help remove excess primary antibody from the embryos. For smaller numbers of embryos one can reduce the amount of antibody solution used. Additionally, when working with very small numbers of embryos one can use Terasaki plates (Greiner Bio-One) to avoid losing embryos during the fixing and staining procedure.
2.4.8 Secondary antibody preparation Remove the blocking buffer following the third wash. PBST can be used to dilute the 2° antibody. Add diluted 2° antibody solution to sample. Incubate at room temperature for 45 min. To stain nuclei and the cell cortex, add DAPI (1:1000) and 6 μL of fluorescein phalloidin (3:100) for approximately 20 min. Longer incubation periods do not increase signal intensity, but instead increase background. There is an excellent discussion on some of the challenges associated with using phalloidin to stain F-actin in Strickland et al. (2004). Remove 2° antibody solution. Wash embryos with 1 PBS for 15 min on ice. Repeat the wash step two more times, for a total of 3 washes.
2.4.9 Mounting and observing specimen Each lab has their own methods for mounting fixed specimens for observation. Fixed samples can be mounted on slides, cover slipped and sealed with clear nail polish. The eggs and embryos should be protected from being distorted by using either clay “legs” or strips of coverslip glass (cut using a diamond knife) placed between the slide and the coverslip. The Dsh staining can be seen as a crescent at the vegetal pole (Fig. 3B). The pool of Dsh protein that accumulates at the vegetal pole is tightly embedded in the vegetal cortex and will remain attached even after isolation of the vegetal cortex (Fig. 3C).
2.5 VISUALIZING THE VEGETAL CORTICAL DOMAIN One exciting recent discovery in sea urchins is that oocytes and eggs have a distinct cytoarchitectural domain, termed the vegetal cortical domain (VCD), that is located at the vegetal pole cortex (Fig. 3; Peng & Wikramanayake, 2013). Microsurgical excision of the VCD blocks activation of the Wnt/β-catenin pathway at the vegetal pole of early embryos and leads to embryos that do not form endomesodermal tissues (Croce et al., 2011). Hence, the VCD appears to specify AV polarity of the egg and plays a critical role in activating Wnt/β-catenin signal transduction in vegetal pole blastomeres (Peng, Wang, & Wikramanayake, 2017; Peng & Wikramanayake, 2013). Moreover, the VCD has been shown to accumulate Disheveled (Dsh), a central regulatory protein in the Wnt signaling pathway (Fig. 3; Peng & Wikramanayake, 2013). In the sea
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urchin embryo, the Wnt/β-catenin pathway plays a critical role in specifying endomesoderm in cleavage stage embryos and the Dsh protein is required for the asymmetric activation of the pathway at the vegetal pole (Logan et al., 1999; Weitzel et al., 2004; Wikramanayake et al., 1998). The Dsh protein is expressed throughout the cytoplasm of the egg and in all cells of the early embryo. However, it is a differentially modified form of Dsh that accumulates at the VCD of unfertilized eggs and produces the domain inherited by the vegetal cells that nuclearize β-catenin in the early embryo (Peng & Wikramanayake, 2013). Staining of isolated egg cortices has shown that Dsh is embedded in the VCD confirming that the sea urchin egg cortex has a cytoarchitectural asymmetry at the vegetal pole (Fig. 3C), that appears to function as a scaffold for the localized activation of Wnt signaling (Peng & Wikramanayake, 2013). The ability to isolate large numbers of egg cortices and the availability of excellent anti-sea urchin Dsh antibodies facilitate the identification of the molecular basis in the VCD of sea urchins and the mechanism for the asymmetric activation of Wnt/β-catenin signaling at the vegetal pole of early embryos.
2.6 VISUALIZING THE VEGETAL CORTEX BY IMMUNOCYTOCHEMISTRY 2.6.1 Overview Visualization of the VCD can be achieved through immunofluorescent staining of Dsh using anti-Dsh antibodies or by microinjection of an mRNA coding for Dsh fused to a fluorescent protein (Peng & Wikramanayake, 2013; Weitzel et al., 2004). Three affinity-purified anti-Dsh polyclonal antibodies have been generated against three distinct epitopes on the L. variegatus Dsh protein (Peng & Wikramanayake, 2013). A His-tagged fusion protein of amino acids 1–101 of LvDsh protein including the DIX domain was used as one antigen to immunize rabbits. In addition, polyclonal antibodies were also produced using synthetic peptides corresponding to epitopes at the N-terminal (NH2-CASVTTDTRGDSQLPPERTG-COOH) and C-terminal (NH2CMVPMMPRQLGSVPEDLSGS-COOH) regions of Dsh as antigens. These polyclonal antibodies were affinity-purified using the immunizing antigens by Bethyl Labs (Montgomery, TX). The antibodies generated against the DIX domain, the N-terminal epitope, and the C-terminal epitope are referred to as anti-SUDshDIX, anti-SUDsh-N, and anti-SUDsh-C, respectively. All three antibodies have been successfully used in all sea urchin species tested thus far including S. purpuratus, L. variegatus, L. pictus, Paracentrotus lividus, Hemicentrotus pulcherrimus, and others. The anti-SUDshDIX antibody also cross reacts with Dsh proteins from other species including the sea anemone Nematostella vectensis (Lee, Kumburegama, Marlow, Martindale, & Wikramanayake, 2007). The anti-Dsh antibodies are available on request from the Wikramanayake lab. These antibodies will be available for use until they are depleted.
2.6.2 Materials and solutions i. Buffers Use same buffers as detailed in Section 2.4.
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ii. Fixative Use paraformaldehyde as a fixative as detailed in Section 2.4. iii. Postfixation solutions 100% Acetone. iv. Blocking buffer Use blocking buffer as detailed in Section 2.4. v. Antibody dilutions Primary Dsh antibodies stored at 4 °C Anti-SUDsh-C: S. purpuratus 1:400; L. pictus 1:200; L. variegatus 1:200 Anti-SUDsh-N: S. purpuratus 1:200; L. pictus 1:100; L. variegatus 1:100 Anti-SUDshDIX: S. purpuratus 1:400; L. pictus 1:400; L. variegatus 1:400 Secondary antibodies stored at 4 °C Use an antirabbit secondary antibody conjugated with an appropriate fluorophore as mentioned in Section 2.4.2, step e. Specifications regarding cytoskeleton and nuclear labeling can also be referenced in Section 2.4.2, step e. vi. Mounting solutions 50% Glycerol in 1 PBS. 0.1% Sodium azide.
2.6.3 Gamete collection Use same procedures as detailed in Section 2.4.
2.6.4 Prefertilization treatments Use same procedures as detailed in Section 2.4.
2.6.5 Fixation and permeabilization Use same procedures as detailed in Section 2.4.
2.6.6 Postfixation washes and blocking Use same procedures as detailed in Section 2.4.
2.6.7 Primary antibody preparation Remove the blocking buffer from the sample until 100 μL is left. Add 100 μL of diluted 1° antibody solution to the sample. Incubate at room temperature for 1 h. Remove 1° antibody solution. Wash embryos with blocking buffer for 15min on ice. Repeat the wash step two more times for a total of 3 washes. You can use a pipette to aspirate and expel the embryos gently in and out of the pipette tip to help remove excess primary antibody from the embryos. For smaller numbers of embryos one can reduce the amount of antibody solution used. Additionally, when working with very small numbers of embryos one can use Terasaki plates to avoid losing embryos during the fixing and staining procedure.
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2.6.8 Secondary antibody preparation Remove the blocking buffer following the third wash. Add diluted 2° antibody solution to sample. Incubate at room temperature for 45 min. To stain nuclei and the cell cortex add DAPI (1:1000) and 6 μL of fluorescein phalloidin (3:100) for approximately 20 min. Longer incubation periods do not increase signal intensity, but instead increase background. Remove 2° antibody solution. Wash embryos with 1 PBS for 15 min on ice. Repeat the wash step two more times, for a total of 3 washes.
2.6.9 Mounting and observing specimen See Section 2.4.9.
2.7 VISUALIZING THE VEGETAL CORTICAL DOMAIN USING FLUORESCENTLY TAGGED Dsh In marine invertebrate embryos expression of synthetic messenger RNAs has become a useful approach for studying gene function. In sea urchins, methods have been developed to carry out rapid microinjection to express RNAs coding for proteins of interest fused to a fluorescent protein to determine subcellular localization of the protein, and wild-type and various mutant proteins to test for protein function. For further information, reference chapter “Microinjection of oocytes and embryos with synthetic mRNA encoding molecular probes” by von Dassow et al.
2.7.1 Overview Upon fertilization in sea urchin eggs, activation leads to a rapid increase in the translation of proteins. Therefore, mRNAs injected into eggs or early blastomeres are readily translated into their protein products. A cDNA coding for a protein of interest fused to a fluorescent protein can be subcloned into an expression vector, such as pCS2 +, and synthetic mRNA can be transcribed using a specific RNA polymerase. It is important to note that RNases are fairly ubiquitous and if there is any contamination of reagents this will lead to degradation of the RNA. Therefore, all materials, benchtops, and solutions must be RNase-free while working with RNA.
2.7.2 Protamine sulfate-coated dish preparation Materials: 60-mm plastic cell culture dish lids (Fisher Scientific) 1% (w/v) protamine sulfate solution: 0.5 g protamine sulfate salt, ddH2O to 50 mL Gather several 60 mm plastic cell culture dish lids in rows. The lids are used because they are shallower and this makes it easier to position the needle for microinjections. Add enough 1% (w/v) protamine sulfate solution to cover the bottom of the lid. Let them sit for 1 min and then transfer the solution to an additional set of lids, or save the solution in an appropriate container for future use. Wash the coated lids by placing them in a ddH2O filled beaker. When all the dishes have been coated and washed, rinse them one additional time in ddH2O and allow them to dry at room temperature.
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2.7.3 Mouth pipette preparation Materials: 1 mL Tuberculin Slip Tip Syringe (BD Biosciences) Mouthpiece (can be purchased from Drummond Scientific as an aspirator tube assembly, model # 2-000-000) (See text for alternate mouthpieces) Rubber tubing, 1/800 ID, 3/1600 OD (Cole Parmer Instrument Company) Rubber pipette bulbs Mouth pipettes are used to move eggs and embryos during the injection process. These mouth pipettes can be made by connecting a mouthpiece to a glass micropipette using rubber tubing. In order for the glass micropipette to securely fit into the tubing, a pipette adapter must be made. Start at the tip of a 1 mL Tuberculin Slip Tip syringe and cut a length of 3 cm. Cut a rubber pipette bulb approximately 2.5 cm from the open end. Insert the wide end of the syringe tip into the cut end of the rubber pipette bulb. Use lab labeling tape to secure and seal the end of the rubber bulb to the end of the syringe. Next, insert the syringe tip of the pipette adapter into one end of a piece of 1/800 rubber tubing cut to 2–3 ft long. Insert the mouthpiece into the opposite end of the tubing. An alternative mouth pipette can be created by taking the rubber tubing of the dimensions mentioned above and inserting the pointed end of a p200 pipette tip into one end and the pointed end of a p1000 pipette tip into the other open end of the tubing. The glass pipette (see Section 2.7.4) can be inserted into the open end of the p1000 pipette and the other end can be used as the mouth piece.
2.7.4 Injection solutions and micropipette preparation Materials: Micropipette puller (similar to Sutter Instrument P-97 Flaming Brown) 1.0/0.75 mm (OD/ID) thin-wall single-barrel standard borosilicate glass tubing (World Precision Instruments) P2 pipetter 0.2 μm, 4 mm syringe driven filter (Millipore, Billerica, MA) 900 Glass Pasteur pipettes Bunsen burner Diamond knife The mRNA injection solutions are always prepared using nuclease-free water and 100% sterile glycerol. The final concentration of glycerol in injection solution is 40% (v/v) if you are using a continuous flow pressure injection system. It is good practice to filter the RNA solution to remove any particulate material that could clog the injection needles. We typically filter the injection solution using a 0.2 μm, 4 mm syringe driven filter (Millipore, Billerica, MA). Filtered mRNA solutions can be stable for several months at 70 °C. To fill injection needles with RNA solution, use a P2 pipetter and load needles by adding 0.5 μL of injection solution to the open end of the needle. Store filled needles at 4 °C for at least 15 min prior to use to allow the RNA solution to move to the needle tip by capillary action. Needles not in use should
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be stored at 20 °C while injecting. If filled needles need to be stored for a longer period of time (2–3 days) store them at 70 °C. Dejellied eggs are prepared for microinjection by lining them up at the bottom of a protamine sulfate-coated dishes. The eggs can be “rowed” using a mouth pipette. Start with a 900 glass Pasteur pipette. Heat the pipette over a Bunsen burner at the point where the wider bore of the pipette narrows to the smaller bore until it softens. Remove from the flame, and quickly pull the narrow end of the glass pipette horizontally to elongate the glass. The size of the bore of the pipette tip can be controlled by varying the time between removing the hot glass from the flame and beginning the pulling motion to lengthen the glass. If the pipette is pulled too soon, it will break or will form very narrow, unusable pipettes. If the glass is allowed to cool for too long, it will have a large bore size, which will make it difficult to control egg dispensing. Typically, you will want the pipette tip to be just large enough for one egg at a time to fit through the opening, making it easier to control the flow of eggs during mouth pipetting. Cut excess glass off using a diamond knife, leaving a 3–4 cm tip. To smoothen the tip before use, heat very briefly at the base of the flame from a Bunsen burner. Fasten the glass micropipette to the open end of the pipette adapter on the mouth pipette tubing.
2.7.5 Gamete collection Refer to Section 2.4.3.
2.7.6 Removing jelly coat Refer to Chapter 1.
2.7.7 Microinjection of sea urchin zygotes Refer to Chapter 9.
2.7.8 Observation under microscope For live imaging one can use glass bottom dishes similar to those available from MatTek Co. Use of the open dish allows a researcher to easily transfer the imaged sample to another dish for continued culture for later observations. These imaging dishes can be easily crafted by cutting a hole at the bottom of a small Petri dish and attaching a cover slip to the bottom using an adhesive or using vacuum grease. There is an excellent discussion on different ways to mount and image live embryos in Strickland et al. (2004) that readers should consult if they are interested in extended live imaging of embryos.
ACKNOWLEDGMENTS The work in AHW’s lab is supported by grant IOS1257967 from the National Science Foundation.
References
REFERENCES Boveri, T. (1901). Die polarit€at von ovocyte, ei und larve des Strongylocentrotus lividus. € Zoologische Jahrbucher, 14, 630–653. Croce, J., Range, R., Wu, S. Y., Miranda, E., Lhomond, G., Peng, J. C., et al. (2011). Wnt6 activates endoderm in the sea urchin gene regulatory network. Development, 138, 3297–3306. Di Carlo, M., Romancino, D. P., Ortolani, G., Montana, G., & Giudice, G. (1996). “BEP” RNAs and proteins are situated in the animal side of sea urchin unfertilized egg, which can be recognized by female pronuclear localization. Biochemistry and Biophysics Research Communications, 229, 511–517. Emily-Fenouil, F., Ghiglione, C., Lhomond, G., Lepage, T., & Gache, C. (1998). GSK3beta/ shaggy mediates patterning along the animal-vegetal axis of the sea urchin embryo. Development, 125, 2489–2498. Foltz, K., Adams, N., & Runft, L. (2004). Echinoderm eggs and embryos: Procurement and culture. In C. Ettensohn, G. Wray, & G. Wessel (Eds.), Development of sea urchins, ascidians, and other invertebrate deuterostomes: Experimental approaches: Vol. 74. Methods in cell biology (pp. 39–74). San Diego: Elsevier. Goda, M., Inoue, S., & Mabuchi, I. (2009). Calyculin-A induces cleavage in a random plane in unfertilized sea urchin eggs. Biological Bulletin, 216, 40–44. Horstadius, S. (1973). Experimental embryology of echinoderms. Oxford: Clarendon Press. Kidd, P. (1978). The jelly and vitelline coats of the sea urchin egg: New ultrastructural features. Journal of Ultrastructure Research, 64, 204–215. Lee, P. N., Kumburegama, S., Marlow, H. Q., Martindale, M. Q., & Wikramanayake, A. H. (2007). Asymmetric developmental potential along the animal-vegetal axis in the anthozoan cnidarian, Nematostella vectensis, is mediated by Dishevelled. Developmental Biology, 310, 169–186. Logan, C. Y., Miller, J. R., Ferkowicz, M. J., & McClay, D. R. (1999). Nuclear beta-catenin is required to specify vegetal cell fates in the sea urchin embryo. Development, 126, 345–357. Martindale, M. Q. (2005). The evolution of metazoan axial properties. Nature Genetics, 6, 917–927. Martindale, M. Q., & Hejnol, A. (2009). A developmental perspective: Changes in the position of the blastopore during bilaterian evolution. Developmental Cell, 17, 162–174. Maruyama, Y. K., Nakaseko, Y., & Yagi, S. (1985). Localization of cytoplasmic determinants responsible for primary mesenchyme formation and gastrulation in the unfertilized egg of the sea urchin Hemicentrotus pulcherrimus. Journal of Experimental Zoology, 236, 155–163. Miyazaki, A., Kato, K. H., & Nemoto, S. (2005). Role of microtubules and centrosomes in the eccentric relocation of the germinal vesicle upon meiosis reinitiation in sea-cucumber oocytes. Developmental Biology, 280, 237–247. Peng, J. C., Wang, L., & Wikramanayake, A. H. (2017). Origins of anterior-posterior polarity by localized activation of disheveled. Molecular Reproduction and Development, 84, 443. Peng, C. J., & Wikramanayake, A. H. (2013). Differential regulation of disheveled in a novel vegetal cortical domain in sea urchin eggs and embryos: Implications for the localized activation of canonical Wnt signaling. PLos One, 8(11), e80693. Petersen, C. P., & Reddien, P. W. (2009). Wnt signaling and the polarity of the primary body axis. Cell, 139, 1056–1068.
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Range, R. (2014). Specification and positioning of the anterior neuroectoderm in deuterostome embryos. Genesis, 52, 222–234. Schatten, H., Schatten, G., Mazia, D., Balczon, R., & Simerly, C. (1986). Behavior of centrosomes during fertilization and cell division in mouse oocytes and in sea urchin eggs. Proceedings of the National Academy of Sciences of the United States of America, 83, 105–109. Schroeder, T. E. (1980a). Expressions of the prefertilization polar axis in sea urchin eggs. Developmental Biology, 79(2), 428–443. Schroeder, T. E. (1980b). The jelly canal marker of polarity for sea urchin oocytes, eggs, and embryos. Experimental Cell Research, 128(2), 490–494. Smiley, S. (1990). A review of echinoderm oogenesis. Journal of Electron Microscopy Technique, 16, 93–114. Strickland, L., von Dassow, G., Ellenberg, J., Foe, V., Lenart, P., & Burgess, D. (2004). Light microscopy of echinoderm embryos. In C. Ettensohn, G. Wray, & G. Wessel (Eds.), Development of sea urchins, ascidians, and other invertebrate deuterostomes: Experimental approaches: Vol. 74. Methods in cell biology (pp. 39–74). San Diego: Elsevier. Weitzel, H. E., Illies, M. R., Byrum, C. A., Xu, R., Wikramanayake, A. H., & Ettensohn, C. A. (2004). Differential stability of B-catenin along the animal-vegetal axis of the sea urchin embryo mediated by disheveled. Development, 131, 2947–2956. Wessel, G. M., Voronina, E., & Brooks, J. M. (2004). Obtaining and handling echinoderm oocytes. In C. Ettensohn, G. Wray, & G. Wessel (Eds.), Development of sea urchins, ascidians and other invertebrate deuterostomes: experimental approaches: Vol. 74. Methods in cell biology (pp. 87–114). San Diego: Elsevier. Wikramanayake, A. H., Huang, L., & Klein, W. H. (1998). Beta-catenin is required for pattern formation along the animal-vegetal axis in the sea urchin embryo. Proceedings of the National Academy of Sciences of the United States of America, 95, 9343–9348.
GLOSSARY Animal-vegetal (AV) axis A primordial polarity found in oocytes and egg of most metazoans established during oogenesis that influences early pattern formation along the anteriorposterior vegetal axis in bilaterians. Bilaterian An animal displaying bilateral symmetry in their embryonic and adult body plan. Germinal vesicle The nucleus of an oocyte that has been arrested in prophase of meiosis 1 of oogenesis. Microtubule organizing center (MTOC) Structure in eukaryotic cells that nucleates microtubules responsible for cilia and flagella function and for nucleating mitotic and meiotic spindles. Vegetal cortical domain (VCD) A domain located at the vegetal pole cortex of the egg/ embryo where there is an accumulation of a differentially modified form of the Disheveled protein and is required for activation of Wnt signaling. Wnt signaling pathway An evolutionarily conserved signal transduction pathway found in all animals that strongly influences early pattern formation in early embryos.