CHAPTER
Sea urchin embryonic cilia
10
Robert L. Morris*,1, Victor D. Vacquier† *Department of Biology, Wheaton College, Norton, MA, United States Marine Biology Research Division, Scripps Institution of Oceanography, University of California San Diego, San Diego, CA, United States 1 Corresponding author: e-mail address:
[email protected]
†
CHAPTER OUTLINE 1 Introduction......................................................................................................236 2 Growth of Sea Urchin Cilia.................................................................................237 3 Regulation of Transcription and Translation During Cilia Regeneration...................238 4 Manipulation of Cilia Length Control...................................................................239 5 Cilia Retraction Versus Cilia Resorption..............................................................240 6 Isolation of Embryonic Cilia From S. Purpuratus Embryos......................................240 7 Adenylate Kinase and ATP Production in Isolated Embryonic Cilia.........................241 8 Current Research on Sea Urchin Embryo Cilia......................................................242 9 Conclusion.......................................................................................................245 Appendix...............................................................................................................245 A.1 Blocking Ciliogenesis in Sea Urchin Embryos by Antibody Microinjection...245 A.2 Growing “Animalized” Embryos Enriched for Long, Apical Tuft Cilia........246 References............................................................................................................246
Abstract Cilia are exceptionally complicated subcellular structures involved in swimming and developmental signaling, including induction of left-right asymmetry in larval stages. We summarize the history of research on sea urchin embryonic cilia. The high salt method to isolate cilia is presented first; methods to block cilia formation and to lengthen cilia are presented in the appendix. Evidence suggests that regenerated cilia may not be as physiologically perfect as those formed normally during embryogenesis. Sea urchin embryonic cilia are valuable models for studying molecular details of cilia assembly and differentiation as well as gene activation, cell signaling, and pattern formation during development.
Methods in Cell Biology, Volume 150, ISSN 0091-679X, https://doi.org/10.1016/bs.mcb.2018.11.016 © 2019 Elsevier Inc. All rights reserved.
235
236
CHAPTER 10 Sea urchin embryonic cilia
ABBREVIATIONS AK aPKC BMP CB CK DID1 DTT EGTA GFP GSTT IFT PMSF TF TGF-β
adenylate kinase atypical protein kinase C bone morphogenic protein ciliary band creatine kinase dillaoiol isoxazoline derivative 1 dithiothreitol ethylene glycol-bis(β-aminoethyl ether)-N,N,N0 ,N0 -tetraacetic acid green fluorescent protein glutathione transferase theta intraflagellar transport phenylmethylsulfonyl-fluoride transcription factor transforming growth factor-beta
1 INTRODUCTION Sea urchin cilia and flagella have served as powerful experimental models for a halfcentury. Studies of sea urchin sperm flagella led to discoveries such as the sliding filament mechanism of ciliary beating discovered almost 50 years ago and the “switch-inhibition” mechanism of beat formation and propagation (Gibbons & Fronk, 1972; Lin & Nicastro, 2018; Summers & Gibbons, 1971). Yet the diversity of forms and functions of cilia on developing sea urchin embryos, and the ease with which those cilia can be observed, manipulated, isolated, and dissected, makes sea urchin embryonic cilia a fruitful system for studying cilia generally, and the roles of cilia in animal development specifically. All eukaryotic cilia and flagella arise from a basal body that initiates, orients and maintains the growth of a microtubule-based axoneme covered by the plasma membrane. Most aquatic animal embryos are covered with cilia that exhibit a coordinated beat to propel the embryo forward with a circular spin. The axoneme of such motile cilia typically has a “9 + 2” architecture with 9 doublet microtubules that possess dynein motors surrounding 2 singlet microtubules that coordinate axonemal beat. In contrast, the axoneme of immotile sensory, or “primary,” cilia is typically “9 + 0” with 9 doublet microtubules that lack dynein motors and singlet microtubules (Ishikawa & Marshall, 2011; Stephens, 2008). Studies of sea urchin embryonic cilia have continued for approximately 60 years driven in part by the discovery that, if shed from the embryo, they regenerate rapidly and can do so many times (reviewed by Stephens, 1995, 2008). Each cell, or blastomere, of the sea urchin blastula grows one cilium, thus at hatching, there are a few hundred cilia per embryo. Cilia can be isolated by hypertonic treatment of ciliated blastulae, leaving the deciliated embryos motionless, but alive. In 10–20 min, the cilia begin to regenerate, and by 2–3 h both cilia length and embryo swimming have returned. Using current methods to study
2 Growth of sea urchin cilia
gene expression regulation, the regeneration of cilia could be experimentally approached with renewed enthusiasm. Interesting and outstanding questions include: what is the molecular signal that tells each blastomere to regrow a cilium; why is multicellularity required for cilia outgrowth; are there common features of ciliary gene activation during cilia regeneration across taxa; how is ciliary length regulated and how is the turnover of tubulin subunits in the axoneme controlled; are the regenerated cilia functionally identical to the first, normally grown crop? Here we provide an historical perspective for pursuing these and other critical questions uniquely suited to study in sea urchin embryos.
2 GROWTH OF SEA URCHIN CILIA The ability to regenerate cilia on deciliated embryos led to studies of the relative contributions of existing proteins versus newly formed proteins to cilia regeneration. The methods developed to hypertonically shock sea urchin embryos to shed their cilia were adapted from methods to induce deciliation in Tetrahymena (Childs, 1959; Watson & Hopkins, 1962). In one of the first sea urchin reports, using Paracentrotus lividus, blastulae were deciliated by a 3-min exposure to hypertonic seawater containing an extra 29.2 g NaCl/L. When returned to normal seawater and cultured in Actinomycin-D (to block RNA synthesis), or Puromycin (to block protein synthesis), cilia regenerated to their original length. These early results suggested that most of the proteins needed for ciliary regeneration were present as an already made pool that only required transport into and assembly within cilia (Auclair & Siegel, 1966). These results would be refined later (see below). Sea urchin embryos can be deciliated repeatedly and will still develop normally but appear to require multicellularity for reciliation. When Pseudocentrotus depressus blastulae were exposed to 1 M sodium acetate the cilia began detaching in 30 s and deciliation was compete in 3 min. When embryos were returned to normal seawater, nascent cilia began to beat in 50 min and achieved full-length in 2–3 h. P. depressus blastulae could repeat ciliary regeneration 5 times in 10 h and still develop normally into gastrulae and plutei (Iwaikawa, 1967), while gastrulae of the cold-water species Strongylocentrotus droebachiensis can regenerate cilia after 10 cycles of deciliation (Stephens, 1977). When blastulae were fragmented into clusters of cells, they still regenerated cilia, showing that components of the blastocoel are not required for regeneration (Iwaikawa, 1967). Furthermore, when deciliated blastulae were dissociated into single cells, the single cells did not regenerate cilia, but when reaggregated, cell clumps did regenerate cilia, indicating that multicellularity is a prerequisite for ciliogenesis (Amemiya, 1971). Observing the kinetics of cilia regeneration on sea urchin embryos provided clues to the mechanism of ciliogenesis. Burns (1973) showed that the kinetics of regrowth of sea urchin cilia are curvilinear and rates of regrowth decrease with successive rounds of deciliation. Decades before the discovery of intraflagellar transport (IFT) (Kozminski, Johnson, Forscher, & Rosenbaum, 1993), Burns (1973) suggested
237
238
CHAPTER 10 Sea urchin embryonic cilia
that rates of ciliogenesis might slow because the time required for proteins to reach the growing ciliary tip might increase or that the cytoplasmic pool of protein components might decrease. Both of these speculations stand in agreement with our current understanding of IFT and the well supported “balance point” model of cilia growth (Avasthi & Marshall, 2012; Marshall, Qin, Brenni, & Rosenbaum, 2005). The ability to microinject antibodies and mRNAs into sea urchin eggs and embryos allowed the first demonstration that normal cilia assembly on animal cells requires kinesin-2 (Morris & Scholey, 1997). When anti-kinesin-2 antibodies were injected into sea urchin zygotes, the resulting blastulae formed short, immotile cilia with axonemes that lacked the central pair of microtubules (see Appendix for method). The fact that the embryos never formed normal cilia even by the gastrula stage suggested the antibody depleted a non-renewing pre-existing pool of kinesin-2 (Morris & Scholey, 1997). Later studies tracking kinesin-2 by labeling one of its subunits, KAP, with green fluorescent protein (GFP), showed that kinesin-2 localized to basal bodies and to cilia (Morris et al., 2004). After ciliary retraction during mitosis, the KAP-GFP moved to nuclei before nuclear envelope breakdown, indicating that it must go through nuclear pores, and after nuclear envelope reformation, nuclear fluorescence decreased after cilia began to regrow. The hypothesis is presented that KAP may instruct the nuclei to begin reformation of cilia (Morris et al., 2004).
3 REGULATION OF TRANSCRIPTION AND TRANSLATION DURING CILIA REGENERATION Does the curvilinear shape of cilia growth curves during regeneration (Burns, 1973) suggest that a lag in assembly exists during which transcription and/or translation of ciliary protein occur? Experiments on reciliation in the presence of radioactive amino acids suggest this is the case for some cilia proteins. Tubulins and dynein ATPase subunits are the most studied and most abundant proteins in cilia, and recent work shows that there are hundreds of important structural and regulatory proteins awaiting functional discovery (Sigg et al., 2017). Sea urchin embryos possess large pools of dynein isozymes involved in cilia regeneration (Gibbons, Asai, Tang, Hays, & Gibbons, 1994; Stephens, 1977, 1989, 1995, 1997). Despite evidence from Puromycin experiments showing that gastrulae can undergo cilia regeneration without protein synthesis (Auclair & Siegel, 1966), use of radiolabeled amino acids has provided evidence for both increased tubulin and dynein protein synthesis in embryos regenerating cilia (Stephens, 1977). Considerable evidence supports the conclusion that transcription of key ciliary genes is triggered during cilia regeneration. RNA radiolabeling of S. purpuratus embryos deciliated 5 times showed that mRNA for alpha- and beta-tubulin was 2–3 times higher than in controls. Actinomycin-D blocked this mRNA increase as well as the corresponding protein increase, and decreased ciliary regeneration after the third cycle of deciliation indicating that gene transcription was necessary for repeated rounds of regeneration (Merlino, Chamberlain, & Kleinsmith, 1978). Similar
4 Manipulation of cilia length control
experiments in Lytechinus pictus embryos showed that transcription of alpha- and beta-tubulin genes is triggered within 5 min of deciliation (Gong & Brandhorst, 1987). Northern blot analysis showed that mRNA for dynein beta-heavy chain increased rapidly after deciliation (Foltz & Asai, 1990). Spec3 is a transmembrane protein of unknown function on the surface of cilia of ectodermal cells (Eldon, Angerer, Angerer, & Klein, 1987; Eldon et al., 1990). Spec3 mRNA levels increase 50-fold from fertilization to the time of ciliation at the hatching blastula stage, then Spec3 mRNA levels fall before rising again at prism stage when cilia may undergo specialization (Eldon et al., 1987, 1990). Transcription of Spec3 mRNA as well as β1, β2, and β3 tubulin mRNAs all respond to deciliation by exhibiting increased accumulation during regeneration (Eldon et al., 1987; Harlow & Nemer, 1987). It is clear from the above summary of past research that gene activation accompanies cilia regeneration in sea urchin embryos. Following deciliation, the window of 10 min before regenerating cilia buds first become visible, is the period when activity at the gene level must be initiated. The ability to easily restart ciliogenesis in sea urchin embryos provides a system to study how gene expression may control ciliogenesis in an animal model.
4 MANIPULATION OF CILIA LENGTH CONTROL A variety of ionic, protease, and cell signaling treatments can be used to experimentally manipulate cilia length control on sea urchin embryos (Burns, 1979; Horstadius, 1953; Mitsunaga, Fujiwara, Yoshimi, & Yasumasu, 1983; Morris et al., 2015; Norrander, Linck, & Stephens, 1995), and this ability to increase or decrease cilia length set-points has aided studies of the regulation of ciliogenesis. Longer cilia can be grown in embryos “animalized” by treatment with seawater containing 400 μM Zn2+ for 18 h (Harlow & Nemer, 1987) and Northern blots show that β1, β2, and β3 tubulin mRNAs are more abundant in the Zn2+ treated embryos than in controls (Harlow & Nemer, 1987). Additional experiments with Arbacia punctulata blastulae (Burns, 1979) showed that trypsin treatment for 12–20 h resulted in a fast rate of regeneration followed by a slow rate. Inhibiting protein synthesis with Emetine had no effect on the fast phase, but totally blocked the slow phase suggesting that the initial fast phase utilized a pool of previously made ciliary proteins, whereas the later slow phase required de novo protein synthesis (Burns, 1979). Theophyline, an inhibitor of cyclic nucleotide phosphodiesterase, also induces the growth of extralong cilia when Tripneustes gratilla are pre-treated with Zn2+, suggesting a role for increases in cyclic ATP, and/or cyclic GMP, as positive regulators of cilia growth (Stephens, 1994a). An effective method for animalizing embryos to grow extra-long cilia is included in the Appendix. Studies show that changes to cilia length control can be long-lasting (RiedererHenderson & Rosenbum, 1979). In A. punctulata blastulae, cilia are 18–25 μm long. When blastulae were exposed to dilute trypsin or to the lectin concanavalin-A, about 66% of the cilia produced were 50 μm long. With trypsin perturbation, blastulae and
239
240
CHAPTER 10 Sea urchin embryonic cilia
gastrulae grow long cilia while prisms and plutei larvae do not, showing that the mechanisms of cilia length regulation change during development. Most importantly, when the long cilia were detached and the blastulae recultured without trypsin, the blastulae form long cilia again rather than normal length cilia. The simplest explanation for this result is that trypsin destroys a transmembrane signaling system controlling ciliary length (Riederer-Henderson & Rosenbum, 1979). This experiment provides a simple model system in which to study cilia length control mechanisms such as the balance point model (Marshall et al., 2005) in animal cells. One of the most intriguing proteins implicated in cilia length control in sea urchins is tektin-A, a ciliary protein of 55-kDa synthesized during ciliogenesis and bound to the outer doublet microtubule of the axoneme. mRNA levels for this protein increase at ciliogenesis and are elevated above control levels in Zn2+ cultured embryos (Norrander et al., 1995). Culturing S. droebachiensis embryos in radioactive leucine, followed by SDS/PAGE and autoradiography, showed that tektin-A had the highest specific radioactivity of all axonemal proteins. Treatment of embryos with Zn2+ to induce the growth of long cilia showed that synthesis of tektin-A mRNA and protein is proportional to ciliary length, unlike tektins-B and -C which remain steady. The hypothesis was presented that tektin-A could be at least one of the proteins regulating cilia length (Norrander et al., 1995; Stephens, 1989).
5 CILIA RETRACTION VERSUS CILIA RESORPTION Another unique feature of sea urchin embryos as a model for studying the cilia lifecycle in animal cells is the way in which mature cilia are disassembled for mitosis. Although mature sea urchin cilia in interphase exhibit steady-state protein turnover as cilia in other model systems do (Rosenbaum, Moulder, & Ringo, 1969; Stephens, 1994b, 1999, 2001), the monocilia on sea urchin blastomeres are not disassembled and resorbed on the surface as in other cell models (Marshall & Rosenbaum, 2001). Instead, axonemes are fully retracted into the cytoplasm and disassembled there. In Temnopleurus toreumaticus (Masuda & Sato, 1984) and in Lytechinus variegatus (Morris et al., 2004), 2 min before nuclear envelope breakdown, cilia are completely retracted into the cytoplasm in a time-span of seconds. Three minutes after completion of mitosis, the daughter blastomeres begin extending new cilia at the same rate of elongation as the first crop (Masuda & Sato, 1984). After being utilized as a spindle pole during mitosis, the centriole pair is again trafficked to the cell surface for use as a basal body for ciliogenesis (Morris et al., 2004).
6 ISOLATION OF EMBRYONIC CILIA FROM S. purpuratus EMBRYOS This description assumes the reader knows how to obtain adult sea urchins, spawn their gametes and prepare the gametes for fertilization (See chapter “Procuring animals and culturing of eggs and embryos” by Adams et al., of this volume). Eggs are
7 Adenylate kinase and ATP production in isolated embryonic cilia
fertilized and in 15 min washed free of suspended sperm by gentle hand centrifugation and resuspension of the zygote pellet in filtered seawater. Zygotes are cultured at 0.5–1.0% v/v suspension in 16 °C filtered seawater supplemented with both 50 mg/L streptomycin and penicillin. Zygotes are grown in Erlenmeyer beakers stirred with a plastic paddle attached to a 60 rpm clock motor (for detailed description see Stephens, 1986). At the desired time after hatching, ciliated embryos are collected by gentle hand centrifugation in 50 mL tubes and washed twice in filtered seawater. The final embryo pellet is resuspended for 2 min in 10 vol hypertonic seawater (made by adding 29.2 g of NaCl per liter seawater). This “2 seawater” also contains 5 mM benzamidine and 1 mM PMSF to inhibit proteases. The naked embryos are collected by gentle hand centrifugation and the supernatant is saved on ice. The deciliated embryos are recultured in fresh seawater with antibiotics. The cilia-containing supernatant is centrifuged 500 g for 2 min to remove embryo debris. The resultant supernatant is then centrifuged 10 min at 10,000 g to sediment the cilia. Isolated cilia can be demembranated in: 0.04% w/v Triton X-100, 150 mM potassium acetate, 2 mM MgSO4, 2 mM EGTA, 1 mM DTT, 10 mM Tris-HCl, pH 8.0. The demembranated cilia can be washed by centrifugation for 10 min at 10,000 g, resuspended in fresh Triton solution and stored on ice for enzyme assays. Demembranated cilia will beat if ATP is added (Nakano, Kobayashi, Yoshimura, & Shingyoji, 2003).
7 ADENYLATE KINASE AND ATP PRODUCTION IN ISOLATED EMBRYONIC CILIA The ability to isolate embryonic cilia in high quantity and at high purity makes them amenable to functional biochemical analysis of, for example, mechanisms of ATP production in cilia. Because dyneins hydrolyze a vast amount of ATP to power ciliary bending (Chen, Heymann, Fraden, Nicastro, & Dogic, 2015), and because cilia do not contain mitochondria and are too long for diffusion to deliver sufficient ATP from cytoplasmic mitochondria, it is logical that ATP must be produced along a cilium’s entire length. The enzyme adenylate kinase (AK; aka myokinase) uses two ADP to make ATP + AMP. Cloning of this AK from sea urchin sperm flagella (Kinukawa, Nomura, & Vacquier, 2007) and embryo cilia (Kinukawa & Vacquier, 2007a) shows that it contains three catalytic domains (Kinukawa et al., 2007; Kinukawa & Vacquier, 2007b). Sea urchin sperm flagella use both creatine kinase (CK; Tombes & Shapiro, 1985) and AK to rephosphorylate ADP into ATP. Experiments with CK and AK inhibitors show that in sperm flagella, 69% of the rephosphorylation of ADP comes from CK activity and 31% from AK (Kinukawa et al., 2007). However, in isolated embryonic cilia, approximately 93% of the nonmitochondrial rephosphorylation of ADP is from AK (Kinukawa & Vacquier, 2007a). Immunolocalization demonstrates AK presence along the entire length of sperm flagella to which it is tightly bound (Kinukawa et al., 2007). The ability to isolate regenerated cilia has also allowed researchers to compare the biochemical properties of cilia produced from regeneration with normally grown
241
242
CHAPTER 10 Sea urchin embryonic cilia
first-generation cilia. To address this question, AK and ATPase activities were studied in regenerated cilia of S. purpuratus embryos after repeated deciliations. Hatched blastulae were deciliated 3 times with 2 h of regeneration between each deciliation. Coomassie stained gels of isolated cilia proteins showed equal densities of stained dynein heavy chains and tubulins in the original, first regeneration and second regeneration cilia. However, overall amount of AK and specific AK activity decreased progressively in each subsequent 2 h regeneration (Kinukawa & Vacquier, 2007a). Ciliary ATPase activity in each of the consecutive regeneration samples, assumed to be attributable mostly to axonemal dynein activity, also decreased steadily. Because the abundance of dynein heavy chains appeared similar in the three cilia preparations, the decrease in ATPase activity in the two batches of regenerated cilia may reflect progressively poorer assembly of dynein (Kinukawa & Vacquier, 2007a). Furthermore, embryo swimming velocities diminished with each round of deciliation and reciliation. Plots of AK and ATPase activity versus swimming velocity both showed near linear relationships (Kinukawa & Vacquier, 2007a). Extending the time for regeneration between deciliations allowed biochemical and swimming performance to approach, though not quite achieve, that of normally grown first-generation cilia (Kinukawa & Vacquier, 2007a). In summary, these data suggest that although normal cilia length is achieved, the regenerated cilia are not biochemically as perfect as the first crop.
8 CURRENT RESEARCH ON SEA URCHIN EMBRYO CILIA Cilia are remarkably complex organelles with ancient evolutionary origins and a high level of conservation across species. A recent study by Sigg et al. (2017) using mass spectroscopy of isolated sea urchin embryonic cilia indicates that they contain 2222 different proteins, 1012 of which are homologous to mouse ciliary proteins, and a range of ciliary signaling capabilities are conserved across phyla (Sigg et al., 2017). The evolutionary conservation of cilia, combined with the experimental advantages for studying cilia in sea urchin embryos, points to several promising lines of experimentation in the future. Soon after the global ciliogenic event that covers the entire sea urchin blastula with a uniform layer of motile cilia, those cilia begin to differentiate from each other so that by late blastula stage at least three cilia subtypes have emerged (summarized in Morris et al., 2015). These include motile lateral cilia of approximately 15–20 μm that beat with a metachronal wave to propel the embryo (Dunn et al., 2007), motile vegetal cilia of approximately 8 μm on the vegetal plate and invaginating archenteron (Tisler et al., 2016; Warner, McCarthy, Morris, & McClay, 2014), and long immotile apical tuft cilia of >40 μm at the animal pole (Dunn et al., 2007; Jin et al., 2013; Yaguchi, Yaguchi, Wei, et al., 2010). Prisms and plutei differentiate a fourth type called the “ciliary band” cilia around the embryonic region that will be primarily neurogenic in the most advanced pre-metamorphic stage (Barsi, Li, & Davidson, 2015; Costa, Nicosia, Cuttitta, Gianguzza, & Ragusa, 2017; Yaguchi, Yaguchi,
8 Current research on sea urchin embryo cilia
Angerer, Angerer, & Burke, 2010; Yaguchi, Yaguchi, Wei, et al., 2010). Each of these cilia subtypes has been useful for specific lines of experimentation. A recent study utilized the uniformity of sea urchin embryos’ lateral motile cilia to identify a protein essential to coordinating the planar polarity and organized beat pattern of this ciliated epithelium (Mizuno et al., 2017). Calaxin is a calcium-sensing protein associated with outer arm dyneins. Knockdown of calaxin in sea urchin embryos produced an uncoordinated lateral cilia beat pattern which, in turn, disturbed the embryos’ ability to properly orient its basal bodies (Mizuno et al., 2017). It will be interesting to learn if the way ciliary beat helps achieve proper basal body orientation in sea urchin blastulae involves interplay between ciliary beat and the planar cell polarity signaling pathway as is the case in mammalian ependymal cells (Guirao et al., 2010). FoxQ2 is a transcription factor (TF) expressed in the animal plate and knocking down its expression with a morpholino results in an abnormal apical tuft (Yaguchi, Yaguchi, Wei, et al., 2010). The most affected gene is ankAT-1 whose expression in early blastulae is FoxQ2 independent, but becomes FoxQ2 dependent in the mesenchyme blastula stage, and this dependency lasts until the pluteus stage. Knocking down ankAT-1 with a morpholino results in dramatically shorter apical tuft cilia without a change in motility (Yaguchi, Yaguchi, Wei, et al., 2010). Another study discovered a glutathione transferase theta (GSTT) that appears to confer mechanosensitivity to apical tuft cilia (Jin et al., 2013). The GSTT mRNA expression is confined to the animal plate of mesenchyme blastula, gastrula and prism stages. An inhibitor of the GSTT induces bending of apical tuft cilia while not affecting embryo swimming. However, the swimming behavior is different in the inhibited embryos in that they are poorer at changing direction when they strike an object. The results suggest that the GSTT on apical tuft cilia is involved in mechanical reception (Jin et al., 2013). The ciliary band (CB) is a distinct zone of ectoderm between the mouth and aboral ectoderm that remains ciliated into the larval stage when other regions become unciliated. TGF-β morpholino and over-expression experiments were used to show that this signaling protein is involved in positioning the CB and hence neuronal differentiation through its effects on Nodal and BMP expression (Yaguchi, Yaguchi, Angerer, et al., 2010). Whether a direct relationship exists between TGF-β signaling and cilia in the CB as exists in other systems (Clement et al., 2013) remains to be determined. Studies on the gene regulation that establishes the CB showed how the boundaries of the four domains comprising the CB are determined by negative regulation by SoxB1. Each boundary is confined by expression of approximately 10 transcriptional repressors four of which are expressed in all ciliated regions (Barsi et al., 2015). Further work showed that an intron regulatory sequence is required for alpha-tubulin gene expression in the CB and animal pole neurogenic regions (Costa et al., 2017). Given the incredible power of the sea urchin embryo as a model for decoding the gene regulatory network of early development (Peter & Davidson, 2010), one of the most promising avenues of research on sea urchin cilia is in identifying transcription factors (TFs) and structural proteins that control or contribute to ciliogenesis during development. For example, analysis of the gene regulatory state of the CB
243
244
CHAPTER 10 Sea urchin embryonic cilia
(Barsi et al., 2015) or the gene regulation states of animalized and vegetalized embryos (Poustka et al., 2007) could provide clues to understand the TF combinations that create and maintain unique cilia subtypes in different embryonic regions. Cilia serve as hubs of cell-signaling activity in many organisms (Nachury, 2014; Sigg et al., 2017) including during development (Drummond, 2012), and the ability to deciliate sea urchin embryos allows testing of roles for cilia in developmental signaling pathways. By eliminating normal cilia with kinesin-2 knock-downs, Warner et al. (2014) demonstrated that cilia are needed for Hedgehog (Hh) signal transduction in sea urchin embryos as in other deuterostomes (Briscoe & Therond, 2013), but not in protostomes (Warner et al., 2014). In sea urchins, Hh signaling contributes to the development of left-right asymmetry through its effect on Nodal and BMP expression that direct the left coelomic pouch to become the urchin rudiment (Warner, Miranda, & McClay, 2016). These results provide further evidence that as a basal invertebrate deuterostome, the sea urchin is much like vertebrates in the conservation of gene signaling mechanisms involving cilia. Whether ciliary motility is required for sea urchin cilia developmental signaling is an unresolved question. Nodal, a TGF-β ligand superfamily member, is critical in establishing left-right (L-R) asymmetry of the feeding larva so that the rudiment forms on the left side of the archenteron (Su, 2014). High-speed videography of the inner surface of the advancing tip of the archenteron showed that the cilia there, ranging from 4 to 10 μm long, were indeed motile. When those cilia were removed by hypertonic shock at mid- to late-gastrula stage, before the asymmetric Nodal cascade is triggered, Nodal expression was bilateral rather than right-sided, and development was perturbed (Tisler et al., 2016). One study used the pharmacologic agent dillaoiol isoxazoline derivative 1 (DID1) (Semenova, Tsyganov, Yakubov, Kiselyov, & Semenov, 2008) to specifically remove motile cilia of Hemicentrotus pulcherrimus embryos and test their role in L-R axis formation (Takemoto et al., 2016). DID1 treatment resulted in 25% of 8-arm plutei larvae having adult rudiments on the right side, or both left and right sides, whereas 98% of control larvae had rudiments on the left side (Takemoto et al., 2016) supporting the hypothesis that motility of embryonic cilia is critical to L-R axis establishment. Interestingly, a recent study by Warner et al. (2016) challenges the view that cilia motility is necessary because in their experiments, even though cilia elimination blocked Hh signaling, the expression of soxE remained left-sided and was not randomized as would be expected if cilia motility were required for breaking L-R symmetry. The cilia-dependent, symmetrybreaking step in chordates is thought to be either a “one-cilium event” requiring only motile cilia to generate fluid flow for signal transmission, or a “two-cilium event” requiring motile cilia for signal transmission and immotile cilia for signal transduction (Su, 2014). For Hh signaling, sea urchin embryos may use a different one-cilium system where only the signal transduction capability of cilia is necessary (Warner et al., 2016), even though the cilia are motile (Warner et al., 2014). The duality of function that appears to exist in sea urchin cilia—being both motile and sensory—exists in some vertebrate cilia (Shah, Ben-shahar, Moninger, Kline, & Welsh, 2009) and deserves further investigation.
Appendix
Sea urchin embryos continue to reveal important details about cilia assembly. Atypical protein kinase C (aPKC), which is known to function in cell polarity and in the formation of primary cilia in other systems, was seen to play a dramatic role in maintenance of cilia in sea urchin embryos (Prulie`re, Cosson, Chevalier, Sardet, & Chenevert, 2011). Antibodies localized aPKC to the cell cortex, but during transition from the 8- to 64-cell stage it became excluded from the cells of the vegetal pole. At blastula and gastrula stages aPKC localized at the base of cilia to form a distinct ring between the basal body and the axoneme (Prulie`re et al., 2011) strikingly reminiscent of the ciliary “transition zone” (Reiter, Blacque, & Leroux, 2012). Blocking aPKC kinase activity led to morphologically and functionally abnormal immotile cilia similar to those formed after kinesin-2 knockdown. The authors suggested that aPKC might phosphorylate kinesin-2 and thereby regulate IFT to support cilia assembly (Prulie`re et al., 2011).
9 CONCLUSION The cilia of sea urchin embryos have served as a productive model system for answering questions about cilia structure, formation, and maintenance for a halfcentury. In this system, we can easily observe, isolate, regenerate, dissect, and modulate cilia length in a deuterostome animal whose development is well understood. These capabilities, combined with powerful new tools to study gene expression and gene regulation, cell signaling, biochemistry, and ultrastructure, hold tremendous potential to illuminate the formation and functions of these ubiquitous and versatile eukaryotic organelles.
APPENDIX A.1 BLOCKING CILIOGENESIS IN SEA URCHIN EMBRYOS BY ANTIBODY MICROINJECTION Motile cilia can be blocked from growing on Lytechinus pictus and L. variegatus embryos by microinjection of the kinesin-2 function blocking antibody K2.4 into zygotes to prevent IFT (Morris & Scholey, 1997) using the steps summarized here. General methods for microinjecting sea urchin embryos are described in Morris, Brown, Wright, Sharp, Sullivan, and Scholey. 2001. Microinjection methods for analyzing the functions of kinesins in early embryos. Methods in Molecular Biology (Clifton, N.J.), 164, pp. 163–172. To block normal cilia formation, required materials include: K2.4 antibody (Abcam, https://www.abcam.com/kif3a-antibody-k24-ab24626.html); control antibody such as SUK-4 against sea urchin kinesin-1 (Developmental Studies Hybridoma Bank, http://dshb.biology.uiowa.edu/kinesin_2); sterile-filtered sea water supplemented with 10 mM p-aminobenzoic acid, pH to 8.0 (FSW-PABA); and
245
246
CHAPTER 10 Sea urchin embryonic cilia
aspartate injection buffer (AIB): 150 mM Kaspartate, 10 mM Kphosphate, monobasic, anhydrous, pH to 7.2 with KOH, and sterile filtered. Exchange antibodies out of preservative-containing buffers by repeated rounds of concentration-and-dilution into AIB using low-protein-binding ultrafiltration devices. Keep antibody concentrations below 10 mg/mL during preparation to prevent protein precipitation. Use siliconized injection needles to prevent antibodies from binding to needles. Working quickly, fertilize eggs, wash out sperm, transfer eggs to FSW-PABA (to prevent hardening of fertilization envelopes), and begin injections. Fertilization envelopes will strip off as zygotes are loaded into injection chambers. Inject 2.5–5% of cell volume of 2.5–5 mg/mL antibody into zygotes; one such injection of K2.4 will block ciliogenesis throughout development. To be sure that injections block ciliogenesis effectively in each batch of embryos, perform control—then K2.4—then control-injections in each injection round to “book-end” the experimental condition and then monitor all injected embryos for ciliogenesis at blastula stage.
A.2 GROWING “ANIMALIZED” EMBRYOS ENRICHED FOR LONG, APICAL TUFT CILIA Long, immotile cilia like those forming the patch of apical tuft cilia on sea urchin embryos can be formed all over the embryos by a zinc ion treatment that shifts cell development toward apical domain fates (Morris et al., 2015). To grow such “animalized” embryos, quickly fertilize eggs, wash out sperm, and strip fertilization envelopes by passing zygotes through 150-μm Nitex mesh before fertilization envelopes harden. Incubate stripped zygotes at low density (a monolayer or less settled in a Petri dish) in filtered sea water supplemented with 125 mM ZnSO4 at a temperature appropriate to the species. The zinc ion animalization protocol works especially well on Lytechinus pictus. Although zinc ion treatment produces varying degrees of animalization within the same batch of embryos, the animalized phenotype will be visible on a large fraction of the embryos by 24 h post fertilization.
REFERENCES Amemiya, S. (1971). Relationship between cilia formation and cell association in sea urchin embryos. Experimental Cell Research, 64, 227–230. Auclair, W., & Siegel, B. W. (1966). Cilia regeneration in the sea urchin embryo: Evidence for a pool of ciliary proteins. Science, 154, 913–915. Avasthi, P., & Marshall, W. F. (2012). Stages of ciliogenesis and regulation of ciliary length. Differentiation, 83(2), S30–S42. https://doi.org/10.1016/j.diff.2011.11.015. Barsi, J. C., Li, E., & Davidson, E. H. (2015). Geometric control of ciliated band regulatory states in the sea urchin embryo. Development, 142(5), 953–961. https://doi.org/10.1242/ dev.117986. Briscoe, J., & Therond, P. P. (2013). The mechanisms of hedgehog signalling and its roles in development and disease. Nature Reviews Molecular Cell Biology, 14(7), 418–431. https://doi.org/10.1038/nrm3598.
References
Burns, R. G. (1973). Kinetics of the regeneration of sea-urchin cilia. Journal of Cell Science, 13(1), 55–67. Retrieved from http://eutils.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi? dbfrom¼pubmed&id¼4729939&retmode¼ref&cmd¼prlinks. Burns, R. G. (1979). Kinetics of the regeneration of sea-urchin cilia. II. Regeneration of animalized cilia. Journal of Cell Science, 37, 205–215. Retrieved from http://eutils. ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom¼pubmed&id¼479325&retmode¼ref& cmd¼prlinks. Chen, D. T. N., Heymann, M., Fraden, S., Nicastro, D., & Dogic, Z. (2015). ATP consumption of eukaryotic flagella measured at a single-cell level. Biophysical Journal, 109(12), 2562–2573. https://doi.org/10.1016/j.bpj.2015.11.003. Childs, F. M. (1959). The characterization of the cilia of Tetrahymena pyriformis. Experimental Cell Research, 18, 258–267. Clement, C. A., Ajbro, K., Koefoed, K., Vestergaard, M., Veland, I., HenriquesdeJesus, M., et al. (2013). TGF-β signaling is associated with endocytosis at the pocket region of the primary cilium. Cell Reports, 3(6), 1806–1814. https://doi.org/10.1016/j.celrep.2013.05.020. Costa, S., Nicosia, A., Cuttitta, A., Gianguzza, F., & Ragusa, M. A. (2017). An intronic cisregulatory element is crucial for the alpha-tubulin PI-Tuba1a gene activation in the ciliary band and animal pole neurogenic domains during sea urchin development. PLoS One, 12, e0170969. https://doi.org/10.1371/journal.pone.0170969. Drummond, I. A. (2012). Cilia functions in development. Current Opinion in Cell Biology, 24(1), 24–30. https://doi.org/10.1016/j.ceb.2011.12.007. Dunn, E. F., Moy, V. N., Angerer, L. M., Angerer, R. C., Morris, R. L., & Peterson, K. J. (2007). Molecular paleoecology: Using gene regulatory analysis to address the origins of complex life cycles in the late Precambrian. Evolution & Development, 9(1), 10–24. https://doi.org/10.1111/j.1525-142X.2006.00134.x. Eldon, E. D., Angerer, L. M., Angerer, R. C., & Klein, W. H. (1987). Spec3: Embryonic expression of a sea urchin gene whose product is involved in ectodermal ciliogenesis. Genes & Development, 1(10), 1280–1292. https://doi.org/10.1101/gad.1.10.1280. Eldon, E. D., Montpetit, I. C., Nguyen, T., Decker, G., Valdizan, M. C., Klein, W. H., et al. (1990). Localization of the sea urchin spec3 protein to cilia and golgi complexes of embryonic ectoderm cells. Genes & Development, 4(1), 111–122. https://doi.org/10.1101/ gad.4.1.111. Foltz, K. R., & Asai, D. J. (1990). Molecular cloning and expression of sea urchin embryonic ciliary dynein beta heavy chain. Cell Motility and the Cytoskeleton, 16, 33–46. Gibbons, B. H., Asai, D. J., Tang, W.-J. Y., Hays, T. S., & Gibbons, I. R. (1994). Phylogeny and expression of axonemal and cytoplasmic dynein genes in sea urchins. Molecular Biology of the Cell, 5, 57–70. Gibbons, I. R., & Fronk, E. (1972). Some properties of bound and soluble dynein from sea urchin sperm flagella. Journal of Cell Biology, 54, 365–381. available at https://www. ncbi.nlm.nih.gov/pmc/articles/PMC2108873/. Gong, Z., & Brandhorst, B. P. (1987). Stimulation of tubulin gene-transcription by deciliation of sea-urchin embryos. Molecular and Cellular Biology, 7(12), 4238–4246. Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC368105/. Guirao, B., Meunier, A., Mortaud, S., Aguilar, A., Corsi, J.-M., Strehl, L., et al. (2010). Coupling between hydrodynamic forces and planar cell polarity orients mammalian motile cilia. Nature Cell Biology, 12(4), 341–350. Harlow, P., & Nemer, M. (1987). Coordinate and selective beta-tubulin gene expression associated with cilium formation in sea urchin embryos. Genes & Development, 1(10), 1293–1304. https://doi.org/10.1101/gad.1.10.1293.
247
248
CHAPTER 10 Sea urchin embryonic cilia
Horstadius, S. (1953). Vegetalization of the sea-urchin egg by dinitrophenol and animalization by trypsin and ficin. Development, 1(4), 327–348. Retrieved from http://dev.biologists. org/content/1/4/327. Ishikawa, H., & Marshall, W. F. (2011). Ciliogenesis: Building the cell’s antenna. Nature Reviews Molecular Cell Biology, 12(4), 222–234. https://doi.org/10.1038/nrm3085. Iwaikawa, Y. (1967). Regeneration of cilia in the sea urchin embryo. Embryologia, 9, 287–294. Jin, Y., Yaguchi, S., Shiba, K., Yamada, L., Yaguchi, J., Shibata, D., et al. (2013). Glutathione transferase theta in apical ciliary tuft regulates mechanical reception and swimming behavior of sea urchin embryos. Cytoskeleton (Hoboken, N.J.), 70, 453–470. Kinukawa, M., Nomura, M., & Vacquier, V. D. (2007). A sea urchin sperm flagellar adenylate kinase with triplicated catalytic domains. Journal of Biological Chemistry, 282, 2947–2955. Kinukawa, M., & Vacquier, V. D. (2007a). Adenylate kinase in sea urchin embryonic cilia. Cell Motility and the Cytoskeleton, 64, 310–319. Kinukawa, M., & Vacquier, V. D. (2007b). Recombinant sea urchin adenylate kinase. Journal of Biochemistry, 142, 501–506. Kozminski, K. G., Johnson, K. A., Forscher, P., & Rosenbaum, J. L. (1993). A motility in the eukaryotic flagellum unrelated to flagellar beating. Proceedings of the National Academy of Sciences of the United States of America, 90(12), 5519–5523. https://doi.org/10.1073/ pnas.90.12.5519. Lin, J., & Nicastro, D. (2018). Asymmetric distribution and spatial switching of dynein activity generates ciliary motility. Science, 360(6387), eaar1968. https://doi.org/10.1126/science. aar1968. Marshall, W. F., Qin, H., Brenni, M. R., & Rosenbaum, J. L. (2005). Flagellar length control system: Testing a simple model based on intraflagellar transport and turnover. Molecular Biology of the Cell, 16(1), 270–278. Retrieved from http://www.molbiolcell.org/content/ 16/1/270.short. Marshall, W. F., & Rosenbaum, J. L. (2001). Intraflagellar transport balances continuous turnover of outer doublet microtubules: Implications for flagellar length control. Journal of Cell Biology, 155(3), 405–414. https://doi.org/10.1083/jcb.200106141. Masuda, M., & Sato, H. (1984). Reversible resorption of cilia and the centriole cycle in dividing cells of sea urchin blastulae. Zoological Science, 1, 445–462. Merlino, G. T., Chamberlain, J. P., & Kleinsmith, L. J. (1978). Effects of deciliation on tubulin messenger RNA activity in sea urchin embryos. Journal of Biological Chemistry, 253, 7078–7085. Mitsunaga, K., Fujiwara, A., Yoshimi, T., & Yasumasu, I. (1983). Stage specific effects on sea urchin embryogenesis of Zn2+, Li+, several inhibitors of cAMP-phosphodiesterase and inhibitors of protein synthesis. Development, Growth & Differentiation, 25(3), 249–260. Mizuno, K., Shiba, K., Yaguchi, J., Shibata, D., Yaguchi, S., Prulie`re, G., et al. (2017). Calaxin establishes basal body orientation and coordinates movement of monocilia in sea urchin embryos. Scientific Reports, 7(1), 1–10. https://doi.org/10.1038/s41598-01710822-z. Morris, R. L., English, C. N., Lou, J. E., Dufort, F. J., Nordberg, J., Terasaki, M., et al. (2004). Redistribution of the kinesin-II subunit KAP from cilia to nuclei during the mitotic and ciliogenic cycles in sea urchin embryos. Developmental Biology, 274(1), 56–69. Retrieved from http://linkinghub.elsevier.com/retrieve/pii/S0012160604004336. Morris, R. L., Pope, H. W., Sholi, N. A., Williams, L. M., Ettinger, C. R., Beacham, G. M., et al. (2015). Methods for imaging individual cilia in living echinoid embryos. Methods in Cell Biology, 127, 223–241. https://doi.org/10.1016/bs.mcb.2014.12.004.
References
Morris, R. L., & Scholey, J. M. (1997). Heterotrimeric kinesin-II is required for the assembly of motile 9 + 2 ciliary axonemes on sea urchin embryos. Journal of Cell Biology, 138, 1009–1022. Nachury, M. V. (2014). How do cilia organize signalling cascades? Philosophical Transactions of the Royal Society, B: Biological Sciences, 369(1650). 20130465–20130465 https://doi.org/10.1098/rstb.2013.0465. Nakano, I., Kobayashi, T., Yoshimura, M., & Shingyoji, C. (2003). Central-pair-linked regulation of microtubule sliding by calcium in flagellar axonemes. Journal of Cell Science, 116, 1627–1636. Norrander, J. M., Linck, R. W., & Stephens, R. E. (1995). Transcriptional control of tektin A mRNA correlates with cilia development and length determination during sea urchin embryogenesis. Development, 121(6), 1615–1623. Peter, I. S., & Davidson, E. H. (2010). The endoderm gene regulatory network in sea urchin embryos up to mid-blastula stage. Developmental Biology, 340(2), 188–199. https://doi. org/10.1016/j.ydbio.2009.10.037. Poustka, A. J., K€uhn, A., Groth, D., Weise, V., Yaguchi, S., Burke, R. D., et al. (2007). A global view of gene expression in lithium and zinc treated sea urchin embryos: New components of gene regulatory networks. Genome Biology, 8(5), R85. Retrieved from http://genomebiology.com/2007/8/5/R85. Prulie`re, G., Cosson, J., Chevalier, S., Sardet, C., & Chenevert, J. (2011). Atypical protein kinase C controls sea urchin ciliogenesis. Molecular Biology of the Cell, 22(12), 2042–2053. Retrieved from http://eutils.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom¼pubmed& id¼21508313&retmode¼ref&cmd¼prlinks. Reiter, J. F., Blacque, O. E., & Leroux, M. R. (2012). The base of the cilium: Roles for transition fibres and the transition zone in ciliary formation, maintenance and compartmentalization. EMBO Reports, 13(7), 608–618. https://doi.org/10.1038/embor.2012.73. Riederer-Henderson, M. A., & Rosenbum, J. L. (1979). Ciliary elongation of Arbacia punctulata induced by trypsin. Developmental Biology, 70, 500–509. Rosenbaum, J. L., Moulder, J. E., & Ringo, D. L. (1969). Flagellar elongation and shortening in Chlamydomonas. The use of cycloheximide and colchicine to study the synthesis and assembly of flagellar proteins. The Journal of Cell Biology, 41(2), 600–619. https://doi. org/10.1083/jcb.41.2.600. Semenova, M. N., Tsyganov, D. V., Yakubov, A. P., Kiselyov, A. S., & Semenov, V. V. (2008). A synthetic derivative of plant allylpolyalkoxybenzenes induces selective loss of motile cilia in sea urchin embryos. ACS Chemical Biology, 3(2), 95–100. Retrieved from http://pubs.acs.org/doi/abs/10.1021/cb700163q. Shah, A. S., Ben-shahar, Y., Moninger, T. O., Kline, J. N., & Welsh, M. J. (2009). Motile cilia of human airway epithelia are chemosensory. Science, 325(5944), 1131–1134. https://doi. org/10.1126/science.1173869. Sigg, M. A., Menchen, T., Lee, C., Johnson, J., Jungnickel, M. K., Choksi, S. P., et al. (2017). Evolutionary proteomics uncovers ancient associations of cilia with signaling pathways. Developmental Cell, 43(6), 744–762.e11. https://doi.org/10.1016/j.devcel. 2017.11.014. Stephens, R. E. (1977). Differential protein synthesis and utilization during cilia formation in sea urchin embryos. Developmental Biology, 61, 311–329. Stephens, R. E. (1986). Isolation of embryonic cilia and sperm flagella. Methods in Cell Biology, 27, 217–227. Stephens, R. E. (1989). Quantal tektin synthesis and ciliary length in sea urchin embryos. Journal of Cell Science, 92, 403–413.
249
250
CHAPTER 10 Sea urchin embryonic cilia
Stephens, R. E. (1994a). Rapid induction of a hyperciliated phenotype in zinc-arrested sea urchin embryos by theophylline. Journal of Experimental Zoology, 269, 106–115. Stephens, R. E. (1994b). Tubulin and tektin in sea urchin embryonic cilia: Pathways of protein incorporation during turnover and regeneration. Journal of Cell Science, 107, 683–692. Stephens, R. E. (1995). Ciliogenesis in sea urchin embryos—A subroutine in the program of development. BioEssays, 17, 331–340. Stephens, R. E. (1997). Synthesis and turnover of embryonic sea urchin ciliary protein during selective inhibition of tubulin synthesis and assembly. Molecular Biology of the Cell, 8, 2187–2198. Stephens, R. E. (1999). Turnover of tubulin in ciliary outer doublet microtubules. Cell Structure and Function, 24, 413–418. Stephens, R. E. (2001). Ciliary protein turnover continues in the presence of inhibitors of golgi function: Evidence of membrane protein pools and unconventional intracellular membrane dynamics. Journal of Experimental Zoology, 289, 335–349. Stephens, R. E. (2008). Ciliogenesis, ciliary function and selective isolation. ACS Chemical Biology, 3(2), 84–86. Available at http://pubs.acs.org/doi/abs/10.1021/cb8000217. Su, Y. H. (2014). Telling left from right: Left-right asymmetric controls in sea urchins. Genesis, 52(3), 269–278. https://doi.org/10.1002/dvg.22739. Summers, K. E., & Gibbons, I. R. (1971). Adenosine triphosphate-induced sliding of tubules in trypsin-treated flagella of sea-urchin sperm. Proceedings of the National Academy of Sciences of the United States of America, 68(12), 3092–3096. https://doi.org/10.1073/pnas. 68.12.3092. Takemoto, A., Miyamoto, T., Simono, F., Kurogi, N., Shirae-Kurabayashi, M., Awazu, A., et al. (2016). Cilia play a role in breaking left–right symmetry of the sea urchin embryo. Genes to Cells, 21(6), 568–578. https://doi.org/10.1111/gtc.12362. Tisler, M., Wetzel, F., Mantino, S., Kremnyov, S., Thumberger, T., Schweickert, A., et al. (2016). Cilia are required for asymmetric nodal induction in the sea urchin embryo. BMC Developmental Biology, 16(1), 1–12. https://doi.org/10.1186/s12861-016-0128-7. Tombes, R. M., & Shapiro, B. M. (1985). Metabolite channeling: A phosphocreatine shuttle to mediate high energy phosphate transport between sperm mitochondrion and tail. Cell, 41, 325–334. Warner, J. F., McCarthy, A. M., Morris, R. L., & McClay, D. R. (2014). Hedgehog signaling requires motile cilia in the sea urchin. Molecular Biology and Evolution, 31(1), 18–22. https://doi.org/10.1093/molbev/mst176. Warner, J. F., Miranda, E. L., & McClay, D. R. (2016). Contribution of hedgehog signaling to the establishment of left- right asymmetry in the sea urchin. Developmental Biology, 411(2), 314–324. https://doi.org/10.1016/j.ydbio.2016.02.008. Watson, M. R., & Hopkins, J. M. (1962). Isolated cilia from Tetrahymena pyriformis. Experimental Cell Research, 28, 280–295. Yaguchi, S., Yaguchi, J., Angerer, R. C., Angerer, L. M., & Burke, R. D. (2010). TGF-β signaling positions the ciliary band and patterns neurons in the sea urchin embryo. Developmental Biology, 347, 71–81. Yaguchi, S., Yaguchi, J., Wei, Z., Shiba, K., Angerer, L. M., & Inaba, K. (2010). AnkAT-1 is a novel gene mediating the apical tuft formation in the sea urchin embryo. Developmental Biology, 348, 67–75.