Retinoic Acid and Nitric Oxide Promote Cell Proliferation and Differentially Induce Neuronal Differentiation in Vitro in the Cnidarian Renilla Koellikeri

Retinoic Acid and Nitric Oxide Promote Cell Proliferation and Differentially Induce Neuronal Differentiation In Vitro in the Cnidarian Renilla koellikeri

Djoyce Estephane, Michel Anctil De´ partement de sciences biologiques and Centre de recherche en sciences neurologiques, Universite´ de Montre´ al, Case postale 6128, Succursale Centre-ville, Montre´ al, Que´ bec, Canada, H3C 3J7

Received 26 January 2010; revised 8 July 2010; accepted 9 July 2010

ABSTRACT: Retinoic acid (RA) and nitric oxide cell density. NO donors also induce cell proliferation in (NO) are known to promote neuronal development in polylysine-coated dishes, but induce neuronal differen- both vertebrates and invertebrates. Retinoic acid tiation and neurite outgrowth in uncoated dishes. No receptors appear to be present in cnidarians and NO other cell type undergoes differentiation in the pres- plays various physiological roles in several cnidarians, ence of NO. These observations suggest that in the sea but there is as yet no evidence that these agents have a pansy (1) cell adhesion promotes proliferation without role in neural development in this basal metazoan phy- morphogenesis and this proliferation is modulated pos- lum. We used primary cultures of cells from the sea itively by 9-cis RA and NO, (2) 9-cis RA and NO differ- pansy Renilla koellikeri to investigate the involvement entially induce neuronal differentiation in nonadherent of these signaling molecules in cnidarian cell differen- cells while repressing proliferation, and (3) the involve- tiation. We found that 9-cis RA induce cell prolifera- ment of RA and NO in neuronal differentiation tion in dose- and time-dependent manners in dishes appeared early during the evolutionary emergence of coated with polylysine from the onset of culture. Cells nervous systems. ' 2010 Wiley Periodicals, Inc. Develop in cultures exposed to RA in dishes devoid of polylysine Neurobiol 70: 842–852, 2010 were observed to differentiate into epithelium-associ- Keywords: cnidarian; retinoic acid; nitric oxide; ated cells, including sensory cells, without net gain in neuronal differentiation

INTRODUCTION 1996; Galliot et al., 2006; Fritzenwanker et al., 2007), our understanding of mechanisms underlying neuro- Cnidarians constitute the first animal phylum in genesis and neuronal specification in cnidarians is which representatives possess an organized nervous limited. In hydra nerve cells are known to derive system and neurally supported behaviors. While ex- from a group of stem cells called the interstitial cell perimental models such as hydra and the starlet sea system (Bode, 1996). Cells committed to the neuronal anemone (Nematostella vectensis) led to significant pathway are arrested at the G2 phase and need a sig- advances for cnidarian developmental biology (Bode, nal to divide and differentiate (Schaller et al., 1989). A native neuropeptide, Hym355, was reported to act as a signaling molecule positively regulating neuronal Correspondence to: M. Anctil ([email protected]). Contract grant sponsor: NSERC of Canada; contract grant num- differentiation in hydra (Takahashi et al., 2000). In ber: 6447-06. contrast, native peptides released by epithelial cells ' 2010 Wiley Periodicals, Inc. have inhibitory effects on neuronal differentiation Published online 15 July 2010 in Wiley Online Library (wileyonlinelibrary.com). (Takahashi et al., 1997; Bosch and Fujisawa, 2001). DOI 10.1002/dneu.20824 These findings beg the question: is cnidarian neuronal 842 Neuronal Differentiation in R. koellikeri 843 differentiation regulated by uniquely evolved signal- ronal differentiation (Peunova and Enikolopov, 1995; ing molecules, or are there other categories of signal- Packer et al., 2003; Gibbs, 2003; Estrada and ing systems found in more complex animals that are Murillo-Carretero, 2005). Gaseous transmisson using already involved in differentiation of cnidarian nerve NO appears to be widespread in the animal kingdom cells? (Moroz, 2001). In insects and molluscs NO is known Two signaling molecules that were reported to be to regulate neurite extension and neuronal migration important for neuronal development in vertebrates (Seidel and Bicker, 2000; Haase and Bicker, 2003; are retinoic acid (RA) and nitric oxide (NO). RA, a Trimm and Rehder, 2004). Although NO was shown physiologically active derivative of vitamin A, is to modulate muscle contraction and nitric oxide syn- known to play a variety of roles in mammalian neuro- thase (NOS) was found to be present in neurons of genesis, neuronal differentiation and patterning cnidarians (Moroz et al., 2004; Anctil et al., 2005), (Sidell et al., 1983; Andrews, 1984; Maden et al., there is no evidence so far for its involvement in cni- 1996; Liu et al., 2001; Wilson et al., 2004; Maden, darian neuronal development. 2007 for review). Two forms of RA, all-trans and 9- The evidence of the presence of retinoic acid and cis RA, exert their actions through retinoic acid nitric oxide signaling systems in cnidarians prompted receptors (RAR) or retinoid X receptors (RXR), us to ask whether they participate in neurogenesis in members of class II receptors in the nuclear receptor this phylum. We used cell culture techniques to superfamily (Allenby et al., 1993). Other members of investigate the role of RA and NO in cell prolifera- Class II nuclear receptors, orphan COUP-TF recep- tion and differentiation, and especially in the process tors, are known to repress RA-induced neuronal dif- of neuronal differentiation, in the sea pansy Renilla ferentiation in a mammalian cell line (Neuman et al., ko¨llikeri. We found that these agents affect cell pro- 1995). Because hydra homologs of COUP-TF recep- liferation and neuronal differentiation differently tors are expressed in a hydra neuron population dur- in adhesion-permissive and nonadhesive culture con- ing neurogenesis and are also able to repress RAR/ ditions. RXR-induced transactivation in mammalian cells (Gauchat et al., 2004), a role for RA in cnidarian neuronal development can be envisaged. Recent studies have produced the first direct evidence of induction MATERIALS AND METHODS of neurite outgrowth by RA in an invertebrate, the mollusc Lymnaea stagnalis (Dmetrichuk et al., 2006). Animals Therefore, widening investigations may lead to find- Colonies of Renilla ko¨llikeri were purchased from, and ings of a generalized role for RA in invertebrate neu- shipped to Montreal by Marinus Scientific (Garden Grove, ronal differentiation. A hypothesized physiological CA, USA). They were kept at 168C in a 120-L tank filled role for RA in cnidarians was bolstered by (1) evi- with filtered artificial sea water (ASW, Instant Ocean). Sea dence that retinoids affect body pattern formation in a water osmolarity was adjusted to 1100 mOsm and pH to 8. hydrozoan (Mu¨ller, 1984) and (2) the identification of Colonies were maintained under a 12 h:12 h light-dark a RXR-like receptor in the jellyfish Tripedalia cysto- cycle and they were kept unfed. They were processed within three to four days of their arrival. phora showing significant similarity with the human RXR alpha receptor (Kostrouch et al., 1998). More recently, report of the presence of immunoreactive RXR in interstitial cells and neurons of two anthozo- Cell Culture ans, the sea pansy Renilla ko¨llikeri and the staghorn Sea pansy colonies were progressively anesthetized in a solu- coral Acropora millepora, adds support for a role of tion of 0.37 mol/L magnesium chloride. Autozooid polyps RA in cnidarian neuronal differentiation (Bouzaiene were excised from the colony, sliced extensively into minute et al., 2007). fragments and large tissue pieces were discarded. The The other signaling molecule of interest, NO, is a remaining small tissue pieces and cell agglomerates were gaseous molecule involved in many different physio- dissociated at room temperature in 10 mL of culture medium logical processes, including cell proliferation and dif- to which 3.1 mmol/L papain and 3.2 mmol/L dithiothreitol were added for 90 min with agitation. The culture medium ferentiation, smooth muscle contraction, apoptosis, contained 23 mmol/L NaCl, 0.63 mmol/L KCl, 10 mmol/L macrophage activity and neurotransmission (Krume- MgCl2, 1.1 mmol/L CaCl2, 3.69 mmol/L Na2SO4,2.38 nacker and Murad, 2006; Tota and Trimmer, 2007). It mmol/L HEPES, and 0.09 mmol/L gentamycin. It was milli- is known to play a direct role in inhibiting mamma- pore filtered and pH was adjusted to 8. This was followed by lian neurogenesis during neuronal differentiation but three washes with fresh culture medium by centrifugation with few exceptions it is not involved directly in neu- with a VWR microcentrifuge at 1500 rpm.

Developmental Neurobiology 844 Estephane and Anctil

To grow dissociated cells 35-mm Nunclon culture other anthozoan cells (Fautin and Mariscal, 1991). Ele- dishes with a bottom grid were used. For some of the ments failing to match these criteria were discarded from experiments dishes were first coated with 1 mg/mL poly-L- the counts. The position of the selected area in each culture lysine and allowed to dry overnight. Dissociated cells were dish at the beginning of treatment was scored and cell den- plated at a density of 5 3 103 cells per dish (1 mL of cell sity was recorded daily in that same area over the duration suspension). Cell cultures were maintained at 118Cinan of treatment (6–8 days) for each dish. Average density val- Echo Therm 30 Chilling incubator. Cells were allowed to ues from five separate control and experimental cultures grow to 90% confluency over a period of 8–10 days post- were then recorded and the means 6 standard error of plating. means computed. The rates of cell proliferation and cell differentiation were analyzed and displayed using Graph Pad Prism. Bonferroni corrected unpaired t tests were performed Assays of Retinoic Acid and Nitric Oxide to assess significance of differences between cell densities at various doses of active agents or at different days after The two isomers of retinoic acid (RA), 9-cis and all-trans, treatment. and the NO donors, S-nitroso-N-acetylpenicillamine (SNAP) and amino-3-morpholinyl-1,2,3-oxadiazolium chloride (SIN-1), were purchased from Sigma-Aldrich (Canada). Stock solutions of retinoic acid were prepared RESULTS using 100% ethanol as solvent to obtain a concentration of 1 mmol/L. NO donors were dissolved in dimethyl sulfoxide Cell Growth in Culture (DMSO) to a stock concentration of 0.9 mmol/L. To test the effect of retinoic acid on cultures of dissoci- When cultured on polylysine-coated or uncoated ated cells, various volumes of stock solutions of RA were dishes, untreated cells rapidly dedifferentiated into added to dishes containing one day old cultures to obtain round or ovoid shapes and for the most part remained the desired final concentration. For NO donors, 100 lLof thus undifferentiated while their population grew stock solutions was diluted in 5 mL of culture medium to over the useful life of the culture (up to 10 days). In 3 À2 obtain a concentration of 2 10 mmol/L and different polylysine-coated dishes, cell density of untreated volumes of this dilution were added to one-day old cultures cultures rose steadily to reach a fivefold increase by to obtain the desired final concentration. Cultures remained exposed to the test substances for their remaining life (7–10 Days 8–10 [Fig. 1(A)], compared with a twofold days). For controls, untreated cultures prepared from the increase only in uncoated dishes [Fig. 3(A)]. same animal as the treated cultures contained the same In contrast to cells grown in polylysine-coated amount of vehicle, ethanol or DMSO, present in the treated dishes, some of the cells grown in uncoated dishes, cultures. In addition, degraded NO donor solutions were while remaining round or slighly oval, exhibited intra- used as additional controls to ensure that no by-product was cellular morphologies associated with known antho- responsible for observed effects when there was no more zoan cell types. The most outstanding example in this NO producing ability. study is the epitheliomuscular and bioluminescent cells After treatments, the cultures were maintained in the in- of the sea pansy, characterized when dissociated by an 8 cubator at 11 C in the dark. The cultures were examined excentric cytoplasm and a large vacuole-like pool of daily with a Nikon Eclipse TE300 inverted microscope disorganized myofibrils [Fig. 2(A); see Germain and equipped with phase-contrast and with Hoffman modula- tion contrast optics. Images were obtained with a Nikon Anctil, 1988]. Symbiotic algal cells also grew in these Coolpix 4500 digital camera and processed with Corel cultures, but they were easily identified by their brown PHOTO-Paint 12. color and discarded from the cell counts.

Analysis Cell Growth and Morphogenetic Effects of Retinoic Acid To assess the effects of RA or NO donors cell densities and morphologies were compared between control and treated Preliminary results indicated that the effects of the 9- cultures in polylysine-coated and uncoated dishes. In cis and all-trans isomers of RA are similar. Therefore, uncoated dishes, the cells were settled on the dish bottom. only the 9-cis isomer was used in all the experiments 3 Densities were evaluated from viewing ﬁelds under a 20 presented here. When cultures in polylysine-coated objective by counting dedifferentiated and/or redifferenti- dishes were treated with 9-cis RA, cell proliferation ated cells over areas of 2 mm2 using the bottom grid squares of culture dishes as guide. Values for analysis were occurred at an increasingly greater rate than in cell numbers per mm2. Cell types were identiﬁed by match- untreated cultures [Fig. 1(A)]. By the tenth day post- ing their shape, size and, if applicable, intracellular charac- treatment cell density in treated cultures had teristics with illustrated descriptions of sea pansy (Germain increased by three- to fourfold over that of untreated and Anctil, 1988; Pani et al., 1994; Pernet et al., 2004) and cultures. The increase in cell density was readily

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centration of 1 lmol/L and cell density rose exponen- tially at higher concentrations until it tended to level off at 100 lmol/L [Fig. 1(B)]. When cells were cultured in uncoated dishes, they began differentiating into various types of epithelium-associated cells after exposure to RA. In untreated cultures cells show few distinctive morphologies [Fig. 2(A)]. In cultures treated with 10–100 lmol/L 9-cis RA, cell morphologies typical of anthozoan epithelium-associated cells (Fautin and Mariscal, 1991) appear with increasing frequency, especially sensory cells, nematocytes, gland cells, and ciliary motor cells [Fig. 2(B)]. As the density of undifferentiated cells steadily declined over the life of the RA-treated cultures, the density of differentiated cells rose gradually [Fig. 3(A)]. In contrast, no measurable increase in overall cell density was observed, thus indicating that no cell proliferation occurred in RA-treated, uncoated culture dishes. Sen-

Figure 1 Effect of retinoic acid on sea pansy cell prolifer- ationinculturesmaintainedinpoly-L-lysine-coated dishes. A: graph showing the increase of cell density over time in untreated cultures and in cultures treated with 100 lmol/L 9- cis retinoic acid added at culture Day 2. Differences in values between treated and untreated cultures are significant for all sampling days in unpaired t tests (p < 0.001). B: Dose- response curve of the proliferative effect of 9-cis retinoic acid on cell cultures. All between-dose comparisons showed significant differences in cell density in unpaired t tests (p < 0.01). The panel below the graph shows the difference of cell density between representative images of untreated and retinoic acid-treated cultures at Day 6. apparent by visual inspection of six-day-old cultures Figure 2 Micrographs of sea pansy cells in cultures main- [Fig. 1(A)]. No cell differentiation was apparent tained in uncoated dishes. A: Untreated culture at Day 7 in under these conditions. The RA-induced accelerated which only round epitheliomuscular cells (EMC) are identifi- rate of cell proliferation was dose-dependent and the able. B: Culture six days after treatment with 100 lmol/L 9- sigmoid curve of the relationship [Fig. 1(B)] was con- cis retinoic (culture Day 7) in which ciliomotor (CMC), epi- sistent with a receptor-mediated event. A significant theliomuscular (EMC), gland (GC), interstitial (IC), nemato- rise in cell density was apparent at a 9-cis RA con- cyte (NC), and sensory cells (SC) are identifiable.

Developmental Neurobiology 846 Estephane and Anctil

to retinoic acid. However, cell density increased at a lesser rate than in retinoic acid-treated cultures in the effective concentration range of the NO donors (10–100 lmol/L), remaining around double the cell density of untreated cultures throughout the life of the cultures [Fig. 4(A)]. In contrast, NO donors induced the differentiation of an increasingly larger number of cells into neurons in uncoated dishes. Two days after treatment with 90 lmol/L SIN-1 or SNAP only 25% of cultured cells were identiﬁable as neurons, whereas 85% of the cultured cells were recognizable neurons six days

Figure 3 Histograms quantifying the effect of 100 lmol/L 9-cis retinoic acid on sea pansy cell differentiation in cultures maintained in uncoated dishes. A: Changes in cell density of untreated (controls) versus treated (undifferentiated and differentiated cells) cultures over time. Note the rise in cell density in untreated cultures and the reversal of relative cell densities between undifferentiated and differentiated cells in treated cultures. Single and double asterisks represent significant differences in cell density compared with untreated cultures at p < 0.05 and p < 0.01, respectively (unpaired t test); ns, not significant (p > 0.05). B: Relative cell density among various types of identifiable differentiated epithelial cells after six days of treatment with retinoic acid. Densities Figure 4 Effects of nitric oxide (NO) donors on cell pro- of gland cells, nematocytes and ciliomotor cells are all sig- liferation and neuronal differentiation in sea pansy cell cul- nificantly lower than those of sensory cells (unpaired t tests, tures. A: Graph showing the effect of 90 lmol/L SNAP p < 0.01). added at Day 2 on the rise of cell density over time in cul- sory cells constituted the greater share of observed tures maintained in poly-L-lysine-coated dishes. Differences epithelial cells, with densities of other epithelial-like in values between treated and untreated cultures are signifi- < cell types being significantly lower to varying degrees cant for all sampling days in unpaired t tests (p 0.001). l than those of sensory cells [Fig. 3(B)]. B: Histogram showing the effect of 90 mol/L SIN-1 on sea pansy neuronal differentiation in cultures maintained in uncoated dishes. Note the rise in cell density in untreated cultures and the sharp reversal of relative cell densities Cell Growth and Morphogenetic Effects between undifferentiated and neuronal cells in treated cul- of Nitric Oxide Donors tures. Single and double asterisks represent significant differences in cell density compared with untreated cultures at Both NO donors SIN-1 and SNAP enhanced cell p < 0.05 and p < 0.01, respectively (unpaired t test); ns, growth in polylysine-coated dishes in a manner similar not significant (p > 0.05).

Developmental Neurobiology Neuronal Differentiation in R. koellikeri 847 after treatment [Fig. 4(B)]. No cell type other than neu- darian cells and the presence of parasites and sym- ronal was detectable in treated cultures. Cells in biont cells in cnidarian tissues cast doubt on the iden- untreated cultures grew in number similarly to uncoated tity of cells proliferating over long time periods control dishes in retinoic acid experiments [compare (Schmid et al., 1999). This demonstrates the need to Fig. 4(B) with Fig. 3(A)]. Treatments with degraded rely on short-term primary cultures for studies NO donor solutions yielded the same results as in intended to examine pathways and mechanisms of untreated cultures (not shown). cell differentiation such as in this study. Cells in untreated cultures remained largely spheri- The first successful attempt at establishing a pri- cal or ovoid throughout the culture period [Fig. 5(A)] mary culture of cnidarian neurons was made in a jel- whereas the majority of cells displayed a smaller soma lyfish (a hydrozoan) in which nerve-rich tissue can be and grew fine extensions (neurites) in NO-treated cul- found (Przysiezniak and Spencer, 1989). The authors tures [Fig. 5(B)]. The nerve cells were mostly unipolar used a homogenate of the jellyfish’s own extracellular or bipolar and their long neurites showed swellings matrix (ECM) in the abundant mesogleal jelly as sub- (varicosities) along their entire length [Fig. 5(C)]. Mul- strate for adhesion of the cultured neurons and in tipolar neurons were also observed, and the neurites of these conditions the cultures survived for up to two some of these neurons appear to cross over each other, weeks in ASW (Przysiezniak and Spencer, 1989). bifurcate and contact non-neuronal cells [Fig. 5(D)]. They and other authors (Schmid et al., 1999) tested These cells resemble the norepinephrine- and RFa- other substrate media than ECM, including polyly- mide-immunoreactive neurons forming the nerve net sine, but only ECM appeared satisfactory. However, in the mesoglea of the sea pansy, particularly near the while cell survival depends on attachment to endoderm (Pani et al., 1995; Pernet et al., 2004). substrate, cell proliferation was inhibited under such conditions (Schmid et al., 1999). In addition, no anthozoan possesses nerve-rich tissues or can yield sufficient ECM material for practical use as substrate DISCUSSION for cell attachment in culture. Polylysine-coated culture surfaces have been known Our results, using the sea pansy as a cnidarian experi- for several decades to provide an adequate substrate for mental model, show that 9-cis RA and NO donors cell attachment and especially strong adhesion for neu- potentiate cell proliferation and induce differentiation rons without inhibiting neurite elongation (Varon, under different culture conditions. All these agents 1979). Day and Lenhoff (1981) reported that hydra cells enhance cell proliferation but fail to induce morpho- adhered well to polylysine and we were also able to suc- genesis in polylysine-coated dishes. In contrast, while cessfully culture sea pansy cells in dishes coated with 9-cis RA and NO donors both induce neuronal differ- poly-L-lysine, but in agreement with Przysiezniak and entiation in uncoated dishes, they differ in their mor- Spencer (1989) cells failed to undergo neuronal differ- phogenetic products, epithelial (including sensory) entiation under such conditions. A larger concentration cells for RA and exclusively neurons for NO donors. of polylysine (1 mg/mL) than those used for vertebrate Together with previous evidence of RA receptors cell cultures (0.1 mg/mL) was necessary to ensure cell (Bouzaiene et al., 2007) and NO signaling systems adhesion, yet contrary to vertebrate cultured cells (Anctil et al., 2005), these results support a role for RA (Varon, 1979) no apparent toxicity to sea pansy cells and NO in regulating adhesion-dependent cell prolifer- was noted at such a concentration. It is not clear why a ation and adhesion-independent neuronal differentia- high level of polylysine is needed and why sea pansy tion in the sea pansy. It remains to be determined to cells are resistant to its toxic effect at such a level, but which extent and in which manner the observed in vitro the unusual lipid environment of cnidarian cell mem- effects of these morphogens are played out in vivo. branes (Schetz and Anderson, 1993) and the divergent molecular structure of cnidarian ECM proteins (Knack Validation of Cell Culture Method et al., 2008; Magie and Martindale, 2008) may at least partly account for these properties. Several attempts to establish long-term cultures of In our hands dissociated sea pansy cells plated in cnidarian cells have met with obstacles or have alto- either uncoated or polylysine-coated dishes and in the gether failed (Schmid et al., 1999 for review). Frank absence of enriched media or trophic factors were et al. (1994) reported successful establishment of con- able to proliferate while showing little or no evidence tinuous cell cultures and of cell lines in colonial of differentiation within the life time of the cultures. anthozoans, including octocorallians other than the This allowed us to design experiments during which sea pansy, but the notorious in vivo plasticity of cni- morphogenetic agents could be tested and their

Developmental Neurobiology 848 Estephane and Anctil

Figure 5 Micrographs of neurons differentiating six days after treatment with 90 lmol/L SIN-1 in cultures maintained in uncoated dishes. A: Untreated culture at Day 7 in which cells of various sizes possess round or ovoid shapes. B: Treated culture at Day 7 (six days post-treatment) in which numerous neuron soma with long, intersecting neurites are visible and relatively few round cells remain. Scale bar in B applies also to A. C: Enlarged view of a bipolar neuron with elongating, var- icose neurite. D: Enlarged view highlighting two neurons (NC), one of which has a neurite crossing over other neurites (arrow) and bifurcating to make contact with non-neuronal cells (arrowheads). effects on cell growth or morphological phenotype and Weston, 1994; Nabeyrat et al., 1998; Wohl and unambiguously observed. Weiss, 1998) and NO (Ulibarri et al., 1999; Mejia-Gar- cia and Paes-de-Carvalho, 2007) can promote proliferation and spreading of adherent cells under speciﬁc Retinoic Acid and Nitric Oxide Promote conditions. It is clear that substrate adhesion is a deter- Proliferation of Adherent Cells mining factor in the repression of differentiation of sea Our results demonstrate that 9-cis RA and NO potenti- pansy cells exposed to RA or NO, because we have ate cell proliferation without inducing differentiation also shown that cells cultured without substrate coating in sea pansy cells allowed to adhere to polylysine- and showing little or no substrate adhesion respond to coated dish bottoms. This is consistent with studies on these agents by differentiating into speciﬁc cell types vertebrate cell lines showing that both RA (Henion (see below).

Developmental Neurobiology Neuronal Differentiation in R. koellikeri 849

The concentrations of 9-cis RA used in the majority 1993; Raz and Kelley, 1999). In contrast, the NO of experiments in this study are substantially higher results do not concur with the lack of direct involve- than those used in experiments with vertebrate cell cul- ment of nitrergic pathways in mammalian neuronal dif- tures (Henion and Weston, 1994), and therefore appear ferentiation (Estrada and Murillo-Carretero, 2005), but to fall outside the physiological range. The endoge- are consonant with invertebrate studies demonstrating nous levels of retinoids in cnidarians are unknown, so a direct role in neuronal differentiation, especially neu- it is not possible to determine what is the physiological rite outgrowth and navigation (Bicker, 2005). range for these basal metazoans. RA binds in the nano- The induced differentiations occur in a time-de- molar range to a jellyfish RXR fusion protein pendent manner, with a gradual reversal of dominance expressed in bacteria (Kostrouch et al., 1998), but from undifferentiated to differentiated morphologies. It effective concentrations for binding RXR in jellyfish is striking that while control cultures displayed a two- membranes or for eliciting a cell response have not fold increase in density, the size of cell populations been reported. In another basal metazoan, the sponge remained stable during the RA- and NO-induced dif- Suberites domuncula, a canal regression was induced ferentiation processes, suggesting that the morphogens by RA in the 1–50 micromolar range (Wiens et al., inhibit growth while inducing differentiation. This is 2003). Even if the RA concentrations used in our consistent with reports showing that both retinoids experiments could be higher than the endogenous lev- (Sidell, 1981; Sidell et al., 1983) and NO (Peunova and els, healthy indicators such as growth and differentia- Enikolopov, 1995; Kuzin et al., 1996) suppress cell tion, not adverse or deleterious effects, were observed, growth during differentiation of neuronal and other cell thus suggesting that RA was acting physiologically. lines. Termination of cell division is required also in The similar proliferative effects of 9-cis RA and cnidarians to engage in processes of cell differentiation NO donors raise the possibility that RA acts through (Bode, 1996), including neurodifferentiation (Hager the mediation of a nitrergic pathway. RA stimulation and David, 1997). Retinoids appear to coordinate of NO production was previously reported in mamma- growth arrest with neuronal differentiation in mammalian endothelial cells (Achan et al., 2002; Urano et al., lian target cells through distinct RXR/RAR receptor 2005) and in a neuroblastoma cell line (Ghigo et al., signaling pathways (Cheung et al., 1996; Giannini 1998). Even if a functional link between RA and NO et al., 1997). NO, on the other hand, appears to specifi- is discovered, the process by which cell growth is pro- cally mediate the growth suppressing effect of nerve moted and differentiation repressed in our experimen- growth factors (Peunova and Enikolopov, 1995) and is tal conditions remains to be examined. One possibility linked to the onset of differentiation only as a prerequi- is that the proliferative effects of these agents are site. Without a better knowledge of retinoid receptor mediated through trophic factor transduction pathways and nitrergic signaling it will be difficult to unravel the that may also interfere with differentiation signals. mechanisms by which these morphogens affect cell This would require that trophic factors are released proliferation and differentiation in this cnidarian. from the cultured cells in the medium upon adhesion. Although 9-cis RA and NO clearly commit sea pansy There are several candidates identified in cnidarians, cells to different fates, it remains possible that the RA- such as cnidarian homologues of vascular endothelial induced arrest of cell proliferation is mediated by NO. growth factor and receptor (Seipel et al., 2004), fibro- The occurrence of morphogen-induced differentia- blast growth factor (Matus et al., 2007) and insulin tion only in nonadhesive conditions suggests that the growth factor (Steele et al., 1996). absence of adhesion activates signals that make the dedifferentiated cells responsive to the morphogenetic actions of RA and NO. The nature of the Retinoic Acid and Nitric Oxide hypothesized signals is suggested by studies showing Differentially Induce Neuronal that jellyfish striated muscle cells remain differenti- Differentiation in Nonadherent Cells ated when the integrity of their ECM is preserved, but dedifferentiate and undergo transdifferentiation We have shown that sea pansy cells cultured on a non- when the ECM is removed or inactivated (Schmid adhesive substrate differentiate into sensory and other and Reber-Mu¨ller, 1995). Dedifferentiation in jelly- epithelium-associated cells when exposed to 9-cis RA fish involves disassembly of cytoskeletal compo- and selectively into neuronal cells when exposed to nents, hence the round cell shapes in culture, and is NO donors. The RA results are consistent with numer- followed by DNA replication, which the cell prolifer- ous studies demonstrating the role of retinoids in verte- ations we observed are assumed to reflect. Protein ki- brate epithelial differentiation (Shapiro, 1986 for nase C signaling may also be involved (Kurz and review), including sensory epithelia (Lefevre et al., Schmid, 1991). In jellyfish transdifferentiation into

Developmental Neurobiology 850 Estephane and Anctil smooth muscle cells and neurons follows dedifferen- Allenby G, Bocquel MT, Saunders M, Kazmer S, Speck J, tiation and cell division without any further experi- Rosenberger M, Lovey A, et al. 1993. Retinoic acid mental treatment. In the sea pansy cells remain receptors and retinoid X receptors: Interactions with en- largely dedifferentiated and proliferate until exposure dogenous retinoic acids. Proc Natl Acad Sci USA 90:30– to 9-cis RA or NO when redifferentiation is initiated. 34. Anctil M, Poulain I, Pelletier C. 2005. Nitric oxide modu- The differential effects of RA and NO suggest that lates peristaltic muscle activity associated with fluid cir- epithelium-associated cell commitment and meso- culation in the sea pansy Renilla koellikeri. J Exp Biol gleal neuron commitment are subjected to distinct 208:2005–2018. signaling pathways. The majority of epithelium-asso- Andrews PA. 1984. Retinoic acid induces neuronal differ- ciated cells in this study (sensory neurons, nemato- entiation of a cloned human embryonal carcinoma cell cytes, gland cells) are known in hydra to be derived line in vitro. Dev Biol 103:285–293. from interstitial cells, a form of multipotent stem cells Bicker G. 2005. STOP and GO witn NO: Nitric oxide as a (Bode, 1996 for review). In hydra all neurons are regulator of cell motility in simple brains. Bio Essays associated with the epithelial monolayers (ectoderm 27:495–505. and endoderm) and the mesogleal jelly, which is Bode HR. 1996. The interstitial cell lineage of hydra: A sandwiched by the two epithelial layers, is acellular. stem cell system that arose early in evolution. J Cell Sci 109:1155–1164. In contrast, the mesoglea of anthozoans such as the Bosch TCG, Fujisawa T. 2001. Polyp, peptides and pattern- sea pansy is thicker and contains cells including neu- ing. Bio Essays 23:420–427. rons resembling the ganglion cells of hydra (Fautin Bouzaiene M, Angers A, Anctil M. 2007. Immunohisto- and Mariscal, 1991; Pani et al., 1995; Pernet et al., chemical localization of a retinoic acid-like receptor in 2004). Thus RA appears to specifically target intersti- nerve cells of two colonial anthozoans (Cnidaria). Tissue tial cells for commitment to epithelium-associated Cell 39:123–130. cell lineages in the sea pansy, whereas NO selectively Cheung B, Hocker JE, Smith SA, Reichert U, Norris MD, targets precursor cells of mesogleal neurons. Meso- Haber M, Stewart BW, Marshall GM. 1996. Retinoic gleal neurons are known to form nerve nets in the sea acid receptors b and c distinguish retinoid signals for pansy (Pernet et al., 2004) and the neurite extensions growth inhibition and neuritogenesis in human neuro- blastema cells. Biochem Biophys Res Commun of the differentiating mesogleal-like neurons in this 229:349–354. study are seen to cross each other [Fig. 5(D)] as Day RM, Lenhoff HM. 1981. Hydra mesoglea: A model for expected from cnidarian nerve-net organizations investigating epithelial cell-basement membrane interac- (Minobe et al., 1995). tions. Science 211:291–294. Dmetrichuk JM, Carlone RL, Jones TRB, Vesprini ND, Spencer GE. 2006. Detection of endogenous retinoids in Evolutionary Significance the molluscan CNS and characterization of the trophic To our knowledge this is the first report of effects of a and tropic actions of 9-cis retinoic acid on isolated neurons. J Neurosci 28:13014–13024. retinoid and of NO on cell proliferation, neuronal dif- Dmetrichuk JM, Carlone RL, Spencer GE. 2006. Retinoic ferentiation and neurite outgrowth in a cnidarian. This acid induces neurite outgrowth and growth cone turning suggests that RA and NO are important morphogens in in invertebrate neurons. Dev Biol 294:39–49. cnidarians as they are in vertebrates. As neurons are Estrada C, Murillo-Carretero M. 2005. Nitric oxide and considered to have evolved first in cnidarians, the adult neurogenesis in health and disease. Neuroscientist implication is that these morphogens were functionally 11:294–307. integrated into morphogenetic signaling early in the Fautin DG, Mariscal RN. 1991. Cnidaria: Anthozoa. In: evolutionary emergence of nervous systems. Harrison FW, Westfall JA, editors. Microscopic Anat- omy of Invertebrates, Vol. 2: Placozoa, Porifera, Cnida- The authors thank Meriem Bouzaiene for her advice on ria and Ctenophora. New York: Wiley-Liss, pp 267–358. technical matters. Frank U, Rabinowitz C, Rinkevich B. 1994. In vitro establishment of continuous cell cultures and cell lines from ten colonial cnidarians. Mar Biol 120:491–499. Fritzenwanker JH, Genikhovich G, Kraus Y, Technau U. REFERENCES 2007. Early development and axis specification in the sea anemone Nematostella vectensis. Dev Biol 310:264–279. Achan V, Tran CTL, Arrigoni F, Whitley GS, Leiper JM, Galliot B, Miljkovic-Licina M, de Rosa R, Chera S. 2006. Vallance P. 2002. All-trans-Retinoic acid increases nitric Hydra, a niche for cell and developmental plasticity. oxide synthesis by endothelial cells. A role for the induc- Semin Cell Dev Biol 17:492–502. tion of dimethylarginine dimethylaminohydrolase. Circ Gauchat D, Escriva H, Miljkovic-Licina M, Chera S, Lan- Res 90:764–769. glois M-C, Begue A, Laudet V, et al. 2004. The orphan

Developmental Neurobiology Neuronal Differentiation in R. koellikeri 851

COUP-TF nuclear receptors are markers for neurogenesis Maden M, Gale E, Kostetskii I, Zile M. 1996. Vitamin A- from cnidarians to vertebrates. Dev Biol 275:104–123. deficient quail embryos have half a midbrain and other Germain G, Anctil M. 1988. Luminescent activity and ul- neural defects. Curr Biol 6:417–426. trastructural characterization of photocytes dissociated Magie CR, Martindale MQ. 2008. Cell-cell adhesion in the from the coelenterate Renilla ko¨llikeri. Tissue Cell Cnidaria: Insights into the evolution of tissue morpho- 20:701–720. genesis. Biol Bull 214:218–232. Ghigo D, Priotto C, Migliorino D, Geromin D, Franchino Matus DQ, Thomsen GH, Martindale MQ. 2007. FGF sig- C, Todde R, Costamagna C, et al. 1998. Retinoic acid- naling in gastrulation and neural development in Nema- induced differentiation in a human neuroblastoma cell tostella vectensis, an anthozoan cnidarian. Dev Genes line is associated with an increase in nitric oxide synthe- Evol 217:137–148. sis. J Cell Physiol 174:99–106. Mejia-Garcia TA, Paes-de-Carvalho R. 2007. Nitric oxide Giannini G, Dawson MI, Zhang X-K, Thiele CJ. 1997. regulates cell survival in purified cultures of avian retinal Activation of three distinct RXR/RAR heterodimers neurons: Involvement of multiple transduction pathways. induces growth arrest and differentiation of neuroblas- J Neurochem 100:382–394. toma cells. J Biol Chem 272:26693–26701. Minobe S, Koizumi O, Sugiyama T. 1995. Nerve cell dif- Gibbs SM. 2003. Regulation of neuronal proliferation and ferentiation in nerve-free tissue of epithelial hydra from differentiation by nitric oxide. Mol Neurobiol 27:107– precursor cells introduced by grafting. I. Tentacles and 120. hypostome. Dev Biol 172:170–181. Haase A, Bicker G. 2003. Nitric oxide and cyclic nucleo- Moroz LL. 2001. Gaseous transmission across time and tides are regulators of neuronal migration in an insect species. Am Zool 41:304–320. embryo. Development 130:3977–3987. Moroz LL, Meech RW, Sweedler JV, Mackie GO. 2004. Hager G, David CN. 1997. Pattern of differentiated nerve Nitric oxide regulates swimming in the jellyfish Aglantha cells in hydra is determined by precursor migration. De- digitale. J Comp Neurol 471:26–36. velopment 124:569–576. Mu¨ller WA. 1984. Retinoids and pattern formation in a Henion PD, Weston JA. 1994. Retinoic acid selectively hydroid. J Embryol Exp Morph 81:253–271. promotes the survival and proliferation of neurogenic Nabeyrat E, Besnard V, Corroyer S, Cazals V, Cle´ment A. precursors in cultured neural crest cell populations. Dev 1998. Retinoic acid-induced proliferation of lung alveo- Biol 161:243–250. lar epithelial cells: Relation with the IGF system. Am J Knack BA, Iguchi A, Shinzato C, Hayward DC, Ball EE, Physiol 275:L71–L79. Miller DJ. 2008. Unexpected diversity of cnidarian integ- Neuman K, Soosaar A, Nornes HO, Neuman T. 1995. rins: Expression during coral integration. BMC Evol Biol Orphan receptor COUP-TF I antagonizes retinoic acid- 8:136. induced neuronal differentiation. J Neurosci Res 41: Kostrouch Z, Kostrouchova M, Love W, Jannini E, Piati- 39–48. gorsky J, Rall JE. 1998. Retinoic acid X receptor in the Packer MA, Stasiv Y, Benraiss A, Chmielnicki E, Grinberg diploblast, Tripedalia cystophora. Proc Natl Acad Sci A, Westphal H, Goldman SA, et al. 2003. Nitric oxide USA 95:13442–13447. negatively regulates mammalian adult neurogenesis. Krumenacker JS, Murad F. 2006. NO-cGMP signaling in Proc Natl Acad Sci USA 100:9566–9571. development and stem cells. Mol Genet Metab 87:311– Pani AK, Anctil M, Umbriaco D. 1994. Neuronal localiza- 314. tion and evoked release of norepinephrine in the cnidar- Kurz E, Schmid V. 1991. Effects of tumor promoters and di- ian Renilla koellikeri. J Exp Zool 272:1–12. acylglycerol on the transdifferentiation of striated muscle Pernet V, Anctil M, Grimmelikhuijzen CJP. 2004. Antho- cells of the medusa Podocoryne carnea to RFamide posi- RFamide-containing neurons in the primitive nervous tive nerve cells. In: Williams RB, Cornelius PFS, Hughes system of the anthozoan Renilla koellikeri. J Comp Neu- RG, Robson EA, editors. Coelenterate Biology: Recent rol 472:208–220. Research on Cnidaria and Ctenophora. Dordrecht, The Peunova N, Enikolopov G. 1995. Nitric oxide triggers a Netherlands: Kluwer Academic Publishers, pp 11–17. switch to growth arrest during differentiation of neuronal Kuzin B, Roberts I, Peunova N, Enikolopov G. 1996. Nitric cells. Nature 375:68–73. oxide regulates cell proliferation during Drosophila de- Przysiezniak J, Spencer AN. 1989. Primary culture of iden- velopment. Cell 87:639–649. tified neurones from a cnidarian. J Exp Biol 142:97–113. Lefebvre PP, Malgrange B, Staecker H, Moonen G, Van De Raz Y, Kelley MW. 1999. Retinoic acid signaling is neces- Water TR. 1993. Retinoic acid stimulates regeneration of sary for the development of the organ of Corti. Dev Biol mammalian auditory hair cell. Science 260:692–695. 213:180–193. Liu J-P, Laufer E, Jessell TM. 2001. Assigning the posi- Schaller HC, Hoffmeister SA, Dubel S. 1989. Role of tional identity of spinal motor neurons: Rostrocaudal pat- the neuropeptide head activator for growth and devel- terning of Hox-c expression by Fgfs. Gdf11, and reti- opment in hydra and mammals. Development 107: noids Neuron 32:997–1012. 99–107. Maden M. 2007. Retinoic acid in the development, regen- Schetz JA, Anderson PAV. 1993. Investigations of lipid eration and maintenance of the nervous system. Nature components of neurone-enriched membranes of the jelly- Rev Neurosci 8:755–765. fish Cyanea capillata. J Exp Biol 177:23–39.

Developmental Neurobiology 852 Estephane and Anctil

Schmid V, Ono S-I, Reber-Mu¨ller S. 1999. Cell-substrate Takahashi T, Muneoka Y, Lohmann J, Lopez deHaro MS, interactions in Cnidaria. Microsc Res Tech 44:254– Solleder G, Bosch TCG, David CN, et al. 1997. System- 268. atic isolation of peptide signal molecules regulating de- Schmid V, Reber-Mu¨ller S. 1995. Transdifferentiation of velopment in hydra: Lwamide and PW families. Proc isolated striated muscle of jellyfish in vitro: The initiation Natl Acad Sci USA 94:1241–1246. process. Sem Cell Biol 6:109–116. Tota B, Trimmer B, editors. Nitric Oxide. Amsterdam: Seidel C, Bicker G. 2000. Nitric oxide and cGMP influence Elsevier B.V.390 p. axogenesis of antennal pioneer neurons. Development Trimm KR, Rehder V. 2004. Nitric oxide acts as a slow- 127:4541–4549. down and search signal in developing neurites. Eur J Seipel K, Eberhardt M, Mu¨ller P, Pescia E, Yanze N, Neurosci 19:809–818. Schmid V. 2004. Homologs of vascular endothelial Ulibarri JA, Mozdziak PE, Schultz E, Cook C, Best TM. growth factor and receptor, VEGF and VEGFR, in the 1999. Nitric oxide donors, sodium nitroprusside and S- jellyfish Podocoryne camea. Dev Dynamics 231:303– Nitroso-N-acetylpenicillamine, stimulate myoblast pro- 312. lifereation in vitro. In Vitro Cell Dev Biol Anim 35:215– Shapiro SS. 1986. Retinoids and epithelial differentiation. 218. In: Sherman MI, editor. Retinoids and Cell Differentia- Urano A, Sugawara A, Kanatsuka H, Kagechika H, Saito tion. Boca Raton: CRC Press, pp 29–60. A, Sato K, Kudo M, Takeuchi K, Ito S. 2005. Upregula- Sidell N. 1981. Retinoic acid-induced growth inhibition and tion of nitric oxide production in vascular endothelial morphologic differentiation of human neuroblastoma cells by all-trans retinoic acid through the phosphoinosi- cells in vitro. J Natl Cancer Inst 68:589–596. tide 3-kinase/Akt pathway. Circulation 112:727–736. Sidell N, Altman A, Haussler MR. 1983. Effects of retinoic Varon S. 1979. The culture of chick embryo dorsal root acid (RA) on the growth and phenotypic expression of ganglionic cells on polylysine-coated plastic. Neurochem several human neuroblastoma cell lines. Exp Cell Res Res 4:155–173. 148:21–30. Wiens M, Batel R, Korzhev M, Mu¨ller WEG. 2003. Reti- Steele RE, Lieu P, Mai NH, Shenk MA, Sarras MP Jr. noic X receptor and retinoic acid response in the marine 1996. Response to insulin and the expression pattern of a sponge Suberites domuncula. J Exp Biol 206:3261–3271. gene encoding an insulin receptor homologue suggest a Wilson L, Gale E, Chambers D, Maden M. 2004. The role role for an insulin-like molecule in regulating growth and of retinoic acid in the dorsoventral patterning of the spi- patterning in Hydra. Dev Genes Evol 206:247–259. nal cord. Dev Biol 269:433–446. Takahashi T, Koizumi O, Ariura Y, Romanovitch A, Bosch Wohl CA, Weiss S. 1998. Retinoic acid enhances neuronal TCG, Kobayakawa Y, Mohri S, et al. 2000. A novel neu- proliferation and astroglial differentiation in cultures of ropeptide. Hym-355, positively regulates neuron differ- CNS stem cell-derived precursors. J Neurobiol 37:281– entiation in hydra. Development 127:997–1005. 290.

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