Regulation of Metamorphosis by Environmental Cues and Retinoic Acid Signaling in the Lecithotrophic Larvae of the Starfish Astropecten latespinosus.
Many marine invertebrates have biphasic life cycles, with planktonic larval and sessile adult phases (1). Because sessile adults have restricted motility, the settling of larvae in suitable environments during metamorphosis is of critical importance (2). Therefore, planktonic larvae usually possess sensory apparatuses to respond to specific environmental cues (2).
The ancestors of starfish (echinoderms) are believed to have planktotrophic larvae (3-5). Planktotrophic larvae need to be fed to commence metamorphosis; thus, their development proceeds depending on the larval nutritional state (6). Some species change their strategies and develop through lecithotrophic larvae (3-5). In contrast to that of planktotrophic larvae, the development of lecithotrophic larvae proceeds in a cascade-like manner because they do not feed before metamorphosis (4, 5). However, whether these lecithotrophic larvae sense environmental cues to commence metamorphosis is not clear.
After lecithotrophy, some starfish species retain their sensory apparatus, brachiolar arms (4). Many planktotrophic starfish larvae use their brachiolar arms to sense environmental cues about where to settle (7). This suggests that these species have continued to sense environmental cues, even after their transition to lecithotrophy. Some lecithotrophic paxillosidan species, such as Astropecten latespinosus Meissner, 1892, however, do not possess brachiolar arms (8-10).
Here we investigated whether lecithotrophic larvae could sense environmental cues by testing whether larvae of A. latespinosus commenced metamorphosis in seawater containing sand from the habitat of adult specimens. During this experiment, we also investigated when larvae became competent for metamorphosis. Previously, Komatsu (8) stated that larvae began to develop juvenile rudiments at around 30 hours post-fertilization (hpf), after the gastrula elongated along the archenteron (Fig. A1b) (8). Juvenile rudiment development then proceeded until about 72-96 hpf (Fig. A1c, d) (8). Komatsu (8) reported that larvae metamorphosed to juveniles after 75 hpf (Fig. A1e, f), although she did not experimentally examine the time to acquisition of competency (8).
We collected adult specimens of A. latespinosus from Notojima Island, Ishikawa Prefecture, Japan; and we obtained fertilized eggs as described previously (8). We introduced the habitat sand to the wells of the 24-hpf larvae, corresponding to gastrula (n = 50 from 3 batches; Fig. A1a). Then we cultured larvae and every day for one week counted the number of larvae that completed metamorphosis. We considered the ability to induce metamorphosis to have been achieved when the whole larval body had been absorbed and the juvenile rudiment had developed (Fig. A1). For statistical analysis, we used ANOVA to evaluate differences in the effects of the substrate on metamorphosis, as used in our previous work (11).
In the seawater without substrate, larvae did not metamorphose before 72 hpf (Fig. 1; Table A1). Small numbers of larvae (5 of 50 larvae) metamorphosed into juveniles after 96 hpf (Fig. 1; Table A1), although the metamorphosis ratio was less than 50% (21 of 50 larvae; Fig. 1; Table A1). However, when substrates were added to the seawater, small numbers of larvae (7 of 50 larvae) were induced to metamorphose even at 72 hpf (Fig. 1; Table A1). More than 70% of larvae (36 of 50 larvae) metamorphosed after 96 hpf (Fig. 1; Table A1). At 192 hpf, significant differences in the metamorphosis ratios were observed between treatments (P = 0.009, ANOVA). These results indicated that A. latespinosus can sense environmental cues, such as natural sand, to commence metamorphosis. We also found that most of the larvae metamorphosed at 72-96 hpf (Fig. 1), suggesting that they became competent around 72 hpf.
In planktotrophic larvae of starfish, Murabe et al. (7) found that brachiolar arms perform a critical role in receiving environmental cues for metamorphosis (7). Recently, we suggested that retinoic acid (RA) signaling mediated the commencement of the metamorphosis process after settlement (11), through RA synthesis by retinal dehydrogenase (RALDH) and binding to retinoic acid receptor (RAR) and retinoid X receptor (RXR) (12). As shown, we found that paxillosidan larvae also received environmental cues to commence metamorphosis, though they use different apparatuses from brachiolar arms for reception; thus, it is unclear whether RA signaling involves metamorphosis regulation in this group.
Here we examined whether the commencement of metamorphosis was also mediated by RA signaling in A. latespinosus. First, we investigated the effect of exogenous RA treatment of competent larvae (n = 40 from 3 batches). Because more than half of 72-hpf larvae treated with habitat sand completed metamorphosis in 24 hours (Fig. 1), we tested the effect of exogenous RA on 72-hpf larvae. We found that exogenous RA (1 [micro]mol [L.sup.-1]) treatment induced metamorphosis (32 of 40 larvae; Fig. 2a, c; Table A2). The larvae commenced metamorphosis immediately after treatment and completed their transitions to juveniles in 24 hours. In contrast, only 1 out of 40 dimethyl sulfoxide (DMSO)-treated larvae metamorphosed (Fig. 2b, c; Table A2). We observed that the presence of RA significantly affected the metamorphosis ratio (P < 0.001, ANOVA). These results suggest that RA mediates internal signaling to commence the metamorphosis of A. latespinosus.
Additionally, we investigated the effect of exogenous RA treatment of larvae of various ages on metamorphosis in order to test whether RA also affected the timing of larval competence to respond to cues for metamorphosis. We treated 24-and 48-hpf larvae with RA (1 [micro]mol [L.sup.-1]) and counted the number of metamorphosed larvae every 24 hours until 96 hpf (n = 30 and 40 from 3 batches, respectively). We observed that metamorphosis was induced only after 72 hpf in both cases (3 of 30 and 2 of 40 with 24- and 48-hpf initiations, respectively; Fig. 2d, e; Tables A3, A4). Thus, regardless of when the larvae were treated with RA, they responded and metamorphosed at 72 hpf, which is comparable to the stage at which larvae acquire competence to metamorphose during normal development (Figs. 1, 2d, e). Furthermore, at 96 hpf, almost half of the larvae metamorphosed (15 of 30 and 19 of 40 from 3 batches with 24- and 48-hpf initiations, respectively; Fig. 2d, e; Tables A3, A4). We found significant differences at 96 hpf in the batches with 24- and 48-hpf initiations (P = 0.034 and P = 0.019, respectively, ANOVA). These timelines are similar to those induced by a substrate (Fig. 1). These results suggest that RA does not affect the development of competence for metamorphosis, but rather functions as an internal mediator of the signaling to commence metamorphosis when added to competent larvae.
Next, we investigated whether endogenous RA synthesis is required for metamorphosis. To investigate the effect of treatment with DEAB (N,N-diethylaminobenzaldehyde), an RA synthesis inhibitor, on metamorphosis, we treated 72-hpf larvae with DEAB (300 [micro]mol [L.sup.-1]) and natural sand in the experiments described above; and we counted the larvae that had completed metamorphosis 24 hours after treatment (n = 40 from 3 batches). As a control, we treated 72-hpf larvae with DMSO and natural sand. More than half of the DMSO-treated larvae transitioned to juveniles (26 of 40 larvae; Fig. 3b, c; Table A5). In contrast, DEAB treatment decreased the number of metamorphosed larvae (6 of 40 larvae; Fig. 3a, c; Table A5). The metamorphosis ratio was significantly suppressed by DEAB treatment (P = 0.022, ANOVA). We observed particular larval behavior in the DEAB treatment prior to metamorphosis, such as attachment to the substrate with rudiments. Thus, larvae were likely to sense the environmental cue but did not commence metamorphosis. These findings suggest that endogenous RA synthesis is required for the commencement of metamorphosis.
RA binding to RAR is required for RA signaling activation (12). Thus, we investigated the effect of RAR antagonist treatment on metamorphosis to test the hypothesis that RA signaling pathways mediate the metamorphosis process. We treated 72-hpf larvae (n = 40 from 3 batches) with RO41-5253 (RO, Focus Biomolecules, Plymouth Meeting, PA; 1 [micro]mol [L.sup.-1]), RAR antagonist, and the natural sand used above; and we counted the number of metamorphosed larvae after 24 hours. As a control, we treated 72-hpf larvae with DMSO and natural sand. Under the DMSO treatment, 67.5% of larvae (27 of 40 larvae) transitioned to juveniles (Fig. 3e, f; Table A6). In contrast, no larva metamorphosed under the RO treatment (Fig. 3d, f; Table A6). The metamorphosis ratio was significantly repressed by RO treatment (P = 0.008, ANOVA). As we observed with DEAB treatment, larvae also stopped floating and attached to the substrate with rudiments after RO treatment.
As shown previously, exogenous RA treatment induces metamorphosis in 72-hpf larvae (Fig. 2). To support the idea that RA binding to RAR is required for metamorphosis, we examined whether RO treatment blocked metamorphosis induced by RA treatment. We treated 72-hpf larvae (n = 40 from 3 batches) with RA (1 [micro]mol [L.sup.-1]) or RA (1 [micro]mol [L.sup.-1]) plus RO (1 [micro]mol [L.sup.-1]). In the RA-only treatment, 77.5% of larvae (31 of 40 larvae) metamorphosed (Fig. 3g-i; Table A7). Conversely, the RA (1 [micro]mol [L.sup.-1]) plus RO (1 [micro]mol [L.sup.-1]) treatment induced metamorphosis in only 12.5% of larvae (5 of 40 larvae; Fig. 3h, k; Table A7). RO significantly repressed the metamorphosis ratio (P < 0.001, ANOVA). These data suggest that RA signaling activation through RA binding to RAR is required for the commencement of metamorphosis.
We examined the expression patterns of genes involved in RA signaling. We confirmed their orthologies by constructing phylogenic trees (Figs. A2-A5). In conclusion, from our de novo transcriptome, we identified three raldh genes (raldha, raldhb, and raldhc), a single rar, and a single rxr. We also investigated the spatial expression patterns of the three raldh genes, rar, and rxr by whole-mount in situ hybridization of 72-hpf larvae (Fig. 4). We identified the expression of two types of receptor, rar and rxr, in the juvenile rudiment (Fig. 4j-1 and m--o, respectively), as well as that of raldha, raldhb, and raldhc (Fig. 4a-c, d-f, and g-i, respectively). Especially in the juvenile rudiment, all genes were expressed in the epidermis region in a different expression pattern: patchwise expression of raldh genes and broad expression of rar and rxr (Fig. 4c, f, i, 1, o). We also found that all genes except raldhc were expressed in hydrolobes (primordium of primary podia and tube feet; Fig. 4c, f, 1, o). These expression patterns were consistent with the idea that hydrolobes are used for sensation of environmental cues in Paxillosida (13, 14). These data support the conclusion that RA signaling mediates the metamorphosis process in A. latespinosus.
Here we provided the evidence that metamorphosis is triggered by environmental cues in A. latespinosus larvae. When we introduced natural sand from the A. latespinosus habitat, the larvae stopped floating, became attached to the substrate, and commenced metamorphosis (Fig. 1). Furthermore, our data suggested that RA signaling mediated the commencement of metamorphosis upon environmental cue reception. Exogenous RA treatment of competent larvae induced metamorphosis (Fig. 2), and metamorphosis was suppressed by the inhibition of two distinct RA signaling pathways (Fig. 3): RA synthesis (Fig. 3a-c) and RA binding to RAR (Fig. 3d-i). The spatial expression pattern of RA signaling-related genes is consistent with the results described above (Fig. 4). Particularly, overlapping expression of two kinds of receptor (rar and rxr) was observed in juvenile rudiments of competent larvae (Fig. 4j-l and m-o, respectively). It should be noted that we did not examine the gene function analysis in this study. To strengthen our hypothesis, future study should focus on the function of each regulatory component.
Planktonic starfish larvae sense environmental cues for metamorphosis with brachiolar arms (7); but paxillosidan larvae, even those that are planktonic, lack brachiolar arms (4, 14). This absence is regarded as a secondary loss due to the transition to a sandy habitat (15). In this study, we found that metamorphosis of A. latespinosus is induced by culture with natural sand from its habitat, suggesting that paxillosidan larvae also respond to environmental cues for metamorphosis. Despite our findings, how paxillosidan larvae sense environmental cues remains unclear.
Previously, several researchers suggested that tube feet (primary podia) are used as the sensory apparatus for the reception of environmental cues in this group (13, 14). Yet Komatsu (8) and Oguro et al. (16) stated that tube feet did not appear before metamorphosis was mostly completed in A. latespinosus and Astropecten scoparius (8, 16). Whether larvae sense environmental cues with this structure is difficult to judge based on hydrolobe morphology. We suggest that researchers' attention be broadened to juvenile structures in efforts to identify the sensory apparatus for environmental cue detection in paxillosidan species.
Our work was supported by Japan Society for the Promotion of Science KAKENHI (Grant-in-Aid for Scientific Research) grants 15KT0074 and 18H04004. We thank the Ibaraki Prefectural Oarai Aquarium for providing the seawater for culturing of adult starfish.
We deposited the gene sequences (raldha, raldhb, raldhc, rar, and rxr) in the DNA Data Bank of Japan (DDBJ; LC485972, LC485973, LC485974, LC485975, LC485976). We also supply the dataset for phylogenetic analysis in supplementary datasets 1 and 2 (available online). The raw reads of transcriptomes are available from the DDBJ Sequence Reads Archives (DRA008444).
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Table A1 Number of metamorphosed/treated larvae of each batch under seawater with and without substrate Number of metamorphosed /treated larvae Substrate (+) Batch 1 Batch 2 Batch 3 Batch 1 Age (hpf) Well 1 Well 2 Well 1 Well 2 Well 1 Well 1 Well 2 48 0/10 0/10 0/10 0/10 0/10 0/10 0/10 72 2/10 3/10 0/10 1/10 1/10 0/10 0/10 96 8/10 8/10 7/10 8/10 4/10 0/10 1/10 120 8/10 8/10 7/10 8/10 4/10 2/10 2/10 144 8/10 8/10 8/10 8/10 4/10 3/10 2/10 168 8/10 8/10 8/10 8/10 4/10 3/10 4/10 192 8/10 8/10 8/10 8/10 4/10 3/10 4/10 Substrate (--) Age (hpf) Batch 2 Batch 3 Well 1 Well 2 Well 1 48 0/10 0/10 0/10 72 0/10 0/10 0/10 96 2/10 1/10 1/10 120 2/10 3/10 2/10 144 6/10 4/10 4/10 168 6/10 4/10 4/10 192 6/10 4/10 4/10 hpf, hours post-fertilization. Table A2 Number of metamorphosed larvae/treated larvae of each batch in retinoic acid (RA) or dimethyl sulfoxide (DMSO) treatment Number of metamo rphosed/treated larvae Batch 1 Batch 2 Batch 3 Treatment Well 1 Well 2 Well 1 Well 1 RA 8/10 8/10 7/10 9/10 DMSO 1/10 0/10 0/10 0/10 Table A3 Number of metamorphosed larvae/treated larvae of each batch in retinoic acid (RA) or dimethyl sulfoxide (DMSO) treatment in the case that treatment was commenced at 24 hours postfertilization (hpf) Number of metamorphosed/treated larvae RA DMSO Batch 1 Batch 2 Batch 3 Batch 1 Batch 2 Batch 3 Age (hpf) Well 1 Well 1 Well 1 Well 1 Well 1 Well 1 48 0/10 0/10 0/10 0/10 0/10 0/10 72 0/10 1/10 2/10 0/10 0/10 0/10 96 3/10 6/10 6/10 0/10 0/10 1/10 Number of metamorphosed/treated larvae of each batch under seawater with and without substrate Table A4 Number of metamorphosed larvae/treated larvae of each batch in retinoic acid (RA) or dimethyl sulfoxide (DMSO) treatment in the case that treatment was commenced at 48 hours post-fertilization (hpf) Number of metamorphosed/treated larvae RA DMSO Batch 1 Batch 2 Batch 3 Batch 1 Age (hpf) Well 1 Well 2 Well 1 Well 1 Well 1 Well 2 72 1/10 0/10 0/10 1/10 0/10 0/10 96 7/10 2/10 5/10 5/10 0/10 0/10 Batch 2 Batch 3 Age (hpf) Well 1 Well 1 72 0/10 0/10 96 0/10 0/10 Table A5 Number of metamorphosed larvae/treated larvae of each batch in DEAB or dimethyl sulfoxide (DMSO) treatment Number of metamorphosed/treated larvae Batch 1 Batch 2 Batch 3 Treatment Well 1 Well 2 Well 1 Well 1 DEAB 0/10 4/10 2/10 0/10 DMSO 6/10 9/10 8/10 3/10 Table A6 Number of metamorphosed larvae/treated lan'ae of each batch in R041-5253 (RO) or dimethyl sulfoxide (DMSO) treatment Number of metamorphosed/treated larvae Batch 1 Batch 2 Batch 3 Treatment Well 1 Well 2 Well 1 Well 1 RO 0/10 0/10 0/10 0/10 DMSO 8/10 8/10 9/10 2/10 Table A7 Number of metamorphosed larvae/treated larvae of each batch in retinoic acid (RA) or retinoic acid plus R041-5253 (RA+RO) treatment Number of metamorphosed/treated larvae Batch 1 Batch 2 Batch 3 Treatment Well 1 Well 2 Well 1 Well 1 RA 8/10 8/10 9/10 6/10 RA+RO 1/10 2/10 1/10 1/10 Table A8 Sequences of primer for amplification of raldha, raldhb, raldhc, rar, and rxr Forward primer (5'[right arrow]3') raldha gcaaccgatcgtcttcagaaggcacacatt raldhb accatcaatccggcaactggggagaagata raldhc gacggtgatttcttctgctactcccgctac rar gcgttacaccaaggtcccaacaacatgtcc rxr gtaaaggtcggcattctcctgaccagtgct Reverse primer including T3 promoter region (5'[right arrow]3') raldha ATTAACCCTCACTAAAGGGAgcagaaaccacgtcttgtat raldhb ATTAACCCTCACTAAAGGGAatattcataaaacgcacaca raldhc ATTAACCCTCACTAAAGGGAtagttgttgacccagatcac rar ATTAACCCTCACTAAAGGGAtacatggcatgaagtgttga rxr ATTAACCCTCACTAAAGGGAatcagcttgaagaagaagag We used 40-bp reverse primers, including a 20-bp T3 promoter sequence, to synthesize Digoxigenin (Dig)-labeled RNA probes for in situ hybridization. Capital letters indicate the consensus sequence for the T3 promoter. Table A9 Accession numbers for the genes used for phvlogenic analysis Species Gene Accession number Hs, Homo sapiens aldhlal P00352.2 aldhlal 094788 aldhla3 P47895 aldhlbl P30837 aldhl P05091 aldhial P30838 aldh3a2 P51648 aldhibl P43353 aldh3b2 P48448 aldh4al P30038 aldh5al P51649 aldh6al 002252 aldh7al P49419 aldh8al Q9H2A2 aldh9al P49189 Mm. Mus musculus Xt, Xenopus tropicalis aldhlal Q4VBE1 aldhla2 Q9DEX5 aldhlai F7BV06 aldhlbl F7DQF8 aldh2 Q6DJ49 aldh3a2 B1WBI3 aldh3b2 F6X8Y6 aldh4al A4QNJ0 aldh[delta]al F6QFQ2 aldh[beta]al F6SRL8 aldh7al F7BQF6 aldhSal F6UH88 aldh9al F6VC33 Dr, Danio rerio aldhla2 Q90XS8 aldhla3 Q0H2G3 aldh2a Q8QGQ2 aldh2b Q6TH48 aldh3al X1WBM4 aldh3a2a A0A2R8PW97 aldh3a2b E9QH31 aldhSbl Q90ZZ7 aldh4al Q7SY23 aldhSal A0A0R4IIB7 aldh[beta]al Q6DHT4 aldhJal Q803R9 aldhSal Q66I21 aldh9alal Q7ZVB2 aldh9ala2 BOS7W5 aldh9alb Q802W2 Bf, Branchiostoma floridae aldhla_1 C3ZGK4 aldhla_2 C3ZG63 B1. Branchiostoma lanceolatum Ci, Ciona intestinalis aldhlaj AOA1W2WB51 aldhlaj A0A1W5BCT1 aldhlaj A0A1W2WDC1 aldh.2 AOA1W5B7N8 Pm, Polyandrocarpa misakiensis Sk, Saccoglossus kowalevskii aldhla_1 XP_006823779.1 aldhla_2 XP_006822197.1 aldhla_3 XP_002736989.1 aldhla_4 XP_002731204.1 aldhla_5 XP_006824634.1 aldh2 XP_006816163.1 Sp, Strongylocentrotus purpuratus aldh2_1 SPU_007284 aldh2_2 SPU_023801 aldh5al_1 SPU_007492.1 aldh5al_2 SPU_016767.1 aldh6al SPU_026493.1 aldh7 SPU_024895.3a aldh8al1_1 SPU_017403.1 aldh8a_2 SPU_000522.1 aldh9 SPU_002901.3a Pp. Patiria pectinifera raldha LC379260 raldhb LC379261 raldhc LC379262 aldh2 (*) Al, Astropecten latespinosus raldha LC485972 raldhb LC485973 raldhc LC485974 Dm, Drosophila melanogaster Rc, Reishia clavigera Ls, Lymnaea stagnalis Tc, Tripedalia cystophora Species Gene Accession number Hs, Homo sapiens thra P10827 thrb P10828 rara P10276 rarb P10826 rarg P13631 rxra P19793 rxrb P28702 rxrg P48443 Mm. Mus musculus thra P63058 thrb P37242 rara P11416 rarb P22605 rarg P18911 rxra P28700 rxrb P28704 rxrg P37238 Xt, Xenopus tropicalis Dr, Danio rerio thraa Q98867 thrab U3JAT9 thrb Q9PVE4 raraa Q90271 rarab Q7ZTI3 rarga Q91392 rargb A2T928 Bf, Branchiostoma floridae rxr Q8MX78 B1. Branchiostoma lanceolatum rar O18608 Ci, Ciona intestinalis rar Q4H2W1 rxr Q4H2U9 Pm, Polyandrocarpa misakiensis rxr K7ZLP3 Sk, Saccoglossus kowalevskii rar XP_002742241.1 rxr D2XNK4 Sp, Strongylocentrotus purpuratus thr SPIL025239 rar SPU_016523 rxr SPU_028422 Pp. Patiria pectinifera rar LC379258 rxr LC379259 thr (*) Al, Astropecten latespinosus rar LC485975 rxr LC485976 Dm, Drosophila melanogaster usp P20153 Rc, Reishia clavigera rar T2HRZ4 rxr E9RHD8 Ls, Lymnaea stagnalis rar D5LIR6 rxr Q5I7G2 Tc, Tripedalia cystophora rxr 096562 (*) Sequences were not deposited to Databank but are available from supplementary datasets 1 or 2 (available online).
SHUMPEI YAMAKAWA (*), YOSHIAKI MORINO, MASANAO HONDA, AND HIROSHI WADA
Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba 305-8572, Japan
Received 17 April 2019; Accepted 23 August 2019; Published online 10 December 2019.
(*) To whom correspondence should be addressed. Email: shumpei .firstname.lastname@example.org.
Abbreviations: ALDH, aldehyde dehydrogenase; DDBJ, DNA Data Bank of Japan; DEAB, N,N-diethylaminobenzaldehyde; DMSO, dimethyl sulfoxide; hpf, hours post-fertilization; RA, retinoic acid; RALDH, retinoic acid synthesis by retinal dehydrogenase; RAR, retinoic acid receptor; RXR, retinoid X receptor; RO, R041-5253.
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|Author:||Yamakawa, Shumpei; Morino, Yoshiaki; Honda, Masanao; Wada, Hiroshi|
|Publication:||The Biological Bulletin|
|Date:||Dec 1, 2019|
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