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Keywords:

  • cephalopod;
  • reflectin genes;
  • expression pattern;
  • development;
  • iridophores

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. ACKNOWLEDGMENTS
  8. REFERENCES
  9. Supporting Information

Background: In the cuttlefish Sepia officinalis, iridescence is known to play a role in patterning and communication. In iridophores, iridosomes are composed of reflectins, a protein family, which show great diversity in all cephalopod species. Iridosomes are established before hatching, but very little is known about how these cells are established, their distribution in embryos, or the contribution of each reflectin gene to iridosome structures. Results: Six reflectin genes are expressed during the development of iridosomes in Sepia officinalis. We show that they are expressed in numerous parts of the body before hatching. Evidence of the colocalization of two different genes of reflectin was found. Curiously, reflectin mRNA expression was no longer detectable at the time of hatchling, while reflectin proteins were present and gave rise to visible iridescence. Conclusion: These data suggest that several different forms of reflectins are simultaneously used to produce iridescence in S. officinalis and that mRNA production and translation are decoupled in time during iridosome development. Developmental Dynamics 242:550–561, 2013. © 2013 Wiley Periodicals, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. ACKNOWLEDGMENTS
  8. REFERENCES
  9. Supporting Information

Color patterns in animals are common adaptations involved in camouflage and/or in inter and intraspecific communication. Colors are mostly obtained by means of pigment components that absorb specific wavelengths (carotenoids, melanins, or flavins; Fox, 1953; Bandaranayake, 2006) and/or by physical structures with a selective reflectance of incident light (Fox, 1953; Vukusic and Sambles, 2003). Most cephalopods species living in shallow water, such as squids, cuttlefish, and octopuses, exhibit a reduced and internalized shell and their skin has evolved into an elaborate organ for color patterning. This patterning is intimately linked to their behavior and ecological life history (Hanlon and Messenger, 1996). Colors of cephalopods derive from both pigments and physical structures produced and stored by specialized cells: chromatophores, leucophores, and iridophores. Chromatophores are dermic superficial neuromuscular organs made of one folded cell containing yellow, red, or brown pigments that become visible when the cell is stretched out by surrounding muscular cells (Cloney and Foley, 1968; Florey, 1969; Mirow, 1972a; Dubas, 1987; Gaston and Tublitz, 2006). Under the chromatophore layer, leucophores provide a structural white background whereas iridophores are responsible for an iridescence using additional wavelengths (pink, blue, and green) (Mirow, 1972a, 1972b; Hanlon, 1982; Cloney and Brocco, 1983). By operating these cells, cephalopods can create a complex repertoire of body patterns using colors across the entire visible spectrum (Packard and Hochberg, 1977; Hanlon and Messenger, 1988; Chiao and Hanlon, 2001; Hanlon, 2007; Kelman et al., 2007; Hanlon et al., 2009).

In animals, iridescence mostly comes from crystalline nanostructures of guanine or hypoxanthine that act as multilayer reflectors (Fox, 1953; Land, 1972; Vukusic and Sambles, 2003). Interestingly, cephalopods are unique among metazoans because their iridescence is due to protein platelets intercalated with plasma membrane folds. The resulting structure, called an iridosome, presents all the characteristics of a multilayer reflector (Arnold et al., 1974; Hanlon et al., 1990; Tao et al., 2010). Iridosomes reflect light through thin-film interference (Denton and land, 1971; Cooper et al., 1990; Mäthger and Denton, 2001; Holt et al., 2011) and are also able to polarize light (Shashar and Hanlon, 1997; Mäthger and Hanlon, 2006; Chiou et al., 2007). Physiological studies have even shown that individuals are able to control iridescence intensity (Hanlon, 1982; Cooper and Hanlon, 1986; Cooper et al., 1990; Hanlon et al., 1990; Mäthger et al., 2004). Proteins of cephalopod iridophores were first characterized in the light-organ of Euprymna scolopes and called reflectins (Crookes et al., 2004). Six reflectin genes and three reflectin cDNAs have been found in the Euprymna scolopes light organ (Crookes et al., 2004) and in the skin of the squid Loligo pealeii (Izumi et al., 2010), respectively. More recently, the analysis of an expressed sequence tag (EST) library from embryonic stages of Sepia officinalis suggests the expression of at least six different reflectin mRNAs during development (Bassaglia et al. 2012).

Cephalopods develop directly without any larval stage or metamorphosis, and hatching in S. officinalis gives rise to a juvenile morphologically identical to an adult and with a similar necto-benthic ecological life history (Boletzky et al., 2006). It has been demonstrated that cuttlefish hatchlings already possess most of the structures implicated in body patterning and use them as adults do (Hanlon and Messenger, 1988). Iridescence, which indicates the presence of reflectins, is observed in S. officinalis juveniles, which suggests that iridophores are established during embryogenesis (Hanlon and Messenger, 1988, 1996). In juveniles and adults, iridophores are present in eyes, and in the skin of the arms, head, and mantle. Each is supposed to have a specific role in patterning and/or communication (Shashar et al., 2000; Barbosa et al., 2007; Mäthger et al., 2009a, 2009b). The recent confirmation that at least six different reflectin mRNAs exist in S. officinalis raises the question of potential specific properties and of probable tissue-specific expression. In this study, we performed an extensive search for reflectin genes in S. officinalis and we compared their sequences with those of mRNA and contigs from the EST library. We here demonstrated that the diversity of reflectin forms in S. officinalis is not due to splicing events but to six clearly distinguishable genes. The reflectin genes of S. officinalis are highly conserved, which makes their discrimination difficult by in situ hybridization (ISH). Nonetheless, we thoroughly investigated the expression patterns of some reflectin forms and provide a first spatio-temporal description of the iridophore development. Comparisons of in situ hybridizations with significantly different probes do not show any obvious tissue-specific expression which suggests that the evolutionary diversification of reflectin forms is more a question of physical or assembling properties rather than of organ-specific color patterning.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. ACKNOWLEDGMENTS
  8. REFERENCES
  9. Supporting Information

Reflectin Genes in Sepia officinalis

As stated in Bassaglia et al. (2012), six tentative contigs (TCs) of reflectins have been identified in the EST library we previously made from S. officinalis embryos at different developmental stages. To confirm that these TCs correspond to real mRNAs, specific primers were designed (Supp. Table S1, which is available online) and polymerase chain reaction (PCR) amplifications were performed on cDNA obtained from a pool of RNA from stage 20 to 29 S. officinalis embryos (Supp. Table S1). After cloning and sequencing PCR products, numerous sequences of equivalent size were obtained and identified as reflectins by BLAST analysis. We then considered that sequences with a percentage of identity lower than 95% (after conceptual translation) were issued from mRNA of different genes or from differently spliced mRNAs of a same gene. As a result, six different predicted protein sequences of reflectin were identified in Sepia officinalis (Supp. Table S1). We also amplified genomic DNA using the same primers. We obtained six PCR products corresponding to the six mRNA forms, with exactly the same length and with highly similar sequence (see Supp. Fig. S1 for alignments). Therefore, we assume that each of these six DNA sequences are coding parts of six different reflectin genes whose expression leads, without splicing event, to the six mRNA forms we detected. Four reflectin forms—SoRef3, SoRef4, SoRef5, and SoRef6—show the five repeated sequences, including a highly conserved subdomains (SD) [M/FD(X)5MD(X)5MD(X)3/4] as defined by Crookes et al. (2004). The two other forms, SoRef1 and SoRef2, lack the first repeated sequence (Fig. 1).

image

Figure 1. Predicted protein sequences of the six reflectins of S. officinalis. Alignments of reflectin proteins SoRef1-SoRef6 predicted from cDNA sequences. Conserved amino acid residues are highlighted in black. The alignment shows five highly conserved motifs named SD (underlined).

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Evidence of Iridescence in S. officinalis Embryos

We evaluated the occurrence of functional iridophores by visual detection of iridescence, after removing the egg capsule of living embryos. Iridescence was first detected in the eyes of late embryos at stage 27, which is three stages before hatching (Fig. 2A). At stage 29, iridescence also appeared as dots in the skin of the dorsal side of the mantle, of the head and of the arms (Fig. 2B). At stage 30, a higher density of iridophores was visible on the skin of different areas throughout the embryo (Fig. 2C–E). In the skin, iridophores were either isolated or organized as lines or clusters. Iridescent lines, pink or purple, were located on the dorsal side of the arms 1, 2, and 3 (Fig. 2D) and on the lateral side of the mantle, iridophores are gathered into a row of clusters (Fig. 2E,F). Some iridophores were also harbored in the numerous papillae located throughout the dorsal side of the mantle (Fig. 2C). We did not observe iridescence cues on the ventral side of the mantle (data not shown).

image

Figure 2. Evidence of iridescence in living S. officinalis embryos before hatching. A: dorsal view of the right eye of a stage 27 embryo showing faint iridescence (arrow). B: dorsal view of a stage 29 embryo. Iridescence (arrows) is visible in both eyes, on the dorsal side of mantle, on the head and in the arms. C: dorsal view of a stage 30 embryo with iridescence on the dorsal side of the mantle in a multitude of papillae (white arrowheads) and on the head. D: dorsal view of the head of a stage 30 embryo showing lines of pink (black arrowhead) and purple (white arrowhead) iridescence on the arms. E: Lateral view of a stage 30 embryo showing a cluster of iridescent cells in the right lateral side of the mantle. F: Focus on a cluster of iridescent cells at the lateral side of the mantle, stage 30. a, arms; e, eye; f, fin; h, head; m, mantle; y, yolk sac; Ant, anterior; Post, posterior; l, left; r, right; D, dorsal; V, ventral.

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SoRef Expression During Development

To discriminate the expression of different forms of reflectin by ISH, three antisense RNA probes were synthesized from the three most different reflectin sequences: one from SoRef3 (AS-3), one from SoRef5 (AS-5) and one from SoRef6 (AS-6) (Supp. Fig. S1). The percentage of difference of these three probes is between 10 and 20% (Supp. Table S2). Because of the high similarity of the different reflectin sequences, a probability exists that an antisense probe, such as AS-5, will hybridize not only SoRef5 endogenous mRNAs but also other reflectin mRNAs. We, therefore, tested if our antisense probes hybridized with nonspecific reflectin mRNAs using “competitive ISH”. For each antisense probe, we performed three in situ hybridizations either with the antisense probe alone (positive control) or with its corresponding sense probe (S-3, S-5, or S-6; negative control) or with a mix of the two other sense probes (test, Fig. 3). The results showed that AS-3 is not inactivated by S-5 and S-6, which strongly suggests that AS-5 and AS-6 recognize different targets than AS-3 (Fig. 3A–C). However, AS-5 and AS-6 are inactivated by, respectively, S-6 and S-5 (Fig. 3D–I), which suggests that these probes have similar mRNA targets.

image

Figure 3. Test of probe specificity by whole-mount fluorescent in situ hybridization: focus on arms at stage 27. Arrows point out positive cells. A–C: Test of the AS-3 probe. D–F: Test of the AS-5 probe. G–I: Test of the AS-6 probe. The “test” line shows hybridizations using the tested antisense probe with the sense probes of the two other forms. The “positive control” line shows hybridization using the tested antisense probe alone. The “negative control” line shows hybridizations using the tested antisense probe with its corresponding sense probe. Scale bars = 300 μm.

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ISHs were performed using antisense probe AS-3 and AS-5. The staining patterns obtained with the two probes were identical at the embryonic stages tested (25 to 30) of the embryo (Supp. Fig. S2). The expression patterns we observed were always bilateral, so we here describe them without specifying any left or right.

In the mantle skin

SoRef3 and SoRef5 were detected in three areas of the mantle skin. On the dorsal side of the mantle, limited by the fins, reflectin expression started at stage 26 in clusters of cells (Fig. 4A,B,G,H), which progressively grew up. At stages 27–28, reflectin was expressed in more new intercalated clusters and isolated cells, progressively covering the dorsal surface (Figs. 4C,D,I,J, 5D). As expected, these stained areas correspond to areas of iridescent cells in living embryos (Fig. 2B,C). However, staining became less detectable starting at 28/29 (Fig. 4E,K) and was progressively replaced by a whitish color, suggesting that reflectin proteins were produced (Fig. 4F,L).

image

Figure 4. Reflectin expression on the dorsal side of S. officinalis embryos (arms, head and mantle) from stage 25 to 30 as revealed by whole mount in situ hybridization with AS-3. A–F: Dorsal view of the whole embryo, white squares focus on the same area on the mantle surface (magnified in G–L); red squares focus on the same area on the head surface (magnified in M–R). G–L: Reflectin expression in two magnified clusters on the dorsal side of the mantle from stage 25 to 30. Reflectin expression in cells (white arrows) becomes undetectable and seems replaced at stage 29 by a whitish color in the very place where iridescence is detected in living embryos (white arrowheads). M–R: Reflectin expression on the portion of the head skin, following one spot from stage 25 to 30. Reflectin expression in cells (black arrows) becomes undetectable and is replaced by a whitish color at stage 30 where iridescence is detected in living embryos (black arrowhead). Scale bars = 1 mm in A–F, 300 μm in G–R. ant, anterior; Post, posterior; l, left; r, right.

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The lateral sides of the mantle (from the fins to the lateral skin papilla) showed a similar dynamic of reflectin expression (Fig. 5). A row of lateral clusters (lc) appeared at stage 26: first two clusters (Fig. 5A,B), then four clusters at stage 27 (Fig. 5C), five at stage 28 (Fig. 5D), and finally six from stage 29 (Fig. 5E,F). This spatial organization is similar to that observed in the living embryos (Fig. 2E). Sections confirmed in all cases the dermal localization of the positive cells (see Fig. 5G). As on the dorsal side, ISH staining decreased in clusters as soon as a whitish color appeared inside (e.g., from stage 29, for the two first clusters lc1 and lc2, Fig. 5E).

image

Figure 5. Reflectin expression on the left lateral side of the mantle in S. officinalis embryos from stage 25 to 30 as revealed by whole mount in situ hybridization with AS-3. A–F: reflectin expression showing gradual apparition of clusters of positive cells (named lc1 to lc6) and positive isolated cells. G: Transverse section of a stage 27 embryo throughout the lc1 cluster (white dotted-line in C) showing reflectin positive cells under the epithelium. Scale bars = 1 mm. ant, anterior; post, posterior; l, left; r, right; D, dorsal; V, ventral.

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The ventral side of the mantle and the fins were devoid of any reflectin expression during development (data not shown), and which is consistent with the iridescence distribution we observed in living embryos.

In the skin on the head

The skin on the dorsal side of the head mostly showed isolated positive cells and some numerous clustered positive cells (Fig. 4). The expression of reflectin mRNA started at stage 27 (Fig. 4C,O) and corresponded, as expected, to the areas where iridescence appeared in living embryos (Fig. 2). As in the mantle skin, the mRNA expression was transient in positive cells and was followed, from stage 30, by a whitish color likely corresponding to reflectin protein production (Fig. 4F,R). The skin of the ventral side of the head exclusively showed isolated cells expressing reflectin with a similar developmental timing (data not shown).

In the eyes

The first expression of reflectins in the eyes was observed at stage 25 in some cells of the iris (Fig. 6A,G). At the following stages, all cells of the iris expressed reflectins (Fig. 6B,C). This expression progressively decreased from stage 27 to stage 30 (Fig. 6D,E,H) and at stage 30, most iris cells showed iridescence (Fig. 6F). A secondary eye structure, the cornea, which consists of two converging skin folds covering the iris, showed positive reflectin cells from stage 27 to stage 30 (Fig. 6C,D,E,F).

image

Figure 6. Reflectin expression in the right eye of S. officinalis embryos from stage 25 to 30 as shown by whole mount in situ hybridization with AS-3. A–F: Reflectin expression and reflectin protein production during development of eye structures. A: stage 25. B: stage 26. C: stage 27. D: stage 28. E: stage 29. F: stage 30. G,H: Transverse sections of the eye. G: Reflectin positive cells in the iris (arrows) at stage 25 (white dotted-line in A). H: Reflectin positive cells in cornea (black arrows). Faded iris cells appear brown (black arrowheads), other iris cells show a light staining (white arrow) at stage 29 (white dotted-line in E). Scale bars = 500 μm in A–F, 200 μm in G–H. cr, crystalline lens; ir, iris; re, retina; co, cornea. Dorsal is up, ventral is down.

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In the arm skin

The expression of reflectin in arms appeared mostly in isolated dermal cells, but also in some proximal cell clusters (Fig. 7). These expressions are restricted to the outer side of the arms (the suckered inner side is devoid of reflectin expression, Fig. 7G). From stage 26 to stage 29, reflectin expression extended from proximal to distal areas. At stage 30, whitish cells became visible and one longitudinal stripe along each arm appeared except in arms 4 and 5 (Fig. 7F) as observed in living embryos (Fig. 2D). Interestingly, there was no reflectin expression in the arms 4, which are the longest arms specialized in prey capture (tentacles).

image

Figure 7. Reflectin expression on arms in S. officinalis embryos from stage 25 to 30 as revealed by whole mount in situ hybridization with AS-3. A–F: Gradual apparition of numerous positive isolated cells and clusters (white arrows) on the whole arm surface. G: Transverse section of arms stage 29 (white dotted-line in E). Reflectin positive cells are located at the outer side of the arms. Scale bars = 1 mm in A–F, 200 μm in G. su: sucker. Posterior is up. Anterior is down.

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Double in situ hybridization

We then investigated whether or not the mRNAs hybridized by AS-3 and AS-5 were co-expressed at the cellular level. The results of double fluorescent in situ hybridization showed that AS-3 and AS-5 were mostly co-hybridized and were rarely in different cells (see Fig. 8, for arms and eyes). This was true independently of the region of the body.

image

Figure 8. Double fluorescent in situ hybridization on embryonic sections from stage 26. A–C: Arm section. D–F: Eye section. Green staining: hybridization with AS-3 probe (A,D). Magenta staining: hybridization with AS-5 probe (B,E). Overlapping of the two signals appears in white (C,F). White box: detail from box in C. Scale bars = 100 μm. Insert scale bar = 20 μm.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. ACKNOWLEDGMENTS
  8. REFERENCES
  9. Supporting Information

Our careful search for reflectin cDNAs and for their associated genomic coding sequences allows us to confirm that S. officinalis possesses at least six different reflectin genes, which we called SoRef1 to SoRef6 (Supp. Fig. S1). This result is consistent with that of Crookes et al. (2004) who suggested the existence of at least six reflectins in E. scolopes. In both S. officinalis and E. scolopes, reflectin coding sequences show no intron (see comparisons of cDNA and genomic sequences in Supp. Fig. S1; Crookes et al. 2004). Reflectin genes are specific to the cephalopod clade (Crookes at al., 2004) and have been identified in several species, each showing different ecological life histories. The relationships between the diversity of reflectins and their use for patterning should be explored. Extensive genomic studies in other cephalopod species are needed to fully understand the evolution of the reflectin genes. In particular, very few data exist in pelagic species despite the fact that their use of iridescence is among the most sophisticated of cephalopods (Hanlon, 1982). Currently, searching for reflectin mRNAs in the skin of L. pealeii (Izumi et al., 2010) and in the eyes of L. forbesi (Weiss et al., 2005) has only detected three (in L. pealeii) and one (in L. forbesi) different reflectin forms, and no data exist on their reflectin genes.

We know that the six reflectin genes we detected in S. officinalis are expressed during embryogenesis because we found their corresponding mRNAs in our embryonic EST library and through PCRs on embryonic cDNAs. As expected, in situ hybridizations using reflectin forms as antisense probes reliably label the developing iridophores, permitting us to follow the developmental dynamics of iridescent structures. For instance, the territories labeled by AS-3 (antisense probe designed from SoRef3) actually correspond to territories where iridescence is detected in decapsulated embryos (Fig. 2): in the eyes and skin on arms, head, and mantle (Figs. 4-7). Nonetheless, describing the specificity of each of the six different reflectin genes turned out to be a much more detailed endeavor. We first ascertained that our probes could distinguish the highly similar reflectin mRNAs. Comparing the signals obtained with these probes in ISH provided good evidence that reflectin mRNAs may not be specifically distributed among tissues and organs but rather expressed in all tissues (see eye and arm in Fig. 8). This result is consistent with previous studies in which different reflectins were identified in a single organ: six forms were found in the light organ in E. scolopes (Crookes et al., 2004) and three forms in the skin of L. pealeii (Izumi et al., 2010). At the tissue level, our analysis of co-expression even demonstrates that mRNAs of different reflectins can be expressed in a single cell at the same time (Fig. 8). As multilayer reflectors in iridophores are made of reflectin proteins self-organized in platelets (Kramer et al., 2007), it is possible that several different reflectin forms assemble into a single iridophore. In S. officinalis, the six reflectin sequences do not have the same number of repeated sequences (4 in SoRef1 and SoRef2; 5 in SoRef3, 4, 5, 6). These differences at the level of the sequence may have an influence on the tertiary and quaternary structure of the proteins and on the optical properties they confer to the iridophores in which they are assembled. A more extensive study about platelet composition should be therefore conducted to confirm if the evolutionary diversification of the reflectin genes into six forms is actually linked to selection on their optic or assembling properties.

This study shows for the first time the dynamic expression of reflectin genes during the embryonic development of a cephalopod. Expression of reflectin mRNA was observed in the later stages of development: the earliest expression was detected at stage 25, in the eyes, five stages before hatching. Although we thought that expression would last through hatching and be maintained in juveniles, we observed that it was actually transient. In most of the tissues concerned, reflectin positive cells were generally positive during three stages and then started to exhibit a whitish color, likely due to increasing production of reflectin proteins. We conclude from this result that the juvenile pool of reflectin is mostly produced before hatching and that maintenance of reflectin expression seems undetectable (Figs. 4F, 5F, 6F, 7F). Interestingly, this developmental process is probably reactivated during postembryonic development, because late juveniles and adults exhibit new iridophores, absent in hatchlings. For instance, we were unable to detect any reflectin expression on the ventral side of the mantle in embryos, which is consistent with the observation of Hanlon and Messenger (1988), who noted that this area shows reflective properties in S. officinalis adults but not in juveniles. Similarly, the fourth arm pair (future tentacles) never show expression of reflectin mRNA during development, while iridescence is visible on the tentacular club in adults (Hanlon, unpublished personal communication).

Mechanisms controlling the spatial and temporal distribution of iridophores at the organ and at the cellular level are currently totally unknown. Iridophore development has only been studied in vertebrates where the role of a melanization inhibitor factor (MIF) has been shown (Bagnara and Fukuzawa, 1990). Here, in S. officinalis, we remain ignorant of the molecular mechanisms that lead to isolated positive cells and to clusters, lines, or layers. The fact that these structures are already present and active before hatching suggests that the presence of iridophores is crucial for the initial camouflage patterns, yielding a better survival rate of the hatchling in a hostile environment. With regard to the iridescent patterning, it must be determined how new iridophores appear: they could be progeny of positive cells or naive neighboring cells recruited to express reflectin genes. Finally, our results raise the question of the turnover of reflectins throughout the life of the animal. We currently don't know if an iridophore that has stopped producing mRNAs is able to resume reflectin mRNA and protein expression. The iridescent areas can be maintained either by the assembling of new iridosomes in previously established iridophores or by the replacement of iridophores themselves. Our study is a first step in describing reflectin expression in S. officinalis and further questions about reflectin dynamics and distributions will now require more specific protocols to increase our knowledge of this unique cephalopod-specific protein family.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. ACKNOWLEDGMENTS
  8. REFERENCES
  9. Supporting Information

Biological Material

The experimental procedures were carried out in strict compliance with the European Communities Council Directive (86/609/EEC) and followed the French legislation requirements (decree 87/848) regarding the care and use of laboratory animals. All efforts were made to minimize animal suffering and to reduce the number of animals used.

Embryo collection

Eggs from S. officinalis were collected from the marine station of Luc-sur-mer (Caen University, France) between April and September 2011. Eggs were kept during development in oxygenated seawater at 20°C. In these conditions, organogenesis occurred in 2 to 3 weeks, giving rise to a juvenile identical to the adult. Embryos developed inside a chorion enclosed in numerous dark envelopes (capsule), and were sampled daily to assemble a complete collection of morphological steps according to the developmental landmarks established by Lemaire (1970). After removing the capsule and chorion, embryos were fixed and processed for in situ hybridization and RNA extraction.

Adult tissue

The mantle tissues of dead animals from fisheries were stored at −80°C until use.

DNA Extraction of Adult Tissue

DNA extraction was performed with approximately 30 mg of mantle tissue of adult Sepia officinalis, stored at −80°C, using DNeasy Tissue Kit (QIAgen).

RNA Extraction and cDNA Synthesis

Dechorionated embryos of stages 20 to 29 were immersed in RNAlater (Ambion, Austin, TX) after anesthesia by progressive temperature lowering on ice and stored at −20°C until use. Total RNA from each S. officinalis embryos was extracted using Tri Reagent (MRC, Cincinnati, OH). After addition of chloroform/isoamyl alcohol (24:1) and centrifugation at 12,000 rpm for 15 min, RNA was purified with RNeasy Plus mini Kit (Qiagen, Valencia, CA) according to manufacturer's instructions. Total RNA was assembled by mixing 2 μg of total RNA of each S. officinalis embryonic stage. cDNA was synthesized using 2 μg of this total RNA pool and a Omniscript Reverse Transcription First-strand cDNA synthesis Kit (Qiagen). cDNA was stored at −20°C.

Cloning of Reflectin Genes and cDNA

Specific primers (Supp. Table S3) were designed according to the TCs (tentative contigs) obtained from an ESTs library (Genoscope CEA project) built from Sepia embryo cDNA (Bassaglia et al., 2012).

PCRs were performed as follows: an initial step of polymerase activation for 5 min at 92°C; 40 cycles with 1 min denaturation at 92°C, 1 min annealing at 64°C for, 1 min extension at 72°C, and a single final extension step of 5 min at 72°C.

The amplicons were cloned into TOPO 4 vector (Invitrogen, Carlsbad, CA) and sequenced by GATC Biotech (Konstanz, Germany).

Sequence Analysis

All alignments were done using MEGA 5.0 software (Tamura et al., 2011) and percentage identity calculations were done using Jalview 2 (Waterhouse et al., 2009). For each pair of sequences, the best global alignment is found using BLOSUM62 as a scoring matrix. The scores reported are the raw scores. The sequences are aligned using a dynamic programming technique and using the following gap penalties: Gap open: 12, Gap extend: 2. The Sepia officinalis reflectins sequences were registered to GenBank (Supp. Table S4).

Whole-Mount In Situ Hybridization

Embryo fixation

Capsule and chorion were removed in seawater and embryos were anesthetized by progressive lowering the temperature on ice. Embryos at stage 24 to 30 were selected and fixed in 3.7% paraformaldehyde (PFA) in filtered seawater at room temperature, for 2 to 3 hr (depending on the stage). After four rinses in phosphate buffered saline (PBS) at room temperature, embryos were placed in 30% glycerol/PBS for 2 hr at room temperature, and transferred in 50% glycerol overnight at 4°C. They were stored at −20°C until use.

RNA probes

RNA probes AS-3, S-3 (from SoRef3), AS-5, S-5 (from SoRef5), and AS-6, S-6 (from SoRef6) were generated from cloned PCR products (Supp. Table S5), by in vitro transcription using digoxigenin-11-UTP (Dig RNA labeling mix kit, Roche, Meylan, France) or fluorescein-12-UTP (Fluorescein RNA labeling mix kit, Roche, Meylan, France). Antisense and sense probes were obtained with T3 or T7 polymerase (Roche). RNA probes were purified by cold precipitation with lithium chloride and anhydrous alcohol. Sense RNA probes were used as negative controls.

NBT-BCIP in situ hybridization

ISH was performed using at least three embryos per stage and controls were done for each stage. All steps were performed at room temperature unless stated otherwise. After five rinses in PTW (PBS, 0.1% Tween 20), embryos were permeabilized by proteinase K (10 μg/ml in PTW for 20 min) and post-fixed 1 hr in 3.7% PFA. Embryos were then prehybridized for 6 hr at 65°C in hybridization solution (SH) (50% formamide, 5X standard saline citrate [SSC], 0.1% Tween 20, 1% sodium dodecyl sulfate) containing 25 μg/ml heparin and 100 μg/ml baker's yeast tRNA (Roche). Hybridization with RNA probes (300 ng/ml) were done in hybridization buffer (SH, 33 μg/ml heparin, 400 μg/ml tRNA) at 65°C overnight. Unbound probes were removed by 4 washes in SH, one in 25% 2X SSC in SH, one in 50% 2X SSC in SH, one in 75% 2X SSC in SH, one in 2X SSC, each 10 min at 65°C. All washing solutions were prewarmed at 65°C.

Hybridized probes were detected by an anti-digoxigenin antibodies coupled with alkaline phosphatase (Roche). After a saturation step in 4% blocking reagent (Roche) and 15% fetal bovine serum in MABT (Maleic acid 100 mM and NaCl 150 mM, pH 7.5, 1% Tween20) for 1 hr at room temperature, embryos were incubated overnight at 4°C with antibodies diluted 1:2,000 in MABT containing 1% blocking reagent (Roche) and 5% fetal bovine serum. Embryos were washed six times in MABT for 10 min, and bound antibodies were revealed using NBT-BCIP (nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate; Roche) substrate in alkaline phosphatase solution (Tris-HCl pH 9.5 100 mM, NaCl 100 mM, MgCl2 50 mM, 0.1% Tween20) prepared fresh and containing 1 mM levamisol, at room temperature for 3 hr. The reaction was stopped by washing in PTW solution. Embryos were post-fixed 24 hr in 3.7% PFA in PBS.

Fluorescent in situ hybridization

In situ hybridization was performed using at least 3 embryos per stage and controls were done for each stage. Hybridization and washes were as described before, but RNA probes were detected by anti-digoxigenin antibodies coupled with horseradish peroxidase (POD) (Roche). Inactivation of endogenous peroxidase activity was done by incubation in PTW with 3% hydrogen peroxide for 30 min at room temperature. After 1 wash in MABT for 5 min, a saturation step in 4% blocking reagent (Roche) and 15% fetal bovine serum in MABT for 1 hr at room temperature was performed. Then, embryos were incubated overnight at 4°C with antibodies diluted 1:500 in MABT containing 1% blocking reagent (Roche) and 5% fetal bovine serum. Embryos were washed three times in PTW for 10 min, and bound antibodies were revealed using fluorescein isothiocyanate (FITC) -tyramide (synthesized in the laboratory) diluted 1:200 in PTW containing 0.001% hydrogen peroxide at room temperature for 45 min in the dark.

The reaction was stopped by washing in PTW solution and embryos were post-fixed 24 hr in 3.7% PFA in PBS.

Double Fluorescent In Situ Hybridization on Cryostat Sections

Cryosection

Embryos were embedded in gelatin/saccharose phosphate solution (phosphate buffer 0.12 M, 12% saccharose, 7.5% gelatin) and then rapidly frozen in liquid isopentane at −80°C. They were cut in 20 μm sections on a Microm Microtech cryostat (HM560).

Double fluorescent in situ hybridization

ISH was performed using sections at different levels of 3 embryos (stage 26) in independent experiments. Controls were done for each manipulation. This protocol was adapted from Braissant and Wahli (1998). After defrosting 30 min at room temperature, sections were incubated for 2 × 15 min in PBS containing 0.1% active DEPC (Sigma), and equilibrated for 15 min in 5X SSC. The sections were then prehybridized for 2 hr at 65°C in the hybridization buffer (50% formamide, 5X SSC, salmon sperm DNA 40 μg/ml, 5X Denhardt's reagent, 10% dextran sulphate). A mix of digoxigenin and fluorescein labeled probes was added to hybridization buffer (each 300 ng/ml). Hybridization was carried out at 65°c overnight. Prehybridization and hybridization were performed in a humidified chamber saturated with a 5X SSC/50% formamide solution to avoid evaporation. After incubation, the sections were washed for 30 min in 2X SSC, 1 hr in 2X SSC, 1 hr in 0.1X SSC at 65°C. All washing solutions were prewarmed at 65°C. Inactivation of endogenous peroxidase activity was performed in PTW with 3% hydrogen peroxide for 30 min at room temperature. After 1 wash in MABT for 5 min, a saturation step in 4% blocking reagent (Roche) and 15% fetal bovine serum in MABT for 1 hr at room temperature was performed. Then, sections were incubated overnight at 4°C with POD-coupled anti-digoxigenin antibodies (Roche) diluted 1:500 in MABT containing 1% blocking reagent (Roche) and 5% fetal bovine serum. Sections were washed three times in PTW for 10 min, and bound antibodies were revealed using Cy3-tyramide diluted 1:200 in PTW containing 0.001% of hydrogen peroxide, at room temperature for 45 min, in the dark. Future steps take place in the dark. The revelation was stopped and any residual peroxidase activity was quenched by washing in PTW with 3% hydrogen peroxide for 30 min. After a new step of saturation for 1 hr, incubation with anti-Fluorescein antibodies coupled with POD, was performed overnight at 4°C. After washing off unbound antibodies, the second color step was performed with FITC-tyramide diluted 1:200 in PTW containing 0.001% hydrogen peroxide at room temperature for 45 min. After washing, the sections were mounted in Mowiol.

Observation and Imaging

Embryos were observed with a Leica M16 2F binocular stereomicroscope and sections were observed with a Leica DMLB microscope. Color pictures were made with a Coolsnap, color camera, and fluorescent pictures were made with a Qimaging Retiga 2000R black and white camera and colorized in Adobe Photoshop CS (Adobe Systems, San Jose, CA).

ACKNOWLEDGMENTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. ACKNOWLEDGMENTS
  8. REFERENCES
  9. Supporting Information

We thank J. Henry and L. Dickel (University of Caen), S. Henry, L. Lévèque and X. Bailly (Roscoff Biology Station - UPMC) for providing biological materials. We thank R. Hanlon for unpublished information. We thank Annabelle Aish (SPN - MNHN) and Caroline B. Albertin (University of Chicago) for helpful English corrections on the article. The Sepia EST library was obtained in collaboration with Corinne Da Silva and Julie Poulain (CEA Genoscope).

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  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. ACKNOWLEDGMENTS
  8. REFERENCES
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. ACKNOWLEDGMENTS
  8. REFERENCES
  9. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
dvdy23938-sup-0001-suppfig1.tif481KFig. S1 Alignments of corresponding S. officinalis reflectin proteins sequences predicted from cDNA and genomic DNA (gDNA). RNA probes synthesized for ISH are shaded in dark grey.
dvdy23938-sup-0001-suppfig2.tif2060KFig. S2 Reflectin expression in S. officinalis embryos as whole-mount in situ hybridization, with probe AS-3 from SoRef3 (A) and probe AS-5 from SoRef5 (B). Stage 27. Scale bars=2mm. Dorsal view. Anterior is up. Posterior is down.
dvdy23938-sup-0001-suppinfo.doc70KSupplementary Information

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