Dev Genes Evol (2015) 225:253–257 DOI 10.1007/s00427-015-0511-6

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A GATA2/3 gene potentially involved in larval shell formation of the Pacific oyster Crassostrea gigas Gang Liu 1,2 & Pin Huan 1 & Baozhong Liu 1,3

Received: 26 April 2015 / Accepted: 1 July 2015 / Published online: 11 July 2015 # Springer-Verlag Berlin Heidelberg 2015

Abstract Shells are one of the most notable features of the majority of mollusks. In addition, the shell is also considered a key characteristic during molluscan evolution and development. However, although the morphological changes during larval shell formation have been well described, the underlying molecular mechanisms remain poorly understood. In this study, we focused on the potential involvement of a GATA gene in shell formation because GATA genes are often downstream genes of BMP (bone morphogenetic protein) signaling pathways, which have been suggested to participate in molluscan shell formation. In the Pacific oyster Crassostrea gigas, we observed that the expression of a GATA2/3 homolog (cgi-gata2/3) was clearly restricted to the edge of the shell field in early larval stages (trochophore and D-veliger). This expression pattern supports the notion that cgi-gata2/3 gene plays conserved roles in bilaterian ectodermal development. It is possible that cgi-gata2/3 is one shell-formation gene under the regulation of BMP signaling pathways. In addition, cgigata2/3 was also detected in the ventral side of embryos. The expression of cgi-gata2/3 away from the shell field may be Communicated by David A. Weisblat Electronic supplementary material The online version of this article (doi:10.1007/s00427-015-0511-6) contains supplementary material, which is available to authorized users. * Baozhong Liu [email protected] 1

Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, 7 Nanhai Road, Qingdao 266071, China

2

University of Chinese Academy of Sciences, Beijing, China

3

National and Local Joint Engineering Laboratory of Ecological Mariculture, Qingdao, China

involved in hematopoiesis. Our results provide fundamental support for studies into the molecular mechanisms of larval shell formation and the functions of molluscan GATA genes. Keywords GATA . Oyster . Shell formation . Hematopoiesis

Introduction The GATA family is a group of transcription factors widely distributed in multicellular organisms (Patient and McGhee 2002). A GATA transcription factor possesses two zinc finger domains, which contain the highly conserved sequence CysX2-Cys-X17-Cys-X2-Cys. GATA transcription factor can bind to the DNA sequence (T/A) GATA (A/G) through their zinc finger domains (Patient and McGhee 2002) and then regulate the transcription of target genes. The GATA family is typically divided into two subfamilies, GATA1/2/3 and GATA4/5/6. GATA1/2/3 members are mostly related to hematopoiesis and the development of nervous systems (Orkin 2000), although other possible functions have been discussed (Neave et al. 1995; Patient and McGhee 2002). GATA4/5/6 transcription factors are mainly expressed in mesodermal and endodermal organs (such as the heart, intestines, blood vessels, and urogenital system) (Patient and McGhee 2002). Shells, having various shapes and colors, are one of the most notable features of the majority of mollusks. Furthermore, the shell is considered to be a key characteristic during molluscan evolution (Knoll 2003). Therefore, a comprehensive understanding of shell formation is important to understand the evolution and biology of mollusks. However, although the morphological changes during larval shell formation have been well described (Silberfeld and Gros 2006), the underlying molecular mechanisms remain poorly understood. Several genes such as decapentaplegic (dpp),

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engrailed, and tyrosinase have been suggested to be involved in shell formation (Nederbragt et al. 2002; Huan et al. 2013), but the current information is still too fragmentary to obtain a comprehensive understanding. Studies on the effects of BMP (bone morphogenetic protein) signaling during shell formation are still very nascent, but it has been proposed that dpp, a homolog of vertebrate BMP2/4, regulates larval shell formation in mollusks, although its precise roles in bivalve versus gastropod species may be different (Nederbragt et al. 2002; Kin et al. 2009). The genes responding to dpp signaling during this process have not been investigated, but because GATA2 has been identified as a direct target of BMP signaling (Dalgin et al. 2007), we wondered whether GATA genes are involved in molluscan shell formation. In this study, we focused on cgi-gata2/3, a homolog of GATA2/3 gene, and investigated its expression patterns during the early development from oocyte to the D-veliger stage. The results suggested that this gene potentially plays a role in shell formation and hematopoiesis during embryonic development of Crassostrea gigas.

Material and methods Animals and sample collection Sexually mature adults of C. gigas were obtained from a local market in Qingdao, China. Artificial fertilization was conducted as described previously (Huan et al. 2012). Oocytes, embryos, and larvae at different time points in the first 24 h postfertilization (hpf) were collected. The samples were immersed in RNAlater Stabilization Solution (Ambion, USA) at 4 °C for 24 h and then stored at −20 °C until RNA extraction. For in situ hybridization, samples were fixed in 4 % paraformaldehyde at 4 °C overnight, transferred to methanol, and stored at −20 °C until further use. RNA extraction and reverse transcription Total RNA was extracted from the samples stored in RNAlater using the High Pure RNA Tissue Kit (Roche, America). Reverse transcription was performed as described previously (Huan et al. 2012). Cloning of cgi-gata2/3 and sequence analysis A predicted GATA gene (GenBank: EKC26787.1) was retrieved from the whole genome sequence database of C. gigas (OysterDB, http://www.oysterdb.com). The open reading frame (ORF) of the gene was confirmed using two primers (cgi-gata2/3-orf-f: 5′-ATGGAAACAG AACAGCAACA-3′, cgi-gata2/3-orf-r: 5′-TTAGGCCA

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TGGCTCCGATCATA-3′), and the gene was designated as cgi-gata2/3. Basic characters of the deduced peptide were analyzed using ExPASy tools (http://web.expasy. o r g / c o m p u t e _ p i / ) a n d S M A RT ( h t t p : / / s m a r t . emblheidelberg.de/). In order to assign the orthology of cgi-gata2/3, a phylogenetic analysis was performed using the neighborjoining (NJ) method with the MEGA 6.0 package (www. megasoftware.net/). The tree was based on 27 full-length amino acid sequences including genes representing all six sub-families of the GATA family. These genes came from representative vertebrates and invertebrate species, such as Homo sapiens, Mus musculus, Danio rerio, and Drosophila melanogaster. Bootstrap test of 1000 replicates were calculated for each node of the tree. Whole mount in situ hybridization Two primers, cgi-gata2/3-wmish-f (5′-CATCTCTTCCTT CATCTCCA-3′) and cgi-gata2/3-wmish-r (5′-AGGTCG GACATAACCATTC-3′), were used to obtain a 534-bp cDNA fragment of cgi-gata2/3. The purified PCR product was cloned into the pGEM-T vector (Promega, America) and certified through sequencing. Linearized recombinant plasmids were used as template for in vitro transcription. Digoxigenin-labeled sense and antisense probes were synthesized using DIG RNA Labeling Mixture (Roche, USA). Whole mount in situ hybridization (WMISH) was conducted as described previously (Maures and Duan 2002).

Results and discussion Characterization of cgi-gata2/3 Only a shortened variant of the gene was amplified from the C. gigas larval cDNA. Compared to the sequence in OysterDB, the sequence we obtained lacked a 210-bp fragment (supplemental file Fig. S1). It is possible that our fragment is a result of alternative splicing in C. gigas larva or that the sequence from OysterDB was assembled incorrectly. The ORF we obtained consisted of 1347 bp and encoded a peptide with 448 amino acids (supplemental file Fig. S2). The theoretical molecular weight of the deduced peptide was 48.22 kDa, and the predicted isoelectric point was 9.54. No signal peptide was predicted in Cgi-GATA2/3, indicating it is not a secretory protein. Two conserved zinc finger domains were revealed by SMART (Fig. 1). Eight cysteine residues, which are crucial in binding to zinc ion (Patient and McGhee 2002), were detected in the highly conserved zinc finger domains (Fig. 1).

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Fig. 1 Conserved zinc finger domains of Cgi-GATA2/3. In the highly conserved zinc finger domains, eight cysteine (asterisks) residues were observed

Multi-alignment revealed that these two zinc finger domains were conserved in vertebrates and invertebrates, including mollusk, arthropod, fish, and mammal (Fig. 1). Detailed analysis revealed that cgi-gata2/3 gene contains other essential sites for its functions, such as the nuclear localization signal (supplemental file Fig. S2). Fig. 2 An NJ tree of 27 representative GATA genes. CgiGATA2/3 (arrow) fell into the GATA2/3 sub-clade, which includes vertebrate (triangles), arthropod (squares), and Lophotrochozoa (circles) clades. The numbers at the nodes are bootstrap support percentages with 1000 replicates

Phylogenetic analysis A phylogenetic tree was constructed using cgi-gata2/3 and 26 other representative GATA genes from both vertebrates and invertebrates. The result clearly reveals that members of the GATA family are divided into two clades of GATA1/2/3 and GATA4/5/6 (Fig. 2). This result is in accordance with a

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Dev Genes Evol (2015) 225:253–257

Fig. 3 Expression pattern of cgi-gata2/3 mRNA at 6–12 hpf. a–d Dorsal view, anterior at the top. e–f Lateral view, ventral at the left. g–h Anterior view, ventral at the top. Cgi-gata2/3 mRNA was first detected in the edge of the shell field (indicated by the wide dashed lines) at 6 hpf, and this pattern persisted in all subsequent stages (a–f). At 10 hpf and 12 hpf, cgi-

gata2/3 mRNA was also detected on the ventral side of embryos (triangles in g and h). At 12 hpf, expression at the edge of the shell field and that of the ventral side overlapped (h). SF shell field, A anterior, P posterior, V ventral, D dorsal. Bar=50 μm

previous report (Patient and McGhee 2002). The GATA4/5/6 clade included GATA4, GATA5, and GATA6 from vertebrates

and pannier from arthropods. The GATA1/2/3 clade further clustered into two sub-clades of GATA1 and GATA2/3. Cgi-

Fig. 4 Expression pattern of cgi-gata2/3 mRNA at 14–24 hpf. a–f Lateral view, ventral at the left and anterior at the top. Color reactions were performed for equal amounts of time in the sense and antisense groups. Cgi-gata2/3 mRNA continued to be expressed in the edge of shell field. At these stages, expression on the ventral side was difficult to discriminate because the larval body became covered by the expanded shell, and expression surrounding the shell field overlapped partially with

other expression domains. However, we believe that the expression originating in ventral domains at 10 hpf persists in these older stages (triangles). Non-specific staining was obvious in the hinge and/or surrounding the shell field at these stages, but it is significantly lighter than the robust signal generated from antisense probes. H hinge. Bar= 50 μm

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GATA2/3 fell into the Lophotrochozoa clade of the GATA2/3 group, certifying its orthology. Expression of cgi-gata2/3 during early development The spatiotemporal expression of cgi-gata2/3 during early development was investigated using WMISH. The earliest detectable expression of cgi-gata2/3 occurred in early gastrulas (6 hpf). Here, cgi-gata2/3 expression was clearly restricted to the edge of the shell field (Fig. 3a–d), and this pattern was observed up to 24 hpf (Fig. 4a–c). In D-veligers beginning at 14 hpf, unspecific staining was observed in the hinge region and the edge of shell field (Fig. 4). Nevertheless, staining intensity with antisense probes (Fig. 4a–c) is noticeably stronger than with sense probes (Fig. 4d–f); we therefore believe that much of it reflects authentic cgi-gata2/3 expression. In addition, there were two regions expressing cgi-gata2/3 on the ventral side of embryos beginning at 10 hpf (Fig. 3g), and this expression was still distinguishable at 12 hpf (Fig. 3h). After 14 hpf, it was difficult to discriminate the signals on the ventral side because the shell field expanded to the ventral side. However, it is likely that the ventral expression domain arising at 10 hpf persists in its expression at least until 24 hpf (Fig. 4a–c). The association of cgi-gata2/3 expression with the shell field suggests that it may participate in shell formation. The shell field is derived from the dorsal ectoderm of the mollusk embryo (Wilt 2005). In multicellular animals, it has been revealed that gata2 and gata3 are involved in ectodermal development (Patient and McGhee 2002). Therefore, our results indicate that cgi-gata2/3 may play conserved roles in ectodermal development of mollusks. On the other hand, because it has been proposed that different compartments develop within the dorsal ectoderm of molluscan embryos, we do not deny the possibility that cgi-gata2/3 is involved in other aspects of ectoderm formation rather than shell formation (Nederbragt et al. 2002). If cgi-gata2/3 is involved in shell formation, its relationship to BMP signaling would be an interesting area of further research, as BMP signaling pathways have been proposed to be involved in shell formation (Nederbragt et al. 2002; Kin et al. 2009). Moreover, gata2 is often a direct target gene downstream of BMPs in animal development (Dalgin et al. 2007), so it is possible that cgi-gata2/3 is a downstream gene of BMP signaling pathways and may participate in the shell formation of C. gigas. Besides high levels of expression in the edge of shell field, cgi-gata2/3 was also detected on the ventral side of embryos (Figs. 3g–h and 4). GATA1/2/3 plays critical roles in hematopoiesis of zebrafish, Xenopus, etc. (Patient and McGhee 2002). In the scallop Chlamys farreri, CfGATA, a GATA1/2/ 3 homolog, plays roles in hemocyte production (Yue et al. 2014). We predicted that, similar to CfGATA, cgi-gata2/3 is involved in hematopoiesis of C. gigas during embryonic

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development. In order to verify this hypothesis, molecular markers related to hematopoiesis are needed and functional studies should be performed. In summary, we investigated the expression patterns of cgigata2/3 during the early development of C. gigas. Our results indicate that this gene may be involved in shell formation and hematopoiesis during embryonic development. These results enrich the knowledge of the molecular mechanisms that potentially control shell formation as well as hematopoiesis in mollusks. Further study is needed to test our hypothesis. For example, RNAi assays are necessary to verify the function of cgi-gata2/3. In addition, research into the relationship between cgi-gata2/3 and BMP signaling pathways will be an important issue in the future. Acknowledgments This work was financially supported by the National Natural Science Foundation of China (31472265).

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3 gene potentially involved in larval shell formation of the Pacific oyster Crassostrea gigas.

Shells are one of the most notable features of the majority of mollusks. In addition, the shell is also considered a key characteristic during mollusc...
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