Expression of the Tetraspanin Family Members Tspan3, Tspan4, Tspan5

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Gene Expression Patterns 13 (2013) 1–11

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Gene Expression Patterns

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Expression of the tetraspanin family members Tspan3, Tspan4, Tspan5 and Tspan7 during Xenopus laevis embryonic development ⇑ ⇑ Jubin Kashef a, , Tanja Diana a,b, Michael Oelgeschläger d,1, Irina Nazarenko b,c,

a Zoological Institute, Department of Cell and Developmental Biology, Karlsruhe Institute of Technology, Karlsruhe, Germany b Institute of Toxicology and Genetics, Karlsruhe Institute of Technology, Karlsruhe, Germany c Institute of Environmental Health Sciences, University Medical Centre Freiburg, Freiburg, Germany d Max-Planck-Institute of Immunobiology, Freiburg, Germany

article info abstract

Article history: Tetraspanins comprise a large family of integral membrane proteins involved in the regulation of cell Received 25 April 2012 adhesion, migration and fusion. In humans it consists of 33 members divided in four subfamilies. Here, Received in revised form 1 August 2012 we examined the spatial and temporal gene expression of four related tetraspanins during the embryonic Accepted 2 August 2012 development of Xenopus laevis by quantitative real-time PCR and in situ hybridization: Tspan3 (encoded Available online 24 August 2012 by the gene Tm4sf8 gene) Tspan4 (encoded by the gene Tm4sf7), Tspan5 (encoded by the gene Tm4sf9) and Tspan7 (encoded by the gene Tm4sf2). These genes appeared first in the vertebrates during the evo- Keywords: lution and are conserved across different species. In humans, they were associated with several diseases Tetraspanins such as sclerosis, mental retardation and cancer; however their physiological role remained unclear. This Tspans Tm4sf work provides a comprehensive comparative analysis of the expression of these tetraspanins during the Neural crest development of X. laevis. The more closely related tetraspanins Tspan3, Tspan4 and Tspan7 exhibited very Cell migration similar spatial expression patterns, albeit differing in their temporal occurrence. The corresponding tran- Cancer scripts were found in the dorsal animal ectoderm at blastula stage. At early tailbud stages (stage 26) the Xenopus laevis genes were expressed in the migrating cranial neural crest located in the somites, developing eye, brain, and in otic vesicles. In contrast, Tspan5 appeared first at later stages of development and was detected prominently in the notochord. These data support close relatedness of Tspan3, Tspan4 and Tspan7. The expression of these tetraspanins in the cells with a high migratory potential, e.g. neural crest cells, suggests their role in the regulation of migration processes, characteristic for tetraspanin family members, during development. Similarity of the expression profiles might indicate at least partial functional redundancy, which is in concordance with earlier findings of tissue-limited or absent phenotypes in the knock- down studies of tetraspanins family members performed. Ó 2012 Elsevier B.V. Open access under CC BY-NC-ND license.

Tetraspanins represent an ancient family of small transmem- loop, and two extracellular loops, a small (SEL) and a large (LEL) brane proteins that appeared already in protists during evolution one (Fig. 1)(Hemler, 2005). The large extracellular loop comprised (Garcia-Espana et al., 2008). In humans this family is represented of approximately 120 amino acids plays a central role in the estab- by 33 members that are expressed in different tissues and play a lishment of protein–protein interactions (Seigneuret et al., 2001; role in the regulation of cell migration, adhesion and fusion during Kitadokoro et al., 2001). It consists of the variable part and a con- physiological and pathological processes (Charrin et al., 2009). Tet- served head domain. Examination of the crystal structure of the tetraspanins are characterized by a specific and evolutionarily con- raspanin CD81 and phylogenetic analysis of other tetraspanin served protein structure consisting of four trans-membrane proteins revealed three strictly conserved elements in the head do- domains, intracellular N- and C-termini, a very short intracellular main: two disulfide bridges, one of those built by cysteines within an invariable CCG domain, and several highly conserved glycine, proline and tyrosine residues (Supplemental materials)(Kitadokoro ⇑ Corresponding authors. Addresses: Zoological Institute, Department of Cell and et al., 2001). Altogether, these elements provide a specific interface Developmental Biology, Fritz Haber Weg 2, D-76131 Karlsruhe, Germany (J. Kashef), for protein–protein interactions, in particular for binding of inte- Department of Environmental Health Sciences, University Medical Centre Freiburg, grins (Rubinstein, 2011; Berditchevski, 2001). Additionally, almost Breisacherstr. 115b, D-79109 Freiburg, Germany. Tel.: +49 76127082100 (I. Nazarenko). all tetraspanins are modified by palmitoylation of the membrane- E-mail addresses: [email protected] (J. Kashef), irina.nazarenko@uniklinik- proximal cysteine residues, which contribute to the establishment freiburg.de (I. Nazarenko). of tetraspanin–tetraspanin interactions (Charrin et al., 2002). 1 Present address: Federal Institute for Risk Assessment, Berlin, Germany.

1567-133X Ó 2012 Elsevier B.V. Open access under CC BY-NC-ND license. http://dx.doi.org/10.1016/j.gep.2012.08.001 2 J. Kashef et al. / Gene Expression Patterns 13 (2013) 1–11

Fig. 1. Schematic representation of the tetraspanin protein structure. Tetraspanins possess a very characteristic for the members of this family proteins structure. These small proteins of approximately 230–280 amino acids harbor 4 transmembrane domains (red), intracellular N- and C-termini and a small intracellular domain (blue), and two extracellular domains: a small extracellular loop (SEL) and a large extracellular loop (LEL) (green).

Due the their ability to bind one another and other surface pro- other TM4SF members (CD9 and CD81) and integrin alpha3beta1 teins, tetraspanins organize microdomains, referred as tetra- and alpha6beta1 (Tachibana et al., 1997). However, little further spanin-enriched microdomains (TEMs) or tetraspanin web that investigations have been performed since then. It is known that contribute to the membrane compartmentalization (Rubinstein, Tspan4 is expressed in a variety of tissues, and albeit it is absent 2011). Albeit the role and the mechanisms of TEMs functions are in the human cerebral cortex and cerebellum (Tachibana et al., not completely understood, available data support that TEMs rep- 1997), it was detected in the chicken nervous system including resent dynamic organizers of the cell membrane, bringing together brain, spinal cord and dorsal root ganglia (Perron and Bixby, interacting partners, for instance integrin alpha3beta1 and lami- 1999). Interestingly, the Puccetti group demonstrated that Tspan4 nin-511 (Yamada et al., 2008), and serving as a platform for the shares some homology with the UL94 epitope of human cytomeg- transmission of signals to intracellular molecules, such as ERK/ alovirus protein and is highly expressed in endothelial cells (Lunar- MAPK (Carloni et al., 2004) and PKC alpha (Wang et al., 2007). di et al., 2000, 2006). Furthermore, nonfunctional antibodies The function of some tetraspanins, e.g. of CD151, CD9, CD81, against Tspan4 from memory B cells of patients with systemic scle- CD82 and CD63 was intensively investigated during the last years. rosis have been proposed as promising novel therapeutic agents for Their role in the regulation of central biological processes is well this disease (Traggiai et al., 2010). established, in particular for CD9 and CD81 in sperm–egg fusion The tetraspanin Tspan5 (Tm4sf9) was identified using substrac- (Rubinstein et al., 2006a; Berditchevski, 2001; Le Naour et al., tive hybridization by Garciá-Frigola et al., as a gene related to the 2000); CD9, CD151 in pathogen infection (Monk and Partridge, development of the murine cerebral cortex (Garcia-Frigola et al., 2012); CD9, CD151, and TM4SF12 in the regulation of vascular func- 2000). Further analysis revealed that cerebella Purkinje cells spe- tions (Bailey et al., 2011); CD9, CD63 in immune response (Jones cifically express Tspan5 at distinct stages of differentiation in the et al., 2011; Tarrant et al., 2003), CD151, CD81, CD82, Tspan5 and mouse brain (Juenger et al., 2008). Interestingly, Iwai et al. demon- Tspan8 in carcinogenesis (Romanska and Berditchevski, 2011; strated that Tspan5 and Tspan13/NET-6 play reciprocal role in the Kanetaka et al., 2003; Lazo, 2007; Le Naour et al., 2006; Murayama osteoclastogenesis, whereby Tspan5 supports osteoclasts matura- et al., 2008; Sadej et al., 2010; Takahashi et al., 2007), and CD63 and tion (Iwai et al., 2007). Tspan8 in exosome activity (Nazarenko et al., 2010; Hemler, 2003; The function of Tspan7 (Tm4sf2) has not being assessed up to Petersen et al., 2011). However, there are some tetraspanins, which now in many details. However, several groups described a correla- are still poorly investigated and their function remains elusive. tion between Tspan7 mutations and non-specific X-linked mental Here, we analyzed the closely related tetraspanins Tspan3 retardation (Dunn et al., 2010; Holinski-Feder et al., 1999; De (Tm4sf8), Tspan4 (Tm4sf7), Tspan5 (Tm4sf9) and Tspan7 (Tm4sf2) et al., 2002; Gomot et al., 2002; Maranduba et al., 2004; Noor during the embryonic development in Xenopus laevis. Albeit the et al., 2009; Piton et al., 2011). Furthermore Tspan7 seems to play orthologs of these tetraspanins were found in Drosophila melano- a role in spermatogenesis, being expressed in spermatozoids and gaster and Caenorhabditis elegans, corresponding homologs ap- associating with SPAG11B (Radhakrishnan et al., 2009). Recently peared first in vertebrata during the evolution. Although our it was identified as a potential prognostic marker for clear-cell re- knowledge about their biological function is still very limited, nal cell carcinoma (Wuttig et al., 2011). studies conducted up to now demonstrate that their deregulation Summarizing, the initial studies provide evidences that these might be associated with various diseases. tetraspanins are expressed in distinct tissues in the adult organism, In particular, up-regulation of Tspan3 (Tm4sf8) has been associ- play important roles in certain pathological processes, and might ated with Wilms tumor (Maschietto et al., 2011) and chronic fati- have a potential as targets in diagnostic and therapeutic fields. gue syndrome (Aspler et al., 2008); furthermore, it is localized in However the physiological role of these proteins is still unclear the region of the chromosome 15 involved in the chromosomal and needs to be investigated. Additionally, there is no data avail- re-localization t(1;15)(p22;q22) that frequently occurs in prostate able allowing a direct comparison of the expression profiles of cancer (Strefford et al., 2006). The developmental expression and these related tetraspanins, In the current study, we provide a com- physiological function of Tspan3 in the brain has been recently as- prehensive comparative analysis of the expression profiles of the sessed (Tiwari-Woodruff et al., 2004). It interacts with claudin-11, tetraspanins Tspan3 (Tm4sf8), Tspan4 (Tm4sf7) Tspan7 (Tm4sf2), forming a complex with the integrin beta1 in oligodendrocytes and Tspan5 (Tm4sf9) during the development in X. laevis as a model (Tiwari-Woodruff et al., 2001). However, nothing is known about organism. Because of the difference between gene and protein the expression and the role of Tspan3 in other organs. nomenclatures, we will refer further in the manuscript to protein The tetraspanin Tspan4 (Tm4sf7), was cloned in the early 1990s designations commonly used in the community, to avoid any by the Hemler group as NAG-2, a protein interacting with two confusion. J. Kashef et al. / Gene Expression Patterns 13 (2013) 1–11 3

1. Results Table 2 Conservation of tetraspanins through the evolution between Xenopus laevis and Homo sapience. 1.1. Phylogenetic analysis of the tetraspanins Tspan3, Tspan4, Tspan5 and Tspan7 Xenopus laevis Homo sapience Similarity (%) Identity (%) To define the evolutionary relationship between the tetraspa- Tspan3 91 84 nins of our interest, amino acid sequences of the corresponding Tspan4 80 65 homologs from different species were aligned using ClustalW algo- Tspan5 98 95 rithm (Table 1, Supplemental materials). The alignment demon- Tspan7 94 87 strated that the tetraspanins remained nearly invariant during All calculations were performed using sequences recruited from the NCBI protein evolution, with the highest identity score of 95% between X. laevis data base. Sequences were aligned and compared with the help of NCBI Blast ser- and Homo sapience for Tspan5 and the lowest identity score (65%) vice http://blast.ncbi.nlm.nih.gov/Blast.cgi. for Tspan4 (Table 2). Comparison of the amino acid sequences of different orthologs revealed the highest identity score of 34% between Tspan4 and Tspan5; followed by 32% identity between Table 3 Similarity and identity of Tspan3, Tspan4, Tspan5 and Tspan7 protein sequences (%) Tspan3 and Tspan7, 29% between Tspan4 and Tspan7, 28% between calculated using NCBI Blast service http://blast.ncbi.nlm.nih.gov/Blast.cgi. Tspan3 and Tspan4, and 22% between Tspan3 and Tspan5 (Table 3). The phylogenetic analysis was performed with the ClustalW2 algo- Tspan3 Tspan4 Tspan5 rithm using Neighbor-joining clustering methods. The phylogram Similarity Identity Similarity Identity Similarity Identity tree, reflecting the relative amount of evolutionary changes, con- Tspan3 100 100 firms close relationship between homologues. Furthermore, it Tspan4 47 28 clearly indicates that Tspan3 and Tspan7 are the most closely re- Tspan5 41 22 52 34 Tspan7 48 32 53 29 47 25 lated orthologs followed by Tspan4. Altogether, Tspan3, Tspan4 and Tspan7 build a subgroup distantly related to Tspan5 (Fig. 2).

1.2. Developmental expression of Tspan3 arrow) and ventral (red arrow) mesoderm (Fig. 4C and D). At late neurula stage transcripts of Tspan3 could be detected in more dor- The temporal expression pattern of Tspan3 during X. laevis sal and anterior structures like the optic cups (OC) and in the neu- development was ﬁrst analyzed by RT-PCR (Fig. 3). The highest lev- ral folds (NF) (Fig. 4E and F). At stage 25 Tspan3 is prominently els of the Tspan3 mRNA were detected maternally until onset of expressed in the developing eye, brain, otic vesicle (OV) and in gastrulation. During neurulation Tspan3 was expressed at lower the migrating cranial neural crest (CNC). Additionally, we observed levels but at tailbud stages transcripts increased again with excep- transcripts in somites (SO) and in the pre-somitic mesoderm (PSM) tion of stage 35. (Fig. 4G). At stage 32 Tspan3 continued to be expressed in the eye, Further, we analyzed the spatial expression pattern of Tspan3 brain, OV, CNC and SO. Interestingly, we also detected transcripts by whole-mount in situ hybridization at different embryonic in the trunk neural crest (TNC) (Fig. 4H). Horizontal vibratome sec- stages. At blastula Tspan3 is expressed in the dorsal animal ecto- tions displayed the expression of Tspan3 in CNC (Fig. 4X and J), derm (Fig. 4A). At gastrula stage Tspan3 transcripts are found in whereas transverse vibratome sections showed transcripts in the dorsal marginal zone (DMZ) (Fig. 4B). Semi-sections of these TNC and SO (Fig. 4K). The sense probe did not show any staining embryos revealed that Tspan3 is expressed in the dorsal (black (Fig. 4I).

Table 1 List of the examined proteins Tspan3, Tspan4, Tspan5 and Tspan7, corresponding NCBI numbers and encoding genes in different species included into phylogenetic analysis.

Protein name Alias name NCBI Nos. Gene name Species Tspan3 OAP-1 NP_001020709.1 Tm4sf8 Danio rerio NP_001088397 Xenopus laevis NP_001017023.1 Xenopis (Silurana) tropicalis AAH43072.1 Mus musculus NP_001005547.1 Rattus norvegicus AAH11206.1 Homo sapience Tspan4 NAG-2 NP_001002348.1 Tm4sf7 Danio rerio NP_001002348.1 Xenopus laevis AAI67361.1 Xenopis (Silurana) tropicalis AAH83070.1 Mus musculus NP_001013088.1 Rattus norvegicus AAH19314.1 Homo sapience Tspan5 NP_001006047.1 Tm4sf9 Danio rerio NP_001089436 Xenopus laevis NP_001072198.1 Xenopis (Silurana) tropicalis AAH58695.1 Mus musculus AAQ11226.1 Rattus norvegicus AAH09704.1 Homo sapience Tspan7 NP_001124290.1 Tm4sf2 Danio rerio NP_001080292 Xenopus laevis NP_989350.1 Xenopis (Silurana) tropicalis CAM23357.1 Mus musculus NP_001102285.1 Rattus norvegicus AAH18036.1 Homo sapience 4 J. Kashef et al. / Gene Expression Patterns 13 (2013) 1–11

Fig. 2. Phylogenetic analysis of the tetraspanins Tspan3, Tspan4, Tspan5 and Tspan7. The phylogram was generated using ClustalW2 algorithm on the EBI public server http://www.ebi.ac.uk/Tools/phylogeny/clustalw2_phylogeny/ and includes Danio rerio, Xenopus laevis, Xenopus tropicalis, Mus musculus, Rattus norvegicus and Homo sapiens homologs of the tetraspanins analyzed.

100 Tspan3

40 normalized to ODC relative expression in % 20

0 stage 2 5 810 1214162025303538

Fig. 3. Real-time RT-PCR analysis of Tspan3 expression in different embryonic stages. The total RNA was isolated from the group of 10 embryos on the corresponding stage of development. Samples were normalized to levels of ornithine decarboxylase (ODC). Error bars indicate standard error.

1.3. Developmental expression of Tspan4 Tspan3. At blastula stage transcripts of Tspan4 were detected in the dorsal animal ectoderm (Fig. 6A). During gastrulation Next we aimed to analyze the temporal expression pattern of Tspan4 was expressed in the dorsal marginal zone (DMZ) Tspan4 during development. First, the expression levels of the (Fig. 6B) and in the dorsal (black arrow) and ventral (red arrow) Tspan4 mRNA were assessed on different stages of X. laevis mesoderm shown by semi-sections (Fig. 6C and D). At late neu- development using real-time RT-PCR (Fig. 5). Tspan4 was weakly rula stage we detected Tspan4 expression in the optic cups and expressed until end of gastrulation. From neurulation onwards in the neural folds (Fig. 6E and F). At stage 26 Tspan4 was transcripts of Tspan4 continuously increased during further strongly expressed in the developing eye, brain, otic vesicle development. Whole-mount in situ hybridization of Tspan4 re- (OV) and in the migrating cranial neural crest (CNC), somites vealed a similar spatial expression pattern in comparison to (SO) and in the pre-somitic mesoderm (PSM) (Fig. 6G). By stage J. Kashef et al. / Gene Expression Patterns 13 (2013) 1–11 5

Fig. 4. Expression of Tspan3 during Xenopus development. (A, B and E–H) Whole mount in situ hybridization of Tspan3 encoding gene Tm4sf8 on stage 8–32 Xenopus embryos as indicated. (I) Control in situ hybridization with sense probe for Tm4sf8 at stage 32. Vegetal views (A–C), anterior view (E), dorsal view, anterior to up (F), lateral views, anterior to left (G–I). (C) Schematic representation of a stage 11.5 embryo modiﬁed from Nieuwkoop and Faber (1967). (D) Semi-sections of embryos shown in C as indicated. (J and K) Vibratome sections of embryos shown in H as indicated. CNC = cranial neural crest, DMZ = dorsal marginal zone, NF = neural fold, OC = optic cup, OV = otic vesicle, PSM = pre-somitic mesoderm, SO = somites, TNC = trunk neural crest. Scale bar 450 lm (A–I) and 100 lm (J and K).

31, staining was located in the developing eye, brain and CNC, lyzed the spatial expression pattern of Tspan7 by whole-mount which we observed additionally on horizontal vibratome sections in situ hybridization on different embryonic stages. At blastula (Fig. 6H and J). Furthermore, at stage 31 Tspan4 was expressed stage transcripts of Tspan7 are mostly detected in the dorsal ani- in the OV, SO and trunk neural crest (TNC) as shown on vertical mal ectoderm (Fig. 8A). During gastrulation Tspan7 is expressed vibratome sections (Fig. 6H and K). No staining was detected ubiquitous with exception of the blastoporus (Fig. 8B). Semi-sec- with the sense probe (Fig. 6I). tions of these embryos revealed that Tspan7 is expressed in the dorsal mesoderm (black arrow), prospective neuroectoderm (red 1.4. Developmental expression of Tspan7 arrow) and in the ectoderm (black arrow head) (Fig. 8C and D). At late neurula stage we detected Tspan7 expression in the optic Using real-time RT-PCR we next analyzed the temporal expres- cups and in the neural folds (Fig. 8E and F). At stage 26 Tspan7 sion pattern of Tspan7 during X. laevis development (Fig. 7). Tspan7 was strongly expressed in the developing eye, brain, otic vesicle was weakly expressed until end of neurulation. From stage 20 on- (OV), in the migrating cranial neural crest (CNC), somites (SO) wards transcripts of Tspan7 strongly increased until stage 30 to and in the pre-somitic mesoderm (PSM) (Fig. 8G). By stage 31, dramatically decrease again during further development. We ana- staining is located in the developing eye, brain, CNC, OV and SO 6 J. Kashef et al. / Gene Expression Patterns 13 (2013) 1–11

112

100 Tspan4

40 normalized to ODC relative expression in % 20

0 stage 2 5 810 1214162025303538

Fig. 5. Real-time RT-PCR analysis of Tspan4 expression in different embryonic stages. The total RNA was isolated from the group of 10 embryos on the corresponding stage of development. Samples were normalized to levels of ornithine decarboxylase (ODC). Error bars indicate standard error.

(Fig. 8H). Interestingly, vertical vibratome sections revealed were shown for Tspan5 and Tspan33 in the regulation of Notch sig- expression of Tspan7 in the notochord (NO) (Fig. 8J). No staining naling (Dunn et al., 2010), for CD63 and Tssc6 in the regulation of was detected with the sense probe (Fig. 8I). cellular immunity (Gartlan et al., 2010), as well as for CD9 and CD81 in the regulation of cell fusion between sperm and egg (Rubinstein et al., 2006b) or between mononuclear phagocytes 1.5. Predominant expression of Tspan5 in the notochord (Takeda et al., 2003). This feature of tetraspanins seems being per- sistent through evolution since the removal of a single tetraspanin To study the temporal expression of Tspan5 we first performed does not allow to obtain strong phenotypical changes in mamma- real-time RT-PCR analysis on RNA preparations from different lian organisms, C. elegans (Moribe et al., 2004)orD. melanogaster stages of X. laevis embryogenesis. In contrast to the tetraspanins (Fradkin et al., 2002). To uncover a role of distinct members of described above, the Tspan5 transcripts were not detectable until the tetraspanin family in physiological processes, the identification stage 30 and then increased during further development (Fig. 9). of closely related members by phylogenetic analysis and compara- The spatial expression pattern of Tspan5 was analyzed by whole- tive assessment of the expression patterns, followed by a combina- mount in situ hybridization on different embryonic stages. Corre- torial functional analysis of the closely related members will sponding to the real-time RT-PCR analysis, Tspan5-specific signal probably be required. was obtained first after stage 30. Interestingly, at stage 32 Tspan5 Here, we examined the expression profiles of four yet little was exclusively expressed in the notochord as shown in transver- investigated members of the tetraspanin family during the embry- sal vibratome sections (Fig. 10A–C). The sense probe in the same onic development using X. laevis as a model organism. A compara- stage did not show any staining (Fig. 10D). By stages 34 and 37, tive analysis of the amino acid sequences of the corresponding we detected expression of Tspan5 in addition to the NO in the homologs among different species revealed high conservation up developing eye, brain (BR), branchial arches (BA) and otic vesicle to 95% between X. laevis and Homo sapiens. This invariance of pro- (OV) (Fig. 10E and F). tein sequences suggests that also their respective functions might remain conserved between vertebrate species. For instance even 2. Discussion on the level of orthologs, functional relatedness was reported for human tetraspanin Tspan5 and its C. elegans ortholog TSP12, both A deregulation of the expression or the functions of tetraspa- being implicated in the regulation of Notch signaling and acting on nins frequently correlates with various human diseases such as the gamma-secretase cleavage step (Dunn et al., 2010). Therefore, cancer (Romanska and Berditchevski, 2011), immune disorders we expect that the overlapping expression profiles of the tetraspa- (Gnanasekar et al., 2007; Petersen et al., 2011), or vascular dys- nins analyzed in this study might also be significantly similar be- function (Barreiro et al., 2005; Kobayashi et al., 2000), supporting tween different species and are highly relevant for the their essential role in the homeostasis. However, the physiological deciphering of tetraspanin functions in future works. functions of the majority of tetraspanins in the adult organism as In contrast to the distantly related tetraspanin Tspan5, the three well as during embryonic development are still poorly understood. closely related tetraspanins Tspan3, Tspan4 and Tspan7 displayed One of the significant restrictions for the assessment of these func- nearly identical spatial expression profiles during early embryonic tions is absence of a pronounced phenotype by the existing knock- development demonstrating a clear correlation of phylogenetic out animal models (Rubinstein et al., 2006a; Le Naour et al., 2000; distance and similarity in expression profiles. The corresponding Wright et al., 2004; Sachs et al., 2006; Cowin et al., 2006; van Spriel transcripts were detected primarily in the dorsal animal ectoderm et al., 2009), which can be explained by a certain degree of func- during blastula, followed by the expression in the dorsal and ven- tional redundancy characteristic for tetraspanins (Rubinstein, tral mesoderm at gastrula stage, and in the optic cups and neural 2011; Huang et al., 2005). For example, complementary effects folds at late neurulation. The persist expression in the neural crest, J. Kashef et al. / Gene Expression Patterns 13 (2013) 1–11 7

Fig. 6. Expression of Tspan4 during Xenopus development. (A, B and E–H) Whole mount in situ hybridization of Tspan4 encoding gene Tm4sf7 on stage 8–31 Xenopus embryos as indicated. (I) Control in situ hybridization with sense probe for Tm4sf7 at stage 31. Vegetal views (A–C), anterior view (E), dorsal view, anterior to up (F), lateral views, anterior to left (G–I). (C) Schematic representation of a stage 11.5 embryo modiﬁed from Nieuwkoop and Faber (1967). (D) Semi-sections of embryos shown in C as indicated. (J and K) Vibratome sections of embryos shown in H as indicated. CNC = cranial neural crest, DMZ = dorsal marginal zone, NF = neural fold, OC = optic cup, OV = otic vesicle, PSM = pre-somitic mesoderm, SO = somites, TNC = trunk neural crest. Scale bar 450 lm (A–I) and 100 lm (J and K). a multipotent and highly migratory cell population unique for ver- throughout all stages, Tspan7 was expressed at the maximal level tebrates (Kuriyama and Mayor, 2008) suggest a potential involve- during the stages 25–30 and was subsequently down-regulated ment of these tetraspanins in the regulation of migratory activity during organogenesis. In contrast, Tspan4 exhibited a contin- of neural crest cells during early development. It is unclear if they ued increase of the expression level from stage 2 to stage 38. also might be involved in the regulation of the epithelial–mesen- Additionally, moderate differences in the expression patterns chymal transition, described for the TM4SF5 family member in became apparent from stage 31/32 onwards. Although all three hepatocarcinomas (Lee et al., 2008). These data show that addi- tetraspanins were expressed in somites, Tspan3 was preferably ex- tionally to the tetraspanin CD9 (Oka et al., 2002), also other tetra- pressed in cranial neural crest and only weakly in trunk neural spanins are expressed in progenitor cells, like the neural crest, Tspan4 revealed much stronger expression in trunk neural crest. Profound functional investigations will be required to assess crest, and Tspan7 transcripts were additionally detected in the their yet unknown functional role in the regulation of stem cell notochord. These differences suggest that the tetraspanins if act fate. complementary during the early embryogenesis, might also exhibit Interestingly, despite of the similarity of expression patterns, individual functions at later stages and in the adult organism. To the tetraspanins analyzed revealed signiﬁcant differences in the test for compensatory effects, it might be interesting to test in fu- mRNA levels at different stages of embryonic development. ture studies, if these late expression pattern are affected upon Whereas Tspan3 transcripts were detectable at similar levels depletion of single members of this closely related tetraspanin sub- 8 J. Kashef et al. / Gene Expression Patterns 13 (2013) 1–11

100 Tspan7

40 normalized to ODC relative expression in % 20

0 stage 2 5 810 1214162025303538

Fig. 7. Real-time RT-PCR analysis of Tspan7 expression in different embryonic stages. The total RNA was isolated from the group of 10 embryos on the corresponding stage of development. Samples were normalized to levels of ornithine decarboxylase (ODC). Error bars indicate standard error.

Fig. 8. Expression of Tspan7 during Xenopus development. (A, B and E–H) Whole mount in situ hybridization of Tspan7 encoding gene Tm4sf2 on stage 8–31 Xenopus embryos as indicated. (I) Control in situ hybridization with sense probe for Tm4sf2 at stage 31. Vegetal views (A–C), anterior view (E), dorsal view, anterior to up (F), lateral views, anterior to left (G–I). (C) Schematic representation of a stage 11.5 embryo modiﬁed from Nieuwkoop and Faber (1967). (D) Semi-sections of embryos shown in C as indicated. (J) Vibratome section of embryo shown in H as indicated. CNC = cranial neural crest, NF = neural fold, OC = optic cup, OV = otic vesicle, PSM = pre-somitic mesoderm, SO = somites, NO = notochord. Scale bar 450 lm (A–I) and 100 lm (J). J. Kashef et al. / Gene Expression Patterns 13 (2013) 1–11 9

100 Tspan5

40 normalized to ODC relative expression in % 20

0 stage 2 5 810 1214162025303538

Fig. 9. Real-time RT-PCR analysis of Tspan5 expression in different embryonic stages. The total RNA was isolated from the group of 10 embryos on the corresponding stage of development. Samples were normalized to levels of ornithine decarboxylase (ODC). Error bars indicate standard error.

Fig. 10. Expression of Tspan5 during Xenopus development. (A) Whole-mount in situ hybridization for Tspan5 encoding gene Tm4sf9 on Xenopus embryos at stage 32, 34 and 37, lateral view, anterior to left. (B) Transverse vibratome section corresponding to the line indicated in panel B. NT, neural tube; NC, notochord. (C) Crop area corresponding to the dashed square indicated in panel C. (D) Whole-mount in situ hybridization for sense Tm4sf9 at stage 32 as negative control, lateral view, anterior to left. NT = neural tube, N0 = notochord, BR = brain, OV = otic vesicle, BA = branchial arches. Scale bar 450 lm (A, D, E and F) and 100 lm (B and C). family. In contrast to the Tspan3, Tspan4 and Tspan7 tetraspanins the described function of Tspan5 in osteoclasts maturation (Iwai described above, Tspan5 revealed a very speciﬁc expression pat- et al., 2007). At the late stages, expression on Tspan5 in the brain tern restricted to the notochord in early stages, suggesting a poten- was detected, supporting the data observed in the mouse by Gar- tial distinct role of this particular tetraspanin in the formation of cía-Frigola et al. and suggesting a role of this tetraspanin in brain axial structures of the adult organism, which is in concert with development (Garcia-Frigola et al., 2000). 10 J. Kashef et al. / Gene Expression Patterns 13 (2013) 1–11

2.1. Conclusions mounted in Mowiol (Sigma) and imaged with a Leica DM5000 B microscope. Our analysis revealed similar spatial expression patterns of three closely related tetraspanins Tspan3, Tspan4, and Tspan7 dur- Acknowledgements ing embryonic development, suggesting their role in the regulation of the fate of different embryonic cell populations. Similarity of We thank D. Wedlich (Karlsruhe Institute of Technology (KIT), their expression profiles suggests at least partial redundancy of Germany), J. Sleeman (University of Heidelberg, Centre for Biomed- functions and should be taken into account in further icine and Medical Technology Mannheim (CBTM), Germany), Mar- investigations. co Horb (Institut de Recherches Cliniques de Montreal (IRCM), Canada) for support; and C. Winter for technical assistance. J.K. 3. Experimental procedures Young Investigator Group received financial support from the ‘‘Concept for the Future’’ of the KIT within the framework of the 3.1. Cloning of the Tm4sf cDNA constructs German Excellence Initiative. I.N. was supported by the European Social Fund and by the Ministry Of Science, Research and the Arts The cDNA clones encoding Xenopus laievis Tm4sf8 (Tspan3) Baden-Württemberg. This work was funded by a start-up grant of (clone IMAGE: 6641220), Tm4sf7 (Tspan4) (clone IMAGE: the Young Investigator Network support from the ‘‘Concept for the 4963216), Tm4sf9 (Tspan5) (clone IMAGE: 4683897) and Tm4sf2 Future’’ of the KIT (I.N., J.K.). (Tspan-7) (clone IMAGE: 5571618) were obtained from RZPD (IM- AGE Consortium; Lennon et al., 1996) . The sequence and orienta- Appendix A. Supplementary data tion of the cDNA in the respective pCMV-Sport6 vectors was verified by SP6 and T7 sequencing and comparison with the full- Supplementary data associated with this article can be found, in length cDNA sequences for X. laevis Tm4sf8 (Tspan3) (Unigene the online version, at http://dx.doi.org/10.1016/j.gep.2012.08.001. Xl.23773), Tm4sf7 (Tspan4) (Unigene Xl.77008), Tm4sf9 (Tspan5) (Unigene Xl.21892) and Tm4sf2 (Tspan-7) (Unigene Xl.5298). For in situ analysis the cDNAs were cut with Sal1 and transcribed with References T7 polymerase. Aspler, A.L., Bolshin, C., Vernon, S.D., Broderick, G., 2008. Evidence of inflammatory immune signaling in chronic fatigue syndrome: a pilot study of gene expression in peripheral blood. Behav. Brain Funct. 4, 44. 3.2. Real-time PCR Bailey, R.L., Herbert, J.M., Khan, K., Heath, V.L., Bicknell, R., Tomlinson, M.G., 2011. The emerging role of tetraspanin microdomains on endothelial cells. Biochem. Soc. Trans. 39, 1667–1673. Total RNA was extracted from groups of 10 embryos of the rec- Barreiro, O., Yanez-Mo, M., Sala-Valdes, M., Gutierrez-Lopez, M.D., Ovalle, S., ommended stage by using TriZOL Reagent (Invitrogen), according Higginbottom, A., Monk, P.N., Cabanas, C., Sanchez-Madrid, F., 2005. Endothelial to the instructions of the manufacturer. 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