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First published online August 8, 2003
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Retinoid X receptor and retinoic acid response in the marine sponge Suberites domuncula

Matthias Wiens1, Renato Batel2, Michael Korzhev1 and Werner E. G. Müller1,*

1 Institut für Physiologische Chemie, Abteilung Angewandte Molekularbiologie, Universität, Duesbergweg 6, D-55099 Mainz, Germany
2 Center for Marine Research, `Ruder Boskovic' Institute, HR-52210 Rovinj, Croatia



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  		Fig. 1. Effect of retinoic acid on whole sponges and primmorphs. (A) Untreated specimens of S. domuncula. (B,C) Induction of gemmules in animals by retinoic acid (50 µmol l–1). (B) Gemmules formed on a shell of the snail Trunculariopsis trunculus (incubation: 10 days) after retinoic acid treatment. (C) Group of gemmules arranged on the grooves of a shell. (D–G) Induction of canals in primmorphs. (D) Primmorphs formed in the absence of any organic matrix in the culture dish. The 3D-aggregates remained round and without canals. (E) Formation of canals by incubation of cells for 5 days on non-coated plates followed by 10 days on plates coated with galectin matrix. (F) Primmorphs with canals formed after for 5 days on non-coated plates followed by 10 days on poly-L-lysine plates. (G) Higher magnification of the canal system (> <) in the primmorphs developed for 20 days on the galectin matrix following 5 days incubation on non-coated plates. (H–L) Effect of retinoic acid on the canal system in primmorphs. (H,I) Incubation of canal-containing primmorphs (formed after a total incubation period of 15 days), cultured on the galectin matrix for 10 days, + 1 µmol l–1 of retinoic acid for an additional 5 days. (J–L) Incubation of the canal-containing primmorphs at the higher concentration of retinoic acid (50 µmol l–1) for the same period of time. For further details, see Materials and methods. Scale bars: 10 mm (except for C and G, 5 mm).

 


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  		Fig. 2. Extent of nucleosome formation in primmorphs, determined by photometric immunoassay. Primmorphs were grown for the initial period of 5 days in the non-attached manner, followed by an additional 3 days (total 8 days), 10 days (total 15 days) or 15 days (total 20 days) on galectin-coated culture dishes. The cultures remained either non-treated or were treated with 1 µmol l–1 or 50 µmol l–1 of retinoic acid (RA) for the last 2 days of incubation. Then the cells (100–200 mg protein) were lysed and the released nucleosomes were quantitated immunochemically as described in Materials and methods. The amount of colored product was measured spectrophotometrically. The absorbance values were correlated to 50 ng of DNA (present in the samples before lysis). Values are means ± S.D., from five parallel determinations for each sample, and show the amount of nucleosomes released in primmorphs grown on the galectin matrix for 3 days (white bars); 10 days (gray bars) and 15 days (black bars).

 


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  		Fig. 3. The S. domuncula retinoid X receptor (RXR). The deduced RXR from S. domuncula, RXR_SUBDO, was aligned with other most similar sequences from human, retinoid X receptor gamma (RXRg_HUMAN; accession number NP_008848), C. elegans, hormone receptor/zinc finger protein (HR-ZF_CAEEL; NP_491103) and D. melanogaster, hepatocyte nuclear factor 4 (HNF-4_DROME; S36218), as well as the partial sequence from a coral (Acropora millepora), the nuclear receptor AmNR8 (AMNR8_ACRMI; AAL29201). Residues conserved (similar or related with respect to their similar physico-chemical properties) in all sequences are shown in white on black and those occurring in at least three sequences in black on gray. The characteristic DNA-binding domain, the two zinc finger modules (#++#) with the conserved cysteine residues (C), are marked. The DNA-binding domain is provided with the conserved residues of the P-box as well as of the D-box (marked). In addition, the borders of the putative secondary structures within the ligand-binding site, the ten {alpha}-helices (~H~) and the ß-turn, have been adopted from Giguère (1999Go). The nuclear receptor signature (NRS) and the activator function-2 (AF2) are indicated.

 


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  		Fig. 4. Phylogenetic relationships of the S. domuncula RXR, RXR_SUBDO, with the related sequences from the coral Acropora millepora nuclear receptors TLL (TLL_ACROMI; AF323680), NR7 (NR7_ACROMI; AF323687) and NR8 (NR8_ACROMI; AF323688), Cnidaria Tripedalia cystophora retinoic acid X receptor (RXR_TRICYS; AF091121), Platyhelminthes Schistosoma mansoni retinoic acid receptor RXR (RXR_SCHMA; AF094759) and S. mansoni retinoid X receptor RXR-2 mRNA (RXR2_SCHMA; AF129816). In addition, relationships are shown with sequences from the Protostomia: D. melanogaster, seven-up protein (svp) type 1 (SVP_DROME; M28863), tailless (TLL_DROME; M34639), mRNA for XR2C ultraspiracle gene (USP_DROME; X53417), D. melanogaster nuclear receptor XR78E/F (XR78E_DROME; U31517); insects Locusta migratoria RXR mRNA (RXR_LOCMI; AF136372) and Tenebrio molitor THR6 (nuclear receptor) gene (TR2_TENMO; AJ005765); and from Deuterostomia: Danio rerio svp 46 mRNA for steroid receptor homologue (SVP46_DARE; X70300); human upstream promoter transcription factor (COUP-TF1_HUMAN; X58241), v-erbA related ear-2 gene (EAR2_HUMAN; X12794), the steroid hormone receptor hERR2 (ERR_HUMAN; X51417), hepatocyte nuclear factor 4 (HNF4_HUMAN; X76930), photoreceptor-specific nuclear receptor (PNR) (PNR_HUMAN; AF121129), receptor of retinoic acid-alpha (RAR{alpha}_HUMAN; X06614), retinoic acid receptor-like protein (RXR{alpha}_HUMAN; X52773), steroidogenic factor 1 mRNA (SF1_HUMAN; U76388), tailless gene homologue (TLX_HUMAN; Y13276) and the steroid receptor (TR2-11) (TR2_HUMAN; M29960); from Mus musculus germ cell nuclear factor (GCNF1_MOUSE; U14666) as well as the orphan steroid hormone receptor from Strongylocentrotus purpuratus (SHR2_STROPU; U38281). The different sequences were classified according to the unified proposition (NRNC, 1999Go). The tree was constructed as described in Materials and methods and remained unrooted. The numbers at the nodes indicate the level of confidence (%) for the branches as determined by bootstrap analysis (100 bootstrap replicates). The scale bar indicates an evolutionary distance of 0.1 aa substitutions per position in the sequence. Numbers in parentheses refer to the respective subfamilies/groups.

 


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  		Fig. 5. Expression of selected genes in canal-forming primmorphs in response to retinoic acid. Primmorphs were first incubated for 5 days on non-coated dishes and then, in order to induce canals, on galectin-coated dishes for 10 days. Subsequently, these primmorphs remained either untreated for 5 days (C; lane a) or were treated with 1 µmol l–1 (lane b), 3 µmol l–1 (lane c), or 50 µmol l–1 of retinoic acid (lane d) as indicated. RNA was then extracted and 5 µg of total RNA per lane were size-separated. After blot transfer, hybridization was performed using the following probes: SDRXR (the putative sponge retinoid X receptor), SDCASPR (S. domuncula caspase), SDLIM4 (sponge LIM/homeodomain protein) or the sponge SDCYP4 (CYP4A related cytochrome P-450 protein). The levels of expression of the respective genes were estimated by northern blot and the intensities of the different transcript bands were determined relative to the intensity of the controls (cultivated for a total of 15 days in solution).

 

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© The Company of Biologists Ltd 2003