Molecular evidence for the presence of a developmental gene in the lowest animals: identification of a homeobox-like gene in the marine sponge Geodia cydonium

ELSEVIER

Mechanisms of Ageing and Development 77 (1994) 43-54

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Molecular evidence for the presence of a developmental gene in the lowest animals identification of a homeobox like gene in the marine sponge Geodia cydonium Michael Kruse a, Andreja Mikoc b, Helena Cetkovic b, Vera Gamulin b, Baruch Rinkevich c, Isabel M. Miiller a, Werner E.G. Mfiller *a alnstitut f~r Physiologische Chemie, Abteilung Angewandte Molekularbiologie, Universitiit, Duesbergweg 6, D-55099 Mainz, Germany blnstitute Ruder Boskovic, Departmentfor Molecular Genetics, 41001 Zagreb, Croatia Clsrael Oceanographic and Limnological Research, National Institute of Oceanography, Tel Shikmona, P.O. Box 8030, Haifa 31080, Israel Received I July 1994; revision received 12 August 1994; accepted 23 September 1994

Abstract

During the development of higher animals, morphogenetic programs are switched on which are frequently controlled by homeotic genes. Until now these genes have not been identified in the lowest animals, the marine sponges. Since sponges show (i) an antero-posterior and/or dorso-ventral axis during embryogenesis and (ii) a complex differentiation pattern during spicula formation, we hypothesized that in sponges homeotic genes - - if present - - are also involved in the control of these processes. Therefore, we searched for homeobox or homeobox-like sequences in the marine sponge Geodia cydonium. Here we describe a homeobox-like sequence from these animals; it was isolated from a cDNA library of an adult specimen. The deduced amino acid sequence of the complete homeodomain shares over 70% similarity with other homeodomain sequences, including those from hydra, insects and vertebrates. These data indicate that the sponge homeodomain-like sequence is similar with respect to structure to those of other animals and may suggest that the sponge homeodomainlike sequence(s) might function during developmental processes and/or during spiculogenesis in a similar manner to that known for higher animals. Abbreviations." aa, amino acid(s); nt, nucleotide(s). * Corresponding author. 0047-6374/94/$07.00 © 1994 Elsevier Science ireland Ltd. All rights reserved SSD1 0047-6374(94)01502-D

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M. Kruse et al. / Mech. Ageing Dev. 77 (1994) 43-54

Keywords: Homeodomain; Development; Sponge; Geodia cydonium; Phylogeny; Evolution; Monophylogeny; Cell adhesion

1. Introduction

Marine sponges are the simplest multicellular animals. They have acquired the structural elements for controlled cell adhesion, during an evolution o f ~ 800 million years [1]. Adult sponges do not contain distinct organs, special digestive apparatus or an excretion system; a nerve system is also lacking. Therefore, until recently, it was assumed that the sponge cells are to a great extent independent in their function and behave like protozoan cells. To prove the assumption that sponge cells, like those from higher animals and unlike individual totipotent cells from protists, are provided with specific control elements for a cell-type specific gene expression present both on the cell surface and on the genetic apparatus, molecular biological approaches were applied, cDNAs with the following elements have been isolated and characterized which may establish a circuit - - linking sensors for mechanochemical changes on and in the cell surface with transcription factors - - allowing a cell-type specific restriction in gene expression: (i) morphoregulatory molecules - - adhesion molecules/receptors, and (ii) molecules controlling the expression of the genes coding for the latter ones, e.g. for homeodomain-containing proteins. The first cDNA sequences encoding two recognition proteins from the marine sponge Geodia cydonium, lectin [2], and a receptor tyrosine kinase [3], already show high homology to mammalian galactose-specific binding proteins and to immunoglobulin, respectively. In the present study, we asked the question if sponges also contain nuclear transcription factors, which (i) show homology to those of higher invertebrates and/or vertebrates and (ii) could hint at the existence of transcriptional regulators involved in cellular differentiation. Based on earlier findings which showed that during the development of sponges and especially during embryogenesis, these animals show a antero-posterior organization pattern as well as controlled processes during spiculogenesis [4], we hypothesized that proteins containing homeodomain or homeodomain-like sequences might control cell identities in particular with regard to spatial domains or cell lineages. The typical homeobox consists of a 183-base pair sequence encoding a trihelical DNA binding motif, the homeodomain [5,6]. Homeobox-containing genes have been isolated first from Drosophila melanogaster [5,6] and later from vertebrates and other invertebrates [7]. Among lower invertebrates, homeobox-containing genes have been identified in the hydra Chlorohydra viridissima [8], the planaria Dugesia tigrina [9], the nematode Caenorhabditis elegans [10], the ascidian Ciona intestinalis [11], and recently in the freshwater sponge Ephydatiafluviatilis [121. Here, we describe for the first time the nucleotide sequence of the cDNA from a marine sponge, G. cydonium, comprising the complete homeobox-like sequence (SHOX) These data (i) suggest that proteins containing homeodomain-like sequences, which are encoded by homeobox-like genes, might be involved in the con-

hi. Kruse et al./Mech. Ageing Dev. 77 (1994) 43-54

45

trol of the development of sponges and/or in the 'organogenesis' in those animals and (ii) shed new light on the generally accepted 'low' organization level in these organisms. 2. Materials and methods

2.1. Materials Restriction endonucleases were obtained from New England BioLabs (Beverley, MA); h-Zap TM kit, Giga pack II Gold packaging extract and cloning vectors from Stratagene (La Jolla, CA); sequencing kit (sequenase version 2.0) from USB (Cleveland, OH); T 3 and T 7 sequencing primers from Pharmacia (Uppsala). 2.2. Sponge and sponge components Live specimens of G. cydonium (Demospongiae) were collected near Rovinj (Croatia). The material was immediately frozen in liquid nitrogen until use. A cDNA library from the sponge G. cydonium (an adult animal) was constructed in h-Zap TM as described [2]. 2.3. Plaque screening Screening of the sponge h-ZAP cDNA library [2] was performed under low stringency hybridization conditions using a [32p] end-labelled 50-'Guessmer' oligodeoxyribonucleotide as a probe [8]. The conditions were as follows - - Prehybridization (2 hr) and hybridization (over night): at 42°C with 35% formamide, 1% blocking reagent (Boehringer), 5 x 55C, 0.1% N-laurylsarcosine, 0.02% SDS; washes: 6 x SSC and 0.1% SDS twice for 10 min at room temperature, 2 x SSC and 0.1% SDS twice at room temperature. Five positive plaques were purified. The cDNA inserts were subsequently subcloned into the plasmid vector pBluescript SK-. 2.4. DNA sequencing The DNA for sequencing was isolated by alkaline lysis according to Sambrook et al. [13]. DsDNA was sequenced by the dideoxy chain termination method [14]. Computer analysis was performed by the PC/GENE 1991 [15] programmes. The predicted secondary structure of the deduced amino acid (aa) sequence was determined according to Chou and Fasman [16]. 3. Results and discussion

3.1. Homeobox-like sequence The cDNA library from the sponge G. cydonium was screened with a degenerated probe. Because the sponge codon usage is not yet well known, we have selected the 'guessmer' oligodeoxyribonucleotide which had been successfully applied for the isolation of homeobox genes in the hydra Chlorohydra viridissima [8]. The cDNA from G. cydonium coding for homeodomain-like protein was isolated and sequenced. The deduced aa sequence of the sponge homeodomain (Fig. 1) contains the characteristic u-helix (aa 11-26 in SHOX; Fig. 1) - - turn (27-29) - - t~-helix

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Fig. 1. Nucleotide sequence ofG. c y d o n i u m c D N A containing the homeobox-like segment (SHOX) (first row; S H O X [nt]) and the decoded aa sequence (second row). Nucleotide sequence comprising the complete homeobox-like sequence spans from nt 145 to nt 333 [aa 4 9 - l I l; underlined). The numbers on the margin given in the third row refer to the aa of the sponge homeodomain-like sequence ( S H O X [aa]). The predicted helix-turn-helix sequence as well as the bipartite

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M. Kruse et al. / Mech. Ageing Dev. 77 (1994) 43-54

47

Table 1 Comparison between deduced polypeptide of homeodomain-like sequence from the marine sponge G. cydonium SHOX and homeodomains from the other organisms: freshwater sponge (proxl), hydra (cnox3), tunicate (eimsh), planaria (Dth-1), nematode (unc86), insect (Antp) and (BarH 1) as well as chicken (Chox3) Homeodomain

proxl (E. fluviatilis) cnox3 C. viridissima cimsh C. intestinalis Dth-I D. tigrina unc86 C. elegans Antp D. melanogaster BarHl D. melanogaster Chox-3 (chicken)

SHOX (G. cydonium) aa % identity

aa % similarity

9 16 14 14

67 61 59 61

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64

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64 69 56

Further details to the source of the homeodomain sequence from other animals are given in legend to Fig. 2.

( 3 0 - 4 0 ) - turn ( 4 1 - 4 4 ) - a-helix (45-55) sequence [5,17] at positions identical to those in other known homeodomains [6]. In addition, the bipartite nuclear targeting sequence, required for nuclear uptake of the protein [18] is present and is located at the N-terminus of the homeodomain (Fig. 1). The deduced aa-sequence of sponge homeodomain shares highest homology with the D. melanogaster BarH1 homeodomain (16% identity and 69% similarity) which functions as a developmental switch required for the promotion of limb development above the evolutionary ground-state of body wall [19]; this sequence is grouped into the dll (distal-less) homeodomain family [20]. The homology to the hydra homeodomain cnox3 was also intriguingly high [8] with 16% identical and 61% similar aa (Fig. 2; Table 1). cnox3 was found to be expressed during the late phase of head regeneration of the hydra Chlorohydra viridissima [8]. Homologies to the homeodomains from the tunicate Ciona intestinalis (cimsh), the planaria Dugesia tigrina (Dth-1), the nematode Caenorhabditis elegans (unc86), the insect D. melanogaster (Antp) and the chicken (Chox3) are lower. Comparably low is the homology of the homeodomainlike sequence from the marine sponge to the homeodomain from the freshwater sponge Ephydatia fluviatilis (proxl) [12,21] with only 9% identical aa (Fig. 2 and Table 1). From an earlier report, it is known that the hydra homeodomain cnox3 contains Tyr at position 50 in the third o~-helix instead of Phe, otherwise found in homeodomains [8]. In the G. cydonium homeodomain, this aa is replaced by the Tyrrelated aa Arg. For the comparison shown in Fig. 2, one gap was inserted into the sponge sequence for optimal alignment. If this gap was omitted, the sponge SHOX aa-sequence matches with the homeodomain consensus pattern [6,15] [LIVMFY]-x(5)-[LIVM_C]- x(4)[IV_C]-[RKQ_E]-x-[W_RI-x(8)-[RKQ];the sponge aa residues are underlined. As an example, the sponge sequence is compared with that of the chicken (Fig. 3). One deviation should be mentioned; the aa Trp, usually present in a conserved manner in

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Fig. 2. Alignment of deduced G. ~;vdonium homeodomain-like sequence (SHOX) with the related sequences from freshwater sponge, Ephydatiafluviatilis (proxl) [I 2], hydra Chloronydra viridissirna (cnox3) [8], tunicate Ciona intestinalis (cimsh) [11], planaria Dugesia tigrina (Dth-I) [9], nematode Caenorhabditis elegans (unc86) [10], D. melanogaster (Antp) 125] and (BarHl) [211 as well as chicken Gallus gallus (Chox3) [26]. The gap is marked [ - - ]; complete consensus is indicated with asterisks and related aa [classified according to Taylor [27]] are indicated by dots. Above the sponge sequence the spectrum of related aa residues observed in the other homeodomains is listed.

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M. Kruse et al./ Mech. Ageing Dev. 77 (1994) 43-54

homeodomain sequences from other animals is replaced in the sponge sequence by the related aa Arg. Therefore, we termed the sponge nt sequence homeobox-like and the deduced aa sequence homeodomain-like. A dendrogram of the multiple alignments including both vertebrate and invertebrate homeodomain containing sequences with that of the sponge shows that the marine sponge sequence is only distantly related to the domain of the freshwater sponge (Fig. 4).

3.2. Guessmer oligodeoxyribonucleotide probe The mixed oligodeoxyribonucleotide, 'guessmer', used as a probe for screening the eDNA library from G. cydonium was operationally termed guessmer-1 and -2 (Fig. 5). At three positions, inosine was chosen as 'neutral' base. A nucleotide (nt) sequence alignment revealed that the highest identity between the guessmer oligodeoxyribonucleotide probe used and the sponge eDNA was - against our expectation - - approximately 150 nt downstream of the end of the homeobox-like sequence. As shown in Fig. 5, two overlapping regions between the probe guessmer-1 and -2 and the sponge eDNA were identified (i) at nt 465-506 (63% identity) and (ii) at nt 478-523 (76%). The degree of identity is low with respect to mathematical calculations published by Lathe [22]. It this report, it had been recommended that a probe should have at least a 76% homology with the authentic gene. One possible explanation for the fact that the eDNA coding for the homeodomainlike protein was detected in the library with this guessmer-probe might be due to the observation that guessmer-1 and -2 bind to overlapping regions of the sponge eDNA resulting in an overall identity of 84% (Fig. 5). 4. Conclusion

Previously published sequence data of genes from the marine sponge G. cydonium revealed that the two adhesion molecules, the lectin(s) [2] and the receptor tyrosine kinase [3], display on the deduced polypeptide level high homology to the corresponding proteins from vertebrates. This strongly indicates that the kingdom Animalia is a monophyletic group of multicellular organisms. By calculations following the procedure described by Kimura [23], which is based on aa substitutions per site per time (the average rate of nonsynonymous substitutions in DNA has been set to be 0.9 × 10 -9 per site and year [1]), the time when sponge lectin and tyrosine kinase started to diverge from the respective common ancestral genes was estimated [1,3]. These data revealed that the oldest multiceUular animals, the sponges, diverged from the other animals approximately 800 million years ago [1]. This figure matches well with the paleontological data. In the present study, we report that the marine sponge also contains a gene with a homeobox-like sequence suggesting that the development of these simple organisms is likewise controlled on the transcriptional level by a homeodomain-containing factor(s) as known from animals which possess organs. In addition, this finding implies that sponges, the simplest animals, are already provided with structural elements required for the establishment of signal transduction pathways involved in cellular dif-

Fig. 4. Dendrogam of 13 homeodomain sequences (deduced aa). The analysis was performed with the CLUSTAL program [15]. The explanation of the ~ quences is given in the legend to Fig. 2.

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M. Kruse et al. /Mech. Ageing Dev. 77 (1994) 43-54

53

ferentiation, with adhesion molecules/receptors on the cell surface and with transcription factors, containing homeodomain-like sequences. It is tempting to assume that the programs allowing a controlled development to an organized animal with specialized cells are basically the same or very similar in all multicellular organisms. Considering the recent cloning data using the sponge system, we propose that the evolution from unicellular to multicellular animals was a singular event which could take place after the acquisition of receptor-mediated signal transduction pathways including phosphatidyl-inositide cascade, tyrosine kinases, etc. We have recently shown that the formation and maintenance of a functional aquiferous system in sponges depends upon the presence of a classical soluble morphogen, the retinoic acid [24]. Hence, these animals do contain not only molecules involved in an intracellular signaling as known from higher invertebrates and vertebrates but also morphogen(s) hitherto also only known from higher animals and which are required for an intercellular signaling allowing the development of organisms composed of functionally arranged differentiated cells. In conclusion, data from adhesion molecules/receptors and homeodomain-like sequence of the marine sponge G. cydonium support the assumption that all multicellular organisms forming the kingdom Animalia fall into one monophyletic group. Furthermore, the data indicate that during the transition period in the evolution from the Protists to the Animalia, the morphoregulatory molecules, the cell adhesion molecules/receptors, as well as the homeodomain proteins have been developed - very likely simultaneously.

Acknowledgements We thank Dr B. Galliot (EMBL - - Heidelberg) for advice. This work was supported by grants from the Deutsche Forschungsgemeinschaft (SFB 169; A11) and the German-Israel Foundation for Scientific Research and Development (no. 1-154034.11/90). The EMBL accession number of the nt sequence comprising the homeoboxlike sequence of Geodia cydonium SHOX is X 79054.

References [l] W.E.G. Mfiller, H.C. Schrfder, H. Sch~icke, I.M Mfiller and V. Gamulin, Phylogenetic relationship of adhesion proteins and ubiquitin from the marine sponge Geodia cydonium. Endocytobiosis, (1994) in press. [2] K. Pfeifer, M. Haasemann, V. Gamulin, H. Bretting, F. Fahrenholz and W.E.G. Miiller, S-type lectins occur also in invertebrates: high conservation of the carbohydrate recognition domain in the lectin genes from the marine sponge Geodia cydonium. Glyeobiology, 3 (1993) 179-184. [3] H. Sch/icke, H.C. Schr6der, I.M. Miiller, W.E.G. Mfiller, V. Gamulin and B. Rinkevich, The immunoglobulin superfamily includes members from the lowest invertebrates to the highest vertebrates. lmmunol. Today, 15 (1994) 497-498. 14] R. Borojevic, I~tude experimentale de la diff6rentiation des cellules de 1'6ponge au cours de son d6velopment. Dev. Biol., 14 (1966) 130-153. [5] M.P. Scott, J.W. Tamkun and G.W. Hartzell, The structure and function of the homeodomain. Biochim. Biophys. Acta, 989 (1989)25-48. [6] W.J. Gehring, The homeobox in perspective. Trends Biochem. Sci., 17 (1992) 277-280.

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[7] F.R. Schubert, K. Nieselt-Struwe and P. Gruss, The antennapedia-type homeobox genes have evolved from three precursors separately early in metazoan evolution. Proe. Natl. A earl Sei. USA, 80 (i 993) 143-147. [8] M. Schummer, 1. Scheurlen, C. Schaller and B. Galliot, HOM/HOX homeobox genes arc present in hydra (Chlorohydra viridissima) and are differentially expressed during regeneration. EMBO J., 1 (1992) 1815-1823. [9] J. Garcia-Fernandez, J. Baguna and E. Salo, Planarian homcobox genes: cloning, sequence analysis and expression. Proc. Natl. Acad Sci. USA, 88 (1991) 7338-7342. [10] M. Finney, G. Ruvkun and H.R. Horvitz, The C elegans cell lineage and differentiation gene unc86 encodes a protein with a homeodomain and extended similarity to transription factors. Cell, 55 (1988) 757-769. [11] P.W.H. Holland, Cloning and evolutionary analysis of msh-like homeobox genes from mouse, zebrafish and ascidian. Gene, 98 (1991) 253-257. [12] M. Scimiya, K. Ishiguro, K. Miura and Y. Watanabe, Homeobox-containing genes in the most primitive metazoa, the sponges. Eur. J. Bioehem., 221 (1994) 219-225. [13] J. Sambrook, E.F. Fritsch and T. Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Lab. Press, Cold Spring Harbor, 1989. [14] F. Sanger, S. Nicklen and A.R. Coulson, DNA sequencing with chain-terminating inhibitors. Proe. Natl. Aead. Sci. USA, 74 (1977) 5463-5467. [I 5] InteiliGenetics, PC/Gene: User and Reference Manual, Release 6.5, l ntelliGenetics, Mountain View, CA, 1991. [16] P.Y. Chou and G.D. Fasman, Prediction of the secondary structure of proteins from their amino acid sequence. Adv. Enzymol., 47 (1978) 45-147. [17] J. Gamier, D.J. Osguthorp¢ and B. Robson, Analysis of the accuracy and implications of simpk methods for predicting the secondary structure of globular proteins. J. Mol. Biol., 120 (1978) 97-120. [18] C. Dingwall and R.A. Laskey, Protein import into the cell nucleus. Annu. Rev. Cell Biol., 2 (1986) 367-390. [19] S.M. Cohen, G. Br6nner, F. Kiittner, F. Jiirgens and H. J/ickle, Distal-less encodes a homeodomain protein required for limb development in Drosophila. Nature, 338 (1989) 432-434. [20] T. Kojima, S. ishimaru, S. Higashijiama, E. Takayama, H. Akimaru, M. Sone, Y. Emori and K. Saigo, Identification of a different-type homvobox gone, BarHl, possibly causing Bar(B) and Om(ID) mutations in Drosophila. Proc. Natl. Acad. Sei. USA, 88 (1991) 4343-4347. [21] C. Coutinho, S. Vissers and G. Van de Vyver, Evidence of homeobox genes in tbe freshwater sponge Ephydatiafluviatilis. In R.W.M v. Soest, T.M.G.v. Kempcn and J.C. Braekman (eds.), Sponges in Time and Space, Balkema Press, Rotterdam, 1994, pp. 47-54. [22] R. Lathe, Synthetic oligonucleotide probes deduced from amino acid sequence data: theoretical and practical considerations. J. MoL Biol.. 183 (1985) 1-12. [23] M. Kimura, The Natural Theory of Molecular Evolution, Cambridge University Press, Cambridge, 1983. [24] G. lmsiecke, R. Borojcvic and W.E.G. Miiller, Retinoic acid acts as a morphogen in freshwater sponges. lnvertehr. Reprod. Dee. (1994)in press. [25] E. Boncinelli, A. Simeone, D. Acampora and F. Mavilio, HOX gene activation by rctinoic acid. Trends Genet., 7 (1991) 329-334. [26] Z. Rangini, A. Frumkin, G. Shani, M Guttmann, H. Eyal-Giladi, Y. Gruenbaum and A. Feinsod, The chicken homeo box genes CHoxl and CHox3: cloning, sequencing and expression during embryogenesis. Gene, 76 (1989) 61-74. [27] W.R. Taylor, Protein structure and prediction. In M.J. Bishop and C.J. Rawlings (eds.), Nucleic Acids and Protein Sequences Analysis, IRL Press, Oxford, 1987, pp. 285-322.