Conservation of Brachyury, Mef2 and Snail in the Myogenic Lineage Of

Developmental Biology 244, 372–384 (2002) doi:10.1006/dbio.2002.0616, available online at http://www.idealibrary.com on

View metadata, citation and similar papers at core.ac.uk brought to you by CORE Conservation of Brachyury, Mef2, and Snail in theprovided by Elsevier - Publisher Connector Myogenic Lineage of Jellyﬁsh: A Connection to the Mesoderm of Bilateria

Ju¨rg Spring, Nathalie Yanze, Christoph Jo¨sch, Arnoud M. Middel, Brigitte Winninger, and Volker Schmid1 Institute of Zoology, University of Basel, Biocenter/Pharmacenter, Klingelbergstrasse 50, CH-4056 Basel, Switzerland

One major difference between simple metazoans such as cnidarians and all the bilaterian animals is thought to involve the invention of mesoderm. The terms diploblasts and triploblasts are therefore, often used to group prebilaterian and bilaterian animals, respectively. However, jellyfish contain well developed striated and smooth muscle tissues that derive from the entocodon, a mesoderm-like tissue formed during medusa development. We investigated the hypothesis, that the entocodon could be homologous to the third germ layer of bilaterians by analyzing the structures and expression patterns of the homologues of Brachyury, Mef2, and Snail in the jellyfish Podocoryne carnea. These are regulatory genes from the T-box, MADS-box and zinc finger families known to play important roles in bilaterian mesoderm patterning and muscle differentiation. The sequence and expression data demonstrate that the genes are structurally and functionally conserved and even more similar to humans or other deuterostomes than to protostome model organisms such as Drosophila or Caenorhabditis elegans. Based on these data we conclude that the common ancestor of the cnidarians and bilaterians not only shared genes that play a role in regulating myogenesis but already used them to develop and differentiate muscle systems similar to those of triploblasts. © 2002 Elsevier Science (USA) Key Words: cnidaria; mesoderm; evolution; myogenesis; regulatory genes.

INTRODUCTION complex. Most of the differences are found in the medusa bell which not only can carry complicated sense organs Podocoryne carnea is a typical representative of the class such as lens eyes, statocysts, and nerve rings but also Hydrozoa. With few exceptions, hydrozoans are marine and consists of two nonmyoepithelial cell layers and addition- exhibit a life cycle which consists of the free swimming ally a third layer of epithelial mononucleated striated planula larva, the sessile polyp and the sexual stage, the muscle cells. In the entire phylum, the planula larva and medusa which is formed from polyps through budding (Fig. the polyp lack these medusa-specific cell types and sense 1). In some hydrozoans, the medusa generation has become organs. secondarily reduced (Boero and Bouillon, 1987; Bouillon, In bilaterians, the striated and smooth muscle tissues are 1994). It is generally assumed that all cnidarians are formed in general a derivative of the third or middle germ layer, the of an outer and an inner layer of multifunctional myoepi- mesoderm. In the hydrozoan jellyfish, the striated muscle is thelial cells. The other cell types, interstitial cells, nerve a derivative of the entocodon, a tissue layer which separates cells or nematocytes are interspersed in either of the two from the ectodermal layer early in medusa development layers. Therefore, Cnidaria are classified as diploblasts or (Ku¨hn, 1910). The entocodon is located between the distal simple bilayered animals. While this classification accu- ectodermal and the endodermal tissue, and is separated rately describes the basic tissue organization of the planula from both layers by an extracellular matrix (Boelsterli, larva and the polyp, the anatomy of the medusa is more 1977). Entocodon cells in early bud stages are of embryonic appearance and highly proliferative (Schmid, 1972; Boel- 1 To whom correspondence should be addressed. Fax: 41-61-267- sterli, 1977). Later the entocodon forms a cavity, which 16-27. E-mail: [email protected]. finally connects to the outside by the developing velar

FIG. 1. Life cycle of the hydrozoan jellyﬁsh Podocoryne carnea. The pelagic sexually mature medusa liberates gametes. The embryo develops into a free swimming planula larva, which attaches to the substrate (disc stage) and transforms into a primary polyp. A colony composed of feeding polyps (gastrozoid) and asexually reproducing polyps (gonozoid) is formed from the primary polyp by stolonization and polyp budding. Cross section of the medusa shows: ma, manubrium; ne: nematocytes; go, gonads, v, velum; t, tentacle; ri, ring canal; rc, radial canal; ex, exumbrella; st, striated muscle.

opening. In older bud stages mitotic activity in the bell at different levels in the specification of the mesodermal gradually stops and the outer wall of the entocodon differ- and myogenic lineage of bilaterian animals from Drosoph- entiates into the striated muscle while the inner wall forms ila to vertebrates (Baylies and Michelson, 2001). the smooth muscle of the feeding and sex organ of the To investigate the hypothesis that the entocodon of animal. jellyfish is homologous to the mesoderm of bilaterians we The histology and developmental pattern of muscle for- isolated a Podocoryne homologue of each of the three gene mation in medusa development has lead to the idea that the families and studied structure and expression patterns entocodon could be a mesoderm-like layer (Boero et al., throughout the life cycle and specifically during muscle 1998). Striated myofilaments are usually found in cells development. The results demonstrate that all three genes derived from the mesoderm, however, exceptions are are expressed during myogenic differentiation. Addition- known. Tentacles of entoprocts contain flagellated ectoder- ally, as is true for their bilataterian cognates, they appear to mal epithelia that contain striated myofilaments (Nielson have other functions as well. The data further strengthen and Rostgaard, 1976). This indicates that the appropriate the hypothesis that the common ancestor of cnidarians and structural genes can be activated independent of the germ bilaterians already used the same regulatory and structural layer and that the analysis of the striated muscle-specific genes and comparable developmental patterns to build structural genes alone would not be sufficient. The molecu- muscle systems. lar analysis of muscle development in Podocoryne demon- strated that the structural genes for a tropomyosin and a myosin heavy chain (Schuchert et al., 1993; Gro¨ ger et al., 1999) are structurally and functionally conserved and spe- MATERIALS AND METHODS cific for the striated muscle tissue. Furthermore, the presence of the homeobox gene Otx (Mu¨ ller et al., 1999), a head and gastrulation regulator in bilaterians, in jellyfish striated Animals muscle and the basic helix-loop-helix (bHLH) factor Twist (Spring et al., 2000) during the formation of the entocodon Podocoryne carnea M. Sars (Cnidaria, Hydrozoa, Anthomedusae) colonies are reared in the laboratory as described elsewhere in medusa development, indicated that genes with specific (Schmid, 1979). Medusa parts and buds were obtained by microsur- roles in mesoderm patterning of bilaterians were already gery and the buds were staged according to Frey (1968). To study present in the common ancestor with bilaterians. Next to the distribution of messages we excised different medusa parts by homeodomain and bHLH transcription factors, the best- microsurgery from freshly hatched and anaesthetized medusa studied regulatory genes are members of the T-box, MADS- (3.5% MgCl in sea water). Within 3 to 5 min after excision the box and zinc finger families, such as Brachyury, Mef2, and fragments were used for RNA extractions. Eggs and larval stages Snail, respectively. These three gene families are involved were obtained as described in Reber-Mu¨ ller et al. (1995).

Molecular Cloning and Sequence Comparison described previously (Spring et al., 2000). Elongation factor 1 alpha (EF1a) was used as an endogenous control to compensate for Molecular biology procedures were carried out as de- variations in the quantity and quality of the preparations. Experi- scribed (Mu¨ ller et al., 1999; Spring et al., 2000; Yanze et al., ments were repeated twice on two different mRNA samples. For 2001). Homology PCR for Brachyury, Mef2 and Snail homo- each gene several primer sets were tested and those best suited for logues of Podocoryne was conducted on cDNA and the SYBR Green I technology were selected. Experiments on genomic DNA. A 256 bp Brachyury fragment was amplified Brachyury, Mef2, Snail, and EF1a, respectively, were performed with TF1 5Ј- GG(AGCT) (AC)G(AGCT) (AC)G(AGCT) with the following forward and reverse primers BraFlc 5Ј-GAC ATG TT(CT) CC-3Ј and TR1 5Ј-AA(AGCT) GC(CT) GCA AAA GAA CGT TCT GAC-3Ј and BraRlc 5Ј-AGG ACT GCG TT(AGCT) GC(AG) AA(AGCT) GG-3Ј followed by TF1 and TGG TGA TAC AG-3Ј, MeF2 5Ј-GAG TCC CTA ATA CAT TGA Ј Ј TR2 5Ј-CG(AGCT) GG(CT) TC(AG) TA(CT) TT(AG) TG- GCA TTC-3 and MeR2 5 -GGT ACT TCA TTT CTG TTC GTG Ј Ј Ј 3Ј, a 131 bp Mef2 fragment with MefF1 5Ј-ATG GG(AGCT) GAG-3 , SnF2lc 5 -ACT CGG TAC CAG TCT ACT ATC-3 and SnRlc 5Ј-TGG CGC GAA TTT TTA CCC TTC-3Ј or EF1AF (AC)G(AGCT) AA(AG) AT-3Ј and MefR1 5Ј-CAT (AG)TC 5Ј-ACG TGG TAT GGT TGC CTC TG-3Ј and EF1AR 5Ј-TGA (AGCT)GT (AGCT)(GC)(AT) (AGCT)GC (AG)TA (CT)TG- TAA CGC CAA CGG CTA CG-3Ј. EF1a primers were designed to Ј Ј 3 followed by MefF1 and MefR2 5 –GC(AGT) AT(CT) span an intron, which allowed the detection of putative genomic TC(AG) CA(AG) TC(AG) CA-3Ј from cDNA and a 136 DNA contamination in the samples. bp Snail fragment with SnaF1 5Ј-GG(AGCT) GC(AGCT) Ј Ј (CT)T(AGCT) AA(AG) ATG CA-3 and SnaR3 5 -TG(AGCT) In Situ Hybridization GT(CT) TG(AGCT) A(AG)(AG) TG(AGCT) GC-3Ј followed by SnaF2 5Ј-AC(AGTC) CA(CT) AC(AGTC) (CT)T(AGTC) Embryos and larvae were fixed over night at 4°C in Lavdowsky’s CC(AGCT) TG-3Ј and SnaR2 5Ј-TT(AT) GA(AGCT) fixative supplemented with 0.2% glutaraldehyde. Embryos at dif- C(GT)(AG) TC(AGCT) GC(AG) AA-3Ј on genomic DNA. For ferent cleavage stages, the larvae in 1, 2, 3, 4–5, and 5–7 days old each PCR round standard conditions were used, except, that cohorts were fixed. Whole mount in situ hybridization experiments were performed as described by Gro¨ ger et al., (1999) with slight the annealing temperature was 40°C and the cycle number variations (Spring et al., 2000). DIG-RNA labeled probes were was 20 for the first and 40 cycles for the second round. PCR synthesized from selected stretches excluding highly conserved products of the expected sizes were gel purified with a sequences. The T-box of Brachyury was excluded as well as the Qiaquick column (Qiagen), subcloned in the pCRII-TOPO MADS box and Mef2 domain from Mef2. Those stretches were vector (TOPO TA cloning Dual Promotor kit, Invitrogen), amplified with the primers listed below and subcloned in the sequenced and used as a probe to screen cDNA libraries or pCRII-TOPO vector (TOPO TA cloning Dual Promotor kit, Invitro- to design gene-specific primers for 3Ј and 5Ј RACE experi- gen). BraF4 5Ј-ACG TAC CCT ATT TAC CAA TCG AGC-3Ј and ments as described (Mu¨ ller et al., 1999; Yanze et al., 2001). an oligo-dT primer, MeF3 5Ј-TCT CCA GAA GAT GGT GAT GCT Ј Ј Clones with the complete coding sequences were isolated GCC-3 and MeR4 5 -ACT GTA ACC TGT ATA ACT AGA Ј Ј Ј for Brachyury (1628 bp), Mef2 (1621 bp), and Snail (1108 bp) CGG-3 or SnF3 5 -GTT TGA GAC AGC AAG AAG ATC-3 and SnR1 5Ј-TGC ACG TCC ACA ATA CGA GC-3Ј. and the sequence has been submitted to the DNA databases Hybridization was carried out at 64°C for 16 h with fresh with the accession numbers AJ428494–6, respectively. unfrozen probes. Afterwards specimens were washed gradually and Nucleotide and deduced amino acid sequences were an- stepwise for 20 min in each of the washing buffers and incubated alyzed using the GCG software package. BLAST searches for1hintheblocking buffer at RT followed by2hatRTinthe (Altschul et al., 1997) were performed using the BLAST antibody solution. After this, specimens were carefully washed in network service at the NCBI (http://www.ncbi.nlm. PBST, incubated in TMNT for 20 min followed by color detection. nih.gov). Multiple sequence alignments and phylogenetic The stained samples were washed for 7 days in cold PBST and then trees based on the neighbor joining method were generated fixed at 4°C in 4% paraformaldehyde 0.2% glutaraldehyde over- with Clustal X (Jeanmougin et al., 1998). Alignments of night, dehydrated in alcohol, embedded in paraffin, and sectioned. entire protein sequences were compared to the alignments of the T-box, MADS-box or zinc finger regions alone, RESULTS AND DISCUSSION respectively. Grouping within the Brachyury, Mef2, and Snail family can change depending on the selection of Mesoderm Genes in Diploblasts? species or methods used but the exclusion from other subfamilies such as TBX2, SRF, or Scratch is always sup- Regulatory genes well conserved from Drosophila and ported by high bootstrap values. Caenorhabditis elegans to humans suggest that a common ancestor of bilaterian animals already used the same, sur- prisingly small set of genes to establish the body plan. Relative Quantification of Gene Expression Interestingly, even genes thought to be mesoderm-specific by RT-PCR from investigations in triploblasts could be found in so- The mRNA was extracted using the Dynabeads direct kit (Dy- called diploblasts, animals that should have no mesoderm. nal) and reverse transcribed into single strand cDNA (First strand Furthermore, the Podocoryne homologue of Twist, a cDNA synthesis kit for RT-PCR, Roche). RT-PCR expression mesoderm-specifying gene, is expressed in a mesoderm-like analysis was performed on a LightCycler with the FastStart DNA third cell layer that forms during medusa bud development Master SYBR Green I kit (Roche). Analysis has been processed as (Spring et al., 2000). The only other information on regula-

FIG. 2. High conservation of Podocoryne Brachyury, Mef2 and Snail sequences. (A) Podocoryne Brachyury shows greater similarity in the T-Box to Hydra HyBra1, Drosophila brachyenteron (byn or Trg) and the human paralogues on different chromosomes, Brachyury (T) and TBX19, than to other T-box proteins. C. elegans lacks a clear Brachyury orthologue. (B) Podocoryne Mef2 shares 92% sequence identity with human MEF2A in the MADS-box and Mef2 domain, but little sequence similarity in the rest of the protein. This is also true for the single Drosophila and C. elegans orthologues or the four human paralogues. Other MADS-box proteins lack the Mef2 domain. (C) Podocoryne Snail contains only four zinc fingers like human SNAIL1, while all other listed Snail and Scratch family members have five. SNAIL1, SLUG, and mouse Smuc appear to be vertebrate paralogues on different chromosomes. The Drosophila Snail family members snail, worniu, and escargot, however, appear to be insect-specific tandem duplicates. A single Snail orthologue could be found in place of K02D7.2 on the C. elegans genome, while ces-1 is not the C. elegans Snail but the Scratch orthologue. SNAG domains are absent in Drosophila and C. elegans Snail homologues although they can even be found in Scratch family members. Podocoryne Brachyury, Mef2, and Snail appear to be slightly more similar to human or other deuterostome than to Drosophila or C. elegans sequences. Bootstrap values for 1000 replicates are indicated at stippled branches representative for other subfamilies.

tory gene function during the life cycle of Podocoryne is sophila or C. elegans. However, this observation needs based on the expression patterns of homeobox genes such as further analysis and comparisons to less derived protos- Otx from the Paired-class (Mu¨ ller et al., 1999) or from the tomes. Hox/ParaHox groups (Aerne et al., 1995; Masuda- Brachyury is a member of the family of T-box genes. The Nakagawa et al., 2000; Yanze et al., 2001). Based on deﬁning feature of the family is a conserved sequence multiple sequence alignments of the highly conserved re- coding for a DNA-binding motif that extents across a region gions of the transcription factors Brachyury from the T-box of 180–200 amino acid residues at the N-terminus (Papaio- family, Mef2 from the MADS-box family and Snail from the annou, 2001). Podocoryne Brachyury (Fig. 2A) is a 445 zinc ﬁnger family (Fig. 2), degenerate primers were designed amino acids protein with a highly conserved T-Box most for homology PCR. Initial PCR fragments were sequenced related to other Brachyury homologues within the T-box and extended to full length coding clones by 5Ј and 3Ј RACE family. Sequence similarities are basically restricted to the and screening of cDNA libraries as described (Spring et al., T-box of 172 amino acids in Podocoryne Brachyury that is 2000). The predicted protein sequences of Podocoryne 72% identical to human Brachyury and Hydra HyBra1 Brachyury, Mef2 and Snail are highly conserved, and as (Technau and Bode, 1999) or 67% identical to the Drosoph- previously noticed for Otx and Twist, the Podocoryne ila homologue brachyenteron. Phylogenetic analysis indi- sequences are even slightly more similar to human or other cates that the two human genes T (Brachyury; 6q27) and deuterostome sequences than to protostomes such as Dro- TBX19 (Tpit; 1q23-q24) which are on two different chromo-

FIG. 3. Expression analysis by in situ hybridization of unfertilized eggs, early embryos and planula larvae of Podocoryne. C, I, and O, are blastula stages; D, J, and P are early postgastrula embryos. All stages are positioned with the anterior pole down. Bar is 100 ␮m.

somes are equally similar to single invertebrate Brachyury a true Brachyury orthologue while it contains some addi- homologues, comparable to the situation with a single tional but highly derived family members. invertebrate and multiple vertebrate Hox clusters. Interest- Mef2 belongs to a subgroup of the evolutionary conserved ingly, the completely sequenced genome of C. elegans lacks MADS-box transcription factors that play a critical role in

related to other Mef2 homologues within the MADS-box family (Fig. 2B). Sequence similarities in the C-terminal transactivation domain are hardly recognizable across phyla while the MADS-box and Mef2 domains are very highly conserved. Podocoryne Mef2 is 92% identical to human MEF2A, 90% to C. elegans and 84% to Drosophila Mef2 in these 86 amino acids. Phylogenetic analysis indicates that Podocoryne Mef2 and the three human paralogues MEF2A, C and D on three different chromosomes are more closely related to each other than the highly derived human paral- ogue MEF2B or the protostome homologues from Drosoph- ila and C. elegans. Serum response factor, a distantly related MADS-box factor is also conserved from cnidarians to humans (Hoffmann and Kroiher, 2001). The Snail family of zinc finger transcription factors is most similar to the Scratch family, and both are conserved from Drosophila to humans and clearly distinguishable from all other zinc finger families (Hemavathy et al., 2000; Manzanaras et al., 2001). All members share a very similar C-terminal half, containing four or five zinc fingers of the C2H2 type and a divergent N-terminal portion. Although there is little conservation outside the zinc finger domain, all vertebrate members contain a conserved motive at their extreme N-termini, the so-called SNAG domain. Podocoryne Snail is a 351 amino acid protein with four highly conserved zinc finger motifs and an N-terminal SNAG domain (Fig. 2C). Like human SNAIL1, Podocoryne Snail seems to have lost the first of the five fingers typical for other Snail and Scratch family members. Because the first fingers do not seem to be functional (Pavletich and Pabo, 1993), independent domain loss seems to be more likely than a phylogenetic grouping of Podocoryne and vertebrate Snail with four fingers versus Drosophila Snail family members and vertebrate Slug and Smuc and the Scratch subfamily with five fingers. Indeed, the four Podocoryne zinc fingers are slightly more similar to human SLUG (82% identity) than to human SNAIL1 or Drosophila Snail (75%) and significantly more than to Scratch family members (60%). The SNAG domain is well conserved in Podocoryne Snail; 9 amino acids at the N-terminus first FIG. 4. Expression analysis by in situ hybridization of the feeding described as a transcriptional repressor motif (Grimes et al., polyp (gastrozoid) and the liberated medusa of Podocoryne. go, 1996) are 100% identical to human SLUG, but also to gonads; h, hypostome (mouth part); ma, manubrium; t, tentacle; tb, Dro- tentacle bulb. Arrows point to manubrium lips. Bar is 25 ␮m. Scratch family members such as human SCRT1. In sophila Snail family members the SNAG domain is hardly detectable. In C. elegans a Snail homologue could be found in the genomic clone K02D7 (The C. elegans Se- Drosophila and vertebrate myogenesis. The MADS-box is quencing Consortium, 1998); it has five zinc fingers but involved in DNA binding and dimerization. The signature lacks a SNAG domain. Again, phylogenetic analysis indi- domain of the Mef2 subgroup is the Mef2 domain, a region cates that Podocoryne Snail and three mammalian homo- of 29 conserved amino acid residues which is involved in logues on three different chromosomes are slightly more interactions with other proteins and also influences DNA similar to each other than to protostome homologues from binding at YTA(AT)4TAR sites, a sequence distinct from Drosophila or C. elegans. the binding sites of the other MADS-box family members. Mef2 factors and the bHLH proteins of the MyoD family Medusa Development as a Biphasic Process mediate muscle-specific gene expression (Black and Olson, 1998). Podocoryne Mef2 is a 431 amino acid protein with a Within 10 to 14 h the fertilized egg develops by total and highly conserved MADS-box and a Mef2 domain most equal cleavage into an elongated stereoblastula (Fig. 3).

Gastrulation occurs by immigration of cells at the posterior invaginates, forming with the adjacent layer of the ento- pole, which corresponds to the animal pole of the egg in codon a double tissue layer, the velum primordium (stages hydrozoans (Freeman, 1990). Between 16 to 20 h after 5–6, v in Fig. 5M). At the most distal ends of the radial fertilization the embryo starts to swim with ectodermal canals the tentacle bulbs and tentacles will develop, how- cilia. A few hours later the ectodermal and endodermal ever, without any cellular contribution from the entocodon. myoepithelia and little later all other larval cell types have The striated muscle of the inner side of the velum (Figs. 1 differentiated and swimming becomes directed, with the and 5) derives from the entocodon whereas the smooth enlarged anterior pole first (Fig. 3). Upon appropriate muscle of the outside of the velum originates from the stimuli the larva settles with the anterior pole and trans- ectoderm, this is also true for the smooth muscle of the forms into the primary bilayered polyp (Fig. 1). It is assumed tentacles. Up to stage 5 the entocodon cavity is completely that the posterior pole of the larva forms tentacles and the sealed off from the outside but from stages 6 to 7 on the polyp mouth and the anterior pole the adhesive pad (Bouil- velar opening forms through which the growing tentacles lon, 1994). By stolonization from the polyp base a colony are pushed into the entocodon cavity. This opening process develops which is composed of feeding polyps, named suddenly brings the striated and smooth muscle into an gastrozoids (Fig. 4), and medusa budding polyps, named ectodermal position, a situation that has helped to disguise gonozoids (Fig. 1). Larva and polyp are bilayered and al- the true character of the bell muscle (discussed in Boero et though different in morphology they share principally the al., 1998). The different cell types of the medusa differenti- same cell types. In the mid-body region of the gonozoid ate gradually as development of the various tissues pro- medusa buds of the different developmental stages form a ceeds. The transformation of the late bud stages into a whirl (Fig. 1). The buds were classified according to the functional medusa happens at stage 9. Within few minutes stage of differentiation from 1 to 10 (Frey, 1968; Fig. 5). the mesoglea of the bell swells (Schmid, 1972). The increase Dedifferentiating and immigrating polyp cells form the in volume unfolds the bell tissues. It brings the tentacles in early stages of the budding process (Bra¨ndle, 1971; Boel- their outside position and activates the striated muscle to sterli, 1977). First, an ectodermal thickening appears at the rhythmic contractions, a process that liberates the medusa budding site, then endoderm and ectoderm bulge to form from the mother polyp. stage 1 (Fig. 5). Cells of stage 1 are highly proliferative and have an embryonic, undifferentiated appearance (Boelsterli, Brachyury Expression in Morphogenetically Active 1977; Spring et al., 2000). A little later, cells from the apical Zones ectoderm start to bulge inward into the endodermal layer, forming the entocodon anlage of stage 2 (Fig. 5; brown). Like in Xenopus (Smith et al., 1991), Podocoryne Because the entocodon cells are clearly separate from the Brachyury is a maternal message in Podocoryne. Although ectoderm and endoderm by the formation of an extracellu- the RT-PCR data demonstrate a clear signal in the unfertil- lar matrix (Boelsterli, 1977; m in Fig. 5) they form an own ized egg and the larva (Fig. 6) the signal is only detectable by layer and can be called a third germ layer (Boero et al., in situ hybridization after 2 days in color detection, in the 1998). At stages 3 and 4 (Fig. 5), the entocodon enlarges and egg at the animal pole and is less distinct in cleavage cells the endoderm pushes distally around the entocodon form- (Figs. 3A and 3B). The signal becomes stronger and more ing finally four tubes destined to become the four radial localized at late blastula-early gastrula stage when ingres- canals. At the same time the endoderm projects centrally sion is occurring at the posterior pole (Figs. 3C and 3D). carrying the central entocodon before it to form the primor- This early expression pattern, although much weaker, par- dium of the manubrium (feeding and sex organ; Hyman, allels that of the ParaHox-like gene Cnox4-Pc, a message 1940). During this process the entocodon forms a cavity, the involved in axial patterning of the embryo (Yanze et al., future subumbrellar space (Fig. 5). At stage 4 the 4 radial 2001). The gastrulation focused expression pattern is con- canal tubes (that at stage 3 were connected) become sepa- served to most bilaterians (Holland, 2000; Papaioannou, rated by the growing bud, however, remain connected 2001). After completion of gastrulation no posterior stain- through the de novo formation of a thin monoepithelial cell ing is found. A few hours later the two layers form smooth layer, the subumbrellar plate or gastrodermal lamella (p in muscle myofibrils and nematocytes and nerve cells differ- Fig. 5, cross sections of stages 6 and 7, Figs. 5G, 5N, and 5U). entiate (Gro¨ ger and Schmid, 2001). In larva competent to The outer layer of the entocodon that covers the radial settle with the anterior pole and form the primary polyp, canals and the interradial subumbrellar plate will differen- graded ectodermal staining reappears at the anterior pole, tiate into the striated muscle (Fig. 5; red), the central layer the older the larva the more intense the signal becomes of the entocodon which covers the endoderm of the manu- (Figs. 3E and 3F). Because the anterior pole is the site where brium anlage will differentiate the smooth muscle of the cells first undergo changes of morphology by forming an manubrium (Fig. 5; dark green). How the thin threads of adhesive disk we assume that Brachyury expression is smooth muscle which run on top and perpendicular to the functionally connected to this process. The polyp stage striated muscle over the radial canals differentiate (Fig. 5; shows some endodermal expression at the tip of the hypo- light green) is not known, however, they also differentiate stome where the mouth is localized (Figs. 4A and 6). from the entocodon. The surface of the bud epidermis then Some variable and weak staining is observed in the

© 2002 Elsevier Science (USA). All rights reserved. The Origin of Mesoderm Specification 379 ectodermal and endodermal cells of the dedifferentiated mesoderm. Usually it is downregulated when mesoderm polyp cells, which form the early medusa bud stages (Fig. matures. In the mouse expression persists in the notochord 5A). In stage 2 medusa buds staining is also present in the and tail bud through gestation (Wilson et al., 1993). Expres- developing entocodon (Fig. 5B). The signal becomes stron- sion in notochord precursor cells but not in other cell types, ger and more localized when the 4 radial pockets of the including mesodermal precursors or adult tissues is also entocodon cavities develop (Figs. 5C-5E). It appears that observed in the ascidian Halocynthia roretzi (Yasuo and staining is strong before and shortly after the entocodon Satoh, 1993, 1994), however, in the related larvaceans the cells separate to form the two entocodon layers (cavity, Fig. gene is expressed not only in the notochord but also in parts 5D), the peripheral one which will develop into the striated of the developing endoderm and gut (Bassham and Postleth- muscle and the central one which will form the smooth wait, 2000). Downstream target genes of Brachyury in the muscle of the manubrium. From stages 4 and 5 on, expres- ascidian Ciona appear to effect motorproteins of the cy- sion is mostly confined to the 4 stripes over the 4 radial toskeleton (Di Gregorio and Levine, 1999; Takahashi et al., canals where the differentiating striated muscle layer re- 1999) indicating that the gene could be involved in the mains in connection with the developing smooth muscle establishment of cell motility and the epithelial-to- layer of the manubrium. When the bell unfolds at stage 9, mesenchymal transition during gastrulation. staining ceases and the two layers separate. Some staining The expression pattern of Brachyury indicates that the is seen at the tip of the manubrium where the lips and the gene functions in comparable developmental contexts in manubrial mouth opening form and in the maturing oocyte Podocoryne and bilaterians. Whereas in most bilaterians (Fig. 5H). This manubrial staining persists even in the Brachyury is expressed in a timewise limited developmen- liberated medusa (Fig. 4B). Interestingly, formation of the tal period around gastrulation and early mesoderm specifi- velar opening and of the tentacles seem not to require cation, expression in Podocoryne is present in all life stages Brachyury expression. The tissue organization of the me- but notably strong in planula and medusa development, dusa, especially that of the bell, is extremely fragile and two stages which can be separated by months. In echino- difficult to fix for histology and in situ investigations. derms Brachyury expression can also be biphasic, first Therefore, the expression analysis for all three genes were when the archenteron forms and later when the larva done by RT-PCR on excised medusa fragments (Fig. 6). The prepares for metamorphosis (Peterson et al., 1999). In hy- data in general support the in situ data from the very late drozoans the two-layered diploblastic situation is first medusa bud stages (Figs. 4 and 5). The Brachyury expression formed as a consequence of gastrulation in larval develop- data of excised medusa parts demonstrate the presence of ment and the situation persist into the polyp stage. When message in all peripheral parts of the bell, circular canal and development finally reaches the medusa budding, tentacle bulbs, areas where cell proliferation remains active gastrulation-like processes, notably the formation of the and where the new cells and tissues for the growth of entocodon as a third, mesoderm-like layer will complete tentacles and the bell are generated (Fig. 6). Although development. In this context the expression pattern ectodermal staining is variable throughout all bud stages it of Brachyury supports the idea that gastrulation in decreases in general in later stages, except for stage 9 when Podocoryne is completed only with the development of the the acellular sheath protecting the bud is enzymatically medusa (Spring et al., 2000). disrupted, which probably leads to artifactual noncellular staining (Fig. 5H). In most metazoans the Brachyury gene is expressed in Dynamic Mef2 Expression in Muscle and posterior body parts (Papaioannou, 2001; Technau, 2001). In Nonmuscle Precursors Hydra, which belongs to the same order as Podocoryne, but lacks a free planula larva and medusa stage, the gene is Podocoryne Mef2 is the first member of this family to be expressed in the apical endoderm of the embryo and in the described in cnidarians. The message is present in all life polyp stage in the distal endoderm of the hypostome, the stages (Fig. 6). It is found in the unfertilized egg and in all mouth bearing part of the polyp (Technau and Bode, 1999). cells up to the blastula and gastrula stage without any In Drosophila the message of the Brachyury homologue specific localization (Figs. 3G-I). In the young larva staining brachyenteron is observed in the hindgut area. In asteroids intensifies in the endoderm (Fig. 3J). In the old larvae Mef2 (echinoderms, starfish) the gene is expressed in the late expression is localized in the most anterior part of the blastula stage, during gastrulation at the blastopore ingres- ectoderm, the site where the larva makes first contact with sion area and little later where the stomodeum forms the substrate (Figs. 3K, 3L, and Fig. 6). Whereas the embry- (Shoguchi et al., 1999). Interestingly, in echinoids (echino- onic message distribution could be associated with the derms, sea urchin) Brachyury is expressed again in a second early differentiation of the ectodermal and endodermal phase, in the so-called “set-aside” cells before the develop- myoepithelia the endodermal staining in the younger larva ment of the pentameral organization occurs prior metamor- cannot be interpreted in this way. At this time all the larval phosis (Harada et al., 1995; Peterson et al., 1999). From cell types have already differentiated (Gro¨ ger and Schmid, amphioxus to mouse the Brachyury message is found in 2001) and cell cycle activity has strongly decreased. In the blastopore cells destined to contribute to endoderm and polyp epidermal staining is strong at the mouth region and

strong in the subumbrellar plate cells, and in the developing striated muscle layer (Figs. 5M and N). The RT-PCR data of the excised medusa parts resembles that of Brachyury (Fig. 6). However, staining is not in the endoderm but in the ectodermal gonads (Fig. 4). Compared with bilaterians, Mef2 expression in Podocoryne appears to be conserved in the differentiation of the striated muscle (swimming muscle) and that of the velum. Additionally and unexpect- edly, however, it also appears necessary for development of the two nonmyoepithelial tissue of the medusa bell, the exumbrella and the subumbrellar plate. Interestingly, the Mef2 expression pattern is almost congruent with that of the mesoderm specification factor Twist (Spring et al., 2000), an indication that the two genes could be functionally linked as they are in Drosophila (Baylies and Bate, 1996) and mouse (Spicer et al., 1996). Although the Mef2 genes in bilaterians were initially identified as muscle-specific transcription factors they are also expressed at high levels in neurons, as well as B and T cells and fibroblasts where they transmit signals from the cell membrane to immediate early genes and stress response genes (Black and Olson, 1998). Promoter analysis of Mef2 has placed the gene downstream of other transcription factors important for regulating Drosophila myogenesis. It appears that Mef2 is a direct target of activation by the bHLH factor Twist that is essential for mesoderm formation and myogenesis in Drosophila (Baylies and Bate, 1996; FIG. 6. Expression analysis of Brachyury, Mef2, and Snail mes- Cripps et al., 1998) and mouse (Spicer et al., 1996). Inter- sage in the life cycle of Podocoryne and in medusa and polyp body estingly, in C. elegans Mef2 activity is not essential for parts. Samples are normalized with elongation factor 1 alpha. b. myogenesis and development (Dichoso et al., 2000). column, body column; inter. segment, interradial segment of the medusa. Snail Expression in Undifferentiated Proliferating Cells The sequence of Podocoryne Snail identifies this gene as weak in the tentacles and in the rest of the body parts (Figs. the first cnidarian member of the Snail subgroup of this 4C and 6). superfamily of zinc finger transcription factors. The mes- Throughout all medusa bud stages staining is variable but sage is maternal and present in all life stages (Fig. 6). It is mostly stronger in the distal ectoderm where in later stages localized in the central cytoplasm of the egg (germinal the velum and tentacles are formed (Figs. 5I–M). The vesicle in Figs. 3M and N). In the blastula stage, staining endoderm of the very early stages up to stage 4 stains appears stronger in the basal parts of the cells (Fig. 3O). distinctly (Figs. 5I and K). This is also true for the entocodon After gastrulation the endoderm stains in the 1-day-old at stage 3 and later (Figs. 5K and L), whereas the very young larva (Fig. 3P). However, in the 4-day-old larva this pattern entocodon of stage 2 stains very little (Fig. 5J). In cross has almost completely disappeared (Fig. 3Q).The distinct sections of older stages (Fig. 5N) it is clear that staining is anterior pole staining observed in the older planula larva

FIG. 5. Expression analysis by in situ hybridization of medusa development of Podocoryne. Medusa buds were staged after Frey (1969). Arrow in S points to developing plate tissue. D is a thick overstained section demonstrating Brachyury expression. be, medusa bud ectoderm; ben, medusa bud endoderm; e, entocodon tissue; ec, entocodon cavity; ema, endoderm of the manubrium; ex, exumbrella; go, gonads; m, mesoglea (ECM) separating entocodon tissues from endoderm; ma, manubrium; maa, manubrium anlage; o, oocytes; p, plate cells of the subumbrella; pe, polyp ectoderm; pen, polyp endoderm; rc, radial canal; sm, smooth muscle of the manubrium; sr, smooth muscle over the radial canal; st, striated muscle; t, tentacle; tb, tentacle bulb; v, developing velum. The entocodon is schematized in brown, derivatives of the entocodon, which differentiate into striated muscle in red, smooth muscle of the manubrium in dark green and smooth muscle of the ring canal in light green. The bar corresponds to 13 ␮m in stages 0 and 1, 30 ␮min2,60␮min3–4, 90 ␮m in 5 and 6, and 150 ␮m in stages 8 and 9.

© 2002 Elsevier Science (USA). All rights reserved. 382 Spring et al. with Brachyury and Mef2 was never seen with Snail probes. same genes and developmental patterns to differentiate The fact that these three genes as well as Hox/ParaHox striated and smooth muscle systems. If this were not the (Yanze et al., 2001), Twist (Spring et al., 2000), and Pax case, we would have to argue that similar regulatory and messages (Gro¨ ger et al., 2000) are maternal could be ex- structural genes were recruited independently in cnidarian plained by the rapid cleavage rate if one assumes that this and bilaterian evolution with similar results. If we assume leaves little time for transciptional activity. As is true for that the common ancestor of cnidarians and bilaterians was other genes, Snail is expressed in the ectoderm of the polyp a complex animal with mesoderm-derived striated muscle, hypostome, additionally, however, the signal is also found this ancestor would more closely resemble a medusa than in the tentacle endoderm (Fig. 4E). the polyp of cnidarians. This is in agreement with the idea In the medusa budding process, both, the dedifferentiated that the polyp was added later into the life cycle, a theory endodermal and ectodermal cells of the polyp stain at stage already discussed repeatedly by many life cycle specialists 1, however, staining is always stronger in the endoderm (Brooks, 1886; Woltereck, 1905; Hyman, 1940; Hand, 1959; (Fig. 5P). Whereas some variable ectodermal staining is Boero and Bouillon, 1987; Brusca and Brusca, 1990; Bouil- observed throughout all bud stages, the endodermal stain- lon, 1994). Within the phylum Cnidaria this seems to be a ing decreases in the older bud stages (Figs. 5Q–U). The most problem, since the Anthozoa, a family without medusa prominent staining is confined to the entocodon, from the stage, appear to be basal in relation to the other three first appearance of entocodon cells (Fig. 5Q) to the forma- classes with medusa stages, the Hydrozoa, Scyphozoa, and tion of the striated and smooth muscle layers (Figs. 5R–U). Cubozoa (Bridge et al., 1995). A single event, however, the When cell cycle activity in the presumptive striated muscle loss of the medusa stage in Anthozoa, could explain this layer has ceased in the older bud stages staining disappears difference (Schuchert, 1993). A medusoid ancestor is also and in the adult medusa is only present in the gonads of the more likely when ctenophores are included into the discus- manubrium (Fig. 5V). Besides the cells of the presumptive sion. The phylum Ctenophora appears even basal to the muscle tissues, cells of the developing subumbrellar plate Anthozoa in the study that argues for the ancestry of the stain (Figs. 5S–U). The strong staining in the endoderm of polyp (Bridge et al., 1995), but ctenophores lack a polyp the manubrium and tentacle bulbs of the medusa (Fig. 4F) stage, are more similar to medusae and have muscle cells appears to be an artifact. All Snail expressing cells belong to derived from mesodermal precursor cells (Martindale and mitotically and morphogenetically active tissues and with Henry, 1999). The fact that the striated muscle system the exception of the gonads, contribute either to the striated appears to become ectodermal in medusae when the velar and smooth muscle of the bell or to the nonmuscle layers of opening develops should not argue against the mesoderm- the bell, the exumbrella and plate cells. These dual effects, like origin. In some reduced, sessile, non feeding medusoids promotion and inhibition of myogenesis were already ob- (eumedusoids; Tardent, 1978; Bouillon, 1994), the striated served with Twist (Spring et al., 2000). muscle is used to force the gametes through gonopores, a Genes from the Snail family are crucial for regulating situation that is roughly comparable to the somatopleura- development of the mesoderm and the nervous system in coelom-gonoduct situation in some bilaterians and could both arthropods and chordates (Hemavathy et al., 2000; possibly mirror an ancestral situation (Boero et al., 1998). Manzanares et al., 2001). Most likely they mediate the Therefore, we prefer to think that the last common ancestor epithelial-to-mesenchymal transition (Nieto et al., 1994), a of Cnidaria, Ctenophora, and Bilateria was not a primitive process that allows an epithelial cell to separate from its bilayered planuloid-type of larva (Holland, 2000) but more neighbors and migrate to populate different regions within likely a mesodermate-like metazoan, and more differenti- the embryo. All the examples reported demonstrate that ated in cell type architecture and body organization than is binding of Snail proteins to their target promoters leads to usually envisaged. repression of gene activity and that the gene is mostly active before muscle differentiation occurs. In the jellyfish this is also true, however, activity clearly stretches further ACKNOWLEDGMENTS into muscle development (Figs. 5U and V). We are much indebted to Drs. G. Freeman and T. Holstein who helped to improve this work. This study was supported by the CONCLUSION Treubel-Fonds (J. S.) and the Swiss National Science Foundation. Furthermore, the work was made possible by the greatly appreci- ated support of the administration and staff of the Station Bi- We have used the mesoderm and myogenic cell line ologique de Roscoff (UPMC-CNRS/SDV-INSU) in France. specifying genes Brachyury, Mef2, and Snail to substantiate the hypothesis that the entocodon, the third cell layer formed during medusa bud development, is homologous to REFERENCES the mesoderm of bilaterians. The expression patterns of all these three genes as well as those of Twist (Spring et al., Aerne, B. L., Baader, C. D., and Schmid, V. (1995). Life stage and 2000) and structural genes support the hypothesis that the tissue-specific expression of the homeobox gene cnox1-Pc of the common ancestor of cnidarians and bilaterians used the hydrozoan Podocoryne carnea. Dev. Biol. 169, 547–556.

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