Characterization of taxonomically restricted genes in a phylum-restricted cell type
- Sabine Milde†1,
- Georg Hemmrich†1,
- Friederike Anton-Erxleben1,
- Konstantin Khalturin1,
- Jörg Wittlieb1 and
- Thomas CG Bosch1Email author
© Milde et al.; licensee BioMed Central Ltd. 2009
Received: 9 October 2008
Accepted: 22 January 2009
Published: 22 January 2009
Despite decades of research, the molecular mechanisms responsible for the evolution of morphological diversity remain poorly understood. While current models assume that species-specific morphologies are governed by differential use of conserved genetic regulatory circuits, it is debated whether non-conserved taxonomically restricted genes are also involved in making taxonomically relevant structures. The genomic resources available in Hydra, a member of the early branching animal phylum Cnidaria, provide a unique opportunity to study the molecular evolution of morphological novelties such as the nematocyte, a cell type characteristic of, and unique to, Cnidaria.
We have identified nematocyte-specific genes by suppression subtractive hybridization and find that a considerable portion has no homologues to any sequences in animals outside Hydra. By analyzing the transcripts of these taxonomically restricted genes and mining of the Hydra magnipapillata genome, we find unexpected complexity in gene structure and transcript processing. Transgenic Hydra expressing the green fluorescent protein reporter under control of one of the taxonomically restricted gene promoters recapitulate faithfully the described expression pattern, indicating that promoters of taxonomically restricted genes contain all elements essential for spatial and temporal control mechanisms. Surprisingly, phylogenetic footprinting of this promoter did not reveal any conserved cis-regulatory elements.
Our findings suggest that taxonomically restricted genes are involved in the evolution of morphological novelties such as the cnidarian nematocyte. The transcriptional regulatory network controlling taxonomically restricted gene expression may contain not yet characterized transcription factors or cis-regulatory elements.
Cnidaria represent the simplest animals at the tissue grade of organization. In order to catch prey, cnidarians have evolved a unique "high-tech cellular weaponry"  - the stinging cells (cnidocytes, nematocytes) - single cells able to shoot structures at their target and inject toxic substances into it. Nematocytes are unique to and present in all species of the phylum Cnidaria. Different phylogenetic lines have different nematocyte types [2, 3]. Evolution of cnidarian families appears to be accompanied by expansion of the nematocyte repertoire . In Hydra, four types of nematocytes can be distinguished based on the distinct morphology of the nematocyte capsule: stenotele, desmoneme, holotrichous isorhiza and atrichous isorhiza. Previous work [5, 6] has identified unusually short proteins with a collagen-related domain (minicollagens) as major constituents of the nematocyst capsule wall. Intermolecular disulfide bonds between the cysteine-rich domains of these minicollagens and an additional capsule protein, NOWA, are thought to stabilize the capsule wall . The spines inside the capsules contain spinalin, another protein unrelated to any protein in other animals .
How novel morphological structures evolve is an open and important question. One currently popular view is that since many genes are shared throughout the animal kingdom, animal diversity is largely based on differential use of conserved genes and regulatory circuits [9–11]. However, all genome and expressed sequence tag (EST) projects to date in every taxonomic group studied so far have uncovered a substantial amount of genes that are without known homologues [12, 13]. A previous study  has discovered that a family of such taxonomically restricted 'orphan' genes plays a significant role in controlling phenotypic features referred to as species-specific traits in the genus Hydra. Thus, morphological diversity in closely related species may be generated through changes in the spatial and temporal deployment of genes that are not highly conserved across long evolutionary distances .
We here have chosen an unbiased comparative approach based on suppression subtractive hybridization (SSH) to identify additional nematocyte-specific genes in Hydra. Among those detected, a considerable portion has no homologues in animals outside Hydra. Since they are exclusively restricted to the phylum Cnidaria, they are considered as 'orphans' or 'taxonomically restricted genes' (TRGs) [13–16].
Analysis of these TRGs indicates striking complexity in their genomic organization and transcript processing. In order to understand how such TRGs are regulated, we generated transgenic polyps that express green fluorescent protein (GFP) under control of one of the TRG promoters. Transgenic Hydra recapitulate faithfully the previously described expression pattern, indicating that the promoter contains all elements essential for spatial and temporal control mechanisms. Surprisingly, phylogenetic footprinting of this promoter did not reveal any conserved cis-regulatory elements. This may indicate that the transcriptional regulatory network controlling TRG expression may contain not yet characterized transcription factors or cis-regulatory elements.
Our data provide a detailed genomic description of several taxonomically restricted genes in a basal metazoan, and functional evidence that TRGs are integrated in transcriptional regulatory networks to form functional signaling cascades.
Identification of taxonomically restricted genes expressed in nematocytes
In order to isolate not yet identified genes potentially involved in nematocyte differentiation, we made use of the sf-1 mutant strain of H. magnipapillata, which has temperature-sensitive interstitial stem cells . Interstitial cells are located between the ectodermal epithelial cells and contain both germline and somatic components, giving rise to all nerve cells, gland cells and nematocytes . Treatment for a few hours at the restrictive temperature (28°C) induces quantitative loss of the entire interstitial cell lineage, including nematocytes from the ectodermal epithelium .
Characterization of taxonomically restricted genes expressed in nematocytes
A novel family of minicollagen proteins originates from one genomic locus
Analysis of nb001 transcripts in the EST data bank and the corresponding genomic locus uncovered five different splice variants (Figure 4a, nb001-sv1 to nb001-sv5: CL1Contig4, CL1Contig3, CL1Contig2, CL1Contig1 and CL1Contig5, respectively). In addition, by PCR amplification we could identify four more splice variants (nb001-sv6 to nb001-sv9; Figure 4a). Interestingly, while the first two introns are spliced by conventional splicing sites (GT/AG), additional variants of the transcripts are generated by processing of exon 3. As a result of this process, which may use unconventional 'splicing' sites, various regions of exon 3 are removed.
The resulting nb001 predicted proteins (Figure 4b) indicate domain length variations of the collagen-like domain as well as the proline and cysteine repeats. In contrast to previously reported minicollagens [5, 22], all nb001 variants described here have 19-27 Gly-X-Y repeats instead of 12-16, resulting in an expanded collagen-like domain (Figure 4b). Other nb001 variants are characterized by a shortened praline repeat following the collagen-like domain. Three variants (nb001-sv7 to nb001-sv9) lack both the collagen-like domain and the proline repeats. These variants contain only a single cystein rich domain with an altered cysteine pattern - (CXXX)7-CC, (CXXX)5-CC or (CXXX)2-CC - instead of the conserved (CXXX)4-CC. Northern blot analysis (Figure 4c) shows a strong signal at around 700 bp, indicating the presence of nb001 transcripts corresponding to most of the predicted variants.
Spinalin, a previously identified nematocyte-specific gene is a splice variant derived from a complex genetic locus
Gene duplication contributes to the complexity of nematocyte-specific gene families
Sharing 3' UTRs in some nematocyte specific genes indicates common regulation of different splice variants
How are taxonomically restricted genes regulated?
The 1 kb upstream region of nb001lacks any conserved transcription factor binding sites
How are genes that lack sequence similarity to known genes regulated? In an attempt to unravel the transcriptional regulatory network controlling expression of a TRG, we analyzed the nb001 5' flanking sequence. To identify the 5' regulatory sequence, we used the H. magnipapillata genome data deposited at NCBI. Since nb001 is expressed in a seemingly identical manner across species borders (Figure 3b), we reasoned that sequences important for control of nb001 expression were strongly conserved at the nucleotide level, since their potential for mutation is constrained by their function. As described previously , such evolutionarily conserved cis-regulatory elements can be identified by phylogenetic footprinting.
The 1 kb upstream region of nb001 is essential and sufficient for correct expression in vivo
One of the main challenges in evolutionary biology is to identify the molecular changes that underlie phenotypic differences that are of evolutionary significance . Our results suggest that taxonomically restricted genes are involved in the evolution of morphological novelties such as the cnidarian nematocyst.
The nematocyte, a cnidarian invention, expresses cnidarian-specific genes
The nematocyte is a cell type exclusively restricted to cnidarians and - from an evolutionary perspective - is considered a neuronal sensory cell [30–32]. During evolution, these neuron-like cells obviously became highly diverged and acquired new cytological features such as the nematocysts (capsules). Each nematocyst consists of an inner and outer capsule wall, an inverted tubule armed with long arrays of spines, and an operculum (for a recent review, see ). Development of this cnidarian-specific structure requires complex genetic machinery, consisting of at least two sets of proteins, regulatory transcription factors and structural proteins. One of the few transcription factors identified up to now as being involved in nematocyte differentiation, Hyzic, is a homolog of the Zn-finger transcription factor gene zic/odd-paired. Hyzic is expressed in the early nematocyte differentiation pathway  and may act before, and possibly directly upstream of, Cnash, a homolog of the proneural basic helix-loop helix transcription factor gene achaete-scute.
In contrast to these conserved transcription factors, the downstream structural proteins responsible for putting the nematocysts into shape appear to belong to the group of taxonomically restricted genes. Some of them, such as some minicollagens, spinalin and NOWA, have been reported previously [5, 8, 33]. Interestingly, in addition to nematocysts, novel proteins appear also to be essential components of other structures of the nematocyte, such as the cnidocil, a cnidarian-specific mechanosensory ciliary structure acting as a 'trigger' for discharge of the nematocyst capsule. The central core of the cnidocil contains a protein, nematocilin, that lacks homologues outside Hydra . Two paralogous sequences of nematocilin are present in the Hydra genome and appear to be the result of recent gene duplication. Nematocilin is absent in the anthozoan Nematostella vectensis; it seems, therefore, to be a gene restricted to the class Hydrozoa.
Nematocysts arguably are one of the most complex secretory products produced by an animal cell . How the different nematocyst morphologies evolved is unknown. David and co-workers  have proposed that a diverse set of minicollagen proteins together with a disulfide-linked network of not yet identified fiber-like structures could have been instrumental in the evolution of the different nematocyst morphologies. Our discovery of striking complexity of nematocyte-specific genes at both the genomic and transcriptomic levels may indicate that bundles of protein variants produced by alternative splicing (Figures 4 and 5) and transcription at multiple loci (Figures 6 and 7) contribute to the conformational and structural flexibility of the nematocyst.
Alternative splicing has been proposed as the primary driver of the evolution of phenotypic complexity in mammals [36–38]. While alternative splicing is known to affect more than half of all human genes , it has been unclear whether and to what extent a similar mechanism operates in early branching metazoans. Our finding of numerous splice variants in Hydra, therefore, was surprising and points to a strong conservation of splicing regulation throughout animal evolution.
Taken together, as described here and consistent with previous studies [5, 8, 33], the majority of genes encoding nematocyst components have no homologues in higher metazoans and are unique to the cnidarian lineage.
Transgenic Hydracontribute to understanding regulatory evolution and transcriptional control of TRGs
The finding that the differentiation of a taxon-specific cell type, the nematocyte, involves the expression of taxon-specific genes promises to unveil novel aspects of the evolution of this complex cell type in particular and of species-specific traits in general. The work also raises an important question: how do these novel genes interact with upstream transcriptional regulators? Do they contain binding sites for conserved transcription factors? Or do they require novel transcription factors? We have previously hypothesized  that taxon-specific genes in combination with the rewiring of the genetic networks of conserved regulatory genes accomplish specification of cnidarian morphologies. Here, in order to address this question experimentally, we took advantage of the recent development of transgenic techniques by embryo-microinjection , which offers a rich opportunity to expand research activities in Hydra [13, 39–41]. As expected, transgenic Hydra appear to yield usable insight into the regulatory network controlling expression of genes that lack sequence similarity to known genes. According to the functional analysis of the nb001 promoter (Figure 10), the transcriptional machinery regulating TRG expression may involve not yet identified transcription factors. Alternatively, regulatory elements for conserved transcription factors may be highly diverged in promoters of TRGs and, therefore, not detectable in the present approach. Current efforts are directed towards identification of transcription factors causally involved in control of TRG expression.
Taken together, although certainly much remains to be discovered about the role of TRGs in Hydra, the observations presented here reaffirm the view [12, 13] that taxon-specific genes account for a substantial part of the Hydra genome and may be of profound evolutionary significance both in animals that reach back to the beginnings of metazoan life as well as in more complex organisms.
Materials and methods
Animals and culture conditions
Experiments were carried out with H. vulgaris strain AEP, H. magnipapillata strain 105, and H. magnipapillata strain sf1. Transgenic animals were generated using H. vulgaris strain AEP . Animals were cultured according to standard procedures at 18°C.
Supression subtractive hybridization and cDNA library construction
For SSH, double-stranded cDNA was synthesized using 2 μg of mRNA from the temperature sensitive mutant H. magnipapillata sf1. SSH was performed using PCR-Select™ cDNA Subtraction kit (Clontech, Mountain View, CA, USA) according to the manufacturer's protocol. Two RNA pools were used for subtractive hybridization (Figure 1). Tester double-stranded cDNA was synthesized from mRNA isolated from heat shocked animals free of i-cells and their derivatives. Driver double-stranded cDNA was synthesized from mRNA from untreated polyps containing all cell types. cDNAs were cloned into pGEM-T vector (Promega, Madison, WI, USA) and transformed into DH5 α Escherichia coli cells. Bacterial clones were picked into 384 well plates using Q-Pix roboter and plasmid inserts were sequenced at the Washington University Genome Sequencing Centre (St Louis, MO, USA). Raw sequences were submitted to NCBI dbEST database ([GenBank:CO371734-CO372031], [GenBank:CO373914-CO377781], [GenBank:CO508771-CO510748]).
Gene expression analysis
To analyze gene expression, whole mount in situ hybridization was carried out as described previously . Whole mount double in situ hybridization was performed using DIG- and Biotin-labeled RNA probes simultaneously. Antibody incubation and substrate reactions were carried out consecutively as described previously . NBT/BCIP- and Fast Red substrates were used for probe detection according to the manufacturer's instructions (Roche, Nutley, NJ, USA). Riboprobes were prepared with the Dig- and Biotin- RNA labeling kit according to the manufacturer's instructions (Roche).
RNA-electrophoresis, transfer, probe-labeling, hybridization and detection procedures were carried out according to standard protocols. For primer sequences used for probe amplification, see Additional data file 1.
Access to primer and sequence data
For primer sequences used to amplify full-length sequences and splice variants, see Additional data file 1. For retrieval of sequence data and EST contigs, see Additional data file 2.
Generation of transgenic H. vulgaris AEP expressing nb001:eGFP
Transgenic founder polyps expressing eGFP under control of the nb001 promoter were produced at the University of Kiel Transgenic Hydra Facility . The transgenic construct was made by placing the 1,035 bp nb001 promoter (-1,075 to +65 relative to the transcription initiation site and including the signal peptide of nb001) in front of the reporter gene for eGFP (Figure 10a). The resulting plasmid ligAB was injected into Hydra embryos as described . Out of 64 injected embryos, 21 (32%) hatched, from which two lines contained eGFP-positive nematocytes and no eGFP expression in any other cell type. Initial founder transgenic animals were expanded into a mass culture by clonal propagation by budding.
Fluorescent images were taken on a Zeiss Axioscope fluorescence microscope with an Axiocam (Zeiss) digital camera. Confocal laser microscopy was done using a LEICA TCS SP1 CLS microscope. A Zeiss S420 microscope was used for scanning electron microscopy.
Additional data files
The following additional data are available with the online version of this paper. Additional data file 1 is a table listing all primer sequences used to amplify full length sequences and splice variants of the described Hydra TRGs. Additional data file 2 is a table showing all GenBank accession numbers of full-length sequences and splice variants and sequence IDs for retrieval of EST contig sequences at .
expressed sequence tag
green fluorescent protein
National Centre for Biotechnology Information
suppression subtractive hybridization
taxonomically restricted gene
We thank three anonymous referees for their helpful and constructive comments on the manuscript. We thank the members of the Bosch laboratory for discussion and Antje Thomas and Meike Friedrichsen for excellent technical help, and Jan Lohman, Ingrid Lohman and Sebastian Fraune for valuable comments on a previous version of the manuscript. We are grateful to Holger Zill and René Augustin for assistance with the SSH libraries. Supported in part by grants from the Deutsche Forschungsgemeinschaft, and grants from the DFG Cluster of Excellence programs "The Future Ocean" and "Inflammation at Interfaces" (to TCGB).
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