The expression of a hunchback ortholog in the polychaete annelid Platynereis dumerilii suggests an ancestral role in mesoderm development and neurogenesis
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- Kerner, P., Zelada González, F., Le Gouar, M. et al. Dev Genes Evol (2006) 216: 821. doi:10.1007/s00427-006-0100-9
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Orthologs of the Drosophila gap gene hunchback have been isolated so far only in protostomes. Phylogenetic analysis of recently available genomic data allowed us to confirm that hunchback genes are widely found in protostomes (both lophotrochozoans and ecdysozoans). In contrast, no unequivocal hunchback gene can be found in the genomes of deuterostomes and non-bilaterians. We cloned hunchback in the marine polychaete annelid Platynereis dumerilii and analysed its expression during development. In this species, hunchback displays an expression pattern indicative of a role in mesoderm formation and neurogenesis, and similar to the expression found for hunchback genes in arthropods. These data suggest altogether that these functions are ancestral to protostomes.
KeywordsEvolutionAnnelidPlatynereis dumeriliihunchbackNeurogenesisMesoderm formation
The segmentation gene hunchback (hb) encodes a Cys2–His2 zinc finger transcription factor and plays a pivotal role in controlling pattern formation of the early Drosophila embryo (for review, see Rivera-Pomar and Jäckle 1996). hb belongs to the gap class of segmentation genes, which defines the first zygotic level in the Drosophila segmentation cascade. Gap genes respond directly to the instructive gradients of asymmetrically distributed maternal factors and, in turn, specify the expression domains of both pair-rule and HOX genes (for review, see Rivera-Pomar and Jäckle 1996). Drosophila embryos mutant for hb show a canonical gap phenotype, with deletions of the labial through third thoracic segments and the eighth abdominal segment. Besides its involvement in early development, Drosophila hb is also expressed in specific mesodermal cells and transiently in all neuroblasts of the nervous system (e.g. Patel et al. 2001 and references therein). Together with Krüppel, castor and pdm, hb plays a key role in determining the temporal identity of the successive ganglion mother cells and neurones produced by the neuroblasts (reviewed in Pearson and Doe 2004).
Orthologs of hb have been cloned in various insects (Liu and Kaufman 2004; Mito et al. 2005; Patel et al. 2001; Pultz et al. 2005; Wolff et al. 1995). Expression and functional studies in short or intermediate germ-band insects (in which only the most anterior body regions are specified at the blastoderm stage), such as the milkweed bug Oncopeltus fasciatus (Liu and Kaufman 2004), the grasshopper Schistocerca americana (Patel et al. 2001), the red flour beetle Tribolium castaneum (Wolff et al. 1995) and the cricket Gryllus bimaculatus (Mito et al. 2005), have pointed out two different, probably ancestral, types of function of hb in antero-posterior axis formation. In these species, hb is first expressed in a gap-like domain in the blastoderm embryo and controls the formation and/or the specification of anterior (gnathal and thoracic) segments, in part through regulation of Hox genes. Later in development, hb is expressed in the posterior growth zone and is required for the formation of the segments produced by this growth zone. hb is also expressed in the central nervous system of all these insects, in a way reminiscent to that found in Drosophila, suggesting a conserved function in neural patterning across insects. In addition, in Oncopeltus, Tribolium and Schistocerca, hb seems to have a broad expression in the developing mesoderm during germ-band elongation. Although not found in Drosophila, this may correspond to an additional widely conserved feature of hb genes in insects. hb orthologs have also been recently isolated in a crustacean, the branchiopod Artemia franciscana (Kontarakis et al. 2005), and in a myriapod, the geophilomorph centipede Strigamia maritima (Chipman and Stollewerk 2006). In both cases, hb is expressed in the forming mesoderm and nervous system but does not display an expression suggesting an involvement in axial patterning similar to that found in insects.
One hb ortholog (named hbl-1) has been isolated in the nematode Caenorhabditis elegans, and whereas this gene is not required for early patterning of the embryo, it is involved in the differentiation of the hypodermal cells and their derivatives (Fay et al. 1999). Furthermore, hbl-1 is also expressed in many neurones, as well as in the pharynx (Fay et al. 1999). In addition to arthropods and nematodes that belong to the ecdysozoans, hb orthologs have been isolated in annelids, the leech Helobdella (Iwasa et al. 2000; Savage and Shankland 1996), the polychaete Capitella (Werbrock et al. 2001) and the oligochaete Tubifex (Shimizu and Savage 2002). In these species, hb is expressed in a subset of neurones of the ventral nerve cord, as well as in a very broad domain comprising ectodermal and endodermal cells.
Here we isolated one hb ortholog in the polychaete annelid Platynereis dumerilii, a marine lophotrochozoan representative. Platynereis is considered to display bilaterian ancestral features at the morphological, developmental and genomic levels, making it a useful model for comparative developmental biology (reviewed in Tessmar-Raible and Arendt 2003). The expression pattern of the Platynereis hb ortholog suggests an involvement in neurogenesis and mesoderm formation, similar to that of its orthologs in arthropods. These functions may therefore represent the ancestral roles of hb genes in protostomes. In addition, extensive database searches indicate that bona fide hb genes, i.e. genes with significant sequence similarity to insect hb, can be found in various protostomes, but neither in deuterostomes nor in non-bilaterians.
Materials and methods
Animals and embryos were bred according to the protocol of Fisher and Dorresteijn (http://www.platynereis.de).
Cloning, sequencing, multiple alignments and phylogenetic analysis
A short fragment of Platynereis hb had previously been isolated in a large polymerase chain reaction (PCR) screen for conserved zinc fingers encoding genes (Sommer et al. 1992). The 3′ and 5′ ends of the gene were amplified from cDNA libraries (24 and 48 h post-fecundation embryos) using vector- and gene-specific (non-degenerate) primers (sequences and detailed protocol are available upon request). PCR products were TA cloned into the PCR2.1 vector (Invitrogen) and sequenced on an ABI automated sequencer. Platynereis hunchback has been submitted to the EMBL database (accession number AM232683).
Sequences of cloned hb homologues were gathered by blast searches (Altschul et al. 1997) on the NCBI NR protein database. We also ran blast searches against the sequenced genomes from various metazoans, including the shotgun reads available in the NCBI Trace archive database. A multiple alignment of the retrieved sequences was built using ClustalW (Thompson et al. 1994), and only regions displaying an unequivocal alignment were selected for the phylogenetic analysis. The phylogenetic analysis of these sequences was performed with PHYML using the Whelan and Goldman (WAG) model of amino acid substitutions (Guindon and Gascuel 2003) and 100 bootstraps to assess the statistical reliability of the obtained internal branches.
Reverse transcriptase PCR and in situ hybridization
Reverse transcriptase (RT)-PCR was performed according to standard procedures with gene-specific primers. Sequences and a detailed protocol for the whole-mount RNA in situ hybridizations are available upon request.
Results and discussion
hb genes in metazoans
Several hb genes have been found in insects and annelids (Sommer et al. 1992), but no deuterostome ortholog has been reported to date. Nevertheless, based on a similar arrangement of zinc fingers, it has been suggested that the Ikaros/Helios family of proteins may represent the vertebrate ortholog of Hunchback proteins (discussed in Patel et al. 2001). The Ikaros/Helios proteins compared to the Hb proteins share a very weak sequence similarity; therefore, it is difficult to consider the Ikaros/Helios genes as true hb orthologs.
Isolation of an hb ortholog in Platynereis
We used a short hb fragment reported for the annelid Platynereis dumerilii (Sommer et al. 1992) to design specific primers that allowed us to isolate the full-length cDNA of Platynereis hb (Pdu-hb), by using vector-anchored rapid amplification of cDNA ends (RACE) PCR. This cDNA is 2,418 bp long and encodes a 726-amino-acid protein that contains 6 Cys2–His2 zinc fingers and which displays high sequence similarity with other Hb proteins (Fig. 1). Phylogenetic analysis indicates that Platynereis Hb is most similar to the Helobdella and Lottia genes (Fig. 1). Pdu-Hb contains the four fingers (MF1-4) and the adjacent C box found in all Hb proteins (the nomenclature of the different conserved regions is following Patel et al. 2001), as well as the N-terminal fingers (NF1-2) which are not found in the Hb proteins from some insects, such as dipterans (Fig. 1). The Pdu-Hb protein sequence deduced from our PCR clone lacks the A box (which is only found in insects) and the two C-terminal fingers (CF1-2) found in all other Hb proteins. We tried to isolate these C-terminal fingers by classical RACE PCR or PCR on genomic DNA without success. Nevertheless, we cannot rule out the possible existence of an isoform of Pdu-Hb including these two additional highly conserved fingers.
Expression pattern of Pdu-hb during development
We first used RT-PCR to analyse the expression of Pdu-hb at different developmental stages (Fig. 2b). We found that Pdu-hb is expressed in all larval stages after 15 hpf and is still expressed in juvenile worms. Its expression fades in adults, but then it is upregulated at the time of spawning, particularly in males. Interestingly, since the transcript is not present in unfertilized eggs and in early embryos, we can exclude a maternal contribution of Pdu-hb in early development, in contrast to what is observed in insects. To study in more detail the spatio-temporal expression of Pdu-hb during larval development, we used whole-mount in situ hybridization, using RNA antisense probes.
Comparative analysis of Pdu-hb expression
Studies in various insects have pointed out three main evolutionary conserved functions of hb genes: axial patterning, somatic mesoderm development and neurogenesis (see ‘Introduction’). Recent studies have shown that the latter two functions are probably conserved in non-insect arthropods, such as crustaceans and myriapods (Chipman and Stollewerk 2006; Kontarakis et al. 2005). Our data on Platynereis suggest the presence of these two functions in a lophotrochozoan species, therefore suggesting that they may be ancestral to protostomes. Indeed, we found that Pdu-hb is expressed in early trochophores in the mesodermal bands and that this expression is maintained until late larval stages. The mesodermal bands will give rise to the somatic segmented mesoderm of the juvenile worm. We also detect an expression of Pdu-hb in mesodermal cells belonging to the posterior growth zone of the juvenile worms that correspond to the mesodermal stem cells. Pdu-hb is therefore expressed in the precursors of the somatic segmented mesoderm formed during both the larval and post-larval stages. This is a clear difference with the expression of hb in two other annelids, Helobdella and Capitella (Iwasa et al. 2000; Savage and Shankland 1996; Werbrock et al. 2001). As an expression in the developing mesoderm strikingly similar to that of Platynereis is observed in several arthropods, we suggest that this represents an ancestral feature of hb which has been lost in Helobdella and Capitella. However, we cannot rule out the possibility of a convergent evolution. Further study of hb genes in other lophotrochozoan species, such as molluscs, will help clarify this issue.
We also found an expression highly suggestive of a role of Pdu-hb in the formation of the nervous system: at about 40 hpf, Pdu-hb becomes expressed in a broad ventral ectodermal domain from which the ventral nerve cord will emerge. At this stage, very few neurones are already differentiated, as seen by immunocytochemical detection with antibodies against acetylated tubulin or neurotransmitters, such as FMRFamide and serotonin (unpublished observations). However, Platynereis orthologs of some genes known to be involved in Drosophila and vertebrates neurogenesis, such as elav and neurogenin, are broadly expressed in the ventral ectoderm, in a pattern similar to that of Pdu-hb (P.K. et al., unpublished observations). This strongly suggests that this region of the larva is already fated to produce neural cells and that Pdu-hb is expressed in these putative neural cells, like its arthropod orthologs. The hb gene in two other annelids, the leech Helobdella and the polychaete Capitella, is also expressed in the developing nervous system (Iwasa et al. 2000; Savage and Shankland 1996; Werbrock et al. 2001). An involvement of hb gene in neurogenesis is therefore conserved among protostomes.
The ancestral feature of the roles of hb genes in axial patterning is questionable. In insects, hb first acts as a ‘gap gene’ and controls the formation and/or the specification of the anterior segments. Later, hb is expressed in the posterior growth zone and is required for the formation of more posterior segments that are produced by this growth zone. The available expression patterns of hb in two non-insect arthropods (Artemia and Strigamia) and two annelids (Helobdella and Capitella) are inconsistent with both of these roles, as hb, in these species, is not expressed in a gap-like domain nor in the posterior growth zone. In Platynereis, whereas Pdu-hb is expressed in early larval stages (13–18 hpf), in a large domain of the prospective trunk region, this expression is not restricted to the anterior part of the trunk and therefore not suggestive of a role in the formation and/or the specification of the anterior segments. We also detect an expression of Pdu-hb in juvenile worms, which probably corresponds to the progenitors of the mesodermal part of the post-larval segments. A similar expression has been observed for other Platynereis genes, such as Pdu-nanos, Pdu-vasa, Pdu-PL10 and Pdu-piwi, indicating the existence of posterior stem cells at the origin of both the segmented somatic mesoderm and the germ line (N. Rebscher, F.Z.G. and D.A., submitted manuscript). However, no expression of Pdu-hb is observed in the ectodermal part of the growth zone, arguing against a general role of Pdu-hb in segment formation from the posterior growth zone. This is similar to what has been observed in Artemia, Strigamia, Capitella and Helobdella and would indicate that the involvement of hb genes in the posterior growth of short germ-band insects may be a derived feature of insects.
This work has been supported by the CNRS and the Ministère Français de la Recherche through its ACI ‘Jeunes chercheurs et jeunes chercheuses’ and ACI ‘Biologie et Physiologie du développement’ (to M.V.). F.Z.G. was financially supported during her Ph.D. thesis by the DFG (Deutsche Forschungsgemeinschaft). P.K. holds a ‘Bourse pour Docteur-Ingénieur’ from the CNRS. V.L. was supported by the Belgian Science Policy.