Evolution and expression of BMP genes in flies
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Bone morphogenetic proteins (BMPs) play key roles in development. In Drosophila melanogaster, there are three BMP-encoding genes: decapentaplegic (dpp), glass bottom boat (gbb) and screw (scw). dpp and gbb are found in all groups of insects. In contrast, the origin of scw via duplication of an ancestral gbb homologue is more recent, with new evidence placing it within the Diptera. Recent studies show that scw appeared basal to the Schizophora, since scw orthologues exist in aschizan cyclorrhaphan flies. In order to further localise the origin of scw, we have utilised new genomic resources for the nematoceran moth midge Clogmia albipunctata (Psychodidae). We identified the BMP subclass members dpp and gbb from an early embryonic transcriptome and show that their expression patterns in the blastoderm differ considerably from those seen in cyclorrhaphan flies. Further searches of the genome of C. albipunctata were unable to identify a scw-like gbb duplicate, but confirm the presence of dpp and gbb. Our phylogenetic analysis shows these to be clear orthologues of dpp and gbb from other non-cyclorrhaphan insects, with C. albipunctata gbb branching ancestrally to the cyclorrhaphan gbb/scw split. Furthermore, our analysis suggests that scw is absent from all Nematocera, including the Bibionomorpha. We conclude that the gbb/scw duplication occurred between the separation of the lineage leading to Brachycera and the origin of cyclorrhaphan flies 200–150 Ma ago.
KeywordsBone morphogenetic proteins (BMPs) Phylogenetic analysis Gene duplication Diptera Clogmia albipunctata
Signalling molecules belonging to the transforming growth factor β (TGFβ) group perform key roles in morphological and physiological processes in all metazoan phyla. They have been variously referred to as a superfamily or family depending on the author (Newfeld et al. 1999; Van der Zee et al. 2008). Here, we follow a commonly accepted evolutionary definition of family as a set of genes derived from a single gene present in the common ancestor of the bilaterians. Under this classification, we consider the TGFβ grouping as a class formed of two subclasses: bone morphogenetic proteins (BMPs) and the activin/TGFβ subclass (Newfeld et al. 1999). In Drosophila melanogaster, there are two known families of BMP-encoding genes: decapentaplegic (dpp)—a member of the vertebrate BMP2/4 family—as well as glass bottom boat (gbb) and screw (scw)—members of the BMP5/6/7/8 family (Van der Zee et al. 2008). Dpp plays multiple roles in Drosophila development. One of them is its key role in dorso-ventral (DV) patterning during early embryogenesis (Irish and Gelbart 1987). Scw co-operates with Dpp in this process (Arora et al. 1994). Gbb has several roles at later embryonic stages, as well as in larval and pupal development (Doctor et al. 1992; reviewed in O'Connor et al. 2006). It is weakly expressed at early stages and is not involved in DV patterning. It has been proposed that this temporal distinction might separate otherwise functionally redundant proteins (Fritsch et al. 2010).
Two recent studies have taken the first steps towards this aim. Aschizan cyclorrhaphan species, such as the hoverfly Episyrphus balteatus (Syrphidae), and the scuttle fly Megaselia abdita, have orthologs of dpp, gbb and scw, which show expression patterns similar to the ones in D. melanogaster (Lemke et al. 2011; Rafiqi et al. 2012). This allows us to place the duplication event giving rise to scw at the base of the cyclorrhaphan lineage.
We wanted to further refine the time point of the gbb/scw duplication. No lineages outside the Cyclorrhapha have been shown to have a gbb duplicate identifiable as an orthologue of scw. Here, we describe the BMP gene complement of the moth midge Clogmia albipunctata (Psychodidae). Despite some recent controversy over the placement of Psychodidae (Wiegmann et al. 2011), they are likely to be a sister group of Neodiptera (Brachycera plus Bibionomorpha; Yeates and Wiegmann 1999; Jimenez-Guri et al. 2013). We have found one dpp and one gbb orthologue in C. albipunctata. Using phylogenetic analysis, we have been able to group C. albipunctata dpp and gbb with their orthologues in other lineages. We find that C. albipunctata dpp is a clear member of the dpp gene family, and C. albipunctata gbb branches ancestrally to the cyclorrhaphan gbb/scw split.
Materials and methods
Gene identification and cloning
We searched the early embryonic transcriptome of C. albipunctata (Jimenez-Guri et al. 2013; http://diptex.crg.es) by BLAST using dpp, gbb and scw sequences from D. melanogaster, M. abdita and E. balteatus (retrieved from GenBank). In addition, we searched a preliminary assembly of the C. albipunctata genome (our unpublished data) and the genome of the Hessian fly Mayetiola destructor (Cedidomyiidae, Bibionomorpha; see Fig. 1; genome version 1.0, Baylor College of Medicine Human Genome Sequencing Center: http://www.hgsc.bcm.tmc.edu/content/hessian-fly-genome-project) with these same sequences. PCR primers for C. albipunctata dpp and gbb were designed from transcriptome sequences (dpp-forward CAGTAGAAGGCGTCATAACC, dpp-reverse ACGGAAAAAGAGAGTGAAAAG; gbb-forward ATCTTTATGGCAAAAGGTCTG, gbb-reverse TTTTCGAGACAAAAGAAGAAC). Amplified sequences for C. albipunctata dpp and gbb have been deposited in GenBank (accession numbers KC810051 and KC810052). Fragments were cloned into the PCRII-TOPO vector (Invitrogen) and used to make DIG-labelled riboprobes for in situ hybridisation.
Whole mount in situ hybridisation
Wild-type C. albipunctata embryos were collected at blastoderm and post-gastrulation stages as described in García-Solache et al. (2010). Embryos were heat fixed using a protocol adapted from Rafiqi et al. (2011). In situ hybridisation was performed as described in Jimenez-Guri et al. (2013) and references therein.
Protein sequences of D. melanogaster Dpp, Gbb and Scw were used to identify and collect homologous sequences using the BLAST algorithm available at NCBI (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The sequences were initially aligned using T-Coffee (Notredame et al. 2000). All subsequent steps (editing, re-alignment, substitution model prediction, phylogenetic analysis and tree visualisation) were carried out using the MEGA5 software (Tamura et al. 2011). Phylogenetic analyses were carried out using a maximum likelihood method and the JTT substitution model and MrBayes allowing model jumping (Huelsenbeck and Ronquist 2001; Ronquist and Huelsenbeck 2003). Online Resource 1 contains the amino acid alignment used for phylogenetic analysis in FASTA format.
Results and discussion
BMP subclass members in Clogmia include dpp and gbb, but not scw
The evidence described in the ‘Introduction’ section is consistent with the gbb/scw duplication occurring at the base of the Cyclorrhapha. However, it may have happened even earlier, at any time between the divergence of the culicomorph lineage and the emergence of the Cyclorrhapha (Fig. 1). In this study, we narrow the gap between mosquitoes and cyclorrhaphan flies by describing the BMP complement of the moth midge C. albipunctata (Psychodidae), a dipteran species whose lineage branches basally to the Brachycera, and thus Cyclorrhapha (Jimenez-Guri et al. 2013).
We obtained putative BMP members from the transcriptome and genome of C. albipunctata and identified their relationships by reciprocal BLAST searches. We identified single putative orthologues of dpp and gbb/scw from an early embryonic transcriptome (Jimenez-Guri et al. 2013; Diptex database: http://diptex.crg.es, accession numbers comp946 and comp6316). Next, we searched a preliminary genome assembly for C. albipunctata (coverage >70×, scaffold N50 ∼242 kb; our unpublished data). We identified the same single copies of the putative dpp and gbb/scw orthologues. No additional gbb duplicate is present in the genome. At this point, we cannot rule out the possibility that C. albipunctata has lost a scw-like duplicate secondarily.
Phylogenetic reconstruction of BMP subclass members in flies
Expression patterns of dpp and gbb in C. albipunctata
In the early blastoderm, C. albipunctata gbb is detected in a very broad central domain, which only excludes two small terminal regions at the poles of the embryo (Fig. 3(c, c′)). This pattern also differs from the expression seen in D. melanogaster, where gbb is not detected before gastrulation (Doctor et al. 1992), or M. abdita, where expression is detected only in the dorsal blastoderm, although it is also excluded from the pole regions (Rafiqi et al. 2012). Similar to the expression in M. abdita and D. melanogaster, we detect C. albipunctata gbb transcript at high levels in a complex, segmentally repeated pattern after gastrulation (Fig. 3(d, d′)).
In conclusion, we identified only two members of the BMP subclass of genes in the psychodid moth midge C. albipunctata: one orthologue of dpp and one gene related to gbb. Their expression patterns at blastoderm stage differ significantly from those seen in Cyclorrapha, while expression during germ band extension is equivalent in both groups. No scw-like gbb duplicate could be identified from the early embryonic transcriptome, or the genome of C. albipunctata, and no such duplicate is present in the genome of the Hessian fly (Bibionomorpha) either. Therefore, our phylogenetic analysis suggests that the gbb/scw duplication occurred within the Brachycera. Further research in non-cyclorrhaphan Brachycera (Empidoidea, Asiloidea, Stratiomyomorpha and Tabanomorpha; Wiegmann et al. 2011; see also Fig. 1) will be required to localise the precise origin of scw within the basal branches of the Brachycera.
This research was funded by the MEC/EMBL agreement for the EMBL/CRG Research Unit in Systems Biology, by AGAUR SGR grant 406 and by Grants BFU2009-10184 and BFU2009-09168 from the Spanish Ministry of Science and Innovation (MICINN). EJG is supported by ERASysBio+ Grant P#161 (MODHEART). AAC acknowledges the contribution of an internship by the Caixa Catalunya savings bank, which first brought her into contact with the Jaeger lab. Genome and transcriptome sequences used in this study were acquired, assembled and annotated in collaboration with the Genomics and Bioinformatics Core Facilities at the CRG. We thank Brenda Gavilán for the help with maintaining fly cultures.
Conflict of interest
The authors declare that they have no conflict of interest.
- Rafiqi AM, Lemke S, Schmidt-Ott U (2011) Megaselia abdita: fixing and devitellinizing embryos. Cold Spring Harb Protoc 2011(4):pdb.prot5602. doi: 10.1101/pdb.prot5602
- Wiegmann BM, Trautwein MD, Winkler IS, Barr NB, Kim JW, Lambkin C, Bertone MA, Cassel BK, Bayless KM, Heimberg AM, Wheeler BM, Peterson KJ, Pape T, Sinclair BJ, Skevington JH, Blagoderov V, Caravas J, Kutty SN, Schmidt-Ott U, Kampmeier GE, Thompson FC, Grimaldi DA, Beckenbach AT, Courtney GW, Friedrich M, Meier R, Yeates DK (2011) Episodic radiations in the fly tree of life. Proc Natl Acad Sci U S A 108(14):5690–5695. doi: 10.1073/pnas.1012675108, Epub 2011 Mar 14PubMedCrossRefGoogle Scholar
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