BMP signaling components in embryonic transcriptomes of the hover fly Episyrphus balteatus (Syrphidae)
© Lemke et al; licensee BioMed Central Ltd. 2011
Received: 28 January 2011
Accepted: 31 May 2011
Published: 31 May 2011
In animals, signaling of Bone Morphogenetic Proteins (BMPs) is essential for dorsoventral (DV) patterning of the embryo, but how BMP signaling evolved with changes in embryonic DV differentiation is largely unclear. Based on the extensive knowledge of BMP signaling in Drosophila melanogaster, the morphological diversity of extraembryonic tissues in different fly species provides a comparative system to address this question. The closest relatives of D. melanogaster with clearly distinct DV differentiation are hover flies (Diptera: Syrphidae). The syrphid Episyrphus balteatus is a commercial bio-agent against aphids and has been established as a model organism for developmental studies and chemical ecology. The dorsal blastoderm of E. balteatus gives rise to two extraembryonic tissues (serosa and amnion), whereas in D. melanogaster, the dorsal blastoderm differentiates into a single extraembryonic epithelium (amnioserosa). Recent studies indicate that several BMP signaling components of D. melanogaster, including the BMP ligand Screw (Scw) and other extracellular regulators, evolved in the dipteran lineage through gene duplication and functional divergence. These findings raise the question of whether the complement of BMP signaling components changed with the origin of the amnioserosa.
To search for BMP signaling components in E. balteatus, we generated and analyzed transcriptomes of freshly laid eggs (0-30 minutes) and late blastoderm to early germband extension stages (3-6 hours) using Roche/454 sequencing. We identified putative E. balteatus orthologues of 43% of all annotated D. melanogaster genes, including the genes of all BMP ligands and other BMP signaling components.
The diversification of several BMP signaling components in the dipteran linage of D. melanogaster preceded the origin of the amnioserosa.
[Transcriptome sequence data from this study have been deposited at the NCBI Sequence Read Archive (SRP005289); individually assembled sequences have been deposited at GenBank (JN006969-JN006986).]
Across animals, the Bone Morphogenetic Protein (BMP) signaling pathway plays a major role in specifying the dorsoventral (DV) axis [1, 2]. However, the components of the BMP pathway have been repeatedly modified through lineage specific gene duplications and gene losses [3, 4]. Whether some of these genetic changes correlate with the origin of species-specific morphological traits that develop under the control of the BMP pathway is unknown. Flies (Diptera) provide an excellent opportunity to address this question firstly because the BMP signaling pathway of Drosophila melanogaster has been studied in great detail [5, 6], and secondly because tissue specification presumably under the control of BMP signaling along the DV axis of dipterans has undergone significant change . In D. melanogaster, dorsal blastoderm differentiates into a single extraembryonic epithelium, called amnioserosa, which closes the developing embryo dorsally . This tissue is found in higher cyclorrhaphan flies (Schizophora), but in other dipterans, dorsal blastoderm gives rise to distinct serosal and amniotic epithelia [9–11]. Serosa and amnion develop from an amnioserosal fold at the margins of the gastrulating embryo. The outer cell layer of this fold becomes the serosa, which closes about the embryo. Its inner cell layer detaches from the serosa but retains continuity with the embryo while closing dorsally (lower cyclorrhaphan flies) or ventrally (non-cyclorrhaphan dipterans). The lower cyclorrhaphan syrphids represent the closest relatives of D. melanogaster that have been shown to develop distinct serosa and amnion tissues . Therefore, they are of particular interest in efforts to understand how the origin of the amnioserosa as a new morphology is linked to changes in the underlying developmental gene network.
In previous studies we have characterized the role that the homeobox gene zerknüllt (zen) may have played in the origin of amnioserosa development [reviewed in 7]. The transcription factor Zen is regulated by BMP signaling and essential in serosa specification in non-schizophoran insects and amnioserosa specification in D. melanogaster[9, 12–14]. In lower Cyclorrhapha and more distant relatives of D. melanogaster, zen expression in the serosa is maintained after gastrulation, i. e., when the serosa begins to spread over the embryo , whereas in D. melanogaster zen expression in the amnioserosa is down-regulated immediately after gastrulation . In lower cyclorrhaphan flies, postgastrular down-regulation of zen abrogates serosa development and results in the formation of a single extraembryonic tissue with amniotic gene expression . Thus, the repression of this single transcription factor may account for the morphological tissue reorganization that accompanied the origin of the amnioserosa. However, loss of postgastrular zen expression does not explain, why in lower cyclorrhaphan and non-cyclorrhaphan dipterans the patterning of the dorsal blastoderm results in the specification of two distinct extraembryonic tissues types as opposed to one in schizophoran (i. e. higher cyclorrhaphan) flies such as D. melanogaster.
In D. melanogaster, amnioserosa specification occurs at the dorsal midline and requires peak-levels of BMP activity [14, 16], which are provided through the interaction of two extracellular ligands, Dpp and Scw [17–19]. Both ligands are secreted into the perivitelline space and transported towards the dorsal midline [17, 20–24], where BMP-ligand dimers are released from antagonists to activate a receptor complex and initiate intracellular signaling [17, 20, 25–27]. A cell autonomous autoregulatory loop further increases the BMP signal at the dorsal midline and generates a narrow and sharply delineated domain of BMP peak activity [20, 28]. Dpp is essential for BMP activity and controls the specification of all tissues that develop under the control of this pathway in the early embryo, including amnioserosa and dorsal ectoderm [16, 29]. Scw boosts BMP activity along the dorsal midline and is in particular required for amnioserosa specification .
In other dipterans, the molecular mechanisms that specify the amnion and serosa are not known. Expression studies in a mosquito suggest that a tighter expression of the Dpp antagonist short gastrulation (Sog) leads to broader BMP signaling, which in turn may allow for the specification of two versus one extraembryonic tissue type . Additionally, Scw is absent from the genomes of mosquitoes and other insects, and it has been suggested that its origin may correlate with the origin of the amnioserosa . Several other BMP signaling components of D. melanogaster resulted from gene duplications that have been mapped to the dipteran lineage, while others - known from the BMP pathways of vertebrates - were lost in the lineage leading to D. melanogaster. Here we use embryonic transcriptome data of the hover fly Episyrphus balteatus (Syrphidae) to address the question of whether evolutionary changes in the complement of BMP signaling components occurred in correlation with the origin of the amnioserosa. Specifically, we found that with the possible exception of one gene-duplication (crossveinless/shrew) and one gene loss (DAN), genes encoding known BMP signaling components, including scw, are conserved across the schizophoran boundary of the dipteran tree. Thus, most or all of the gains and losses of BMP signaling genes in the dipteran lineage do not correlate with the origin of the amnioserosa, suggesting that the origin of amnioserosa specification was probably achieved by rearranging the interaction of established factors.
Results and Discussion
Putative Orthologues of 6013 E. balteatus Genes
To identify E. balteatus genes, we pooled sequence data from both libraries and performed reciprocal BLAST against annotated genes of D. melanogaster. Based on reciprocal hits, we identified putative D. melanogaster orthologues of 6013 E. balteatus genes. In total, about 8.1 MB (39%) of the assembled E. balteatus sequence data could be annotated (red line in Figure 1A), corresponding to 43% of annotated D. melanogaster genes. Specifically, we recovered 85% of genes annotated for translational control, over 60% of the genes known to encode gene-specific transcription factors (transcription factor - strict ), 50% of the genes associated with known and putative gene-specific transcription factors (transcription factor - putative ), about 50% of genes associated with structural functions (structure) and enzymes, and 30-40% of genes associated with receptor binding, molecular transporters, and signal transduction (transducer) (Figure 1B).
Assessment of Coverage
To estimate the coverage of developmental genes, we separately mapped the reads from each library back onto all of 14 previously described E. balteatus segmentation genes, which comprise orthologues of bicoid (Eba-bcd), caudal (Eba-cad), nanos (Eba-nos), torso (Eba-tor), orthodenticle (Eba-otd), hunchback (Eba-hb), Krüppel (Eba-Kr), knirps (Eba-kni), giant (Eba-gt), hairy (Eba-h), even-skipped (Eba-eve), zerknüllt (Eba-zen), tailless (Eba-tll), and huckebein (Eba-hkb) [9, 31–33]. The combined maternal and zygotic coverage of all fourteen genes was on average 8.6-fold (Additional file 2), slightly less than the average coverage of the entire assembled transcriptome (~12-fold). However, coverage of 5'UTR sequences (2.0-fold) and 3'UTR sequences (1.6-fold) was considerably lower than the coverage of ORF sequences (12.5-fold). As the CG content of UTRs (20%) was notably lower than the CG content of the ORFs (43%), a systematic bias against AT rich sequences may have been introduced by less efficient annealing of the random hexamer primers during first strand cDNA synthesis. In any case, coverage of our E. balteatus transcriptome data set was high enough to identify at least fragments of genes known to be active during early embryonic development.
BMP Signaling Components in the E. balteatus Transcriptome Database
In D. melanogaster, BMP signaling at the dorsal side of the blastoderm is required to specify the amnioserosa as a single extraembryonic tissue (see Background). Based on mosquito data, it has been suggested that in lower dipterans restricted expression of the BMP antagonist Short gastrulation (Sog) may account for an expanded BMP signaling domain in the dorsal blastoderm, which resolves into serosa and amnion territories . Furthermore, it has been suggested that the complement of BMP signaling components changed in the dipteran lineage in parallel with the origin of the amnioserosa . We used our transcriptome database as a tool to test the latter idea by searching for E. balteatus homologues of specific BMP signaling components of D. melanogaster.
In D. melanogaster, activity of BMP ligands is modulated by Sog [22, 23, 26], which in turn is regulated by the related metalloproteases Tolloid (Tld) and Tolkin (Tok) [25, 49, 50]. We identified E. balteatus homologues of sog as well as of tld. While we were not able to identify an orthologue of tok, the presence of a distinct tld orthologue in E. balteatus suggests that the dipteran gene duplication giving rise to tld and tok occurred before the origin of the amnioserosa (Figure 3D). BMP ligand activity in D. melanogaster is additionally modulated by Twisted gastrulation (Tsg) [51, 52], Crossveinless (Cv) [53, 54], Shrew (Srw) [26, 55], as well as the membrane associated factors Crossveinless-2 (Cv-2) [56, 57], Kekkon 5 (Kek5) , Pentagone (Pent)  and Larval Translucida (Ltl) . We identified E. balteatus homologues of cv-2, kek5 (Figure 3E) as well as tsg, cv and an additional cv paralogue, Eba-cv-like (Figure 3F). Previous studies have suggested that tsg, cv, and srw originated by two successive duplications of a cv-like ancestor in the dipteran lineage . Our gene tree analysis is consistent with this idea, but does not resolve whether Eba-cv-like is orthologous to srw, or whether it is the product of an independent gene duplication in E. balteatus. We did not identify orthologues of pent and ltl in E. balteatus, but putative orthologues of both genes are present in the genome of T. castaneum (data not shown). Thus, all currently known modulators of BMP ligand activity in D. melanogaster may have existed prior to the origin of the amnioserosa.
Putative orthologues of the vertebrate BMP ligands BMP10  and Anti-Dorsalizing Morphogenetic Protein (ADMP) [62–64], as well as the vertebrate BMP inhibitors BAMBI , DAN and Gremlin  have been found in beetles and/or wasps but not in D. melanogaster. Among these, we were able to identify a putative orthologue of DAN in E. balteatus (Figure 3G). The loss of this gene may correlate with the origin of the amnioserosa. However, the function of DAN in insects remains unknown and its potential role in BMP signaling is therefore speculative.
Differences in maternal expression of BMP signaling components in D. melanogaster and E. balteatus
Preparation of Transcriptome Library
Total RNA was prepared by homogenizing embryos in Trizol (Inivtrogen), treated with DNaseI, and enriched for polyA containing transcripts using the Oligotex kit (Qiagen). First-strand cDNA was synthesized from approximately 1 μg of mRNA. Annealing of random hexamer primers (15 mM) was at 25°C for 10 minutes, cDNA was synthesized at 50°C for 1 hour and followed by inactivation of the reverse transcriptase (Superscript, Invitrogen) at 85°C for 5 minutes. Second-strand cDNA was synthesized using the first strand reaction with Klenow DNA Polymerase at 15°C for 1.5 hours, and terminated by the addition of 0.5 M EDTA, pH 8. cDNA was purified using the QIAquick MinElute Reaction Clean-up Kit (Qiagen). cDNA ends were filled in ("polished") using a mix of Klenow DNA polymerase and T4 polynucleotide kinase with dNTPs at 20°C for 30 minutes, after which the reactions were purified again using the QIAquick MinElute Reaction Clean-up Kit. "A" overhangs were created by incubating polished cDNA with 0.2 mM dATP 0.3 U/μl Klenow exo- at 37°C for 30 minutes. cDNA was purified using the QIAquick MinElute Reaction Clean-up Kit (Qiagen), ligated with a mix of AdaptorA and AdaptorB using T4 DNA ligase, and purified again. Adaptors were generated by annealing equimolar amounts of complementary oligos in 2x TNE buffer (20 mM Tris-Cl, pH 8, 0.2 mM EDTA, pH 8, 100 mM NaCl). Oligo sequences for both adaptors were adapted from 454 Sequencing Technical Bulletin No. 004-2009, ordered from Integrated DNA Technologies and HPLC purified. Amplification of the library was performed in triplicate using Platinum Taq DNA polymerase HiFi with AdapterA and AdapterB primers. AdapterA FW and AdapterB FW primers. Pooled volumes of the library were purified using the QIAquick MinElute Reaction Clean-up Kit.
Library Normalization and Fragment Size Selection
Libraries were normalized using the TRIMMER DIRECT cDNA Normalization Kit (Evrogen) and were carried out essentially as described in the user manual. Briefly, 400 ng of each library were suspended in hybridization buffer and split into four tubes. Following 5 hr incubation at 68°C, the aliquots were treated either with 4 units, 2 units, 1 unit, or no duplex-specific nuclease (DSN). After DSN digestion, the normalized cDNA libraries were amplified by PCR. Optimal amplification within the exponential phase of the PCR was determined visually after electrophoresing different amplification runs of the non-DSN treated sample, after which all aliquots were amplified by a total of 20 cycles. The normalization efficiency was assessed by quantitative PCR (qPCR) of hunchback (Eba-hb, 5'-CTCAGCCCGAATCCAAAT/5'-GGTTGTGGGAGTTGATGTTG, amplicon 137 bp), caudal (Eba-cad, 5'-GAAAGAATACTGCACCTCCC/5'-GTCGTTCCGATAGTTGAAGC, amplicon 79 bp), and alpha-tubulin as reference (Eba-tub, 5'-TGAGGCTCGTGAGGATTT/5'-TCACCATCTCCAGAATCCA, amplicon 71 bp). Primer efficiency was estimated from a standard curve using five different template concentrations (12.5-200 ng); all analyses were run simultaneously in triplicates. For both libraries, the normalization with 4 units DSN were chosen for sequencing. Based on optimal cycle numbers for amplification and degree of normalization, these libraries were size fractionated using agarose gel electrophoresis, excising fragments at roughly 500 bp, and then purifying them using the MinElute Gel Extraction Kit (Qiagen) prior to sequencing.
Transcriptome library sequencing was performed on the Roche/454 Life Sciences GS-FLX platform at the Institute for Genomics and Systems Biology's (IGSB) High-Throughput Genome Analysis Core (HGAC) at Argonne National Laboratory according to the Roche GS-FLX XLR70 Titanium emPCR and amplicon sequencing protocols. Each transcriptome library was sequenced on one region of a two region GS-FLX gasket using Roche GS-FLX XLR70 titanium sequencing reagents. An emPCR titration was initially performed on each library to determine the proper bead:library copy ratio that yielded optimal clonal bead percent enrichment to be used in the final bulk XLR70 emPCR reaction.
Sequencing raw data was processed with gsRunProcessor (software release 2.0.00.20) using standard quality filtering and trimming as defined by the default settings. We obtained 417,735 reads with a mean length of 243 nt for the maternal time point and 406,580 reads with a mean length of 278 nt for the zygotic time point, totaling 214.6 MB of sequence data. This raw data set was contaminated with sequences of the pea aphid Acyrthosiphon pisum, because E. balteatus requires aphids for egg deposition, and embryos were collected in batch from leaves heavily infested with aphids. For subsequent analyses, we removed all reads that matched published A. pisum sequences (mRNA + genomic, NCBI, 2009-07-13)  with 95% or higher identity. Removal of A. pisum sequences reduced the total sequence data by about 26% to 158.1 MB and the number of reads from each library by about 22%, resulting in 325,200 reads from the maternal library with a mean read length of 228 nt, and 311,906 reads from the zygotic library with a mean read length of 269 nt. The distribution of read lengths displayed two peaks, one at about 400 nt (360 nt for reads from the zygotic library) and one at less than 100 nt (Additional file 1C,D). The peak at 400 nt corresponded to the expected mean length using the Titanium chemistry. The peak slightly below 100 nt presumably resulted from our cDNA preparation protocol, which had not yet been optimized for the Titanium chemistry at the time of library preparation and lacked, for example, any additional size exclusion steps following gel electrophoreses.
All reads have been deposited as the E. balteatus transcriptome at the NCBI Sequence Read Archive (SRA, SRP005289). Assembly was based on combined SFF sequence files of the maternal and zygotic libraries using Newbler Assembler (software release 2.3). Newbler parameters were default except: minimal identity of 90% in overlaps (-mi 90), overlaps to be at least 30 nt in length (-ml 30), minimal length of contigs to be 100 nt (-l 100). Newbler was run as cDNA assembly (-cdna), which includes processing of contigs that are found to be variants of the same transcript into distinct isotigs. From a total of 637,080 reads, 544,776 reads (85.5%) were assembled (fully assembled reads: 432,160 reads, 67.8%; partially assembled reads: 112,616 reads, 17.7%). The remaining reads were singletons (56,625 reads; 8.9%) or excluded as either originating from repeat regions (221 reads; 0.03%), outliers (23,060 reads; 3.6%), or too short (< 50 base pairs: 12,398 reads; 1.9%) (see Additional file 4 for comparison with Newbler 2.5.3).
Reported isotigs (12,296; 12.9 MB) and singletons of at least 100 nt length (26,862; 7.1 MB; identified from 454ReadStatus.txt) were combined, and reciprocal BLAST searches of the E. balteatus transcriptome were carried out against the translated transcriptome of D. melanogaster (dmel-all-translation-r5.29.faa, flybase.org) using blastx (E. balteatus query against D. melanogaster database) and tblastn (D. melanogaster query against E. balteatus database). Annotation was performed with an e-value threshold of 10 to screen for all putative orthologues ("no cutoff") and with an e-value threshold of 1e-10 to obtain a conservative list of high confidence orthologues ("1e-10") (see Additional file 5 for comparison of annotation based on assemblies with Newbler 2.3 and Newbler 2.5.3). Reciprocal hits of E. balteatus with D. melanogaster were assigned with the same flybase D. melanogaster CG identifiers. Gene ontology terms were assigned based on the current D. melanogaster gene ontology, with the exception of 'transcription factor - strict' and 'transcription factor - putative', which were based on a curated list of genes of known and putative gene specific transcription factors .
To determine coverage levels for individual published E. balteatus genes, associated reads of the maternal and zygotic library sequences were identified by BLAST search (blastn) and assembled with the published sequence using CAP3 with standard parameters . Coverage levels were then calculated for each nucleotide position of the published gene sequences. Fold-coverage in the maternal library was used to approximate maternal expression levels of all annotated E. balteatus genes. The assembled E. balteatus transcriptome (isotigs and singletons) was blasted against all maternal reads (blastn). Coverage of annotated transcripts was then calculated from all reads that matched with at least 95% identity and over the length of at least 50 nt. To approximate levels of gene expression, coverage of each gene was divided by its sequence length and the total RNAseq data of the maternal library (74 MB). Fold-coverage in the 0-2 hr time point of the D. melanogaster development transcriptome was used to approximate maternal expression levels of D. melanogaster genes . Expression data of D. melanogaster genes in 0-2 hrs old embryos was downloaded from modEncode as coverage data mapped onto the entire genome (BC1_plus.wig, BC1_minus.wig). Expression of gene transcripts was retrieved from this genomic map by extracting coverage information of all exons for each annotated gene (BDGP/dm3). To account for potentially mis-annotated exon-intron structures of computationally predicted genes, gene expression was approximated by the coverage of the most strongly expressed exon longer than 500 nt, or by the coverage of the most strongly expressed transcript variant, whichever was higher. To approximate levels of gene expression, coverage of each gene was divided by its sequence length and the total RNAseq data of the 0-2 hr time point (3.4 GB, SRP001696).
Gene Discovery of BMP Signaling Components
A list of D. melanogaster and Tribolium castaneum BMP signaling compounds was matched against assembled and unassembled E. balteatus transcriptome data (tblastn, e-value of 0.001). Identified E. balteatus sequences were assembled in Sequencher, assemblies were manually corrected for ORF frame shifts by alignment with D. melanogaster protein sequence, and sequence orthology was confirmed by reciprocal blast against a D. melanogaster database. To exhaustively search for putative duplicates specific to the E. balteatus lineage, blast searches were repeated using lower cut-offs (increasingly higher e-values) until all newly identified and assembled E. balteatus reads matched a clearly non-orthologous sequence in available insect protein databases. All T. castaneum sequences were retrieved from BeetleBase (ftp://bioinformatics.ksu.edu/pub/BeetleBase/latest/Sequences/Tribolium_Official_Gene_Sequences/mRNA.fa).
Phylogenetic Gene Trees
Protein alignments were created using the Clustal algorithm with standard parameters (MegAlign). When more than half of the aligned sequences carried a gap at a given position, these positions were removed from the alignment. The amino acid substitution model was estimated using AIC in ProtTest ; maximum likelihood trees were calculated using PhyML . Bootstrap values were based on 1000 replicas. Trees were plotted with drawtree (Phylip package)  and the newick-utils package .
Custom scripts (Perl, R) were used to automate blast searches and evaluation, calculate E. balteatus and D. melanogaster gene coverage, and prune sequence alignments. Scripts are available on request. Plots were prepared with gnuplot and finished with Freehand. Assembly, blast searches, and bootstrap analysis were computed on the computer cluster of the Department of Ecology & Evolution at the University of Chicago (http://biocomputing.uchicago.edu).
We thank Lucien Jarymowycz, IT systems manager in the Department of Organismal Biology at the University of Chicago, for his professional help. Funding was provided by NSF grants 0719445 and 0840687 to U. S.-O.
- Niehrs C: On growth and form: a Cartesian coordinate system of Wnt and BMP signaling specifies bilaterian body axes. Development. 2010, 137 (6): 845-857. 10.1242/dev.039651.PubMedView Article
- De Robertis EM: Spemann's organizer and the self-regulation of embryonic fields. Mech Dev. 2009, 126 (11-12): 925-941. 10.1016/j.mod.2009.08.004.PubMed CentralPubMedView Article
- Fritsch C, Lanfear R, Ray RP: Rapid evolution of a novel signalling mechanism by concerted duplication and divergence of a BMP ligand and its extracellular modulators. Dev Genes Evol. 2010, 220 (9-10): 235-250. 10.1007/s00427-010-0341-5.PubMedView Article
- Van der Zee M, da Fonseca RN, Roth S: TGFbeta signaling in Tribolium: vertebrate-like components in a beetle. Dev Genes Evol. 2008, 218 (3-4): 203-213. 10.1007/s00427-007-0179-7.PubMedView Article
- O'Connor MB, Umulis D, Othmer HG, Blair SS: Shaping BMP morphogen gradients in the Drosophila embryo and pupal wing. Development. 2006, 133 (2): 183-193.PubMedView Article
- Umulis D, O'Connor MB, Blair SS: The extracellular regulation of bone morphogenetic protein signaling. Development. 2009, 136 (22): 3715-3728. 10.1242/dev.031534.PubMed CentralPubMedView Article
- Schmidt-Ott U, Rafiqi AM, Lemke S: Hox3/zen and the Evolution of Extraembryonic Epithelia in Insects. Hox Genes' Studies from the 20th to the 21st Century. Edited by: Deutsch J. 2010, Austin, TX: Landes Biosciences
- Campos-Ortega J, Hartenstein V: The embryonic development of Drosophila melanogaster. 1997, Berlin, Heidelberg, New York: Springer Verlag, 2View Article
- Rafiqi AM, Lemke S, Ferguson S, Stauber M, Schmidt-Ott U: Evolutionary origin of the amnioserosa in cyclorrhaphan flies correlates with spatial and temporal expression changes of zen. Proc Natl Acad Sci USA. 2008, 105 (1): 234-239. 10.1073/pnas.0709145105.PubMed CentralPubMedView Article
- Goltsev Y, Rezende G, Vranizan K, Lanzaro G, Valle D, Levine M: Developmental and evolutionary basis for drought tolerance of the Anopheles gambiae embryo. Dev Biol. 2009, 330: 462-470. 10.1016/j.ydbio.2009.02.038.PubMed CentralPubMedView Article
- Goltsev Y, Fuse N, Frasch M, Zinzen RP, Lanzaro G, Levine M: Evolution of the dorsal-ventral patterning network in the mosquito, Anopheles gambiae. Development. 2007, 134 (13): 2415-2424. 10.1242/dev.02863.PubMedView Article
- van der Zee M, Berns N, Roth S: Distinct functions of the Tribolium zerknüllt genes in serosa specification and dorsal closure. Curr Biol. 2005, 15: 624-636. 10.1016/j.cub.2005.02.057.PubMedView Article
- Rushlow C, Levine M: Role of the zerknüllt gene in dorsal-ventral pattern formation in Drosophila. Adv Genet. 1990, 27: 277-307.PubMedView Article
- Ray RP, Arora K, Nüsslein-Volhard C, Gelbart WM: The control of cell fate along the dorsal-ventral axis of the Drosophila embryo. Development. 1991, 113 (1): 35-54.PubMed
- Rafiqi AM, Lemke S, Schmidt-Ott U: Postgastrular zen expression is required to develop distinct amniotic and serosal epithelia in the scuttle fly Megaselia. Dev Biol. 2010, 341 (1): 282-90. 10.1016/j.ydbio.2010.01.040.PubMedView Article
- Ferguson EL, Anderson KV: Decapentaplegic acts as a morphogen to organize dorsal-ventral pattern in the Drosophila embryo. Cell. 1992, 71 (3): 451-461. 10.1016/0092-8674(92)90514-D.PubMedView Article
- Shimmi O, Umulis D, Othmer H, O'Connor MB: Facilitated transport of a Dpp/Scw heterodimer by Sog/Tsg leads to robust patterning of the Drosophila blastoderm embryo. Cell. 2005, 120 (6): 873-886. 10.1016/j.cell.2005.02.009.PubMedView Article
- Padgett RW, St Johnston RD, Gelbart WM: A transcript from a Drosophila pattern gene predicts a protein homologous to the transforming growth factor-beta family. Nature. 1987, 325 (6099): 81-84. 10.1038/325081a0.PubMedView Article
- Arora K, Levine MS, O'Connor MB: The screw gene encodes a ubiquitously expressed member of the TGF-beta family required for specification of dorsal cell fates in the Drosophila embryo. Genes Dev. 1994, 8 (21): 2588-2601. 10.1101/gad.8.21.2588.PubMedView Article
- Wang YC, Ferguson EL: Spatial bistability of Dpp-receptor interactions during Drosophila dorsal-ventral patterning. Nature. 2005, 434 (7030): 229-234. 10.1038/nature03318.PubMedView Article
- Eldar A, Dorfman R, Weiss D, Ashe H, Shilo BZ, Barkai N: Robustness of the BMP morphogen gradient in Drosophila embryonic patterning. Nature. 2002, 419 (6904): 304-308. 10.1038/nature01061.PubMedView Article
- Decotto E, Ferguson EL: A positive role for Short gastrulation in modulating BMP signaling during dorsoventral patterning in the Drosophila embryo. Development. 2001, 128 (19): 3831-3841.PubMed
- Ashe HL, Levine M: Local inhibition and long-range enhancement of Dpp signal transduction by Sog. Nature. 1999, 398 (6726): 427-431. 10.1038/18892.PubMedView Article
- Francois V, Solloway M, O'Neill JW, Emery J, Bier E: Dorsal-ventral patterning of the Drosophila embryo depends on a putative negative growth factor encoded by the short gastrulation gene. Genes Dev. 1994, 8 (21): 2602-2616. 10.1101/gad.8.21.2602.PubMedView Article
- Marqués G, Musacchio M, Shimell MJ, Wünnenberg-Stapleton K, Cho KW, O'Connor MB: Production of a DPP activity gradient in the early Drosophila embryo through the opposing actions of the SOG and TLD proteins. Cell. 1997, 91 (3): 417-426. 10.1016/S0092-8674(00)80425-0.PubMedView Article
- Ferguson EL, Anderson KV: Localized enhancement and repression of the activity of the TGF-beta family member, decapentaplegic, is necessary for dorsal-ventral pattern formation in the Drosophila embryo. Development. 1992, 114 (3): 583-597.PubMed
- Shimell MJ, Ferguson EL, Childs SR, O'Connor MB: The Drosophila dorsal-ventral patterning gene tolloid is related to human bone morphogenetic protein 1. Cell. 1991, 67 (3): 469-481. 10.1016/0092-8674(91)90522-Z.PubMedView Article
- Umulis DM, Serpe M, O'Connor MB, Othmer HG: Robust, bistable patterning of the dorsal surface of the Drosophila embryo. Proc Natl Acad Sci USA. 2006, 103 (31): 11613-11618. 10.1073/pnas.0510398103.PubMed CentralPubMedView Article
- Wharton KA, Ray RP, Gelbart WM: An activity gradient of decapentaplegic is necessary for the specification of dorsal pattern elements in the Drosophila embryo. Development. 1993, 117 (2): 807-822.PubMed
- Pfreundt U, James DP, Tweedie S, Wilson D, Teichmann SA, Adryan B: FlyTF: improved annotation and enhanced functionality of the Drosophila transcription factor database. Nucleic Acids Res. 2010, 38: 443-447. 10.1093/nar/gkp910.View Article
- Lemke S, Busch S, Antonopoulos D, Meyer F, Domanus M, Schmidt-Ott U: Maternal activation of gap genes in the hover fly Episyrphus. Development. 2010, 137 (10): 1709-1719. 10.1242/dev.046649.PubMedView Article
- Lemke S, Schmidt-Ott U: Evidence for a composite anterior determinant in the hover fly Episyrphus balteatus (Syrphidae), a cyclorrhaphan fly with an anterodorsal serosa anlage. Development. 2009, 136: 117-127. 10.1242/dev.030270.PubMedView Article
- Bullock SL, Stauber M, Prell A, Hughes JR, Ish-Horowicz D, Schmidt-Ott U: Differential cytoplasmic mRNA localisation adjusts pair-rule transcription factor activity to cytoarchitecture in dipteran evolution. Development. 2004, 131 (17): 4251-4261. 10.1242/dev.01289.PubMedView Article
- Doctor JS, Jackson PD, Rashka KE, Visalli M, Hoffmann FM: Sequence, biochemical characterization, and developmental expression of a new member of the TGF-beta superfamily in Drosophila melanogaster. Dev Biol. 1992, 151 (2): 491-505. 10.1016/0012-1606(92)90188-M.PubMedView Article
- Xie T, Finelli AL, Padgett RW: The Drosophila saxophone gene: a serine-threonine kinase receptor of the TGF-beta superfamily. Science. 1994, 263 (5154): 1756-1759. 10.1126/science.8134837.PubMedView Article
- Brummel TJ, Twombly V, Marqués G, Wrana JL, Newfeld SJ, Attisano L, Massagué J, O'Connor MB, Gelbart WM: Characterization and relationship of Dpp receptors encoded by the saxophone and thick veins genes in Drosophila. Cell. 1994, 78 (2): 251-261. 10.1016/0092-8674(94)90295-X.PubMedView Article
- Nellen D, Affolter M, Basler K: Receptor serine/threonine kinases implicated in the control of Drosophila body pattern by decapentaplegic. Cell. 1994, 78 (2): 225-237. 10.1016/0092-8674(94)90293-3.PubMedView Article
- Penton A, Chen Y, Staehling-Hampton K, Wrana JL, Attisano L, Szidonya J, Cassill JA, Massagué J, Hoffmann FM: Identification of two bone morphogenetic protein type I receptors in Drosophila and evidence that Brk25D is a decapentaplegic receptor. Cell. 1994, 78 (2): 239-250. 10.1016/0092-8674(94)90294-1.PubMedView Article
- Letsou A, Arora K, Wrana JL, Simin K, Twombly V, Jamal J, Staehling-Hampton K, Hoffmann FM, Gelbart WM, Massagué J: Drosophila Dpp signaling is mediated by the punt gene product: a dual ligand-binding type II receptor of the TGF beta receptor family. Cell. 1995, 80 (6): 899-908. 10.1016/0092-8674(95)90293-7.PubMedView Article
- Ruberte E, Marty T, Nellen D, Affolter M, Basler K: An absolute requirement for both the type II and type I receptors, punt and thick veins, for dpp signaling in vivo. Cell. 1995, 80 (6): 889-897. 10.1016/0092-8674(95)90292-9.PubMedView Article
- Brummel T, Abdollah S, Haerry TE, Shimell MJ, Merriam J, Raftery L, Wrana JL, O'Connor MB: The Drosophila activin receptor baboon signals through dSmad2 and controls cell proliferation but not patterning during larval development. Genes Dev. 1999, 13 (1): 98-111. 10.1101/gad.13.1.98.PubMed CentralPubMedView Article
- Aberle H, Haghighi AP, Fetter RD, McCabe BD, Magalhães TR, Goodman CS: wishful thinking encodes a BMP type II receptor that regulates synaptic growth in Drosophila. Neuron. 2002, 33 (4): 545-558. 10.1016/S0896-6273(02)00589-5.PubMedView Article
- Marqués G, Bao H, Haerry TE, Shimell MJ, Duchek P, Zhang B, O'Connor MB: The Drosophila BMP type II receptor Wishful Thinking regulates neuromuscular synapse morphology and function. Neuron. 2002, 33 (4): 529-543. 10.1016/S0896-6273(02)00595-0.PubMedView Article
- Sekelsky JJ, Newfeld SJ, Raftery LA, Chartoff EH, Gelbart WM: Genetic characterization and cloning of mothers against dpp, a gene required for decapentaplegic function in Drosophila melanogaster. Genetics. 1995, 139 (3): 1347-1358.PubMed CentralPubMed
- Wisotzkey RG, Mehra A, Sutherland DJ, Dobens LL, Liu X, Dohrmann C, Attisano L, Raftery LA: Medea is a Drosophila Smad4 homolog that is differentially required to potentiate DPP responses. Development. 1998, 125 (8): 1433-1445.PubMed
- Hudson JB, Podos SD, Keith K, Simpson SL, Ferguson EL: The Drosophila Medea gene is required downstream of dpp and encodes a functional homolog of human Smad4. Development. 1998, 125 (8): 1407-1420.PubMed
- Das P, Maduzia LL, Wang H, Finelli AL, Cho SH, Smith MM, Padgett RW: The Drosophila gene Medea demonstrates the requirement for different classes of Smads in dpp signaling. Development. 1998, 125 (8): 1519-1528.PubMed
- Henderson KD, Andrew DJ: Identification of a novel Drosophila SMAD on the X chromosome. Biochem Biophys Res Commun. 1998, 252 (1): 195-201. 10.1006/bbrc.1998.9562.PubMedView Article
- Srinivasan S, Rashka KE, Bier E: Creation of a Sog morphogen gradient in the Drosophila embryo. Dev Cell. 2002, 2 (1): 91-101. 10.1016/S1534-5807(01)00097-1.PubMedView Article
- Serpe M, Ralston A, Blair SS, O'Connor MB: Matching catalytic activity to developmental function: tolloid-related processes Sog in order to help specify the posterior crossvein in the Drosophila wing. Development. 2005, 132 (11): 2645-2656. 10.1242/dev.01838.PubMedView Article
- Mason ED, Konrad KD, Webb CD, Marsh JL: Dorsal midline fate in Drosophila embryos requires twisted gastrulation, a gene encoding a secreted protein related to human connective tissue growth factor. Genes Dev. 1994, 8 (13): 1489-1501. 10.1101/gad.8.13.1489.PubMedView Article
- Ross JJ, Shimmi O, Vilmos P, Petryk A, Kim H, Gaudenz K, Hermanson S, Ekker SC, O'Connor MB, Marsh JL: Twisted gastrulation is a conserved extracellular BMP antagonist. Nature. 2001, 410 (6827): 479-483. 10.1038/35068578.PubMedView Article
- Shimmi O, Ralston A, Blair SS, O'Connor MB: The crossveinless gene encodes a new member of the Twisted gastrulation family of BMP-binding proteins which, with Short gastrulation, promotes BMP signaling in the crossveins of the Drosophila wing. Dev Biol. 2005, 282 (1): 70-83. 10.1016/j.ydbio.2005.02.029.PubMedView Article
- Vilmos P, Sousa-Neves R, Lukacsovich T, Marsh JL: crossveinless defines a new family of Twisted-gastrulation-like modulators of bone morphogenetic protein signalling. EMBO Rep. 2005, 6 (3): 262-267. 10.1038/sj.embor.7400347.PubMed CentralPubMedView Article
- Bonds M, Sands J, Poulson W, Harvey C, Von Ohlen T: Genetic screen for regulators of ind expression identifies shrew as encoding a novel twisted gastrulation-like protein involved in Dpp signaling. Dev Dyn. 2007, 236 (12): 3524-3531. 10.1002/dvdy.21360.PubMedView Article
- Serpe M, Umulis D, Ralston A, Chen J, Olson DJ, Avanesov A, Othmer H, O'Connor MB, Blair SS: The BMP-binding protein Crossveinless 2 is a short-range, concentration-dependent, biphasic modulator of BMP signaling in Drosophila. Dev Cell. 2008, 14 (6): 940-953. 10.1016/j.devcel.2008.03.023.PubMed CentralPubMedView Article
- Conley CA, Silburn R, Singer MA, Ralston A, Rohwer-Nutter D, Olson DJ, Gelbart W, Blair SS: Crossveinless 2 contains cysteine-rich domains and is required for high levels of BMP-like activity during the formation of the cross veins in Drosophila. Development. 2000, 127 (18): 3947-3959.PubMed
- Evans TA, Haridas H, Duffy JB: Kekkon5 is an extracellular regulator of BMP signaling. Dev Biol. 2009, 326 (1): 36-46. 10.1016/j.ydbio.2008.10.002.PubMedView Article
- Vuilleumier R, Springhorn A, Patterson L, Koidl S, Hammerschmidt M, Affolter M, Pyrowolakis G: Control of Dpp morphogen signalling by a secreted feedback regulator. Nat Cell Biol. 2010, 12 (6): 611-617. 10.1038/ncb2064.PubMedView Article
- Szuperák M, Salah S, Meyer EJ, Nagarajan U, Ikmi A, Gibson MC: Feedback regulation of Drosophila BMP signaling by the novel extracellular protein Larval Translucida. Development. 2011, 138 (4): 715-724. 10.1242/dev.059477.PubMedView Article
- Chen H, Shi S, Acosta L, Li W, Lu J, Bao S, Chen Z, Yang Z, Schneider MD, Chien KR, et al: BMP10 is essential for maintaining cardiac growth during murine cardiogenesis. Development. 2004, 131 (9): 2219-2231. 10.1242/dev.01094.PubMed CentralPubMedView Article
- Moos M, Wang S, Krinks M: Anti-dorsalizing morphogenetic protein is a novel TGF-beta homolog expressed in the Spemann organizer. Development. 1995, 121 (12): 4293-4301.PubMed
- Joubin K, Stern CD: Molecular interactions continuously define the organizer during the cell movements of gastrulation. Cell. 1999, 98 (5): 559-571. 10.1016/S0092-8674(00)80044-6.PubMedView Article
- Lele Z, Nowak M, Hammerschmidt M: Zebrafish admp is required to restrict the size of the organizer and to promote posterior and ventral development. Dev Dyn. 2001, 222 (4): 681-687. 10.1002/dvdy.1222.PubMedView Article
- Onichtchouk D, Chen YG, Dosch R, Gawantka V, Delius H, Massagué J, Niehrs C: Silencing of TGF-beta signalling by the pseudoreceptor BAMBI. Nature. 1999, 401 (6752): 480-485. 10.1038/46794.PubMedView Article
- Hsu DR, Economides AN, Wang X, Eimon PM, Harland RM: The Xenopus dorsalizing factor Gremlin identifies a novel family of secreted proteins that antagonize BMP activities. Mol Cell. 1998, 1 (5): 673-683. 10.1016/S1097-2765(00)80067-2.PubMedView Article
- Graveley BR, Brooks AN, Carlson JW, Duff MO, Landolin JM, Yang L, Artieri CG, van Baren MJ, Boley N, Booth BW, et al: The developmental transcriptome of Drosophila melanogaster. Nature. 2010
- Consortium TIAG: Genome sequence of the pea aphid Acyrthosiphon pisum. PLoS Biol. 2010, 8 (2): e1000313-10.1371/journal.pbio.1000313.View Article
- Huang X, Madan A: CAP3: A DNA sequence assembly program. Genome Res. 1999, 9: 868-877. 10.1101/gr.9.9.868.PubMed CentralPubMedView Article
- Abascal F, Zardoya R, Posada D: ProtTest: selection of best-fit models of protein evolution. Bioinformatics. 2005, 21: 2104-2105. 10.1093/bioinformatics/bti263.PubMedView Article
- Guindon S, Gascuel O: A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol. 2003, 52 (5): 696-704. 10.1080/10635150390235520.PubMedView Article
- Felsenstein J: PHYLIP (Phylogeny Inference Package) version 3.6. Distributed by the author. Department of Genome Sciences, University of Washington, Seattle. 2005
- Junier T, Zdobnov EM: The Newick utilities: high-throughput phylogenetic tree processing in the Unix shell. Bioinformatics. 2010
- Van der Zee M, da Fonseca RN, Roth S: TGFbeta signaling in Tribolium: vertebrate-like components in a beetle. Dev Genes Evol. 2008, 218 (3-4): 203-213. 10.1007/s00427-007-0179-7.PubMedView Article
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.