The origin of bmp16, a novel Bmp2/4 relative, retained in teleost fish genomes

<p>Abstract</p> <p>Background</p> <p>Whole genome sequences have allowed us to have an overview of the evolution of gene repertoires. The target of the present study, the TGFβ superfamily, contains many genes involved in vertebrate development, and provides an ideal system to explore the relationships between evolution of gene repertoires and that of developmental programs.</p> <p>Results</p> <p>As a result of a bioinformatic survey of sequenced vertebrate genomes, we identified an uncharacterized member of the TGFβ superfamily, designated <it>bmp16</it>, which is confined to teleost fish species. Our molecular phylogenetic study revealed a high affinity of <it>bmp16 </it>to the <it>Bmp2/4 </it>subfamily. Importantly, further analyses based on the maximum-likelihood method unambiguously ruled out the possibility that this teleost-specific gene is a product of teleost-specific genome duplication. This suggests that the absence of a <it>bmp16 </it>ortholog in tetrapods is due to a secondary loss. <it>In situ </it>hybridization showed embryonic expression of the zebrafish <it>bmp16 </it>in the developing swim bladder, heart, tail bud, and ectoderm of pectoral and median fin folds in pharyngula stages, as well as gut-associated expression in 5-day embryos.</p> <p>Conclusion</p> <p>Comparisons of expression patterns revealed (1) the redundancy of <it>bmp16 </it>expression with its homologs in presumably plesiomorphic expression domains, such as the fin fold, heart, and tail bud, which might have permitted its loss in the tetrapod lineage, and (2) the loss of craniofacial expression and gain of swim bladder expression of <it>bmp16 </it>after the gene duplication between <it>Bmp2</it>, <it>-4 </it>and <it>-16</it>. Our findings highlight the importance of documenting secondary changes of gene repertoires and expression patterns in other gene families.</p

Evolution, gene regulation and functional analysis of BMP2 in fish

Bone morphogenetic proteins (BMPs) are multifunctional growth factors belonging to the transforming growth factor β (TGFβ) superfamily with a central role in bone formation and mineralization. BMP2, a founding member of this family, has demonstrated remarkable osteogenic properties and is clinically used to promote bone repair and fracture healing. Lack of basic data on factors regulating BMP2 expression and activity have hampered a better understanding of its role in bone formation and bone-related diseases. The objective of this work was to collect new functional data and determine spatiotemporal expression patterns in a fish system aiming towards a better understanding of BMP2 function and regulation. Transcriptional and post-transcriptional regulation of gilthead seabream BMP2 gene was inferred from luciferase reporter systems. Several bone- and cartilage-related transcription factors (e.g. RUNX3, MEF2c, SOX9 and ETS1) were found to regulate BMP2 transcription, while microRNA 20a was shown to affect stability of the BMP2 transcript and thus the mineralogenic capacity of fish bone-derived host cells. The regulation of BMP2 activity through an interaction with the matrix Gla protein (MGP) was investigated in vitro using BMP responsive elements (BRE) coupled to luciferase reporter gene. Although we demonstrated the functionality of the experimental system in a fish cell line and the activation of BMP signaling pathway by seabream BMP2, no conclusive evidence could be collected on a possible interaction beween MGP and BMP2. The evolutionary relationship among the members of BMP2/4/16 subfamily was inferred from taxonomic and phylogenetic analyses. BMP16 diverged prior to BMP2 and BMP4 and should be the result of an ancient genome duplication that occurred early in vertebrate evolution. Structural and functional data suggested that all three proteins are effectors of the BMP signaling pathway, but expression data revealed different spatiotemporal patterns in teleost fish suggesting distinct mechanisms of regulation. In this work, through the collection of novel data, we provide additional insight into the regulation, the structure and the phylogenetic relationship of BMP2 and its closely related family members.O sistema esquelético confere suporte e proteção ao organismo, permite o armazenamento de minerais e desempenha funções hematopoiéticas. Um dos seus principais componentes é o osso, um tecido conectivo especializado e constituído por uma matriz extracelular extensamente mineralizada. O processo de mineralização envolve mecanismos extremamente complexos que estão sujeitos a um rigoroso controlo a nível molecular, no qual estão envolvidas várias proteínas responsáveis pela diferenciação celular e pela síntese de matriz extracelular. Dentro deste conjunto de proteínas, destacam-se alguns fatores de crescimento essenciais ao mecanismo de mineralização tecidular, como é o caso das proteínas morfogenéticas do osso (BMPs). As BMPs pertencem à superfamília de fatores de crescimento de transformação β (TGFβ) e estão envolvidas em vários processos durante a embriogénese, organogénese, proliferação e diferenciação celular e mecanismos de formação óssea. Atualmente estão descritos e caracterizados mais de vinte membros pertencentes a esta família, que foram divididos em várias subfamílias, de acordo com a semelhança da estrutura primária das suas proteínas. A subfamília BMP2/4/16 à qual pertence a BMP2 foi uma das primeiras a ser identificada e caracterizada. A BMP2 é uma das proteínas que possui uma maior capacidade osteogénica e é, desde há muito tempo, considerada um potencial agente terapêutico para o tratamento de doenças relacionadas com o osso, sendo mesmo utilizada em alguns casos clínicos de fraturas ósseas. O conhecimento dos processos de formação óssea, bem como os mecanismos de regulação da BMP2, são por isso de extrema importância para uma melhor compreensão dos processos subjacentes ao desenvolvimento e progressão de algumas doenças ósseas. Assim, a BMP2 tem sido alvo de vários estudos, quer a nível de conhecimento da sua função, quer a nível de mecanismos de ação e processamento. Sabe-se que a BMP2 é uma proteína secretada para a matriz extracelular onde, através de um mecanismo de sinalização molecular, é responsável pela regulação de vários processos. O mecanismo de sinalização inicia-se quando dímeros de BMP2 se ligam aos respetivos recetores, presentes na superfície da célula, ativando assim uma cascata de sinalização molecular. Através de diferentes intermediários intracelulares, envolvidos na cascata de sinalização, a BMP2 é responsável pela regulação transcricional de vários genes-alvo. No entanto, e apesar dos vários estudos que foram feitos nesta área, o conhecimento existente acerca deste assunto é ainda bastante escasso. Neste sentido, o objetivo principal deste trabalho foi a recolha de novos dados funcionais e estruturais, que permitam uma melhor compreensão da função da BMP2. Para tal, e de modo a complementar o conhecimento existente, utilizámos o peixe como modelo alternativo aos sistemas de mamíferos. O peixe é atualmente reconhecido como um modelo válido para estudos do esqueleto de vertebrados para o qual existem já várias ferramentas que permitem análises in silico, in vitro e in vivo. Este trabalho envolveu o estudo da regulação do gene da BMP2 de dourada, tanto a nível transcricional como a nível pós-transcricional. Numa primeira fase, foram identificados potenciais reguladores transcricionais da BMP2 de dourada, através da análise in silico da região reguladora do gene. Dentro dos potenciais reguladores transcricionais, foram identificados vários fatores de transcrição com funções descritas ao nível do osso e da cartilagem, nomeadamente o RUNX3, SOX9, MEF2C e ETS1, que foram posteriormente testados a nível funcional através de ensaios repórter de luciferase. Em paralelo, no decorrer da caracterização de reguladores pós-transcricionais da BMP2, através da análise da região 3’ não traduzida (3’UTR) do seu mRNA, foi possível identificar um local de ligação para o miR-20a, conservado ao longo da evolução. A fim de melhor compreender os mecanismos de ação da BMP2, neste trabalho investigámos também possíveis parceiros desta proteína. A caraterização da interação entre a BMP2 e a proteína Gla da matriz (MGP), um conhecido inibidor da calcificação, foi avaliada através do uso de um sistema de elementos de resposta às BMPs, acoplado a um gene repórter, a luciferase. Embora tenhamos demonstrado a funcionalidade do sistema através da ativação do mecanismo de sinalização celular pela BMP2 de dourada, não foram obtidos dados conclusivos no que diz respeito à interação entre a BMP2 e a MGP. Finalmente, abordamos o aspecto evolutivo dos membros da subfamília BMP2/4/16 através da análise da sua distribuição taxonómica entre vários organismos vertebrados, bem como as relações filogenéticas existentes entre os vários membros desta subfamília. Foi demonstrado que a BMP16 divergiu antes da BMP2 e BMP4 na linhagem dos vertebrados e foi, provavelmente, o resultado de uma duplicação genómica que terá ocorrido ancestralmente. Dados estruturais sugerem uma conservação funcional das três proteínas, facto que foi confirmado pela capacidade de ativação dos mecanismos de sinalização das BMPs. No entanto, e apesar da conservação ao nível da região codante dos genes das BMP2, BMP4 e BMP16, as regiões não traduzidas são substancialmente diferentes, apontando para uma regulação diferencial dos três genes, como é aliás sugerido pelos distintos padrões de expressão observados para a BMP2, BMP4 e BMP16, tanto em linguado como em peixe zebra. Ao longo deste trabalho foram recolhidos novos dados que permitem uma melhor compreensão da função e regulação da BMP2. Foram igualmente obtidas informações relevantes acerca da filogenia molecular dos membros da subfamília das BMP2/4/16 que contribuíram para uma melhor compreensão e interpretação da complexa história evolutiva desta subfamília. No seu conjunto, os resultados deste trabalho contribuem para uma validação do uso dos peixes como um modelo alternativo na investigação de mecanismos moleculares envolvidos no processo de mineralização tecidular.Universidade do Algarve, Departamento de Ciências Biomédica

Comparative analysis of zebrafish bone morphogenetic proteins 2, 4 and 16: molecular and evolutionary perspectives

BMP2, BMP4 and BMP16 form a subfamily of bone morphogenetic proteins acting as pleiotropic growth factors during development and as bone inducers during osteogenesis. BMP16 is the most recent member of this subfamily and basic data regarding protein structure and function, and spatio-temporal gene expression is still scarce. In this work, insights on BMP16 were provided through the comparative analysis of structural and functional data for zebrafish BMP2a, BMP2b, BMP4 and BMP16 genes and proteins, determined from three-dimensional models, patterns of gene expression during development and in adult tissues, regulation by retinoic acid and capacity to activate BMP-signaling pathway. Structures of Bmp2a, Bmp2b, Bmp4 and Bmp16 were found to be remarkably similar; with residues involved in receptor binding being highly conserved. All proteins could activate the BMP-signaling pathway, suggesting that they share a common function. On the contrary, stage-and tissue-specific expression of bmp2, bmp4 and bmp16 suggested the genes might be differentially regulated (e.g. different transcription factors, enhancers and/or regulatory modules) but also that they are involved in distinct physiological processes, although with the same function. Retinoic acid, a morphogen known to interact with BMP-signaling during bone formation, was shown to downregulate the expression of bmp2, bmp4 and bmp16, although to different extents. Taxonomic and phylogenetic analyses indicated that bmp16 diverged before bmp2 and bmp4, is not restricted to teleost fish lineage as previously reported, and that it probably arose from a whole genomic duplication event that occurred early in vertebrate evolution and disappeared in various tetrapod lineages through independent events

Phylogenetic analysis of the vertebrate Excitatory/Neutral Amino Acid Transporter (SLC1/EAAT) family reveals lineage specific subfamilies

BACKGROUND: The composition and expression of vertebrate gene families is shaped by species specific gene loss in combination with a number of gene and genome duplication events (R1, R2 in all vertebrates, R3 in teleosts) and depends on the ecological and evolutionary context. In this study we analyzed the evolutionary history of the solute carrier 1 (SLC1) gene family. These genes are supposed to be under strong selective pressure (purifying selection) due to their important role in the timely removal of glutamate at the synapse. RESULTS: In a genomic survey where we manually annotated and analyzing sequences from more than 300 SLC1 genes (from more than 40 vertebrate species), we found evidence for an interesting evolutionary history of this gene family. While human and mouse genomes contain 7 SLC1 genes, in prototheria, sauropsida, and amphibia genomes up to 9 and in actinopterygii up to 13 SLC1 genes are present. While some of the additional slc1 genes in ray-finned fishes originated from R3, the increased number of SLC1 genes in prototheria, sauropsida, and amphibia genomes originates from specific genes retained in these lineages.Phylogenetic comparison and microsynteny analyses of the SLC1 genes indicate, that theria genomes evidently lost several SLC1 genes still present in the other lineage. The genes lost in theria group into two new subfamilies of the slc1 gene family which we named slc1a8/eaat6 and slc1a9/eaat7. CONCLUSIONS: The phylogeny of the SLC1/EAAT gene family demonstrates how multiple genome reorganization and duplication events can influence the number of active genes. Inactivation and preservation of specific SLC1 genes led to the complete loss of two subfamilies in extant theria, while other vertebrates have retained at least one member of two newly identified SLC1 subfamilies

Unresolved orthology and peculiar coding sequence properties of lamprey genes: the KCNA gene family as test case

Background:In understanding the evolutionary process of vertebrates, cyclostomes (hagfishes and lamprey) occupy crucial positions. Resolving molecular phylogenetic relationships of cyclostome genes with gnathostomes (jawed vertebrates) genes is indispensable in deciphering both the species tree and gene trees. However, molecular phylogenetic analyses, especially those including lamprey genes, have produced highly discordant results between gene families. To efficiently scrutinize this problem using partial genome assemblies of early vertebrates, we focused on the potassium voltage-gated channel, shaker-related (KCNA) family, whose members are mostly single-exon.Results:Seven sea lamprey KCNA genes as well as six elephant shark genes were identified, and their orthologies to bony vertebrate subgroups were assessed. In contrast to robustly supported orthology of the elephant shark genes to gnathostome subgroups, clear orthology of any sea lamprey gene could not be established. Notably, sea lamprey KCNA sequences displayed unique codon usage pattern and amino acid composition, probably associated with exceptionally high GC-content in their coding regions. This lamprey-specific property of coding sequences was also observed generally for genes outside this gene family.Conclusions:Our results suggest that secondary modifications of sequence properties unique to the lamprey lineage may be one of the factors preventing robust orthology assessments of lamprey genes, which deserves further genome-wide validation. The lamprey lineage-specific alteration of protein-coding sequence properties needs to be taken into consideration in tackling the key questions about early vertebrate evolution

Ancestral and derived attributes of the dlx gene repertoire, cluster structure and expression patterns in an African cichlid fish

<p>Abstract</p> <p>Background</p> <p>Cichlid fishes have undergone rapid, expansive evolutionary radiations that are manifested in the diversification of their trophic morphologies, tooth patterning and coloration. Understanding the molecular mechanisms that underlie the cichlids' unique patterns of evolution requires a thorough examination of genes that pattern the neural crest, from which these diverse phenotypes are derived. Among those genes, the homeobox-containing <it>Dlx </it>gene family is of particular interest since it is involved in the patterning of the brain, jaws and teeth.</p> <p>Results</p> <p>In this study, we characterized the <it>dlx </it>genes of an African cichlid fish, <it>Astatotilapia burtoni</it>, to provide a baseline to later allow cross-species comparison within Cichlidae. We identified seven <it>dlx </it>paralogs (<it>dlx1a</it>, <it>-2a</it>, <it>-4a</it>, <it>-3b</it>, <it>-4b</it>, <it>-5a </it>and <it>-6a</it>), whose orthologies were validated with molecular phylogenetic trees. The intergenic regions of three <it>dlx </it>gene clusters (<it>dlx1a-2a</it>, <it>dlx3b-4b</it>, and <it>dlx5a-6a</it>) were amplified with long PCR. Intensive cross-species comparison revealed a number of conserved non-coding elements (CNEs) that are shared with other percomorph fishes. This analysis highlighted additional lineage-specific gains/losses of CNEs in different teleost fish lineages and a novel CNE that had previously not been identified. Our gene expression analyses revealed overlapping but distinct expression of <it>dlx </it>orthologs in the developing brain and pharyngeal arches. Notably, four of the seven <it>A. burtoni dlx </it>genes, <it>dlx2a</it>, <it>dlx3b</it>, <it>dlx4a </it>and <it>dlx5a</it>, were expressed in the developing pharyngeal teeth.</p> <p>Conclusion</p> <p>This comparative study of the <it>dlx </it>genes of <it>A. burtoni </it>has deepened our knowledge of the diversity of the <it>Dlx </it>gene family, in terms of gene repertoire, expression patterns and non-coding elements. We have identified possible cichlid lineage-specific changes, including losses of a subset of <it>dlx </it>expression domains in the pharyngeal teeth, which will be the targets of future functional studies.</p

Use and efficacy of bone morphogenetic proteins in fracture healing

Signal transduction in aging related disease