Calcareous sponge genomes reveal complex evolution of alpha-carbonic anhydrases and two key biomineralization enzymes

Background: Calcium carbonate biominerals form often complex and beautiful skeletal elements, including coral exoskeletons and mollusc shells. Although the ability to generate these carbonate structures was apparently gained independently during animal evolution, it sometimes involves the same gene families. One of the best-studied of these gene families comprises the a-carbonic anhydrases (CAs), which catalyse the reversible transformation of CO2 to HCO3- and fulfill many physiological functions. Among Porifera -the oldest animal phylum with the ability to produce skeletal elements- only the class of calcareous sponges can build calcitic spicules, which are the extracellular products of specialized cells, the sclerocytes. Little is known about the molecular mechanisms of their synthesis, but inhibition studies suggest an essential role of CAs. In order to gain insight into the evolution and function of CAs in biomineralization of a basal metazoan species, we determined the diversity and expression of CAs in the calcareous sponges Sycon ciliatum and Leucosolenia complicata by means of genomic screening, RNA-Seq and RNA in situ hybridization expression analysis. Active biomineralization was located with calcein-staining. Results: We found that the CA repertoires of two calcareous sponge species are strikingly more complex than those of other sponges. By characterizing their expression patterns, we could link two CAs (one intracellular and one extracellular) to the process of calcite spicule formation in both studied species. The extracellular biomineralizing CAs seem to be of paralogous origin, a finding that advises caution against assuming functional conservation of biomineralizing genes based upon orthology assessment alone. Additionally, calcareous sponges possess acatalytic CAs related to human CAs X and XI, suggesting an ancient origin of these proteins. Phylogenetic analyses including CAs from genomes of all non-bilaterian phyla suggest multiple gene losses and duplications and presence of several CAs in the last common ancestor of metazoans. Conclusions: We identified two key biomineralization enzymes from the CA-family in calcareous sponges and propose their possible interaction in spicule formation. The complex evolutionary history of the CA family is driven by frequent gene diversification and losses. These evolutionary patterns likely facilitated the numerous events of independent recruitment of CAs into biomineralization within Metazoa

Spicule formation in calcareous sponges: Coordinated expression of biomineralization genes and spicule-type specific genes

The ability to form mineral structures under biological control is widespread among animals. In several species, specific proteins have been shown to be involved in biomineralization, but it is uncertain how they influence the shape of the growing biomineral and the resulting skeleton. Calcareous sponges are the only sponges that form calcitic spicules, which, based on the number of rays (actines) are distinguished in diactines, triactines and tetractines. Each actine is formed by only two cells, called sclerocytes. Little is known about biomineralization proteins in calcareous sponges, other than that specific carbonic anhydrases (CAs) have been identified, and that uncharacterized Asx-rich proteins have been isolated from calcitic spicules. By RNA-Seq and RNA in situ hybridization (ISH), we identified five additional biomineralization genes in Sycon ciliatum: two bicarbonate transporters (BCTs) and three Asx-rich extracellular matrix proteins (ARPs). We show that these biomineralization genes are expressed in a coordinated pattern during spicule formation. Furthermore, two of the ARPs are spicule-type specific for triactines and tetractines (ARP1 or SciTriactinin) or diactines (ARP2 or SciDiactinin). Our results suggest that spicule formation is controlled by defined temporal and spatial expression of spicule-type specific sets of biomineralization genes

Wound healing and regeneration in the reef building coral Acropora millepora

Branching scleractinian corals are niche-constructing organisms, providing continuously-growing, structural foundation for spectacularly biodiverse coral reef ecosystems. A large part of their success lies in the ability to quickly regenerate following mechanical damage. Even now, when the corals undergo great decline due to anthropogenic weather and storm extremes, it is surprising how little is known about molecular mechanisms governing regeneration in these iconic organisms. In this study, we used RNA-seq to identify genes involved in the regeneration of Acropora millepora, starting with the initial wound closure up to complete rebuilding of lost structures. Many of the differentially expressed genes we found in the wound healing steps are homologues of genes known to be involved in wound healing and regeneration of bilaterian and other cnidarian species, prominently including multiple components of FGF and Wnt signalling pathways. Comparison between genes involved in wound healing and continuous growth of the colony demonstrates both similarity and distinctiveness of the genetic programmes controlling these processes. A striking example is specific expression of c-Fos, a transcription factor with conserved role in early injury response, during the earliest stages of wound healing of A. millepora. By comparing results obtained in diverse experimental conditions including a closed-loop, recirculating aquarium and a flow-through system of marine station, we have demonstrated feasibility of using zooxanthellate scleractinian corals as experimental models in fundamental biology research, including studies of regeneration

Early divergence, broad distribution, and high diversity of animal chitin synthases

Even though chitin is one of themost abundant biopolymers in nature, current knowledge on chitin formation is largely based only on data from fungi and insects. This study reveals unanticipated broad taxonomic distribution and extensive diversification of chitin synthases (CSs) in Metazoa, shedding newlight on the relevance of chitin in animals and suggesting unforeseen complexity of chitin synthesis in many groups. We uncovered robust orthologs to insect type CSs in several representatives of deuterostomes, which generally are not thought to possess chitin. This suggests a broader distribution and function of chitin in this branch of the animal kingdom. We characterize a new CS type present not only in basal metazoans such as sponges and cnidarians but also in several bilaterian representatives. Themost extensive diversification of CSs took place during emergence of lophotrochozoans, the third large group of protostomes next to arthropods and nematodes, resulting in coexistence of up to ten CS paralogs inmolluscs. Independent fusion to different kinds of myosinmotor domains in fungi and lophotrochozoans points toward high relevance of CS interaction with the cytoskeleton for fine-tuned chitin secretion. Given the fundamental role that chitin plays in themorphology of many animals, the here presented CS diversification revealsmany evolutionary complexities. Our findings strongly suggest a very broad andmultifarious occurrence of chitin and question an ancestral role as cuticular component. The molecular mechanisms underlying regulation of animal chitin synthesis are most likely far more complex and diverse than existing data from insects suggest

Spicule formation in calcareous sponges: Coordinated expression of biomineralization genes and spicule-type specific genes

The ability to form mineral structures under biological control is widespread among animals. In several species, specific proteins have been shown to be involved in biomineralization, but it is uncertain how they influence the shape of the growing biomineral and the resulting skeleton. Calcareous sponges are the only sponges that form calcitic spicules, which, based on the number of rays (actines) are distinguished in diactines, triactines and tetractines. Each actine is formed by only two cells, called sclerocytes. Little is known about biomineralization proteins in calcareous sponges, other than thatspecific carbonic anhydrases (CAs) have been identified, and that uncharacterized Asx-rich proteins have been isolated from calcitic spicules. By RNA-Seq and RNA in situ hybridization (ISH), we identified five additional biomineralization genes in Sycon ciliatum: two bicarbonate transporters (BCTs) and three Asx-rich extracellular matrix proteins (ARPs). We show that these biomineralization genes are expressed in a coordinated pattern during spicule formation. Furthermore, two of the ARPs are spiculetype specific for triactines and tetractines (ARP1 or SciTriactinin) or diactines (ARP2 or SciDiactinin). Our results suggest that spicule formation is controlled by defined temporal and spatial expression of spicule-type specific sets of biomineralization gene

Early evolution of the T-box transcription factor family

Deèelopmental transcription factors are key players in animal multicellularity, being members of the T-box family that are among the most important. Until recently, T-box transcription factors were thought to be exclusièely present in metazoans. Here, we report the presence of T-box genes in seèeral nonmetazoan lineages, including ichthyosporeans, filastereans, and fungi. Our data confirm that Brachyury is the most ancient member of the T-box family and establish that the T-box family dièersified at the onset of Metazoa. Moreoèer, we demonstrate functional conserèation of a homolog of Brachyury of the protist Capsaspora owczarzaki in Xenopus laeèis. By comparing the molecular phenotype of C. owczarzaki Brachyury with that of homologs of early branching metazoans, we define a clear difference between unicellular holozoan and metazoan Brachyury homologs, suggesting that the specificity of Brachyury emerged at the origin of Metazoa. Experimental determination of the binding preferences of the C. owczarzaki Brachyury results in a similar motif to that of metazoan Brachyury and other T-box classes. This finding suggests that functional specificity between different T-box classes is likely achieèed by interaction with alternatièe cofactors, as opposed to differences in binding specificity

Traffic-related pollution (NO2 and CO) and its association with asthma symptoms among 10-11 year old children in Malaysia

Introduction: Children attending schools located close to major traffic sources are exposed to higher indoor air pollution levels due to infiltration of outdoor air pollutants. This study investigates the respiratory health of Malaysian schoolchildren in relation to indoor air pollution in schools and at home. Methods: This cross-sectional study was conducted among 2,164 schoolchildren aged 10-11 years in 35 urban and semi-rural schools in Kuala Lumpur Negeri Sembilan. Parents completed the International Study of Asthma and Allergy in Childhood questionnaire and provided information on the home environment and socio demographic background. Concentrations of NO2 and CO were measured indoors and outdoors of 14 schools. Results: The mean (standard deviation (SD)) indoor exposure to concentrations of air pollutants measured in Malaysian schools were: 42.9 (8.77) μg/m3 of NO2 and 3.16 (2.47) ppm of CO in urban schools and 12.4 (8.79) μg/m3 of NO2 and 0.33 (0.18) ppm of CO respectively for semi-rural schools. The prevalence of asthma was significantly higher among urban compared to semi-rural children. Ever wheeze was more common in urban areas (14.3%) compared to semi-rural areas (10.6%) as was current wheeze (urban 9.1%; semi-rural 6.2%), nocturnal cough (urban 12.0%; semi-rural 7.2%) and most other symptoms. The 3 main predictors of nocturnal cough were the presence of a current smoker at home (OR 1.97; 95% CI: 1.31-2.96), the absence of exhaust system in the kitchen (OR 1.67; 95% CI: 1.03-2.68) and the use of air conditioning (OR 3.10; 95% CI: 1.37-5.71). Self-reported asthma was predicted by the occupation of mothers; managerial positions (OR 1.78; 95% CI: 1.21-2.62), mothers working in the armed forces (OR 1.92; 95% CI: 1.32- 2.78) and fathers with a high school level of education (OR 0.46; 95% CI: 0.26- 0.82) while exposure to SHS of ≥4 hours per day (OR 2.53; 95% CI: 1.55-4.14) explained approximately 6.0% of the likelihood of reporting doctor-diagnosed asthma. Conclusion: This study has identified that many Malaysian schoolchildren experience exposure to air pollution indoors that exceeds WHO guidance limits for NO2 pollution but did not exceed the RMAQG levels. Considering that traffic-related sources largely contribute towards NO2 concentrations, compliance with the standards is not indicative of the reduced health risks for air pollution exposures

Mutation of FIG4 causes neurodegeneration in the pale tremor mouse and patients with CMT4J

Membrane-bound phosphoinositides are signalling molecules that have a key role in vesicle trafficking in eukaryotic cells(1). Proteins that bind specific phosphoinositides mediate interactions between membrane-bounded compartments whose identity is partially encoded by cytoplasmic phospholipid tags. Little is known about the localization and regulation of mammalian phosphatidylinositol-3,5-bisphosphate ( PtdIns( 3,5)P-2), a phospholipid present in small quantities that regulates membrane trafficking in the endosome - lysosome axis in yeast(2). Here we describe a multi-organ disorder with neuronal degeneration in the central nervous system, peripheral neuronopathy and diluted pigmentation in the 'pale tremor' mouse. Positional cloning identified insertion of ETn2 beta ( early transposon 2 beta)(3) into intron 18 of Fig4 (A530089I17Rik), the homologue of a yeast SAC ( suppressor of actin) domain PtdIns(3,5) P-2 5-phosphatase located in the vacuolar membrane. The abnormal concentration of PtdIns( 3,5) P2 in cultured fibroblasts from pale tremor mice demonstrates the conserved biochemical function of mammalian Fig4. The cytoplasm of fibroblasts from pale tremor mice is filled with large vacuoles that are immunoreactive for LAMP-2 (lysosomal-associated membrane protein 2), consistent with dysfunction of the late endosome - lysosome axis. Neonatal neurodegeneration in sensory and autonomic ganglia is followed by loss of neurons from layers four and five of the cortex, deep cerebellar nuclei and other localized brain regions. The sciatic nerve exhibits reduced numbers of large-diameter myelinated axons, slowed nerve conduction velocity and reduced amplitude of compound muscle action potentials. We identified pathogenic mutations of human FIG4 (KIAA0274) on chromosome 6q21 in four unrelated patients with hereditary motor and sensory neuropathy. This novel form of autosomal recessive Charcot - Marie - Tooth disorder is designated CMT4J.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/62835/1/nature05876.pd

Reconstruction of Family-Level Phylogenetic Relationships within Demospongiae (Porifera) Using Nuclear Encoded Housekeeping Genes

Background: Demosponges are challenging for phylogenetic systematics because of their plastic and relatively simple morphologies and many deep divergences between major clades. To improve understanding of the phylogenetic relationships within Demospongiae, we sequenced and analyzed seven nuclear housekeeping genes involved in a variety of cellular functions from a diverse group of sponges. Methodology/Principal Findings: We generated data from each of the four sponge classes (i.e., Calcarea, Demospongiae, Hexactinellida, and Homoscleromorpha), but focused on family-level relationships within demosponges. With data for 21 newly sampled families, our Maximum Likelihood and Bayesian-based approaches recovered previously phylogenetically defined taxa: Keratosap, Myxospongiaep, Spongillidap, Haploscleromorphap (the marine haplosclerids) and Democlaviap. We found conflicting results concerning the relationships of Keratosap and Myxospongiaep to the remaining demosponges, but our results strongly supported a clade of Haploscleromorphap+Spongillidap+Democlaviap. In contrast to hypotheses based on mitochondrial genome and ribosomal data, nuclear housekeeping gene data suggested that freshwater sponges (Spongillidap) are sister to Haploscleromorphap rather than part of Democlaviap. Within Keratosap, we found equivocal results as to the monophyly of Dictyoceratida. Within Myxospongiaep, Chondrosida and Verongida were monophyletic. A well supported clade within Democlaviap, Tetractinellidap, composed of all sampled members of Astrophorina and Spirophorina (including the only lithistid in our analysis), was consistently revealed as the sister group to all other members of Democlaviap. Within Tetractinellidap, we did not recover monophyletic Astrophorina or Spirophorina. Our results also reaffirmed the monophyly of order Poecilosclerida (excluding Desmacellidae and Raspailiidae), and polyphyly of Hadromerida and Halichondrida. Conclusions/Significance: These results, using an independent nuclear gene set, confirmed many hypotheses based on ribosomal and/or mitochondrial genes, and they also identified clades with low statistical support or clades that conflicted with traditional morphological classification. Our results will serve as a basis for future exploration of these outstanding questions using more taxon- and gene-rich datasets