High-precision morphology: bifocal 4D-microscopy enables the comparison of detailed cell lineages of two chordate species separated for more than 525 million years

Background

Understanding the evolution of divergent developmental trajectories requires detailed comparisons of embryologies at appropriate levels. Cell lineages, the accurate visualization of cleavage patterns, tissue fate restrictions, and morphogenetic movements that occur during the development of individual embryos are currently available for few disparate animal taxa, encumbering evolutionarily meaningful comparisons. Tunicates, considered to be close relatives of vertebrates, are marine invertebrates whose fossil record dates back to 525 million years ago. Life-history strategies across this subphylum are radically different, and include biphasic ascidians with free swimming larvae and a sessile adult stage, and the holoplanktonic larvaceans. Despite considerable progress, notably on the molecular level, the exact extent of evolutionary conservation and innovation during embryology remain obscure.

Results

Here, using the innovative technique of bifocal 4D-microscopy, we demonstrate exactly which characteristics in the cell lineages of the ascidian Phallusia mammillata and the larvacean Oikopleura dioica were conserved and which were altered during evolution. Our accurate cell lineage trees in combination with detailed three-dimensional representations clearly identify conserved correspondence in relative cell position, cell identity, and fate restriction in several lines from all prospective larval tissues. At the same time, we precisely pinpoint differences observable at all levels of development. These differences comprise fate restrictions, tissue types, complex morphogenetic movement patterns, numerous cases of heterochronous acceleration in the larvacean embryo, and differences in bilateral symmetry.

Conclusions

Our results demonstrate in extraordinary detail the multitude of developmental levels amenable to evolutionary innovation, including subtle changes in the timing of fate restrictions as well as dramatic alterations in complex morphogenetic movements. We anticipate that the precise spatial and temporal cell lineage data will moreover serve as a high-precision guide to devise experimental investigations of other levels, such as molecular interactions between cells or changes in gene expression underlying the documented structural evolutionary changes. Finally, the quantitative amount of digital high-precision morphological data will enable and necessitate software-based similarity assessments as the basis of homology hypotheses.

     

Metabolic dominance of bivalves predates brachiopod diversity decline by more than 150 million years

Brachiopods and bivalves feed in similar ways and have occupied the same environments through geological time, but brachiopods were far more diverse and abundant in the Palaeozoic whereas bivalves dominate the post-Palaeozoic, suggesting a transition in ecological dominance 250 Ma. However, diversity and abundance data alone may not adequately describe key changes in ecosystem function, such as metabolic activity. Here, we use newly compiled body size data for 6066 genera of bivalves and brachiopods to calculate metabolic rates and revisit this question from the perspective of energy use, finding that bivalves already accounted for a larger share of metabolic activity in Palaeozoic oceans. We also find that the metabolic activity of bivalves has increased by more than two orders of magnitude over this interval, whereas brachiopod metabolic activity has declined by more than 50%. Consequently, the increase in bivalve energy metabolism must have occurred via the acquisition of new food resources rather than through the displacement of brachiopods. The canonical view of a mid-Phanerozoic transition from brachiopod to bivalve dominance results from a focus on taxonomic diversity and numerical abundance as measures of ecological importance. From a metabolic perspective, the oceans have always belonged to the clams.     

A group of bactericidal factors conserved by vertebrates for more than 300 million years

A complement-dependent bactericidal factor (RaRF) specific for the Ra chemotype strains of Salmonella and for the Ra-like strains of other enterobacteria has been found in sera of mice. We show here that the anti-Ra bactericidal factors are present in sera of all species, so far tested, of mammals, birds, reptiles, amphibians, and bony and cartilaginous fish. Certain properties, such as binding specificity, requirement of divalent cations for binding, and sensitivities to heat and reducing agents, of the factor in the representative animals were the same as or similar to those of the mouse RaRF. These results indicate that these factors have been conserved by vertebrates for more than 300 million years as a result of the necessity for the resistance to rough mutants of Gram-negative bacteria.     

HeT-A and TART, two Drosophila retrotransposons with a bona fide role in chromosome structure for more than 60 million years

Drosophila telomeres have been maintained by retrotransposition for at least 60 MY, which predates the separation of extant species of this genus. Studies of D. melanogaster, D. yakuba, and D. virilis show that, in Drosophila, telomeres are composed of two non-LTR retrotransposons, HeT-A and TART. Far from being static, HeT-A and TART evolve faster than Drosophila euchromatic genes. In spite of their high rate of sequence change, HeT-A and TART maintain their basic structures and unusual individual features. The maintenance of their separate identities suggests that HeT-A and TART cooperate either in the process of retrotransposition onto the chromosome end, or in the formation of telomere chromatin by transposed DNA copies. The telomeric retrotransposons and the Drosophila genome constitute an example of a robust symbiotic relationship between mobile elements and the genome.     

On the persistence of supplementary resources in biomedical publications

Background  

Providing for long-term and consistent public access to scientific data is a growing concern in biomedical research. One aspect of this problem can be demonstrated by evaluating the persistence of supplementary data associated with published biomedical papers.     

Patterns of aggregation behaviour in six species of cockroach: comparing two experimental approaches

Cockroaches (Blattaria) are considered universally gregarious insects. In this study, we tested the hypothesis that even related species and/or males and females of the same species may exhibit different aggregation patterns. We compared aggregation behaviour in six cockroach species belonging to three groups: Eublaberus, Blaberus, and Gromphadorhina‐Princisia (all Blaberidae). For comparison, binary choice and free aggregation arena tests were used. The results revealed that blaberid clades clearly differed in their aggregation patterns. Females of Eublaberus species aggregated, whereas only a low aggregation tendency was recorded in conspecific males. Both sexes of Blaberus species aggregated, especially when tested in the arena. The Madagascar hissing cockroaches of the Gromphadorhina‐Princisia clade are not gregarious – the aggregation tendency was entirely absent in males of the two species studied and in females of Princisia vanwaerebeki van Herrewege, and was only weakly demonstrated in females of Gromphadorhina portentosa (Schaum). The observed patterns of aggregation are compared with previous reports concerning social behaviour of blaberid species.     

Identification and characterization of more than 4 million intervarietal SNPs across the group 7 chromosomes of bread wheat

Despite being a major international crop, our understanding of the wheat genome is relatively poor due to its large size and complexity. To gain a greater understanding of wheat genome diversity, we have identified single nucleotide polymorphisms between 16 Australian bread wheat varieties. Whole‐genome shotgun Illumina paired read sequence data were mapped to the draft assemblies of chromosomes 7A, 7B and 7D to identify more than 4 million intervarietal SNPs. SNP density varied between the three genomes, with much greater density observed on the A and B genomes than the D genome. This variation may be a result of substantial gene flow from the tetraploid Triticum turgidum, which possesses A and B genomes, during early co‐cultivation of tetraploid and hexaploid wheat. In addition, we examined SNP density variation along the chromosome syntenic builds and identified genes in low‐density regions which may have been selected during domestication and breeding. This study highlights the impact of evolution and breeding on the bread wheat genome and provides a substantial resource for trait association and crop improvement. All SNP data are publically available on a generic genome browser GBrowse at www.wheatgenome.info .     

Estimating the responsiveness of an instrument using more than two repeated measures

The responsiveness of a measuring instrument is its ability to detect change over time. A commonly used index of responsiveness is the effect size for paired differences. This paper generalizes the effect size for paired differences to more than two repeated observations per subject. The sampling distribution of the generalized responsiveness statistic, Rt, is simulated for a range of plausible parameter values and for a range of sample sizes varying both the number of subjects (n) and the number of observations per subject (t). The coverage properties of confidence intervals constructed by four methods are compared. Confidence intervals based on jackknife estimates of the standard error and bias of Rt have good coverage properties even when n and t are small. The methods are used to determine which of two standard quality-of-life measures is more responsive to improvements in quality of life following surgery for early-stage breast cancer.     

Detection of heteromerization of more than two proteins by sequential BRET-FRET

Identification of higher-order oligomers in the plasma membrane is essential to decode the properties of molecular networks controlling intercellular communication. We combined bioluminescence resonance energy transfer (BRET) and fluorescence resonance energy transfer (FRET) in a technique called sequential BRET-FRET (SRET) that permits identification of heteromers formed by three different proteins. In SRET, the oxidation of a Renilla luciferase (Rluc) substrate by an Rluc fusion protein triggers acceptor excitation of a second fusion protein by BRET and subsequent FRET to a third fusion protein. We describe two variations of SRET that use different Rluc substrates with appropriately paired acceptor fluorescent proteins. Using SRET, we identified complexes of cannabinoid CB(1), dopamine D(2) and adenosine A(2A) receptors in living cells. SRET is an invaluable technique to identify heteromeric complexes of more than two neurotransmitter receptors, which will allow us to better understand how signals are integrated at the molecular level.     

Smaller fields support more butterflies: comparing two neighbouring European countries with different socioeconomic heritage

Changing landscape configuration, a component of landscape heterogeneity, due to varying arable field size represents an attribute of farmland intensification with potentially grave consequences for biodiversity. We recorded butterflies in an intensively farmed region along the border of two European countries with contrasting socioeconomic heritage, one with small farms (Poland), the other with huge farmed units (Czech Republic). Although only the most common generalist species occurred in our records, we observed 2.3× more individuals and 1.9× more species in Poland, and the differences withstood robust statistical controls. Different socioeconomic heritage—in this case, different policies of two communist regimes, one tolerating small family farms and the other expropriating them—influence biodiversity long after the policies that shaped the respective landscapes had been abandoned.     

13C tracer experiments and metabolite balancing for metabolic flux analysis: comparing two approaches

Conventional metabolic flux analysis uses the information gained from determination of measurable fluxes and a steady-state assumption for intracellular metabolites to calculate the metabolic fluxes in a given metabolic network. The determination of intracellular fluxes depends heavily on the correctness of the assumed stoichiometry including the presence of all reactions with a noticeable impact on the model metabolite balances. Determination of fluxes in complex metabolic networks often requires the inclusion of NADH and NADPH balances, which are subject to controversial debate. Transhydrogenation reactions that transfer reduction equivalents from NADH to NADPH or vice versa can usually not be included in the stoichiometric model, because they result in singularities in the stoichiometric matrix. However, it is the NADPH balance that, to a large extent, determines the calculated flux through the pentose phosphate pathway. Hence, wrong assumptions on the presence or activity of transhydrogenation reactions will result in wrong estimations of the intracellular flux distribution. Using 13C tracer experiments and NMR analysis, flux analysis can be performed on the basis of only well established stoichiometric equations and measurements of the labeling state of intracellular metabolites. Neither NADH/NADPH balancing nor assumptions on energy yields need to be included to determine the intracellular fluxes. Because metabolite balancing methods and the use of 13C labeling measurements are two different approaches to the determination of intracellular fluxes, both methods can be used to verify each other or to discuss the origin and significance of deviations in the results. Flux analysis based entirely on metabolite balancing and flux analysis, including labeling information, have been performed independently for a wild-type strain of Aspergillus oryzae producing alpha-amylase. Two different nitrogen sources, NH4+ and NO3-, have been used to investigate the influence of the NADPH requirements on the intracellular flux distribution. The two different approaches to the calculation of fluxes are compared and deviations in the results are discussed. Copyright 1998 John Wiley & Sons, Inc.     

BoLA class I charge heterogeneity reflects the expression of more than two loci

Internationally recognized allo-antisera in lymphocyte microcytotoxicity assays are thought to detect allelic products of a single highly polymorphic class I locus. A recent report suggested that two bovine lymphocyte antigen (BoLA) class I loci are expressed at the protein level. However, 1D-IEF analysis of BoLA class I molecules reveals multi-band patterns which cannot be reconciled with the reported number of loci. The aim of this study was to investigate the origins of the charge diversity of BoLA class I molecules observed using 1D-IEF. BoLA class I molecules appear to be glycosy-lated at a single N-linked position with a complex type carbohydrate moiety which has up to three terminal sialic acid residues. Class I molecules immunoprecipitated from resting bovine PBL are not phosphorylated. Neither modification is responsible for the observed charge heterogeneity. Peptide mapping reveals that different BoLA charge variants have distinct digestion patterns. Furthermore, a number of different polypeptides are associated with each serological specificity. These polypeptides appear to be encoded by different loci which exist in linkage disequilibrium. The number of charge variants with different peptide maps indicates that the BoLA system has a minimum of three class I loci expressed at the protein level.     

Knowledge-based biomedical word sense disambiguation: comparison of approaches

Background  

Word sense disambiguation (WSD) algorithms attempt to select the proper sense of ambiguous terms in text. Resources like the UMLS provide a reference thesaurus to be used to annotate the biomedical literature. Statistical learning approaches have produced good results, but the size of the UMLS makes the production of training data infeasible to cover all the domain.     

Bacterial phytochromes: more than meets the light

Phytochromes are environmental sensors, historically thought of as red/far-red photoreceptors in plants. Their photoperception occurs through a covalently linked tetrapyrrole chromophore, which undergoes a light-dependent conformational change propagated through the protein to a variable output domain. The phytochrome composition is modular, typically consisting of a PAS-GAF-PHY architecture for the N-terminal photosensory core. A collection of three-dimensional structures has uncovered key features, including an unusual figure-of-eight knot, an extension reaching from the PHY domain to the chromophore-binding GAF domain, and a centrally located, long α-helix hypothesized to be crucial for intramolecular signaling. Continuing identification of phytochromes in microbial systems has expanded the assigned sensory abilities of this family out of the red and into the yellow, green, blue, and violet portions of the spectrum. Furthermore, phytochromes acting not as photoreceptors but as redox sensors have been recognized. In addition, architectures other than PAS-GAF-PHY are known, thus revealing phytochromes to be a varied group of sensory receptors evolved to utilize their modular design to perceive a signal and respond accordingly. This review focuses on the structures of bacterial phytochromes and implications for signal transmission. We also discuss the small but growing set of bacterial phytochromes for which a physiological function has been ascertained.     

Re-evolution of lost mandibular teeth in frogs after more than 200 million years, and re-evaluating Dollo's law

Dollo's law states that structures that are evolutionarily lost will not be regained. Recent phylogenetic studies have revealed several potential examples in which Dollo's law seems to be violated, where lost structures appear to have been regained over evolutionary time. However, these examples have recently been questioned and suggested to be methodological artifacts. In this article, I document a striking and incontrovertible phylogenetic example of the re-evolution of a lost, complex structure: mandibular teeth in the frog genus Gastrotheca. I use a time-calibrated phylogeny for 170 amphibian species to show that mandibular teeth were lost in the ancestor of modern frogs at least 230 million years ago (Mya) and have been regained in the last ～ 5-17 My. I review recent studies on trait re-evolution and show that this long period of trait absence prior to re-acquisition is largely unprecedented. I also argue that there are several methodological issues that may cause trait re-evolution to be hardest to detect under those conditions when it is most likely to occur, leading to erroneous failures to reject Dollo's law. Finally, I discuss a mechanism that may facilitate trait re-evolution, and the evolution of mandibular teeth in frogs as an example of developmental constraint.