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Background/Objectives: This study examines the factors associated with U.S. adolescents’ obesity and overweight status. Methods: Using a multivariable logistic regression, we analyzed the data from the 2021 Youth Risk Behavior Surveillance System (YRBSS), comprising 17,232 students. Results: The odds of being obese or overweight were significantly higher (p ≤ 0.05) for the participants who perceived themselves as slightly overweight (AOR, 13.31; 95% CI [11.83, 14.97]) or very overweight (AOR, 39.29; 95% CI [30.12, 51.25]) compared to those who perceived their weight as about right. The participants with significantly higher odds included those aged 14 years (AOR, 2.53) compared to those aged 13 years or younger; male students (AOR, 1.63) compared to female students; and American Indian/Alaska Native and Native Hawaiian/Other Pacific Islander students (AOR, 2.11), Black or African American students (AOR, 2.63), Hispanic/Latino students (AOR, 1.54), and students of multiple races (AOR, 1.56), compared to White students. The odds were also significantly higher for the participants who did not eat breakfast on all seven days of a week (AOR, 1.21) and for the students who did not report their mental health status (AOR, 2.07) compared to those who reported their mental health as mostly or always not good. Conclusions: These findings suggest schools are uniquely positioned to implement strategies for healthier behaviors designed and implemented with a focus on health equity.

FUNAABOR-2 is a popular Ofada rice variety grown in a large area under rainfed upland condition across western states of Nigeria. We used the combination of phenotypic and marker-assisted selection (MAS) to improve grain yield of FUNAABOR-2 under drought stress (DS) at the reproductive stage via introgression of two drought quantitative trait loci (QTLs), qDTY12.1 and qDTY2.3. Foreground selection was carried out using peak markers RM511 and RM250, associated with qDTY12.1 and qDTY2.3, respectively, followed by recombinant selection with RM28099 and RM1261 distally flanking qDTY12.1. Furthermore, BC1F2-derived introgressed lines and their parents were evaluated under DS and non-stress (NS) conditions during the 2015–2016 dry season. Overall reduction of grain yield under DS compared to NS was recorded. Introgressed lines with qDTY12.1 and qDTY2.3 combinations showed higher yield potential compared to lines with single or no QTL under DS, indicating significant positive interactions between the two QTLs under the FUNAABOR-2 genetic background. Pyramiding of qDTY12.1 and qDTY2.3 in the FUNAABOR-2 genetic background led to higher grain yield production under DS and NS. Keywords: drought, foreground selection, introgressed line, peak marker, yield potential, rice, quantitative trait locus

Abstract Background SAGE has been used widely to study the expression of known transcripts, but much less to annotate new transcribed regions. LongSAGE produces tags that are sufficiently long to be reliably mapped to a whole-genome sequence. Here we used this property to study the position of human LongSAGE tags obtained from all public libraries. We focused mainly on tags that do not map to known transcripts. Results Using a published error rate in SAGE libraries, we first removed the tags likely to result from sequencing errors. We then observed that an unexpectedly large number of the remaining tags still did not match the genome sequence. Some of these correspond to parts of human mRNAs, such as polyA tails, junctions between two exons and polymorphic regions of transcripts. Another non-negligible proportion can be attributed to contamination by murine transcripts and to residual sequencing errors. After filtering out our data with these screens to ensure that our dataset is highly reliable, we studied the tags that map once to the genome. 31% of these tags correspond to unannotated transcripts. The others map to known transcribed regions, but many of them (nearly half) are located either in antisense or in new variants of these known transcripts. Conclusion We performed a comprehensive study of all publicly available human LongSAGE tags, and carefully verified the reliability of these data. We found the potential origin of many tags that did not match the human genome sequence. The properties of the remaining tags imply that the level of sequencing error may have been under-estimated. The frequency of tags matching once the genome sequence but not in an annotated exon suggests that the human transcriptome is much more complex than shown by the current human genome annotations, with many new splicing variants and antisense transcripts. SAGE data is appropriate to map new transcripts to the genome, as demonstrated by the high rate of cross-validation of the corresponding tags using other methods.

Mice have evolved a new dental plan with two additional cusps on the upper molar, while hamsters were retaining the ancestral plan. By comparing the dynamics of molar development with transcriptome time series, we found at least three early changes in mouse upper molar development. Together, they redirect spatio-temporal dynamics to ultimately form two additional cusps. The mouse lower molar has undergone much more limited phenotypic evolution. Nevertheless, its developmental trajectory evolved as much as that of the upper molar and co-evolved with it. Among the coevolving changes, some are clearly involved in the new upper molar phenotype. We found a similar level of coevolution in bat limbs. In conclusion, our study reveals how serial organ morphology has adapted through organ-specific developmental changes, as expected, but also through shared changes that have organ-specific effects on the final phenotype. This highlights the important role of developmental system drift in one organ to accommodate adaptation in another., Competing Interests: Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Sex differences in gene expression start at puberty and vary across species and organs.

Regenerating animals have the ability to reproduce body parts that were originally made in the embryo and subsequently lost due to injury. Understanding whether regeneration mirrors development is an open question in most regenerative species. Here, we take a transcriptomics approach to examine whether leg regeneration shows similar temporal patterns of gene expression as leg development in the embryo, in the crustacean Parhyale hawaiensis . We find that leg development in the embryo shows stereotypic temporal patterns of gene expression. In contrast, the dynamics of gene expression during leg regeneration show a higher degree of variation related to the physiology of individual animals. A major driver of this variation is the molting cycle. We dissect the transcriptional signals of individual physiology and regeneration to obtain clearer temporal signals marking distinct phases of leg regeneration. Comparing the transcriptional dynamics of development and regeneration we find that, although the two processes use similar sets of genes, the temporal patterns in which these genes are deployed are different and cannot be systematically aligned.

Changes in interacting cis - and trans -regulatory elements are important candidates for Dobzhansky-Muller hybrid incompatibilities and may contribute to hybrid dysfunction by giving rise to misexpression in hybrids. To gain insight into the molecular mechanisms and determinants of gene expression evolution in natural populations, we analyzed the transcriptome from multiple tissues of two recently diverged Ficedula flycatcher species and their naturally occurring F 1 hybrids. Differential gene expression analysis revealed that the extent of differentiation between species and the set of differentially expressed genes varied across tissues. Common to all tissues, a higher proportion of Z-linked genes than autosomal genes showed differential expression, providing evidence for a fast-Z effect. We further found clear signatures of hybrid misexpression in brain, heart, kidney, and liver. However, while testis showed the highest divergence of gene expression among tissues, it showed no clear signature of misexpression in F 1 hybrids, even though these hybrids were found to be sterile. It is therefore unlikely that incompatibilities between cis - trans regulatory changes explain the observed sterility. Instead, we found evidence that cis -regulatory changes play a significant role in the evolution of gene expression in testis, which illustrates the tissue-specific nature of cis -regulatory evolution bypassing constraints associated with pleiotropic effects of genes., (© 2020 Mugal et al.; Published by Cold Spring Harbor Laboratory Press.)

Do developmental systems preferentially produce certain types of variation that orient phenotypic evolution along preferred directions? At different scales, from the intra-population to the interspecific, the murine first upper molar shows repeated anterior elongation. Using a novel quantitative approach to compare the development of two mouse strains with short or long molars, we identified temporal, spatial and functional differences in tooth signaling center activity, that arise from differential tuning of the activation-inhibition mechanisms underlying tooth patterning. By tracing their fate, we could explain why only the upper first molar reacts via elongation of its anterior part. Despite a lack of genetic variation, individuals of the elongated strain varied in tooth length and the temporal dynamics of their signaling centers, highlighting the intrinsic instability of the upper molar developmental system. Collectively, these results reveal the variational properties of murine molar development that drive morphological evolution along a line of least resistance., Competing Interests: LH, KL, MS, SR, MD, MV, RP, MH, SP No competing interests declared, (© 2020, Hayden et al.)

In evolutionary genomics, researchers have taken an interest in identifying substitutions that subtend convergent phenotypic adaptations. This is a difficult question that requires distinguishing foreground convergent substitutions that are involved in the convergent phenotype from background convergent substitutions. Those may be linked to other adaptations, may be neutral or may be the consequence of mutational biases. Furthermore, there is no generally accepted definition of convergent substitutions. Various methods that use different definitions have been proposed in the literature, resulting in different sets of candidate foreground convergent substitutions. In this article, we first describe the processes that can generate foreground convergent substitutions in coding sequences, separating adaptive from non-adaptive processes. Second, we review methods that have been proposed to detect foreground convergent substitutions in coding sequences and expose the assumptions that underlie them. Finally, we examine their power on simulations of convergent changes-including in the presence of a change in the efficacy of selection-and on empirical alignments. This article is part of the theme issue 'Convergent evolution in the genomics era: new insights and directions'.

Motivation: RNA sequencing (RNA-Seq) is a widely used approach to obtain transcript sequences in non-model organisms, notably for performing comparative analyses. However, current bioinformatic pipelines do not take full advantage of pre-existing reference data in related species for improving RNA-Seq assembly, annotation and gene family reconstruction., Results: We built an automated pipeline named CAARS to combine novel data from RNA-Seq experiments with existing multi-species gene family alignments. RNA-Seq reads are assembled into transcripts by both de novo and assisted assemblies. Then, CAARS incorporates transcripts into gene families, builds gene alignments and trees and uses phylogenetic information to classify the genes as orthologs and paralogs of existing genes. We used CAARS to assemble and annotate RNA-Seq data in rodents and fishes using distantly related genomes as reference, a difficult case for this kind of analysis. We showed CAARS assemblies are more complete and accurate than those assembled by a standard pipeline consisting of de novo assembly coupled with annotation by sequence similarity on a guide species. In addition to annotated transcripts, CAARS provides gene family alignments and trees, annotated with orthology relationships, directly usable for downstream comparative analyses., Availability and Implementation: CAARS is implemented in Python and Ocaml and is freely available at https://github.com/carinerey/caars., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author(s) 2018. Published by Oxford University Press.)

Actinopterygian fishes harbor at least eight distinct pigment cell types, leading to a fascinating diversity of colors. Among this diversity, the cellular origin of the white color appears to be linked to several pigment cell types such as iridophores or leucophores. We used the clownfish Amphiprion ocellaris, which has a color pattern consisting of white bars over a darker body, to characterize the pigment cells that underlie the white hue. We observe by electron microscopy that cells in white bars are similar to iridophores. In addition, the transcriptomic signature of clownfish white bars exhibits similarities with that of zebrafish iridophores. We further show by pharmacological treatments that these cells are necessary for the white color. Among the top differentially expressed genes in white skin, we identified several genes (fhl2a, fhl2b, saiyan, gpnmb, and apoD1a) and show that three of them are expressed in iridophores. Finally, we show by CRISPR/Cas9 mutagenesis that these genes are critical for iridophore development in zebrafish. Our analyses provide clues to the genomic underpinning of color diversity and allow identification of new iridophore genes in fish., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

In the history of life, some phenotypes have been acquired several times independently, through convergent evolution. Recently, lots of genome-scale studies have been devoted to identify nucleotides or amino acids that changed in a convergent manner when the convergent phenotypes evolved. These efforts have had mixed results, probably because of differences in the detection methods, and because of conceptual differences about the definition of a convergent substitution. Some methods contend that substitutions are convergent only if they occur on all branches where the phenotype changed toward the exact same state at a given nucleotide or amino acid position. Others are much looser in their requirements and define a convergent substitution as one that leads the site at which they occur to prefer a phylogeny in which species with the convergent phenotype group together. Here, we suggest to look for convergent shifts in amino acid preferences instead of convergent substitutions to the exact same amino acid. We define as convergent shifts substitutions that occur on all branches where the phenotype changed and such that they correspond to a change in the type of amino acid preferred at this position. We implement the corresponding model into a method named PCOC. We show on simulations that PCOC better recovers convergent shifts than existing methods in terms of sensitivity and specificity. We test it on a plant protein alignment where convergent evolution has been studied in detail and find that our method recovers several previously identified convergent substitutions and proposes credible new candidates., (© The Author(s) 2018. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Asymmetric cell division is essential to generate cellular diversity. In many animal cells, the cleavage plane lies perpendicular to the mitotic spindle, and it is the spindle positioning that dictates the size of the daughter cells. Although some properties of spindle positioning are conserved between distantly related model species and different cell types, little is known of the evolutionary robustness of the mechanisms underlying this event. We recorded the first embryonic division of 42 species of nematodes closely related to Caenorhabditis elegans, which is an excellent model system to study the biophysical properties of asymmetric spindle positioning. Our recordings, corresponding to 128 strains from 27 Caenorhabditis and 15 non-Caenorhabditis species (accessible at http://www.ens-lyon.fr/LBMC/NematodeCell/videos/), constitute a powerful collection of subcellular phenotypes to study the evolution of various cellular processes across species. In the present work, we analyzed our collection to the study of asymmetric spindle positioning. Although all the strains underwent an asymmetric first cell division, they exhibited large intra- and inter-species variations in the degree of cell asymmetry and in several parameters controlling spindle movement, including spindle oscillation, elongation, and displacement. Notably, these parameters changed frequently during evolution with no apparent directionality in the species phylogeny, with the exception of spindle transverse oscillations, which were an evolutionary innovation at the base of the Caenorhabditis genus. These changes were also unrelated to evolutionary variations in embryo size. Importantly, spindle elongation, displacement, and oscillation each evolved independently. This finding contrasts starkly with expectations based on C. elegans studies and reveals previously unrecognized evolutionary changes in spindle mechanics. Collectively, these data demonstrate that, while the essential process of asymmetric cell division has been conserved over the course of nematode evolution, the underlying spindle movement parameters can combine in various ways. Like other developmental processes, asymmetric cell division is subject to system drift.

Synonymous codon usage (SCU) varies widely among human genes. In particular, genes involved in different functional categories display a distinct codon usage, which was interpreted as evidence that SCU is adaptively constrained to optimize translation efficiency in distinct cellular states. We demonstrate here that SCU is not driven by constraints on tRNA abundance, but by large-scale variation in GC-content, caused by meiotic recombination, via the non-adaptive process of GC-biased gene conversion (gBGC). Expression in meiotic cells is associated with a strong decrease in recombination within genes. Differences in SCU among functional categories reflect differences in levels of meiotic transcription, which is linked to variation in recombination and therefore in gBGC. Overall, the gBGC model explains 70% of the variance in SCU among genes. We argue that the strong heterogeneity of SCU induced by gBGC in mammalian genomes precludes any optimization of the tRNA pool to the demand in codon usage.

Background: Comparative transcriptomics can answer many questions in developmental and evolutionary developmental biology. Most transcriptomic studies start by showing global patterns of variation in transcriptomes that differ between species or organs through developmental time. However, little is known about the kinds of expression differences that shape these patterns., Results: We compared transcriptomes during the development of two morphologically distinct serial organs, the upper and lower first molars of the mouse. We found that these two types of teeth largely share the same gene expression dynamics but that three major transcriptomic signatures distinguish them, all of which are shaped by differences in the relative abundance of different cell types. First, lower/upper molar differences are maintained throughout morphogenesis and stem from differences in the relative abundance of mesenchyme and from constant differences in gene expression within tissues. Second, there are clear time-shift differences in the transcriptomes of the two molars related to cusp tissue abundance. Third, the transcriptomes differ most during early-mid crown morphogenesis, corresponding to exaggerated morphogenetic processes in the upper molar involving fewer mitotic cells but more migrating cells. From these findings, we formulate hypotheses about the mechanisms enabling the two molars to reach different phenotypes. We also successfully applied our approach to forelimb and hindlimb development., Conclusions: Gene expression in a complex tissue reflects not only transcriptional regulation but also abundance of different cell types. This knowledge provides valuable insights into the cellular processes underpinning differences in organ development. Our approach should be applicable to most comparative developmental contexts.

Acquisition of new ecological opportunities is a major driver of adaptation and species diversification [1-4]. However, how groups of organisms expand their habitat range is often unclear [3]. We study the Gerromorpha, a monophyletic group of heteropteran insects that occupy a large variety of water surface-associated niches, from small puddles to open oceans [5, 6]. Due to constraints related to fluid dynamics [7-9] and exposure to predation [5, 10], we hypothesize that selection will favor high speed of locomotion in the Gerromorpha that occupy water-air interface niches relative to the ancestral terrestrial life style. Through biomechanical assays and phylogenetic reconstruction, we show that only species that occupy water surface niches can generate high maximum speeds. Basally branching lineages with ancestral mode of locomotion, consisting of tripod gait, achieved increased speed on the water through increasing midleg length, stroke amplitude, and stroke frequency. Derived lineages evolved rowing as a novel mode of locomotion through simultaneous sculling motion almost exclusively of the midlegs. We demonstrate that this change in locomotory behavior significantly reduced the requirement for high stroke frequency and energy expenditure. Furthermore, we show how the evolution of rowing, by reducing stroke frequency, may have eliminated the constraint on body size, which may explain the evolution of larger Gerromorpha. This correlation between the diversity in locomotion behaviors and niche specialization suggests that changes in morphology and behavior may facilitate the invasion and diversification in novel environments., (Copyright © 2016 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The amphipod crustacean Parhyale hawaiensis is a blossoming model system for studies of developmental mechanisms and more recently regeneration. We have sequenced the genome allowing annotation of all key signaling pathways, transcription factors, and non-coding RNAs that will enhance ongoing functional studies. Parhyale is a member of the Malacostraca clade, which includes crustacean food crop species. We analysed the immunity related genes of Parhyale as an important comparative system for these species, where immunity related aquaculture problems have increased as farming has intensified. We also find that Parhyale and other species within Multicrustacea contain the enzyme sets necessary to perform lignocellulose digestion ('wood eating'), suggesting this ability may predate the diversification of this lineage. Our data provide an essential resource for further development of Parhyale as an experimental model. The first malacostracan genome will underpin ongoing comparative work in food crop species and research investigating lignocellulose as an energy source., Competing Interests: The authors declare that no competing interests exist.

Background: Only a handful of signaling pathways are major actors of development and responsible for both the conservation and the diversification of animal morphologies. To explain this twofold nature, gene duplication and enhancer evolution were predominantly put forth as tinkering mechanisms whereas the evolution of alternative isoforms has been, so far, overlooked. We investigate here the role of gain and loss of isoforms using Edaradd, a gene of the Ecodysplasin pathway, implicated in morphological evolution. A previous study had suggested a scenario of isoform gain and loss with an alternative isoform (A) newly gained in mammals but secondarily lost in mouse lineage., Results: For a comprehensive view of A and B Edaradd isoforms history during mammal evolution, we obtained sequences for both isoforms in representative mammals and performed in vitro translations to support functional predictions. We showed that the ancestral B isoform is well conserved, whereas the mammal-specific A isoform was lost at least 7 times independently in terminal lineages throughout mammal phylogeny. Then, to gain insights into the functional relevance of this evolutionary pattern, we compared the biological function of these isoforms: i) In cellulo promoter assays showed that they are transcribed from two alternative promoters, only B exhibiting feedback regulation. ii) RT-PCR in various tissues and ENCODE data suggested that B isoform is systematically expressed whereas A isoform showed a more tissue-specific expression. iii) Both isoforms activated the NF-κB pathway in an in cellulo reporter assay, albeit at different levels and with different dynamics since A isoform exhibited feedback regulation at the protein level. Finally, only B isoform could rescue a zebrafish edaradd knockdown., Conclusions: These results suggest that the newly evolved A isoform enables modulating EDA signaling in specific conditions and with different dynamics. We speculate that during mammal diversification, A isoform regulation may have evolved rapidly, accompanying and possibly supporting the diversity of ectodermal appendages, while B isoform may have ensured essential roles. This study makes the case to pay greater attention to mosaic loss of evolutionarily speaking "young" isoforms as an important mechanism underlying phenotypic diversity and not simply as a manifestation of neutral evolution.

Comparative transcriptomics has become an important tool for revisiting many evo-devo questions and exploring new ones, and its importance is likely to increase in the near future, partly because RNA-seq data open many new possibilities. The aim of this opinion piece is twofold. In the first section, we discuss the particularities of transcriptomic studies in evo-devo, focusing mainly on RNA-seq data. The preliminary processing steps (getting coding sequences as well as expression levels) are challenging, because many studied species do not have a sequenced genome. The next step (interpreting expression differences) is also challenging, due to several issues with interpreting expression levels in complex tissues, managing developmental stages and species heterochronies, and the problem of conceptualizing expression differences. In the second section, we discuss some past and possible future applications of transcriptomic approaches (using microarray or RNA-seq) to three major themes in evo-devo: the evolution of the developmental toolkit, the genetic and developmental basis for phenotypic changes, and the general rules of the evolution of development. We believe that conceptual and technical tools are necessary in order to fully exploit the richness of multispecies transcriptomic time-series data., (© 2014 Wiley Periodicals, Inc.)

In mouse embryonic cells, ligand-activated retinoic acid receptors (RARs) play a key role in inhibiting pluripotency-maintaining genes and activating some major actors of cell differentiation. To investigate the mechanism underlying this dual regulation, we performed joint RAR/RXR ChIP-seq and mRNA-seq time series during the first 48 h of the RA-induced Primitive Endoderm (PrE) differentiation process in F9 embryonal carcinoma (EC) cells. We show here that this dual regulation is associated with RAR/RXR genomic redistribution during the differentiation process. In-depth analysis of RAR/RXR binding sites occupancy dynamics and composition show that in undifferentiated cells, RAR/RXR interact with genomic regions characterized by binding of pluripotency-associated factors and high prevalence of the non-canonical DR0-containing RA response element. By contrast, in differentiated cells, RAR/RXR bound regions are enriched in functional Sox17 binding sites and are characterized with a higher frequency of the canonical DR5 motif. Our data offer an unprecedentedly detailed view on the action of RA in triggering pluripotent cell differentiation and demonstrate that RAR/RXR action is mediated via two different sets of regulatory regions tightly associated with cell differentiation status., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

How can organisms silence deleterious gene loci? A recent study has shed light on a very brute mechanism in a jawless vertebrate: the irreversible deletion of massive chunks of genomic DNA., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Although numerous studies have emphasized the role of microRNAs (miRNAs) in the control of many different cellular processes, they might also exert a profound effect on the macroevolution of animal body plans. It has been hypothesized that, because miRNAs increase genic precision and are continuously being added to metazoan genomes through geologic time, miRNAs might be instrumental for canalization of development and morphological evolution. Nonetheless, an outstanding question remains: how are new miRNAs constantly evolving? To address this question, we assessed the miRNA complements of four deuterostome species, chosen because of their sequenced genomes and well-resolved phylogeny. Our comparative analysis shows that each of these four species is characterized by a unique repertoire of miRNAs, with few instances of miRNA loss. Moreover, we find that almost half of the miRNAs identified in this study are located in intronic regions of protein coding genes, suggesting that new miRNAs might arise from intronic regions in a process we term intronic exaptation. We also show that miRNAs often occur within cotranscribed clusters, and describe the biological function of one of these conserved clusters, the miR-1/miR-133 cluster. Taken together, our work shows that miRNAs can easily emerge within already transcribed regions of DNA, whether it be introns or preexisting clusters of miRNAs and/or miRNAs and protein coding genes, and because of their regulatory roles, these novel players change the structure of gene regulatory networks, with potential macroevolutionary results., (© 2011 Wiley Periodicals, Inc.)

Point mutations result from errors made during DNA replication or repair, so they are usually expected to be homogeneous across all regions of a genome. However, we have found a region of chloroplast DNA in plants related to sweetpea (Lathyrus) whose local point mutation rate is at least 20 times higher than elsewhere in the same molecule. There are very few precedents for such heterogeneity in any genome, and we suspect that the hypermutable region may be subject to an unusual process such as repeated DNA breakage and repair. The region is 1.5 kb long and coincides with a gene, ycf4, whose rate of evolution has increased dramatically. The product of ycf4, a photosystem I assembly protein, is more divergent within the single genus Lathyrus than between cyanobacteria and other angiosperms. Moreover, ycf4 has been lost from the chloroplast genome in Lathyrus odoratus and separately in three other groups of legumes. Each of the four consecutive genes ycf4-psaI-accD-rps16 has been lost in at least one member of the legume "inverted repeat loss" clade, despite the rarity of chloroplast gene losses in angiosperms. We established that accD has relocated to the nucleus in Trifolium species, but were unable to find nuclear copies of ycf4 or psaI in Lathyrus. Our results suggest that, as well as accelerating sequence evolution, localized hypermutation has contributed to the phenomenon of gene loss or relocation to the nucleus.

Hagfish and lampreys are the only living representatives of the jawless vertebrates (agnathans), and compared with jawed vertebrates (gnathostomes), they provide insight into the embryology, genomics, and body plan of the ancestral vertebrate. However, this insight has been obscured by controversy over their interrelationships. Morphological cladistic analyses have identified lampreys and gnathostomes as closest relatives, whereas molecular phylogenetic studies recover a monophyletic Cyclostomata (hagfish and lampreys as closest relatives). Here, we show through deep sequencing of small RNA libraries, coupled with genomic surveys, that Cyclostomata is monophyletic: hagfish and lampreys share 4 unique microRNA families, 15 unique paralogues of more primitive microRNA families, and 22 unique substitutions to the mature gene products. Reanalysis of morphological data reveals that support for cyclostome paraphyly was based largely on incorrect character coding, and a revised dataset is not decisive on the mono- vs. paraphyly of cyclostomes. Furthermore, we show fundamental conservation of microRNA expression patterns among lamprey, hagfish, and gnathostome organs, implying that the role of microRNAs within specific organs is coincident with their appearance within the genome and is conserved through time. Together, these data support the monophyly of cyclostomes and suggest that the last common ancestor of all living vertebrates was a more complex organism than conventionally accepted by comparative morphologists and developmental biologists.

Previous studies of vertebrate development have shown that retinoic acid (RA) signaling at the gastrula stage strongly influences anterior-posterior (A-P) patterning of the neurula and later stages. However, much less is known about the more immediate effects of RA signaling on gene transcription and developmental patterning at the gastrula stage. To investigate the targets of RA signaling during the gastrula stage, we used the basal chordate amphioxus, in which gastrulation involves very minimal tissue movements. First, we determined the effect of altered RA signaling on expression of 42 genes (encoding transcription factors and components of major signaling cascades) known to be expressed in restricted domains along the A-P axis during the gastrula and early neurula stage. Of these 42 genes, the expression domains during gastrulation of only four (Hox1, Hox3, HNF3-1 and Wnt3) were spatially altered by exposure of the embryos to excess RA or to the RA antagonist BMS009. Moreover, blocking protein synthesis with puromycin before adding RA or BMS009 showed that only three of these genes (Hox1, Hox3 and HNF3-1) are direct RA targets at the gastrula stage. From these results we conclude that in the amphioxus gastrula RA signaling primarily acts via regulation of Hox transcription to establish positional identities along the A-P axis and that Hox1, Hox3, HNF3-1 and Wnt3 constitute a basal module of RA action during chordate gastrulation.

Both the monophyly and inter-relationships of the major annelid groups have remained uncertain, despite intensive research on both morphology and molecular sequences. Morphological cladistic analyses indicate that Annelida is monophyletic and consists of two monophyletic groups, the clitellates and polychaetes, whereas molecular phylogenetic analyses suggest that polychaetes are paraphyletic and that sipunculans are crown-group annelids. Both the monophyly of polychaetes and the placement of sipunculans within annelids are in conflict with the annelid fossil record--the former because Cambrian stem taxa are similar to modern polychaetes in possessing biramous parapodia, suggesting that clitellates are derived from polychaetes; the latter because although fossil sipunculans are known from the Early Cambrian, crown-group annelids do not appear until the latest Cambrian. Here we apply a different data source, the presence versus absence of specific microRNAs--genes that encode approximately 22 nucleotide non-coding regulatory RNAs--to the problem of annelid phylogenetics. We show that annelids are monophyletic with respect to sipunculans, and polychaetes are paraphyletic with respect to the clitellate Lumbricus, conclusions that are consistent with the fossil record. Further, sipunculans resolve as the sister group of the annelids, rooting the annelid tree, and revealing the polarity of the morphological change within this diverse lineage of animals.

What determines the recombination rate of a gene? Following the observation that, in humans, imprinted genes have unusually high recombination levels, we ask whether increased recombination is seen for other monoallelically expressed genes and, more generally, how transcriptional properties relate to recombination. We find that monoallelically expressed genes do have high crossover rates and discover a striking negative correlation between within-gene crossover rate and expression breadth. We hypothesise that these findings are possibly symptomatic of a more general, adverse relationship between recombination and transcription in the human genome.

Metazoans are largely made of repeated parts, and metazoan evolution is marked by changes in the number of these parts, called meristic evolution. Understanding the mechanisms associated with meristic changes is thus a critical issue to evolutionary developmental biology. Palatal rugae are sensory ridges regularly arranged on the hard palate of mammals. They develop sequentially following mesio-distal growth of the palate, and activation-inhibition mechanisms very likely control spacing and timing of this sequential addition. In this study, we characterized trends in rugae number evolution among muroid rodents, showing that most species display 8+/-1 rugae, changes by one being very frequent in the phylogeny. We then compared development of three muroid species: mouse (nine rugae), rat (eight), and golden hamster (seven). We showed that palatal growth rate, spacing, and addition rate in mouse/rat were remarkably similar (with respect to the embryo size difference), and that increase to nine rugae in mouse is achieved by postponing the end of the addition process (hypermorphosis). Such a heterochronic shift may be typical of +/-1 variations observed among muroid rodents. In contrast, decrease to seven rugae in golden hamster is attributed to early growth termination (progenesis) of the palate, which correlates with the severe shortening of gestation in this species. Our results provide an experimental support to the intuitive view that heterochronies are especially relevant to meristic evolution of traits that rely on a sequential addition process. We also interpret our results in the light of developmental constraints specifically linked to this kind of process.

As paleopolyploid genomes evolve, the expression profiles of retained gene pairs are expected to diverge. To examine this divergence process on a large scale in a vertebrate system, we compare Xenopus laevis, which has retained approximately 40% of loci in duplicate after a recent whole-genome duplication (WGD), with its unduplicated relative Silurana (Xenopus) tropicalis. This comparison of ingroup pairs to an outgroup allows the direction of change in expression profiles to be inferred for a set of 1,300 X. laevis pairs, relative to their single orthologs in S. tropicalis, across 11 tissues. We identify 68 pairs in which X. laevis is inferred to have undergone a significant reduction of expression in at least two tissues since WGD. Of these pairs, one-third show evidence of subfunctionalization, with decreases in the expression levels of different gene copies in two different tissues. Surprisingly, we find that genes with slow rates of evolution are particularly prone to subfunctionalization, even when the tendency for highly expressed genes to evolve slowly is controlled for. We interpret this result to be an effect of allopolyploidization. We then compare the outcomes of this WGD with an independent one that happened in the teleost fish lineage. We find that if a gene pair was retained in duplicate in X. laevis, the orthologous pair is more likely to have been retained in duplicate in zebrafish, suggesting that similar factors, among them subfunctionalization, determined which gene pairs survived in duplicate after the two WGDs.

Polyploidy has been widely appreciated as an important force in the evolution of plant genomes, but now it is recognized as a common phenomenon throughout eukaryotic evolution. Insight into this process has been gained by analyzing the plant, animal, fungal, and recently protozoan genomes that show evidence of whole genome duplication (a transient doubling of the entire gene repertoire of an organism). Moreover, comparative analyses are revealing the evolutionary processes that occur as multiple related genomes diverge from a shared polyploid ancestor, and in individual genomes that underwent several successive rounds of duplication. Recent research including laboratory studies on synthetic polyploids indicates that genome content and gene expression can change quickly after whole genome duplication and that cross-genome regulatory interactions are important. We have a growing understanding of the relationship between whole genome duplication and speciation. Further, recent studies are providing insights into why some gene pairs survive in duplicate, whereas others do not.

Transposable elements (TEs) are genomic sequences able to replicate themselves, and to move from one chromosomal position to another within the genome. Many TEs contain their own regulatory regions, which means that they may influence the expression of neighboring genes. TEs may also be activated and transcribed in various cancers. We therefore tested whether gene expression in normal and tumor tissues is influenced by the neighboring TEs. To do this, we associated all human genes to the nearest TEs. We analyzed the expression of these genes in normal and tumor tissues using SAGE and EST data, and related this to the presence and type of TEs in their vicinity. We confirmed that TEs tend to be located in antisense orientation relative to their hosting genes. We found that the average number of tissues where a gene is expressed varies depending on the type of TEs located near the gene, and that the difference in expression level between normal and tumor tissues is greatest for genes that host SINE elements. This deregulation increases with the number of SINE copies in the gene vicinity. This suggests that SINE elements might contribute to the cascade of gene deregulation in cancer cells.

The whole genome duplication that occurred in ray-finned fish coincided with the radiation of teleost species; consequently, these two phenomena have often been linked. Using the Tetraodon and zebrafish complete genome sequences, we tested a molecular hypothesis that can relate whole genome duplication to speciation in teleosts. We estimate that thousands of genes that remained duplicated when Tetraodon and zebrafish diverged underwent reciprocal loss subsequently in these two species, probably contributing to reproductive isolation between them.

It is now clear that a whole-genome duplication (WGD) occurred at the base of the teleost fish lineage. Like the other anciently polyploid genomes investigated so far, teleost genomes now behave like diploids with chromosomes forming pairs at meiosis. The diploidization process is currently poorly understood. It is associated with many gene deletions, such that one of the duplicates is lost at most loci and has also been proposed to coincide with an increase in genomic instability. Here we ask whether WGD is a determinant of the genomic rearrangement rate in teleosts. We study variability of the rates of rearrangement along a vertebrate phylogenetic tree, composed of 3 tetrapods (human, chicken, and mouse) and 3 teleost fishes (zebrafish, Tetraodon, and Takifugu), whose complete genome sequences are available. We devise a simple parsimony method for counting rearrangements, which takes into account various methodological complications caused by the WGD and the subsequent gene losses. We show that there does appear to be an increase in rearrangement rate after WGD, but that there is also a great deal of additional variability in rearrangement rates across species.

Gene order is not random with regard to gene expression in mammals: coexpressed genes, and in particular housekeeping genes, are clustered along chromosomes more often than expected by chance. To understand the origin of these clusters and to quantify the impact of this phenomenon on genome organization, we analyzed clusters of coexpressed genes in the human and mouse genomes. We show that neighboring genes experience continuous concerted expression changes during evolution, which leads to the formation of coexpressed gene clusters. The pattern of expression within these clusters evolves more slowly than the genomic average. Moreover, by studying gene order evolution, we show that some clusters are maintained by natural selection and, therefore, have a functional significance. However, we also demonstrate that some coexpressed gene clusters are the result of neutral coevolution effects, as illustrated by the clustering of genes escaping inactivation on the X chromosome. Moreover, we show that, although statistically significant, constraints on gene orders have a limited impact on mammalian genome organization, affecting only 3-5% of the pool of human and murine genes. It had been hypothesized that coexpressed gene clusters might correspond to large chromatin domains. In contradiction, we find that most of these clusters contain only 2 genes whose coexpression may be due to transcriptional read-through or the activity of bidirectional promoters.

It has been proposed that the synonymous codon usage of human tissue-specific genes was under selective pressure to modulate the expression of proteins by codon-mediated translational control (Plotkin, J. B., H. Robins, and A. J. Levine. 2004. Tissue-specific codon usage and the expression of human genes. Proc. Natl. Acad. Sci. USA 101:12588-12591.) To test this model, we analyzed by internal correspondence analysis the codon usage of 2,126 human tissue-specific genes expressed in 18 different tissues. We confirm that synonymous codon usage differs significantly between the tissues. However, the effect is very weak: the variability of synonymous codon usage between tissues represents only 2.3% of the total codon usage variability. Moreover, this variability is directly linked to isochore-scale (>100 kb) variability of GC-content that affect both coding and introns or intergenic regions. This demonstrates that variations of synonymous codon usage between tissue-specific genes expressed in different tissues are due to regional variations of substitution patterns and not to translational selection.

Mammalian chromosomes are characterized by large-scale variations of DNA base composition (the so-called isochores). In contradiction with previous studies, Lercher et al. (Hum. Mol. Genet., 12, 2411, 2003) recently reported a strong correlation between gene expression breadth and GC-content, suggesting that there might be a selective pressure favoring the concentration of housekeeping genes in GC-rich isochores. We reassessed this issue by examining in human and mouse the correlation between gene expression and GC-content, using different measures of gene expression (EST, SAGE and microarray) and different measures of GC-content. We show that correlations between GC-content and expression are very weak, and may vary according to the method used to measure expression. Such weak correlations have a very low predictive value. The strong correlations reported by Lercher et al. (2003) are because of the fact that they measured variables over neighboring genes windows. We show here that using gene windows artificially enhances the correlation. The assertion that the expression of a given gene depends on the GC-content of the region where it is located is therefore not supported by the data.

Transcriptome analyses have revealed that a large proportion of the human genome is transcribed. However, many of these transcripts might be functionless. To distinguish functional transcription units (FTUs) from spurious transcripts, we searched for the hallmarks of selective pressure against mutations that impair transcription. We analyzed the distribution of transposable elements, which are counter selected within FTUs. We show that these features are sufficiently informative to predict whether a sequence is transcribed and, if transcribed, in which orientation. Our results indicate that FTUs constitute at least 50% of the genome and that approximately one-third of these transcripts apparently do not encode proteins.