Comparative Genome-Wide Analysis of the Malate Dehydrogenase Gene Families in Cotton

Malate dehydrogenases (MDHs) play crucial roles in the physiological processes of plant growth and development. In this study, 13 and 25 MDH genes were identified from Gossypium raimondii and Gossypium hirsutum, respectively. Using these and 13 previously reported Gossypium arboretum MDH genes, a comparative molecular analysis between identified MDH genes from G. raimondii, G. hirsutum, and G. arboretum was performed. Based on multiple sequence alignments, cotton MDHs were divided into five subgroups: mitochondrial MDH, peroxisomal MDH, plastidial MDH, chloroplastic MDH and cytoplasmic MDH. Almost all of the MDHs within the same subgroup shared similar gene structure, amino acid sequence, and conserved motifs in their functional domains. An analysis of chromosomal localization suggested that segmental duplication played a major role in the expansion of cotton MDH gene families. Additionally, a selective pressure analysis indicated that purifying selection acted as a vital force in the evolution of MDH gene families in cotton. Meanwhile, an expression analysis showed the distinct expression profiles of GhMDHs in different vegetative tissues and at different fiber developmental stages, suggesting the functional diversification of these genes in cotton growth and fiber development. Finally, a promoter analysis indicated redundant but typical cis-regulatory elements for the potential functions and stress activity of many MDH genes. This study provides fundamental information for a better understanding of cotton MDH gene families and aids in functional analyses of the MDH genes in cotton fiber development.     

Hox genes in digit development and evolution

Homeobox genes located in the 5' part of the HoxA and HoxD complexes are required for proliferation of skeletal progenitor cells of the vertebrate limb. Specific combinations of gene products determine the length of the upper arm (genes belonging to groups 9 and 10), the lower arm (groups 10, 11 and 12) and the digits (groups 11, 12 and 13). In these different domains, individual gene products quantitatively contribute to an overall protein dose, with predominant roles for groups 11 and 13. Quantitative reduction in the gene dose in each set results in truncations of the corresponding anatomical regions. The physical order of the genes in the HoxA and HoxD complexes, as well as a unidirectional sequence in gene activation, allow for completion of the process in a precise order, which in turn makes possible the sequential outgrowth of the respective primordia. While the skeletal patterns of upper and lower limb are relatively stable throughout the tetrapods, more variation is seen in the digits. Molecular analysis of the underlying regulatory processes promises further exciting insights into the genetic control of development, pathology and the course of evolution.     

Batrachochytrium dendrobatidis Infection and Lethal Chytridiomycosis in Caecilian Amphibians (Gymnophiona)

Batrachochytrium dendrobatidis (Bd) is commonly termed the ‘amphibian chytrid fungus’ but thus far has been documented to be a pathogen of only batrachian amphibians (anurans and caudatans). It is not proven to infect the limbless, generally poorly known, and mostly soil-dwelling caecilians (Gymnophiona). We conducted the largest qPCR survey of Bd in caecilians to date, for more than 200 field-swabbed specimens from five countries in Africa and South America, representing nearly 20 species, 12 genera, and 8 families. Positive results were recovered for 58 specimens from Tanzania and Cameroon (4 families, 6 genera, 6+ species). Quantities of Bd were not exceptionally high, with genomic equivalent (GE) values of 0.052–17.339. In addition, we report the first evidence of lethal chytridiomycosis in caecilians. Mortality in captive (wild-caught, commercial pet trade) Geotrypetes seraphini was associated with GE scores similar to those we detected for field-swabbed, wild animals.     

Genetic Interactions Between Shox2 and Hox Genes During the Regional Growth and Development of the Mouse Limb

The growth and development of the vertebrate limb relies on homeobox genes of the Hox and Shox families, with their independent mutation often giving dose-dependent effects. Here we investigate whether Shox2 and Hox genes function together during mouse limb development by modulating their relative dosage and examining the limb for nonadditive effects on growth. Using double mRNA fluorescence in situ hybridization (FISH) in single embryos, we first show that Shox2 and Hox genes have associated spatial expression dynamics, with Shox2 expression restricted to the proximal limb along with Hoxd9 and Hoxa11 expression, juxtaposing the distal expression of Hoxa13 and Hoxd13. By generating mice with all possible dosage combinations of mutant Shox2 alleles and HoxA/D cluster deletions, we then show that their coordinated proximal limb expression is critical to generate normally proportioned limb segments. These epistatic interactions tune limb length, where Shox2 underexpression enhances, and Shox2 overexpression suppresses, Hox-mutant phenotypes. Disruption of either Shox2 or Hox genes leads to a similar reduction in Runx2 expression in the developing humerus, suggesting their concerted action drives cartilage maturation during normal development. While we furthermore provide evidence that Hox gene function influences Shox2 expression, this regulation is limited in extent and is unlikely on its own to be a major explanation for their genetic interaction. Given the similar effect of human SHOX mutations on regional limb growth, Shox and Hox genes may generally function as genetic interaction partners during the growth and development of the proximal vertebrate limb.     

Expression of Hoxa-11 and Hoxa-13 in the pectoral fin of a basal ray-finned fish, Polyodon spathula: implications for the origin of tetrapod limbs

Summary Paleontological and anatomical evidence suggests that the autopodium (hand or foot) is a novel feature that distinguishes limbs from fins, while the upper and lower limb (stylopod and zeugopod) are homologous to parts of the sarcopterygian paired fins. In tetrapod limb development Hoxa-11 plays a key role in differentiating the lower limb and Hoxa-13 plays a key role in differentiating the autopodium. It is thus important to determine the ancestral functions of these genes in order to understand the developmental genetic changes that led to the origin of the tetrapod autopodium. In particular it is important to understand which features of gene expression are derived in tetrapods and which are ancestral in bony fishes. To address these questions we cloned and sequenced the Hoxa-11 and Hoxa-13 genes from the North American paddlefish, Polyodon spathula, a basal ray-finned fish that has a pectoral fin morphology resembling that of primitive bony fishes ancestral to the tetrapod lineage. Sequence analysis of these genes shows that they are not orthologous to the duplicated zebrafish and fugu genes. This implies that the paddlefish has not duplicated its HoxA cluster, unlike zebrafish and fugu. The expression of Hoxa-11 and Hoxa-13 in the pectoral fins shows two main phases: an early phase in which Hoxa-11 is expressed proximally and Hoxa-13 is expressed distally, and a later phase in which Hoxa-11 and Hoxa-13 broadly overlap in the distal mesenchyme of the fin bud but are absent in the proximal fin bud. Hence the distal polarity of Hoxa-13 expression seen in tetrapods is likely to be an ancestral feature of paired appendage development. The main difference in HoxA gene expression between fin and limb development is that in tetrapods (with the exception of newts) Hoxa-11 expression is suppressed by Hoxa-13 in the distal limb bud mesenchyme. There is, however, a short period of limb bud development where Hoxa-11 and Hoxa-13 overlap similarly to the late expression seen in zebrafish and paddlefish. We conclude that the early expression pattern in tetrapods is similar to that seen in late fin development and that the local exclusion by Hoxa-13 of Hoxa-11 from the distal limb bud is a derived feature of limb developmental regulation.     

Dimerization Is Essential for 14-3-3�� Stability and Function in Vivo

Members of the conserved 14-3-3 protein family spontaneously self-assemble as homo- and heterodimers via conserved sequences in the first four (αA-αD) of the nine helices that comprise them. Dimeric 14-3-3s bind conserved motifs in diverse protein targets involved in multiple essential cellular processes including signaling, intracellular trafficking, cell cycle regulation, and modulation of enzymatic activities. However, recent mostly in vitro evidence has emerged, suggesting functional and regulatory roles for monomeric 14-3-3s. We capitalized on the simplicity of the 14-3-3 family in Drosophila to investigate in vivo 14-3-3ζ monomer properties and functionality. We report that dimerization is essential for the stability and function of 14-3-3ζ in neurons. Moreover, we reveal the contribution of conserved amino acids in helices A and D to homo- and heterodimerization and their functional consequences on the viability of animals devoid of endogenous 14-3-3ζ. Finally, we present evidence suggesting endogenous homeostatic adjustment of the levels of the second family member in Drosophila, D14-3-3ϵ, to transgenic monomeric and dimerization-competent 14-3-3ζ.     

Repeated modification of early limb morphogenesis programmes underlies the convergence of relative limb length in Anolis lizards

The independent evolution of similar morphologies has long been a subject of considerable interest to biologists. Does phenotypic convergence reflect the primacy of natural selection, or does development set the course of evolution by channelling variation in certain directions? Here, we examine the ontogenetic origins of relative limb length variation among Anolis lizard habitat specialists to address whether convergent phenotypes have arisen through convergent developmental trajectories. Despite the numerous developmental processes that could potentially contribute to variation in adult limb length, our analyses reveal that, in Anolis lizards, such variation is repeatedly the result of changes occurring very early in development, prior to formation of the cartilaginous long bone anlagen.     

A network of conserved co-occurring motifs for the regulation of alternative splicing

Cis-acting short sequence motifs play important roles in alternative splicing. It is now possible to identify such sequence motifs as conserved sequence patterns in genome sequence alignments. Here, we report the systematic search for motifs in the neighboring introns of alternatively spliced exons by using comparative analysis of mammalian genome alignments. We identified 11 conserved sequence motifs that might be involved in the regulation of alternative splicing. These motifs are not only significantly overrepresented near alternatively spliced exons, but they also co-occur with each other, thus, forming a network of cis-elements, likely to be the basis for context-dependent regulation. Based on this finding, we applied the motif co-occurrence to predict alternatively skipped exons. We verified exon skipping in 29 cases out of 118 predictions (25%) by EST and mRNA sequences in the databases. For the predictions not verified by the database sequences, we confirmed exon skipping in 10 additional cases by using both RT–PCR experiments and the publicly available RNA-Seq data. These results indicate that even more alternative splicing events will be found with the progress of large-scale and high-throughput analyses for various tissue samples and developmental stages.     

Regressive evolution of the arthropod tritocerebral segment linked to functional divergence of the Hox gene labial

The intercalary segment is a limbless version of the tritocerebral segment and is present in the head of all insects, whereas other extant arthropods have retained limbs on their tritocerebral segment (e.g. the pedipalp limbs in spiders). The evolutionary origin of limb loss on the intercalary segment has puzzled zoologists for over a century. Here we show that an intercalary segment-like phenotype can be created in spiders by interfering with the function of the Hox gene labial. This links the origin of the intercalary segment to a functional change in labial. We show that in the spider Parasteatoda tepidariorum the labial gene has two functions: one function in head tissue maintenance that is conserved between spiders and insects, and a second function in pedipalp limb promotion and specification, which is only present in spiders. These results imply that labial was originally crucial for limb formation on the tritocerebral segment, but that it has lost this particular subfunction in the insect ancestor, resulting in limb loss on the intercalary segment. Such loss of a subfunction is a way to avoid adverse pleiotropic effects normally associated with mutations in developmental genes, and may thus be a common mechanism to accelerate regressive evolution.     

WNT/β-Catenin Signalling and Epithelial Patterning in the Homoscleromorph Sponge Oscarella

Sponges branch basally in the metazoan phylogenetic tree and are thus well positioned to provide insights into the evolution of mechanisms controlling animal development, likely to remain active in adult sponges. Of the four sponge clades, the Homoscleromorpha are of particular interest as they alone show the “true” epithelial organization seen in other metazoan phyla (the Eumetazoa). We have examined the deployment in sponges of Wnt signalling pathway components, since this pathway is an important regulator of many developmental patterning processes. We identified a reduced repertoire of three divergent Wnt ligand genes in the recently-sequenced Amphimedon queenslandica (demosponge) genome and two Wnts from our EST collection from the homoscleromorph Oscarella lobularis, along with well-conserved genes for intracellular pathway components (β-catenin, GSK3β). Remarkably, the two O. lobularis Wnt genes showed complementary expression patterns in relation to the evenly spaced ostia (canal openings) of the exopinacoderm (ectoderm), highly reminiscent of Wnt expression during skin appendage formation in vertebrates. Furthermore, experimental activation of the Wnt/β-catenin pathway using GSK3β inhibitors provoked formation of ectopic ostia, as has been shown for epithelial appendages in Eumetazoa. We thus suggest that deployment of Wnt signalling is a common and perhaps ancient feature of metazoan epithelial patterning and morphogenesis.     

BAC transgenic analysis reveals enhancers sufficient for Hoxa13 and neighborhood gene expression in mouse embryonic distal limbs and genital bud

SUMMARY We previously demonstrated that a ∼1 Mb domain of genes upstream of and including Hoxa13 is co-expressed in the developing mouse limbs and genitalia. A highly conserved non-coding sequence, mmA13CNS, was shown to be insufficient in transgenic mice to direct precise Hoxa13 -like expression in the limb buds or genital bud, although some LacZ expression from the transgene was reproducibly found in these tissues. In this report, we used β -globin minimal promoter LacZ recombinant BAC transgenes encompassing mmA13CNS to identify a single critical region involved in mouse Hoxa13 -like embryonic genital bud expression. By analyzing the expression patterns of these overlapping BAC clones in transgenic mice, we show that at least two sequences remote to the HoxA cluster are required collectively to drive Hoxa13 -like expression in developing distal limbs. Given that the paralogous posterior HoxD and neighboring genes have been shown to be under the influence of long-range distal limb and genital bud enhancers, we hypothesize that both long-range enhancers have one ancestral origin, which diverged in both sequence and function after the HoxA/D cluster duplication.     

Functional Divergence and Evolutionary Dynamics of the Putative AAAP Gene Family in Brassica rapa

The amino acid/auxin permease (AAAP) protein family is ubiquitously present in almost all eukaryotic species and functions in various aspects of growth and development. To investigate the evolution of AAAP proteins, here 83 AAAP genes in Brassica rapa were identified, and their sequence features, and evolutionary relationships were analyzed using in silico methods. According to the phylogenetic analysis, the AAAP genes of B. rapa are divided into six clades, and these clades share relatively similar sequence features, including gene structures, conserved motifs, and domain organizations. Synteny mapping strongly suggested that segmental duplications could be responsible for the expansion of this family. Adaptive evolution analysis demonstrated that most of AAAP proteins were subject to purifying selection. However, the site Tyr²⁵⁷ on eight AAAP proteins from clade 2b underwent significant positive selection. Functional divergent analysis showed that type I functional divergence coefficients (θ _I ) were significantly greater than zero in six pair-wise comparisons. However, functional divergence sites (Q _k ?>?0.95) found only in the AAAP I/II and AAAP I/III comparisons were localized mainly to the trans-membrane (TM) regions, suggesting highly divergent TM structures between these groups might be associated with group-specific functions. Our results could provide a valuable clue for further investigations of the evolutionary history and biological functions of the AAAP genes in B. rapa.     

Whole genome sequencing of Luxi Black Head sheep for screening selection signatures associated with important traits

ObjectiveLuxi Black Head sheep (LBH) is the first crossbreed specialized for meat production and was developed by crossbreeding Black Head Dorper sheep (DP) and Small Tailed Han sheep (STH) in the farming areas of northern China. Research on the genomic variations and selection signatures of LBH caused by continuous artificial selection is of great significance for identifying the genetic mechanisms of important traits of sheep and for the continuous breeding of LBH.MethodsWe explored the genetic relationships of LBH, DP, and several Mongolian sheep breeds by constructing phylogenetic tree, principal component analysis and linkage disequilibrium analysis. In addition, we analysed 29 whole genomes of sheep. The genome-wide selection signatures have been scanned with four methods: heterozygosity (H_P), fixation index (F_ST), cross-population extended haplotype homozygosity (XP-EHH) and the nucleotide diversity (θ_π) ratio.ResultsThe genetic relationships analysis showed that LBH appeared to be an independent cluster closer to DP. The candidate signatures of positive selection in sheep genome revealed candidate genes for developmental process (HoxA gene cluster, BCL2L11, TSHR), immunity (CXCL6, CXCL1, SKAP2, PTK6, MST1R), growth (PDGFD, FGF18, SRF, SOCS2), and reproduction (BCAS3, TRIM24, ASTL, FNDC3A). Moreover, two signalling pathways closely related to reproduction, the thyroid hormone signalling pathway and the oxytocin signalling pathway, were detected.ConclusionThe selective sweep analysis of LBH genome revealed candidate genes and signalling pathways associated with developmental process, immunity, growth, and reproduction. Our findings provide a valuable resource for sheep breeding and insight into the mechanisms of artificial selection.     

Microarray Analysis of microRNA Expression during Axolotl Limb Regeneration

Among vertebrates, salamanders stand out for their remarkable capacity to quickly regrow a myriad of tissues and organs after injury or amputation. The limb regeneration process in axolotls (Ambystoma mexicanum) has been well studied for decades at the cell-tissue level. While several developmental genes are known to be reactivated during this epimorphic process, less is known about the role of microRNAs in urodele amphibian limb regeneration. Given the compelling evidence that many microRNAs tightly regulate cell fate and morphogenetic processes through development and adulthood by modulating the expression (or re-expression) of developmental genes, we investigated the possibility that microRNA levels change during limb regeneration. Using two different microarray platforms to compare the axolotl microRNA expression between mid-bud limb regenerating blastemas and non-regenerating stump tissues, we found that miR-21 was overexpressed in mid-bud blastemas compared to stump tissue. Mature A. mexicanum (“Amex”) miR-21 was detected in axolotl RNA by Northern blot and differential expression of Amex-miR-21 in blastema versus stump was confirmed by quantitative RT-PCR. We identified the Amex Jagged1 as a putative target gene for miR-21 during salamander limb regeneration. We cloned the full length 3′UTR of Amex-Jag1, and our in vitro assays demonstrated that its single miR-21 target recognition site is functional and essential for the response of the Jagged1 gene to miR-21 levels. Our findings pave the road for advanced in vivo functional assays aimed to clarify how microRNAs such as miR-21, often linked to pathogenic cell growth, might be modulating the redeployment of developmental genes such as Jagged1 during regenerative processes.