Gene expression analysis of the Xenopus laevis early limb bud proximodistal axis.

BACKGROUND: Limb buds develop as bilateral outgrowths of the lateral plate mesoderm and are patterned along three axes. Current models of proximal to distal patterning of early amniote limb buds suggest that two signals, a distal organizing signal from the apical epithelial ridge (AER, Fgfs) and an opposing proximal (retinoic acid [RA]) act early on pattern this axis. RESULTS: Transcriptional analysis of stage 51 Xenopus laevis hindlimb buds sectioned along the proximal-distal axis showed that the distal region is distinct from the rest of the limb. Expression of capn8.3, a novel calpain, was located in cells immediately flanking the AER. The Wnt antagonist Dkk1 was AER-specific in Xenopus limbs. Two transcription factors, sall1 and zic5, were expressed in distal mesenchyme. Zic5 has no described association with limb development. We also describe expression of two proximal genes, gata5 and tnn, not previously associated with limb development. Differentially expressed genes were associated with Fgf, Wnt, and RA signaling as well as differential cell adhesion and proliferation. CONCLUSIONS: We identify new candidate genes for early proximodistal limb patterning. Our analysis of RA-regulated genes supports a role for transient RA gradients in early limb bud in proximal-to-distal patterning in this anamniote model organism.

A non-coding genetic variant associated with abdominal aortic aneurysm alters ERG gene regulation.

Abdominal aortic aneurysm (AAA) is a major cause of sudden death in the elderly. While AAA has some overlapping genetic and environmental risk factors with atherosclerosis, there are substantial differences, and AAA-specific medication is lacking. A recent meta-analysis of genome-wide association studies has identified four novel single-nucleotide polymorphisms (SNPs) specifically associated with AAA. Here, we investigated the gene regulatory function for one of four non-coding SNPs associated with AAA, rs2836411, which is located in an intron of the ERG gene. Rs2836411 resides within a >70 kb super-enhancer that has high levels of H3K27ac and H3K4me1 in vascular endothelial and haematopoietic cell types. Enhancer luciferase assays in cell lines showed that the risk allele significantly alters enhancer activity. The risk allele also correlates with reduced ERG expression in aortic and other vascular tissues. To identify whether rs2836411 directly contacts the promoters of ERG and/or of genes further away, we performed allele-specific circular chromosome conformation capture sequencing. In vascular endothelial cells, which express ERG, the SNP region interacts highly within the super-enhancer, while in vascular smooth muscle cells, which do not express ERG, the interactions are distributed across a wider region that includes neighbouring genes. Furthermore, the risk allele has fewer interactions within the super-enhancer compared to the protective allele. In conclusion, our results indicate that rs2836411 likely affects ERG expression by altering enhancer activity and changing local chromatin interactions. ERG is involved in vascular development, angiogenesis, and inflammation in atherosclerosis; therefore mechanistically, rs2836411 could contribute to AAA by modulating ERG levels.

Association of Prostaglandin Use for Cervical Ripening with Mode of Delivery in Small for Gestational Age versus Non-Small for Gestational Age Neonates.

OBJECTIVE: Prostaglandins (PGs) use for cervical ripening with small for gestational age (SGA) fetuses is controversial since it remains uncertain if use increases the chance of cesarean delivery (CD). We aimed to assess the association between PG use for cervical ripening and mode of delivery between SGA and appropriate for gestational age (AGA) neonates. STUDY DESIGN: Secondary analysis of the Nulliparous Pregnancy Outcomes Study: Monitoring Mothers-to-Be (nuMoM2b), a prospective observational cohort study of 10,038 nulliparas. We included women undergoing induction with nonanomalous fetuses in the cephalic presentation. Women with >2 cm cervical dilation or prior uterine scar were excluded. We assessed the association of PG use with CD among women with SGA and AGA neonates. SGA was defined as birth weight <10th percentile for gestational age and sex. Multivariable logistic regression was used to adjust for potential confounders and test for interaction. Secondary outcomes included adverse neonatal outcomes, indication for CD, maternal hemorrhage, and chorioamnionitis. RESULTS: Among 2,353 women eligible, PGs were used in 54.8%, SGA occurred in 15.1%, and 35.0% had CD. The association between PG use and CD differed significantly (interaction p = 0.018) for SGA versus AGA neonates; CD occurred more often in SGA neonates exposed to PGs than not (35 vs. 22%, p = 0.009). PG use was not associated with CD among AGA neonates (36 vs. 36%, p = 0.8). This effect remained significant when adjusting for body mass index, race/ethnicity, and cervical dilation. Among SGA neonates, CD for "nonreassuring fetal status" was similar between PG groups. Among SGA neonates, PG use was not associated with adverse neonatal outcomes or postpartum hemorrhage but had a higher rate of chorioamnionitis (7.0 vs. 2.1%, p = 0.048). CONCLUSION: PG use was associated with a higher rate of CD in SGA but not AGA neonates; however, further studies are needed before PG use is discouraged with SGA neonates. KEY POINTS: · PGs are commonly used for cervical ripening.. · PG use was associated with increased risk of cesarean delivery in SGA neonates.. · PG use was not associated with adverse neonatal outcomes..

Fungal endophyte infection of ryegrass reprograms host metabolism and alters development.

Beneficial associations between plants and microbes play an important role in both natural and agricultural ecosystems. For example, associations between fungi of the genus Epichloë, and cool-season grasses are known for their ability to increase resistance to insect pests, fungal pathogens and drought. However, little is known about the molecular changes induced by endophyte infection. To study the impact of endophyte infection, we compared the expression profiles, based on RNA sequencing, of perennial ryegrass infected with Epichloë festucae with noninfected plants. We show that infection causes dramatic changes in the expression of over one third of host genes. This is in stark contrast to mycorrhizal associations, where substantially fewer changes in host gene expression are observed, and is more similar to pathogenic interactions. We reveal that endophyte infection triggers reprogramming of host metabolism, favouring secondary metabolism at a cost to primary metabolism. Infection also induces changes in host development, particularly trichome formation and cell wall biogenesis. Importantly, this work sheds light on the mechanisms underlying enhanced resistance to drought and super-infection by fungal pathogens provided by fungal endophyte infection. Finally, our study reveals that not all beneficial plant-microbe associations behave the same in terms of their effects on the host.

Developmentally regulated HEART STOPPER, a mitochondrially targeted L18 ribosomal protein gene, is required for cell division, differentiation, and seed development in Arabidopsis.

Evidence is presented for the role of a mitochondrial ribosomal (mitoribosomal) L18 protein in cell division, differentiation, and seed development after the characterization of a recessive mutant, heart stopper (hes). The hes mutant produced uncellularized endosperm and embryos arrested at the late globular stage. The mutant embryos differentiated partially on rescue medium with some forming callus. HES (At1g08845) encodes a mitochondrially targeted member of a highly diverged L18 ribosomal protein family. The substitution of a conserved amino residue in the hes mutant potentially perturbs mitoribosomal function via altered binding of 5S rRNA and/or influences the stability of the 50S ribosomal subunit, affecting mRNA binding and translation. Consistent with this, marker genes for mitochondrial dysfunction were up-regulated in the mutant. The slow growth of the endosperm and embryo indicates a defect in cell cycle progression, which is evidenced by the down-regulation of cell cycle genes. The down-regulation of other genes such as EMBRYO DEFECTIVE genes links the mitochondria to the regulation of many aspects of seed development. HES expression is developmentally regulated, being preferentially expressed in tissues with active cell division and differentiation, including developing embryos and the root tips. The divergence of the L18 family, the tissue type restricted expression of HES, and the failure of other L18 members to complement the hes phenotype suggest that the L18 proteins are involved in modulating development. This is likely via heterogeneous mitoribosomes containing different L18 members, which may result in differential mitochondrial functions in response to different physiological situations during development.

The role of the MCM2-7 helicase complex during Arabidopsis seed development.

The MINICHROMOSOME MAINTENANCE 2-7 (MCM2-7) complex, a ring-shaped heterohexamer, unwinds the DNA double helix ahead of the other replication machinery. Although there is evidence that individual components might have other roles, the essential nature of the MCM2-7 complex in DNA replication has made it difficult to uncover these. Here, we present a detailed analysis of Arabidopsis thaliana mcm2-7 mutants and reveal phenotypic differences. The MCM2-7 genes are coordinately expressed during development, although MCM7 is expressed at a higher level in the egg cell. Consistent with a role in the egg cell, heterozygous mcm7 mutants resulted in frequent ovule abortion, a phenotype that does not occur in other mcm mutants. All mutants showed a maternal effect, whereby seeds inheriting a maternal mutant allele occasionally aborted later in seed development with defects in embryo patterning, endosperm nuclear size, and cellularization, a phenotype that is variable between subunit mutants. We provide evidence that this maternal effect is due to the necessity of a maternal store of MCM protein in the central cell that is sufficient for maintaining seed viability and size in the absence of de novo MCM transcription. Reducing MCM levels using endosperm-specific RNAi constructs resulted in the up-regulation of DNA repair transcripts, consistent with the current hypothesis that excess MCM2-7 complexes are loaded during G1 phase, and are required during S phase to overcome replicative stress or DNA damage. Overall, this study demonstrates the importance of the MCM2-7 subunits during seed development and suggests that there are functional differences between the subunits.

Attenuation of bone morphogenetic protein signaling during amphibian limb development results in the generation of stage-specific defects.

The vertebrate limb is one of the most intensively studied organs in the field of developmental biology. Limb development in tetrapod vertebrates is highly conserved and dependent on the interaction of several important molecular pathways. The bone morphogenetic protein (BMP) signaling cascade is one of these pathways and has been shown to be crucial for several aspects of limb development. Here, we have used a Xenopus laevis transgenic line, in which expression of the inhibitor Noggin is under the control of the heat-shock promoter hsp70 to examine the effects of attenuation of BMP signaling at different stages of limb development. Remarkably different phenotypes were produced at different stages, illustrating the varied roles of BMP in development of the limb. Very early limb buds appeared to be refractory to the effects of BMP attenuation, developing normally in most cases. Ectopic limbs were produced by overexpression of Noggin corresponding to a brief window of limb development at about stage 49/50, as recently described by Christen et al. (2012). Attenuation of BMP signaling in stage 51 or 52 tadpoles lead to a reduction in the number of digits formed, resulting in hypodactyly or ectrodactyly, as well as occasional defects in the more proximal tibia-fibula. Finally, inhibition at stage 54 (paddle stage) led to the formation of dramatically shortened digits resulting from loss of distal phalanges. Transcriptome analysis has revealed the possibility that more Noggin-sensitive members of the BMP family could be involved in limb development than previously suspected. Our analysis demonstrates the usefulness of heat-shock-driven gene expression as an effective method for inhibiting a developmental pathway at different times during limb development.

RNA-seq analysis of synchronized developing pollen isolated from a single anther.

Pollen development, from unicellular microspores to anthesis, is a complex process involving the coordinated specification, differentiation and functions of different cell types. Key to understanding this development is identifying the genes expressed at precise stages of development. However, transcriptomic studies on pollen prior to anthesis are complicated by the inaccessible nature of pollen developing in the anther and the resistant pollen wall. To assist with understanding gene expression during pollen development we have developed a protocol to perform RNA-Seq on pollen isolated from a single anther (SA RNA-Seq). The protocol involves removing pollen from a single anther for analysis and viewing the remaining pollen to determine the developmental stage. The isolated pollen is chemically lysed and mRNA isolated from the lysate using an oligo-dT column before library preparation. Here, we report on the development and testing of our method and the generation of a transcriptome for three stages of pollen development from Arabidopsis (Arabidopsis thaliana) and two stages from male kiwifruit (Actinidia chinensis). This protocol enables the transcriptome of precise developmental stages of pollen to be analyzed, and uses a small number of plants, potentially facilitating studies that require a range of treatments or the analysis of the first generation of transgenic plants.

Next-Generation Bisulfite Sequencing for Targeted DNA Methylation Analysis.

Bisulfite sequencing is the "gold-standard" technique for DNA methylation analysis. By combining bisulfite sequencing with high-throughput, next-generation sequencing technology, we can document methylation from many thousands of individual reads (equivalent to alleles or "cells"), for multiple target regions and from many samples simultaneously. Here, we describe a next-generation bisulfite-sequencing assay for targeted DNA methylation analysis which offers scope for the simultaneous interrogation of multiple genomic loci across numerous samples.

Circular Chromosome Conformation Capture Sequencing (4C-Seq ) in Primary Adherent Cells.

The three-dimensional structure of the genome is highly organized and is an important aspect of gene regulation. Chromatin interactions can be identified using chromosome conformation capture-based techniques, which rely on proximity ligation. Of these techniques, circular chromosome conformation capture sequencing (4C-seq) is used to identify all chromatin interactions occurring with a single chromosomal location (one versus all). Here we describe a 4C-seq protocol that has been optimized for primary adherent cells, for which the first digestion step is inefficient using standard 4C-seq protocols. It can, however, also be applied to other cell or tissue types. This protocol utilizes a standard DNA library preparation method using a commercial kit, and includes a description of the data processing steps.