A putative de novo evolved gene required for spermatid chromatin condensation in Drosophila melanogaster

Comparative genomics has enabled the identification of genes that potentially evolved de novo from non-coding sequences. Many such genes are expressed in male reproductive tissues, but their functions remain poorly understood. To address this, we conducted a functional genetic screen of over 40 putative de novo genes with testis-enriched expression in Drosophila melanogaster and identified one gene, atlas, required for male fertility. Detailed genetic and cytological analyses showed that atlas is required for proper chromatin condensation during the final stages of spermatogenesis. Atlas protein is expressed in spermatid nuclei and facilitates the transition from histone- to protamine-based chromatin packaging. Complementary evolutionary analyses revealed the complex evolutionary history of atlas. The protein-coding portion of the gene likely arose at the base of the Drosophila genus on the X chromosome but was unlikely to be essential, as it was then lost in several independent lineages. Within the last ~15 million years, however, the gene moved to an autosome, where it fused with a conserved non-coding RNA and evolved a non-redundant role in male fertility. Altogether, this study provides insight into the integration of novel genes into biological processes, the links between genomic innovation and functional evolution, and the genetic control of a fundamental developmental process, gametogenesis.     

Subcellular localization of the APOBEC3 proteins during mitosis and implications for genomic DNA deamination

Humans have seven APOBEC3 DNA cytosine deaminases. The activity of these enzymes allows them to restrict a variety of retroviruses and retrotransposons, but may also cause pro-mutagenic genomic uracil lesions. During interphase the APOBEC3 proteins have different subcellular localizations: cell-wide, cytoplasmic or nuclear. This implies that only a subset of APOBEC3s have contact with nuclear DNA. However, during mitosis, the nuclear envelope breaks down and cytoplasmic proteins may enter what was formerly a privileged zone. To address the hypothesis that all APOBEC3 proteins have access to genomic DNA, we analyzed the localization of the APOBEC3 proteins during mitosis. We show that APOBEC3A, APOBEC3C and APOBEC3H are excluded from condensed chromosomes, but become cell-wide during telophase. However, APOBEC3B, APOBEC3D, APOBEC3F and APOBEC3G are excluded from chromatin throughout mitosis. After mitosis, APOBEC3B becomes nuclear, and APOBEC3D, APOBEC3F and APOBEC3G become cytoplasmic. Both structural motifs as well as size may be factors in regulating chromatin exclusion. Deaminase activity was not dependent on cell cycle phase. We also analyzed APOBEC3-induced cell cycle perturbations as a measure of each enzyme’s capacity to inflict genomic DNA damage. AID, APOBEC3A and APOBEC3B altered the cell cycle profile, and, unexpectedly, APOBEC3D also caused changes. We conclude that several APOBEC3 family members have access to the nuclear compartment and can impede the cell cycle, most likely through DNA deamination and the ensuing DNA damage response. Such genomic damage may contribute to carcinogenesis, as demonstrated by AID in B cell cancers and, recently, APOBEC3B in breast cancers.     

Long‐term dynamics of mycorrhizal root tips in a loblolly pine forest grown with free‐air CO2 enrichment and soil N fertilization for 6 years

Large‐scale, long‐term FACE (Free‐Air CO₂ enrichment) experiments indicate that increases in atmospheric CO₂ concentrations will influence forest C cycling in unpredictable ways. It has been recently suggested that responses of mycorrhizal fungi could determine whether forest net primary productivity (NPP) is increased by elevated CO₂ over long time periods and if forests soils will function as sources or sinks of C in the future. We studied the dynamic responses of ectomycorrhizae to N fertilization and atmospheric CO₂ enrichment at the Duke FACE experiment using minirhizotrons over a 6 year period (2005–2010). Stimulation of mycorrhizal production by elevated CO₂ was observed during only 1 (2007) of 6 years. This increased the standing crop of mycorrhizal tips during 2007 and 2008; during 2008, significantly higher mortality returned standing crop to ambient levels for the remainder of the experiment. It is therefore unlikely that increased production of mycorrhizal tips can explain the lack of progressive nitrogen limitations and associated increases in N uptake observed in CO₂‐enriched plots at this site. Fertilization generally decreased tree reliance on mycorrhizae as tip production declined with the addition of nitrogen as has been shown in many other studies. Annual NPP of mycorrhizal tips was greatest during years with warm January temperatures and during years with cool spring temperatures. A 2 °C increase in average late spring temperatures (May and June) decreased annual production of mycorrhizal root tip length by 50%. This has important implications for ecosystem function in a warmer world in addition to potential for forest soils to sequester atmospheric C.     

Soil enzyme activities and organic matter composition in a turfgrass chronosequence

Highly managed turfgrass systems accumulate considerable soil organic C, which supports a diverse and robust soil microbial community. Degradation of this soil organic C is mediated by a suite of soil enzymes. The relationship between these enzyme activities and the quality of soil organic C is central to understanding the dynamics of soil organic matter. We examined the activities of several soil enzymes involved in microbial C acquisition, including β-glucosidase, N-acetyl-β-glucosaminidase, cellulase, chitinase, and phenol oxidase, and characterized the chemical composition of soil organic matter using Fourier transform infrared spectroscopy (FTIR) in a turfgrass chronosequence (1–95 years old) and adjacent native pines. Non-metric multidimensional scaling analysis showed that the chemical composition of soil organic matter varied with turf age and land use (turf versus pines). Using the polysaccharide peak (1,060 cm⁻¹) as a reference, both aliphatic (2,930 cm⁻¹) and carboxylic (1,650 and 1,380 cm⁻¹) compounds increased with turf age, indicating that soil organic matter became more recalcitrant. Soil enzyme activities per unit soil mass increased with turf age and were correlated to soil C content. Most soil enzyme activities in native pines were similar to those in young turf, but the cellulase activity was similar to or greater than the activity in old turfgrass systems. On a soil C basis, however, the activities of N-acetyl-β-glucosaminidase and cellulase decreased with turf age; this reduction was correlated to the relative changes in the chemical composition of soil organic matter. We observed that the chemical composition of soil organic matter was significantly correlated with the enzyme activity profile when expressed per unit microbial biomass C, but not per unit soil organic C. Our results suggest that chemical composition of soil organic matter changes with turf age and this change partially determines the relative abundance of C-degrading soil enzymes, likely through the influence on microbial community composition.     

An analysis of the energetic reward offered by field bean (Vicia faba) flowers: Nectar,pollen, and operative force

Global consumption of crops with a yield that is dependent on animal pollinators is growing, with greater areas planted each year. However, the floral traits that influence pollinator visitation are not usually the focus of breeding programmes, and therefore, it is likely that yield improvements may be made by optimizing floral traits to enhance pollinator visitation rates. We investigated the variation present in the floral reward of the bee‐pollinated crop Vicia faba (field bean). We examined the genetic potential for breeding flowers with a greater reward into current commercial varieties and used bee behavioral experiments to gain insight into the optimal nectar concentration to maximize bee preference. There was a large range of variation in the amount of pollen and nectar reward of flowers in the genotypes investigated. Bee behavioral experiments using nectar sugar concentrations found in V. faba lines suggest that Bombus terrestris prefers 55% w/w sugar solution over 40% w/w, but has no preference between 55% w/w and 68% w/w sugar solution. We provide a first indication of the force required to open V. faba flowers. Our results provide a valuable starting point toward breeding for varieties with optimized floral reward. Field studies are now needed to verify whether the genetic potential for breeding more rewarding flowers can translate into higher yield and yield stability.     

Role of GSK-3β in the osteogenic differentiation of palatal mesenchyme

Introduction

The function of Glycogen Synthase Kinases 3β (GSK-3β) has previously been shown to be necessary for normal secondary palate development. Using GSK-3ß null mouse embryos, we examine the potential coordinate roles of Wnt and Hedgehog signaling on palatal ossification.

Methods

Palates were harvested from GSK-3β, embryonic days 15.0–18.5 (e15.0–e18.5), and e15.5 Indian Hedgehog (Ihh) null embryos, and their wild-type littermates. The phenotype of GSK-3β null embryos was analyzed with skeletal whole mount and pentachrome stains. Spatiotemporal regulation of osteogenic gene expression, in addition to Wnt and Hedgehog signaling activity, were examined in vivo on GSK-3β and Ihh +/+ and −/− e15.5 embryos using in situ hybridization and immunohistochemistry. To corroborate these results, expression of the same molecular targets were assessed by qRT-PCR of e15.5 palates, or e13.5 palate cultures treated with both Wnt and Hedgehog agonists and anatagonists.

Results

GSK-3β null embryos displayed a 48 percent decrease (*p<0.05) in palatine bone formation compared to wild-type littermates. GSK-3β null embryos also exhibited decreased osteogenic gene expression that was associated with increased Wnt and decreased Hedgehog signaling. e13.5 palate culture studies demonstrated that Wnt signaling negatively regulates both osteogenic gene expression and Hedgehog signaling activity, while inhibition of Wnt signaling augments both osteogenic gene expression and Hedgehog signaling activity. In addition, no differences in Wnt signaling activity were noted in Ihh null embryos, suggesting that canonical Wnt may be upstream of Hedgehog in secondary palate development. Lastly, we found that GSK-3β −/− palate cultures were “rescued” with the Wnt inhibitor, Dkk-1.

Conclusions

Here, we identify a critical role for GSK-3β in palatogenesis through its direct regulation of canonical Wnt signaling. These findings shed light on critical developmental pathways involved in palatogenesis and may lead to novel molecular targets to prevent cleft palate formation.