Fasting/refeeding: an experimental model to study the impact of early thermal manipulation on hepatic metabolism in mule ducks.

An increase in egg incubation temperature was previously shown to enhance the metabolism of mule ducks and increase liver fattening after overfeeding, through a metabolic programming mechanism. Here, we examined whether fasting (F) followed by refeeding (RF) in 11-wk-old mule ducks could become an accelerated model to study the mechanisms of metabolic programming following embryonic thermal manipulation. This study investigated the hepatic response of mule ducks subjected to 23 h of fasting and 1 h of refeeding, in control or thermally programmed animals (with an increase of 1°C, 16 h per day from days 13 to 27 of embryogenesis). Liver weight and energy composition, hepatocyte structure, plasma parameters, and gene expression levels were measured at 1, 2, and 4 h after RF. All these parameters were strongly affected by RF, whereas significant impacts of embryonic programming were measured in cell size (+1 µm on average), lipid composition (+4.2% of saturated fatty acids 4 h after the meal), and relative gene expressions (including HK1, SCD1, ELOVL6, and FASN). In addition to confirming previously identified molecular targets of thermal manipulation, this study revealed new ones, thanks to kinetic sampling after RF. Finally, the detailed description of the impact of the F/RF challenge on the liver structure, composition, and gene expression, but also on plasma parameters allowed us to draw a parallel with these same traits measured during overfeeding. This comparative analysis suggests that this protocol could become a pertinent model to study the mechanisms involved in embryonic liver thermal programming, without overfeeding.

Two repeated motifs enriched within some enhancers and origins of replication are bound by SETMAR isoforms in human colon cells.

Setmar is a gene specific to simian genomes. The function(s) of its isoforms are poorly understood and their existence in healthy tissues remains to be validated. Here we profiled SETMAR expression and its genome-wide binding landscape in colon tissue. We found isoforms V3 and V6 in healthy and tumour colon tissues as well as incell lines. In two colorectal cell lines SETMAR binds to several thousand Hsmar1 and MADE1 terminal ends, transposons mostly located in non-genic regions of active chromatin including in enhancers. It also binds to a 12-bp motifs similar to an inner motif in Hsmar1 and MADE1 terminal ends. This motif is interspersed throughout the genome and is enriched in GC-rich regions as well as in CpG islands that contain constitutive replication origins. It is also found in enhancers other than those associated with Hsmar1 and MADE1. The role of SETMAR in the expression of genes, DNA replication and in DNA repair are discussed.

Embryonic thermal manipulation impacts the postnatal transcriptome response of heat-challenged Japanese quails.

BACKGROUND: The thermal-manipulation (TM) during egg incubation is a cyclic exposure to hot or cold temperatures during embryogenesis that is associated to long-lasting effects on growth performance, physiology, metabolism and temperature tolerance in birds. An increase of the incubation temperature of Japanese quail eggs affected the embryonic and post-hatch survival, growth, surface temperatures and blood characteristics potentially related to thermoregulation capacities. To gain new insights in the molecular basis of TM in quails, we investigated by RNA-seq the hypothalamus transcriptome of 35 days-old male and female quails that were treated by TM or not (C, control) during embryogenesis and that were exposed (HC) or not (RT) to a 36 °C heat challenge for 7 h before sampling. RESULTS: For males, 76, 27, 47 and 0 genes were differentially expressed in the CHC vs. CRT, CRT vs. TMRT, TMHC vs. TMRT and CHC vs. TMHC comparisons, respectively. For females, 17, 0, 342 and 1 genes were differentially expressed within the same respective comparisons. Inter-individual variability of gene expression response was observed particularly when comparing RT and HC female animals. The differential expression of several genes was corroborated by RT-qPCR analysis. Gene Ontology functional analysis of the differentially expressed genes showed a prevalent enrichment of terms related to cellular responses to stimuli and gene expression regulation in both sexes. Gene Ontology terms related to the membrane transport, the endoplasmic reticulum and mitochondrial functions as well as DNA metabolism and repair were also identified in specific comparisons and sexes. CONCLUSIONS: TM had little to no effect on the regulation of gene expression in the hypothalamus of 35 days-old Japanese quails. However, the consequences of TM on gene expression were revealed by the HC, with sex-specific and common functions altered. The effects of the HC on gene expression were most prominent in TM females with a ~ 20-fold increase of the number of differentially expressed genes, suggesting that TM may enhance the gene response during challenging conditions in female quail hypothalamus. TM may also promote new cellular strategies in females to help coping to the adverse conditions as illustrated by the identification of differentially expressed genes related to the mitochondrial and heat-response functions.

Variations in genome size between wild and domesticated lineages of fowls belonging to the Gallus gallus species.

Efforts to elucidate the causes of biological differences between wild fowls and their domesticated relatives, the chicken, have to date mainly focused on the identification of single nucleotide mutations. Other types of genomic variations have however been demonstrated to be important in avian evolution and associated to variations in phenotype. They include several types of sequences duplicated in tandem that can vary in their repetition number. Here we report on genome size differences between the red jungle fowl and several domestic chicken breeds and selected lines. Sequences duplicated in tandem such as rDNA, telomere repeats, satellite DNA and segmental duplications were found to have been significantly re-shaped during domestication and subsequently by human-mediated selection. We discuss the extent to which changes in genome organization that occurred during domestication agree with the hypothesis that domesticated animal genomes have been shaped by evolutionary forces aiming to adapt them to anthropized environments.

An Assessment of Fixed and Native Chromatin Preparation Methods to Study Histone Post-Translational Modifications at a Whole Genome Scale in Skeletal Muscle Tissue.

BACKGROUND: Genomic loci associated with histone marks are typically analyzed by immunoprecipitation of the chromatin followed by quantitative-PCR (ChIP-qPCR) or high throughput sequencing (ChIP-seq). Chromatin can be either cross-linked (X-ChIP) or used in the native state (N-ChIP). Cross-linking of DNA and proteins helps stabilizing their interactions before analysis. Despite X-ChIP is the most commonly used method, muscle tissue fixation is known to be relatively inefficient. Moreover, no protocol described a simple and reliable preparation of skeletal muscle chromatin of sufficient quality for subsequent high-throughput sequencing. Here we aimed to set-up and compare both chromatin preparation methods for a genome-wide analysis of H3K27me3, a broad-peak histone mark, using chicken P. major muscle tissue. RESULTS: Fixed and unfixed chromatin were prepared from chicken muscle tissues (Pectoralis major). Chromatin fixation, shearing by sonication or digestion and immunoprecipitation performed equivalently. High-quality Illumina reads were obtained (q30 > 93%). The bioinformatic analysis of the data was performed using epic, a tool based on SICER, and MACS2. Forty millions of reads were analyzed for both X-ChIP-seq and N-ChIP-seq experiments. Surprisingly, H3K27me3 X-ChIP-seq analysis led to the identification of only 2000 enriched regions compared to about 15,000 regions identified in the case of N-ChIP-seq. N-ChIP-seq peaks were more consistent between replicates compared to X-ChIP-seq. Higher N-ChIP-seq enrichments were confirmed by ChIP-qPCR at the PAX5 and SOX2 loci known to be enriched for H3K27me3 in myotubes and at the loci of common regions of enrichment identified in this study. CONCLUSIONS: Our findings suggest that the preparation of muscle chromatin for ChIP-seq in cross-linked conditions can compromise the systematic analysis of broad histone marks. Therefore, native chromatin preparation should be preferred to cross-linking when a ChIP experiment has to be performed on skeletal muscle tissue, particularly when a broad source signal is considered.

Embryonic environment and transgenerational effects in quail.

BACKGROUND: Environmental exposures, for instance to chemicals, are known to impact plant and animal phenotypes on the long term, sometimes across several generations. Such transgenerational phenotypes were shown to be promoted by epigenetic alterations such as DNA methylation, an epigenetic mark involved in the regulation of gene expression. However, it is yet unknown whether transgenerational epigenetic inheritance of altered phenotypes exists in birds. The purpose of this study was to develop an avian model to investigate whether changes to the embryonic environment had a transgenerational effect that could alter the phenotypes of third-generation offspring. Given its impact on the mammalian epigenome and the reproductive system in birds, genistein was used as an environment stressor. RESULTS: We compared several third-generation phenotypes of two quail "epilines", which were obtained from genistein-injected eggs (Epi+) or from untreated eggs (Epi-) from the same founders. A "mirrored" crossing strategy was used to minimize between-line genetic variability by maintaining similar ancestor contributions across generations in each line. Three generations after genistein treatment, a significant difference in the sexual maturity of the females, which, after three generations, could not be attributed to direct maternal effects, was observed between the lines, with Epi+ females starting to lay eggs later. Adult body weight was significantly affected by genistein treatment applied in a previous generation, and a significant interaction between line and sex was observed for body weight at 3 weeks. Behavioral traits, such as evaluating the birds' reaction to social isolation, were also significantly affected by genistein treatment. Yet, global methylation analyses revealed no significant difference between the epilines. CONCLUSIONS: These findings demonstrate that embryonic environment affects the phenotype of offspring three generations later in quail. While one cannot rule out the existence of some initial genetic variability between the lines, the mirrored animal design should have minimized its effects, and thus, the observed differences in animals of the third generation may be attributed, at least partly, to transgenerational epigenetic phenomena.

Thermal manipulation of the chicken embryo triggers differential gene expression in response to a later heat challenge.

BACKGROUND: Meat type chickens have limited capacities to cope with high environmental temperatures, this sometimes leading to mortality on farms and subsequent economic losses. A strategy to alleviate this problem is to enhance adaptive capacities to face heat exposure using thermal manipulation (TM) during embryogenesis. This strategy was shown to improve thermotolerance during their life span. The aim of this study was to determine the effects of TM (39.5 °C, 12 h/24 vs 37.8 °C from d7 to d16 of embryogenesis) and of a subsequent heat challenge (32 °C for 5 h) applied on d34 on gene expression in the Pectoralis major muscle (PM). A chicken gene expression microarray (8 × 60 K) was used to compare muscle gene expression profiles of Control (C characterized by relatively high body temperatures, Tb) and TM chickens (characterized by a relatively low Tb) reared at 21 °C and at 32 °C (CHC and TMHC, respectively) in a dye-swap design with four comparisons and 8 broilers per treatment. Real-time quantitative PCR (RT-qPCR) was subsequently performed to validate differential expression in each comparison. Gene ontology, clustering and network building strategies were then used to identify pathways affected by TM and heat challenge. RESULTS: Among the genes differentially expressed (DE) in the PM (1.5 % of total probes), 28 were found to be differentially expressed between C and TM, 128 between CHC and C, and 759 between TMHC and TM. No DE gene was found between TMHC and CHC broilers. The majority of DE genes analyzed by RT-qPCR were validated. In the TM/C comparison, DE genes were involved in energy metabolism and mitochondrial function, cell proliferation, vascularization and muscle growth; when comparing heat-exposed chickens to their own controls, TM broilers developed more specific pathways than C, especially involving genes related to metabolism, stress response, vascularization, anti-apoptotic and epigenetic processes. CONCLUSIONS: This study improved the understanding of the long-term effects of TM on PM muscle. TM broilers displaying low Tb may have lower metabolic intensity in the muscle, resulting in decreased metabolic heat production, whereas modifications in vascularization may enhance heat loss. These specific changes could in part explain the better adaptation of TM broilers to heat.

Thermal conditioning of quail embryos has transgenerational and reversible long-term effects.

BACKGROUND: In the current context of global warming, thermal manipulation of avian embryos has received increasing attention as a strategy to promote heat tolerance in avian species by simply increasing the egg incubation temperature. However, because of their likely epigenetic origin, thermal manipulation effects may last more than one generation with consequences for the poultry industry. In this work, a multigenerational and transgenerational analysis of thermal manipulation during embryogenesis was performed to uncover the long-term effects of such procedure. RESULTS: Thermal manipulation repeated during 4 generations had an effect on hatchability, body weight, and weight of eggs laid in Japanese quails, with some effects increasing in importance over generations. Moreover, the effects on body weight and egg weight could be transmitted transgenerationally, suggesting non-genetic inheritance mechanisms. This hypothesis is reinforced by the observed reversion of the effect on growth after five unexposed generations. Interestingly, a beneficial effect of thermal manipulation on heat tolerance was observed a few days after hatching, but this effect was not transgenerational. CONCLUSIONS: Our multigenerational study showed that thermal conditioning of quail embryos has a beneficial effect on post-hatch heat tolerance hampered by transgenerational but reversible defects on growth. Assuming that no genetic variability underlies these changes, this study provides the first demonstration of epigenetic inheritance of traits induced by environmental temperature modification associated with long-term impacts in an avian species.

Prenatal maternal stress is associated with behavioural and epigenetic changes in Japanese quail.

Prenatal maternal stress (PMS) influences many facets of offspring's phenotype including morphology, behaviour and cognitive abilities. Recent research suggested that PMS also induced epigenetic modifications. In the present study, we analysed, in the Japanese quail, the effects of PMS on the emotional reactivity and cognitive abilities of the F1 offspring. We also investigated in the hippocampus, the paraventricular hypothalamic nucleus and subnuclei of the arcopallium/amygdala the level of two histone post-translational modifications, H3K4me2 and H3K27me3, known to be impacted by stress. We found that PMS does not affect F1 quail's learning abilities but increases their emotional reactivity. Moreover, we demonstrated that PMS induced an increased density of H3K27me3 positive cells, in the hippocampus, paraventricular hypothalamic nucleus and dorsal nucleus of the amygdala, but not variations of H3K4me2. As these brain regions are involved in the control of vertebrates' emotional responses, the effect of PMS on the epigenetic mark H3K27me3 could possibly be a mechanism involved in the behavioural effects we observed in F1 quail.

Microbiota and stress: a loop that impacts memory.

Chronic stress and the gut microbiota appear to comprise a feed-forward loop, which contributes to the development of depressive disorders. Evidence suggests that memory can also be impaired by either chronic stress or microbiota imbalance. However, it remains to be established whether these could be a part of an integrated loop model and be responsible for memory impairments. To shed light on this, we used a two-pronged approach in Japanese quail: first stress-induced alterations in gut microbiota were characterized, then we tested whether this altered microbiota could affect brain and memory function when transferred to a germ-free host. The cecal microbiota of chronically stressed quails was found to be significantly different from that of unstressed individuals with lower α and ß diversities and increased Bacteroidetes abundance largely represented by the Alistipes genus, a well-known stress target in rodents and humans. The transfer of this altered microbiota into germ-free quails decreased their spatial and cue-based memory abilities as previously demonstrated in the stressed donors. The recipients also displayed increased anxiety-like behavior, reduced basal plasma corticosterone levels and differential gene expression in the brain. Furthermore, cecal microbiota transfer from a chronically stressed individual was sufficient to mimic the adverse impact of chronic stress on memory in recipient hosts and this action may be related to the Alistipes genus. Our results provide evidence of a feed-forward loop system linking the microbiota-gut-brain axis to stress and memory function and suggest that maintaining a healthy microbiota could help alleviate memory impairments linked to chronic stress.

Perinuclear distribution of heterochromatin in developing C. elegans embryos.

Specific nuclear domains are nonrandomly positioned within the nuclear space, and this preferential positioning has been shown to play an important role in genome activity and stability. Well-known examples include the organization of repetitive DNA in telomere clusters or in the chromocenter of Drosophila and mammalian cells, which may provide a means to control the availability of general repressors, such as the heterochromatin protein 1 (HP1). We have specifically characterized the intranuclear positioning of in vivo fluorescence of the Caenorhabditis elegans HP1 homologue HPL-2 as a marker for heterochromatin domains in developing embryos. For this purpose, the wavelet transform modulus maxima (WTMM) segmentation method was generalized and adapted to segment the small embryonic cell nuclei in three dimensions. The implementation of a radial distribution algorithm revealed a preferential perinuclear positioning of HPL-2 fluorescence in wild-type embryos compared with the diffuse and homogeneous nuclear fluorescence observed in the lin-13 mutants. For all other genotypes analyzed, the quantitative analysis highlighted various degrees of preferential HPL-2 positioning at the nuclear periphery, which directly correlates with the number of HPL-2 foci previously counted on 2D projections. Using a probabilistic 3D cell nuclear model, we found that any two nuclei having the same number of foci, but with a different 3D probabilistic positioning scheme, can have significantly different counts in the 2D maximum projection, thus showing the deceptive limitations of using techniques of 2D maximum projection foci counts. By this approach, a strong perinuclear positioning of HPL-2 foci was brought into light upon inactivation of conserved chromatin-associated proteins, including the HAT cofactor TRAPP.

HPL-2/HP1 prevents inappropriate vulval induction in Caenorhabditis elegans by acting in both HYP7 and vulval precursor cells.

A current model for Caenorhabditis elegans vulval cell fate specification is that SynMuv genes act redundantly in the hyp7 hypodermal syncytium to repress the LIN-3/EGF inducer and prevent ectopic vulval induction of vulva precursor cells (VPCs). Here we show that the SynMuv gene hpl-2/HP1 has an additional function in VPCs, where it may act through target genes including LIN-39/Hox.

Embryonic thermal manipulation has short and long-term effects on the development and the physiology of the Japanese quail.

In vertebrates, the embryonic environment is known to affect the development and the health of individuals. In broiler chickens, the thermal-manipulation (TM) of eggs during the incubation period was shown to improve heat tolerance at slaughter age (35 days of age) in association with several modifications at the molecular, metabolic and physiological levels. However, little is known about the Japanese quail (Coturnix japonica), a closely related avian species widely used as a laboratory animal model and farmed for its meat and eggs. Here we developed and characterized a TM procedure (39.5°C and 65% relative humidity, 12 h/d, from days 0 to 13 of incubation) in quails by analyzing its short and long-term effects on zootechnical, physiological and metabolic parameters. Heat-tolerance was tested by a heat challenge (36°C for 7h) at 35 days of age. TM significantly reduced the hatching rate of the animals and increased mortality during the first four weeks of life. At hatching, TM animals were heavier than controls, but lighter at 25 days of age for both sexes. Thirty-five days after hatching, TM decreased the surface temperature of the shank in females, suggesting a modulation of the blood flow to maintain the internal temperature. TM also increased blood partial pressure and oxygen saturation percentage at 35 days of age in females, suggesting a long-term modulation of the respiration physiology. Quails physiologically responded to the heat challenge, with a modification of several hematologic and metabolic parameters, including an increase in plasma corticosterone concentration. Several physiological parameters such as beak surface temperature and blood sodium concentration revealed that TM birds responded differently to the heat challenge compared to controls. Altogether, this first comprehensive characterization of TM in Japanese quail showed durable effects that may affect the response of TM quails to heat.

Identification of Reference Genes for Quantitative Gene Expression Studies in Three Tissues of Japanese Quail.

RT-qPCR is the gold standard for candidate gene expression analysis. However, the interpretation of RT-qPCR results depends on the proper use of internal controls, i.e., reference genes. Japanese quail is an agronomic species also used as a laboratory model, but little is known about RT-qPCR reference genes for this species. Thus, we investigated 10 putative reference genes (ACTB, GAPDH, PGK1, RPS7, RPS8, RPL19, RPL32, SDHA, TBP and YWHAZ) in three different female and male quail tissues (liver, brain and pectoral muscle). Gene expression stability was evaluated with three different algorithms: geNorm, NormFinder and BestKeeper. For each tissue, a suitable set of reference genes was defined and validated by a differential analysis of gene expression between females and males (CCNH in brain and RPL19 in pectoral muscle). Collectively, our study led to the identification of suitable reference genes in liver, brain and pectoral muscle for Japanese quail, along with recommendations for the identification of reference gene sets for this species.

Thermal Manipulation During Embryogenesis Impacts H3K4me3 and H3K27me3 Histone Marks in Chicken Hypothalamus.

Changes in gene activity through epigenetic alterations induced by early environmental challenges during embryogenesis are known to impact the phenotype, health, and disease risk of animals. Learning how environmental cues translate into persisting epigenetic memory may open new doors to improve robustness and resilience of developing animals. It has previously been shown that the heat tolerance of male broiler chickens was improved by cyclically elevating egg incubation temperature. The embryonic thermal manipulation enhanced gene expression response in muscle (P. major) when animals were heat challenged at slaughter age, 35 days post-hatch. However, the molecular mechanisms underlying this phenomenon remain unknown. Here, we investigated the genome-wide distribution, in hypothalamus and muscle tissues, of two histone post-translational modifications, H3K4me3 and H3K27me3, known to contribute to environmental memory in eukaryotes. We found 785 H3K4me3 and 148 H3K27me3 differential peaks in the hypothalamus, encompassing genes involved in neurodevelopmental, metabolic, and gene regulation functions. Interestingly, few differences were identified in the muscle tissue for which differential gene expression was previously described. These results demonstrate that the response to embryonic thermal manipulation (TM) in chicken is mediated, at least in part, by epigenetic changes in the hypothalamus that may contribute to the later-life thermal acclimation.

SHOOT GROWTH1 maintains Arabidopsis epigenomes by regulating IBM1.

Maintaining correct DNA and histone methylation patterns is essential for the development of all eukaryotes. In Arabidopsis, we identified SHOOT GROWTH1 (SG1), a novel protein involved in the control of gene methylation. SG1 contains both a Bromo-Adjacent Homology (BAH) domain found in several chromatin regulators and an RNA-Recognition Motif (RRM). The sg1 mutations are associated with drastic pleiotropic phenotypes. The mutants degenerate after few generations and are similar to mutants of the histone demethylase INCREASE IN BONSAI METHYLATION1 (IBM1). A methylome analysis of sg1 mutants revealed a large number of gene bodies hypermethylated in the cytosine CHG context, associated with an increase in di-methylation of lysine 9 on histone H3 tail (H3K9me2), an epigenetic mark normally found in silenced transposons. The sg1 phenotype is suppressed by mutations in genes encoding the DNA methyltransferase CHROMOMETHYLASE3 (CMT3) or the histone methyltransferase KRYPTONITE (KYP), indicating that SG1 functions antagonistically to CMT3 or KYP. We further show that the IBM1 transcript is not correctly processed in sg1, and that the functional IBM1 transcript complements sg1. Altogether, our results suggest a function for SG1 in the maintenance of genome integrity by regulating IBM1.

Lessons from eQTL mapping studies: non-coding regions and their role behind natural phenotypic variation in plants.

Even if considerable progress has been achieved towards the understanding of natural variation in plant systems, the contribution of transcript abundance variation to phenotypic diversity remains unappreciated. Over the last decade, efforts to characterise the genome-wide expression variation in natural accessions, structured populations and hybrids have improved our knowledge of the contribution of non-coding polymorphisms to gene expression regulation. Moreover, new studies are helping to unravel the role of expression polymorphisms and their orchestrated performance. Recent advances involving classical linkage analysis, GWAS and improved eQTL mapping strategies will provide a greater resolution to determine the genetic variants shaping the broad diversity in plant systems.

Quantitative modulation of polycomb silencing underlies natural variation in vernalization.

Arabidopsis thaliana accessions have adapted to growth in a wide range of climates. Variation in flowering and alignment of vernalization response with winter length are central to this adaptation. Vernalization involves the epigenetic silencing of the floral repressor FLC via a conserved Polycomb (PRC2) mechanism involving trimethylation of Lys(27) on histone H3 (H3K27me3). We found that variation for response to winter length maps to cis polymorphism within FLC. A rare combination of four polymorphisms localized around the nucleation region of a PHD-Polycomb complex determines a need for longer cold. Chromatin immunoprecipitation experiments indicate that these polymorphisms influence the accumulation of H3K27me3 in Arabidopsis accession Lov-1, both at the nucleation site and over the gene body. Quantitative modulation of chromatin silencing through cis variation may be a general mechanism contributing to evolutionary change.

Protocol: A simple phenol-based method for 96-well extraction of high quality RNA from Arabidopsis.

BACKGROUND: Many experiments in modern plant molecular biology require the processing of large numbers of samples for a variety of applications from mutant screens to the analysis of natural variants. A severe bottleneck to many such analyses is the acquisition of good yields of high quality RNA suitable for use in sensitive downstream applications such as real time quantitative reverse-transcription-polymerase chain reaction (real time qRT-PCR). Although several commercial kits are available for high-throughput RNA extraction in 96-well format, only one non-kit method has been described in the literature using the commercial reagent TRIZOL. RESULTS: We describe an unusual phenomenon when using TRIZOL reagent with young Arabidopsis seedlings. This prompted us to develop a high-throughput RNA extraction protocol (HTP96) adapted from a well established phenol:chloroform-LiCl method (P:C-L) that is cheap, reliable and requires no specialist equipment. With this protocol 192 high quality RNA samples can be prepared in 96-well format in three hours (less than 1 minute per sample) with less than 1% loss of samples. We demonstrate that the RNA derived from this protocol is of high quality and suitable for use in real time qRT-PCR assays. CONCLUSION: The development of the HTP96 protocol has vastly increased our sample throughput, allowing us to fully exploit the large sample capacity of modern real time qRT-PCR thermocyclers, now commonplace in many labs, and develop an effective high-throughput gene expression platform. We propose that the HTP96 protocol will significantly benefit any plant scientist with the task of obtaining hundreds of high quality RNA extractions.

Unique and redundant functions of C. elegans HP1 proteins in post-embryonic development.

HP1 proteins are essential components of heterochromatin and contribute to the transcriptional repression of euchromatic genes. Although most species contain more than one HP1 family member which differ in their chromosomal distribution, it is not known to what extent the activity of these different family members is redundant or specific in a developmental context. C. elegans has two HP1 homologues, HPL-1 and HPL-2. While HPL-2 functions in vulval and germline development, no function has so far been attributed to HPL-1. Here we report the characterization of an hpl-1 null allele. We show that while the absence of hpl-1 alone results in no obvious phenotype, hpl-1;hpl-2 double mutants show synthetic, temperature sensitive phenotypes including larval lethality and severe defects in the development of the somatic gonad. Furthermore, we find that hpl-1 has an unexpected role in vulval development by acting redundantly with hpl-2, but not other genes previously implicated in vulval development. Localization studies show that like HPL-2, HPL-1 is a ubiquitously expressed nuclear protein. However, HPL-1 and HPL-2 localization does not completely overlap. Our results show that HPL-1 and HPL-2 play both unique and redundant functions in post-embryonic development.