Birger Scholz

Faced with inequality: chicken do not have a general dosage compensation of sex-linked genes

BackgroundThe contrasting dose of sex chromosomes in males and females potentially introduces a large-scale imbalance in levels of gene expression between sexes, and between sex chromosomes and autosomes. In many organisms, dosage compensation has thus evolved to equalize sex-linked gene expression in males and females. In mammals this is achieved by X chromosome inactivation and in flies and worms by up- or down-regulation of X-linked expression, respectively. While otherwise widespread in systems with heteromorphic sex chromosomes, the case of dosage compensation in birds (males ZZ, females ZW) remains an unsolved enigma.ResultsHere, we use a microarray approach to show that male chicken embryos generally express higher levels of Z-linked genes than female birds, both in soma and in gonads. The distribution of male-to-female fold-change values for Z chromosome genes is wide and has a mean of 1.4–1.6, which is consistent with absence of dosage compensation and sex-specific feedback regulation of gene expression at individual loci. Intriguingly, without global dosage compensation, the female chicken has significantly lower expression levels of Z-linked compared to autosomal genes, which is not the case in male birds.ConclusionThe pronounced sex difference in gene expression is likely to contribute to sexual dimorphism among birds, and potentially has implication to avian sex determination. Importantly, this report, together with a recent study of sex-biased expression in somatic tissue of chicken, demonstrates the first example of an organism with a lack of global dosage compensation, providing an unexpected case of a viable system with large-scale imbalance in gene expression between sexes.

Development and Evaluation of Normalization Methods for Label-free Relative Quantification of Endogenous Peptides

The performances of 10 different normalization methods on data of endogenous brain peptides produced with label-free nano-LC-MS were evaluated. Data sets originating from three different species (mouse, rat, and Japanese quail), each consisting of 35–45 individual LC-MS analyses, were used in the study. Each sample set contained both technical and biological replicates, and the LC-MS analyses were performed in a randomized block fashion. Peptides in all three data sets were found to display LC-MS analysis order-dependent bias. Global normalization methods will only to some extent correct this type of bias. Only the novel normalization procedure RegrRun (linear regression followed by analysis order normalization) corrected for this type of bias. The RegrRun procedure performed the best of the normalization methods tested and decreased the median S.D. by 43% on average compared with raw data. This method also produced the smallest fraction of peptides with interblock differences while producing the largest fraction of differentially expressed peaks between treatment groups in all three data sets. Linear regression normalization (Regr) performed second best and decreased median S.D. by 38% on average compared with raw data. All other examined methods reduced median S.D. by 20–30% on average compared with raw data.

Proteomic Evaluation of Neonatal Exposure to 2,2′,4,4′,5-Pentabromodiphenyl Ether

Exposure to the brominated flame retardant 2,2′,4,4′,5-pentabromodiphenyl ether (PBDE-99) during the brain growth spurt disrupts normal brain development in mice and results in disturbed spontaneous behavior in adulthood. The neurodevelopmental toxicity of PBDE-99 has been reported to affect the cholinergic and catecholaminergic systems. In this study we use a proteomics approach to study the early effect of PBDE-99 in two distinct regions of the neonatal mouse brain, the striatum and the hippocampus. A single oral dose of PBDE-99 (12 mg/kg body weight) or vehicle was administered to male NMRI mice on neonatal day 10, and the striatum and the hippocampus were isolated. Using two-dimensional fluorescence difference gel electrophoresis (2D-DIGE), we found 40 and 56 protein spots with significantly (p < 0.01) altered levels in the striatum and the hippocampus, respectively. We used matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI–ToF–MS) to determine the protein identity of 11 spots from the striatum and 10 from the hippocampus. We found that the levels of proteins involved in neurodegeneration and neuroplasticity (e.g., Gap-43/neuromodulin, stathmin) were typically altered in the striatum, and proteins involved in metabolism and energy production [e.g., α-enolase; γ-enolase; ATP synthase, H+ transporting, mitochondrial F1 complex, β subunit (Atp5b); and α-synuclein] were typically altered in the hippocampus. Interestingly, many of the identified proteins have been linked to protein kinase C signaling. In conclusion, we identify responses to early exposure to PBDE-99 that could contribute to persistent neurotoxic effects. This study also shows the usefulness of proteomics to identify potential biomarkers of developmental neurotoxicity of organohalogen compounds.

Sex-dependent gene expression in early brain development of chicken embryos

BackgroundDifferentiation of the brain during development leads to sexually dimorphic adult reproductive behavior and other neural sex dimorphisms. Genetic mechanisms independent of steroid hormones produced by the gonads have recently been suggested to partly explain these dimorphisms.ResultsUsing cDNA microarrays and real-time PCR we found gene expression differences between the male and female embryonic brain (or whole head) that may be independent of morphological differentiation of the gonads. Genes located on the sex chromosomes (ZZ in males and ZW in females) were common among the differentially expressed genes, several of which (WPKCI-8, HINT, MHM non-coding RNA) have previously been implicated in avian sex determination. A majority of the identified genes were more highly expressed in males. Three of these genes (CDK7, CCNH and BTF2-P44) encode subunits of the transcription factor IIH complex, indicating a role for this complex in neuronal differentiation.ConclusionIn conclusion, this study provides novel insights into sexually dimorphic gene expression in the embryonic chicken brain and its possible involvement in sex differentiation of the nervous system in birds.

Striatal Proteomic Analysis Suggests that First L-Dopa Dose Equates to Chronic Exposure

L-3,4-dihydroxypheylalanine (L-dopa)-induced dyskinesia represent a debilitating complication of therapy for Parkinsons disease (PD) that result from a progressive sensitization through repeated L-dopa exposures. The MPTP macaque model was used to study the proteome in dopamine-depleted striatum with and without subsequent acute and chronic L-dopa treatment using two-dimensional difference in-gel electrophoresis (2D-DIGE) and mass spectrometry. The present data suggest that the dopamine-depleted striatum is so sensitive to de novo L-dopa treatment that the first ever administration alone would be able (i) to induce rapid post-translational modification-based proteomic changes that are specific to this first exposure and (ii), possibly, lead to irreversible protein level changes that would be not further modified by chronic L-dopa treatment. The apparent equivalence between first and chronic L-dopa administration suggests that priming would be the direct consequence of dopamine loss, the first L-dopa administrations only exacerbating the sensitization process but not inducing it.

Impact of Temperature Dependent Sampling Procedures in Proteomics and Peptidomics – A Characterization of the Liver and Pancreas Post Mortem Degradome

Little is known about the nature of post mortem degradation of proteins and peptides on a global level, the so-called degradome. This is especially true for nonneural tissues. Degradome properties in relation to sampling procedures on different tissues are of great importance for the studies of, for instance, post translational modifications and/or the establishment of clinical biobanks. Here, snap freezing of fresh (<2 min post mortem time) mouse liver and pancreas tissue is compared with rapid heat stabilization with regard to effects on the proteome (using two-dimensional differential in-gel electrophoresis) and peptidome (using label free liquid chromatography). We report several proteins and peptides that exhibit heightened degradation sensitivity, for instance superoxide dismutase in liver, and peptidyl-prolyl cis-trans isomerase and insulin C-peptides in pancreas. Tissue sampling based on snap freezing produces a greater amount of degradation products and lower levels of endogenous peptides than rapid heat stabilization. We also demonstrate that solely snap freezing related degradation can be attenuated by subsequent heat stabilization. We conclude that tissue sampling involving a rapid heat stabilization step is preferable to freezing with regard to proteomic and peptidomic sample quality.

In Vitro Neurotoxicity of PBDE-99 : Immediate and Concentration-Dependent Effects on Protein Expression in Cerebral Cortex Cells

Polybrominated diphenyl ethers (PBDEs) are commonly used flame retardants in various consumer products. Pre- and postnatal exposure to congeners of PBDEs disrupts normal brain development in rodents. Two-dimensional difference gel electrophoresis (2D-DIGE) was used to analyze concentration-dependent differences in protein expression in cultured cortical cells isolated from rat fetuses (GD 21) after 24 h exposure to PBDE-99 (3, 10, or 30 microM). Changes on a post-translational level were studied using a 1 h exposure to 30 microM PBDE-99. The effects of 24 h exposure to 3 and 30 microM PBDE-99 on mRNA levels were measured using oligonucleotide microarrays. A total of 62, 46, and 443 proteins were differentially expressed compared to controls after 24 h of exposure to 3, 10, and 30 microM PDBE-99, respectively. Of these, 48, 43, and 238 proteins were successfully identified, respectively. We propose that the biological effects of low-concentration PBDE-99 exposure are fundamentally different than effects of high-concentration exposure. Low-dose PBDE-99 exposure induced marked effects on cytoskeletal proteins, which was not correlated to cytotoxicity or major morphological effects, suggesting that other more regulatory aspects of cytoskeletal functions may be affected. Interestingly, 0.3 and 3 microM, but not 10 or 30 microM increased the expression of phosphorylated (active) Gap43, perhaps reflecting effects on neurite extension processes.

The impact of biosampling procedures on molecular data interpretation

The separation between biological and technical variation without extensive use of technical replicates is often challenging, particularly in the context of different forms of protein and peptide modifications. Biosampling procedures in the research laboratory are easier to conduct within a shorter time frame and under controlled conditions as compared with clinical sampling, with the latter often having issues of reproducibility. But is the research laboratory biosampling really less variable? Biosampling introduces within minutes rapid tissue-specific changes in the cellular microenvironment, thus inducing a range of different pathways associated with cell survival. Biosampling involves hypoxia and, depending on the circumstances, hypothermia, circumstances for which there are evolutionarily conserved defense strategies in the range of species and also are relevant for the range of biomedical conditions. It remains unclear to what extent such adaptive processes are reflected in different biosampling procedures or how important they are for the definition of sample quality. Lately, an increasing number of comparative studies on different biosampling approaches, post-mortem effects and pre-sampling biological state, have investigated such immediate early biosampling effects. Commonalities between biosampling effects and a range of ischemia/reperfusion- and hypometabolism/anoxia-associated biological phenomena indicate that even small variations in post-sampling time intervals are likely to introduce a set of nonrandom and tissue-specific effects of experimental importance (both in vivo and in vitro). This review integrates the information provided by these comparative studies and discusses how an adaptive biological perspective in biosampling procedures may be relevant for sample quality issues.

Effects of the Histone Deacetylase Inhibitor Valproic Acid on Human Pericytes In Vitro

Microvascular pericytes are of key importance in neoformation of blood vessels, in stabilization of newly formed vessels as well as maintenance of angiostasis in resting tissues. Furthermore, pericytes are capable of differentiating into pro-fibrotic collagen type I producing fibroblasts. The present study investigates the effects of the histone deacetylase (HDAC) inhibitor valproic acid (VPA) on pericyte proliferation, cell viability, migration and differentiation. The results show that HDAC inhibition through exposure of pericytes to VPA in vitro causes the inhibition of pericyte proliferation and migration with no effect on cell viability. Pericyte exposure to the potent HDAC inhibitor Trichostatin A caused similar effects on pericyte proliferation, migration and cell viability. HDAC inhibition also inhibited pericyte differentiation into collagen type I producing fibroblasts. Given the importance of pericytes in blood vessel biology a qPCR array focusing on the expression of mRNAs coding for proteins that regulate angiogenesis was performed. The results showed that HDAC inhibition promoted transcription of genes involved in vessel stabilization/maturation in human microvascular pericytes. The present in vitro study demonstrates that VPA influences several aspects of microvascular pericyte biology and suggests an alternative mechanism by which HDAC inhibition affects blood vessels. The results raise the possibility that HDAC inhibition inhibits angiogenesis partly through promoting a pericyte phenotype associated with stabilization/maturation of blood vessels.

Neuropeptidomic analysis of the embryonic Japanese quail diencephalon

BackgroundEndogenous peptides such as neuropeptides are involved in numerous biological processes in the fully developed brain but very little is known about their role in brain development. Japanese quail is a commonly used bird model for studying sexual dimorphic brain development, especially adult male copulatory behavior in relation to manipulations of the embryonic endocrine system. This study uses a label-free liquid chromatography mass spectrometry approach to analyze the influence of age (embryonic days 12 vs 17), sex and embryonic day 3 ethinylestradiol exposure on the expression of multiple endogenous peptides in the developing diencephalon.ResultsWe identified a total of 65 peptides whereof 38 were sufficiently present in all groups for statistical analysis. Age was the most defining variable in the data and sex had the least impact. Most identified peptides were more highly expressed in embryonic day 17. The top candidates for EE2 exposure and sex effects were neuropeptide K (downregulated by EE2 in males and females), gastrin-releasing peptide (more highly expressed in control and EE2 exposed males) and gonadotropin-inhibiting hormone related protein 2 (more highly expressed in control males and displaying interaction effects between age and sex). We also report a new potential secretogranin-2 derived neuropeptide and previously unknown phosphorylations in the C-terminal flanking protachykinin 1 neuropeptide.ConclusionsThis study is the first larger study on endogenous peptides in the developing brain and implies a previously unknown role for a number of neuropeptides in middle to late avian embryogenesis. It demonstrates the power of label-free liquid chromatography mass spectrometry to analyze the expression of multiple endogenous peptides and the potential to detect new putative peptide candidates in a developmental model.