Creatine supports propagation and promotes neuronal differentiation of inner ear progenitor cells

Long-term propagation of inner ear-derived progenitor/stem cells beyond the third generation and differentiation into inner ear cell types has been shown to be feasible, but challenging. We investigated whether the known neuroprotective guanidine compound creatine (Cr) promotes propagation of inner ear progenitor/stem cells as mitogen-expanded neurosphere cultures judged from the formation of spheres over passages. In addition, we studied whether Cr alone or in combination with brain-derived neurotrophic factor (BDNF) promotes neuronal differentiation of inner ear progenitors. For this purpose, early postnatal rat spiral ganglia, utricle, and organ of Corti-derived progenitors were grown as floating spheres in the absence (controls) or presence of Cr (5 mM) from passage 3 onward. Similarly, dissociated sphere-derived cultures were differentiated for 14 days in the presence or absence of Cr (5 mM) and spiral ganglia sphere-derived cultures in a combination of Cr with the neurotrophin BDNF (50 ng/ml). We found that the cumulative total number of spheres over all passages was significantly higher after Cr supplementation as compared with controls in all the three inner ear cultures. In contrast, sphere sizes were not affected by the administration of Cr. Administration of Cr during differentiation of spiral ganglia cells resulted in a significantly higher density of β-III-tubulin-positive cells compared with controls, whereas densities of myosin VIIa-positive cells in cultures of utricle and organ of Corti were not affected by the treatment. Importantly, a combination of Cr with the neurotrophin BDNF resulted in further significantly increased densities of β-III-tubulin-positive cells in cultures of spiral ganglia cells as compared with single treatments. In sum, Cr promoted continuing propagation of rat inner ear-derived progenitor cells and supported specifically in combination with BDNF the differentiation of neuronal cell types from spiral ganglion-derived spheres.

CEBPA-dependent HK3 and KLF5 expression in primary AML and during AML differentiation

The basic leucine zipper transcription factor CCAAT/enhancer binding protein alpha (CEBPA) codes for a critical regulator during neutrophil differentiation. Aberrant expression or function of this protein contributes to the development of acute myeloid leukemia (AML). In this study, we identified two novel unrelated CEBPA target genes, the glycolytic enzyme hexokinase 3 (HK3) and the krüppel-like factor 5 (KLF5) transcription factor, by comparing gene profiles in two cohorts of CEBPA wild-type and mutant AML patients. In addition, we found CEBPA-dependent activation of HK3 and KLF5 transcription during all-trans retinoic acid (ATRA) mediated neutrophil differentiation of acute promyelocytic leukemia (APL) cells. Moreover, we observed direct regulation of HK3 by CEBPA, whereas our data suggest an indirect regulation of KLF5 by this transcription factor. Altogether, our data provide an explanation for low HK3 and KLF5 expression in particular AML subtype and establish these genes as novel CEBPA targets during neutrophil differentiation.

A mutation in the SUV39H2 gene in Labrador Retrievers with hereditary nasal parakeratosis (HNPK) provides insights into the epigenetics of keratinocyte differentiation.

Hereditary nasal parakeratosis (HNPK), an inherited monogenic autosomal recessive skin disorder, leads to crusts and fissures on the nasal planum of Labrador Retrievers. We performed a genome-wide association study (GWAS) using 13 HNPK cases and 23 controls. We obtained a single strong association signal on chromosome 2 (p(raw) = 4.4×10⁻¹⁴). The analysis of shared haplotypes among the 13 cases defined a critical interval of 1.6 Mb with 25 predicted genes. We re-sequenced the genome of one case at 38× coverage and detected 3 non-synonymous variants in the critical interval with respect to the reference genome assembly. We genotyped these variants in larger cohorts of dogs and only one was perfectly associated with the HNPK phenotype in a cohort of more than 500 dogs. This candidate causative variant is a missense variant in the SUV39H2 gene encoding a histone 3 lysine 9 (H3K9) methyltransferase, which mediates chromatin silencing. The variant c.972T>G is predicted to change an evolutionary conserved asparagine into a lysine in the catalytically active domain of the enzyme (p.N324K). We further studied the histopathological alterations in the epidermis in vivo. Our data suggest that the HNPK phenotype is not caused by hyperproliferation, but rather delayed terminal differentiation of keratinocytes. Thus, our data provide evidence that SUV39H2 is involved in the epigenetic regulation of keratinocyte differentiation ensuring proper stratification and tight sealing of the mammalian epidermis.

Analysis of protein expression in zebrafish during gonad differentiation by targeted proteomics

The molecular mechanisms governing sex determination and differentiation in the zebrafish (Danio rerio) are not fully understood. To gain more insights into the function of specific genes in these complex processes, the expression of multiple candidates needs to be assessed, preferably on the protein level. Here, we developed a targeted proteomics method based on selected reaction monitoring (SRM) to study the candidate sex-related proteins in zebrafish which were selected based on a global proteomics analysis of adult gonads and representational difference analysis of male and female DNA, as well as on published information on zebrafish and other vertebrates. We employed the developed SRM protocols to acquire time-resolved protein expression profiles during the gonad differentiation period in vas::EGFP transgenic zebrafish. Evidence on protein expression was obtained for the first time for several candidate genes previously studied only on the mRNA level or suggested by bioinformatic predictions. Tuba1b (tubulin alpha 1b), initially included in the study as one of the potential housekeeping proteins, was found to be preferentially expressed in the adult testis with nearly absent expression in the ovary. The revealed changes in protein expression patterns associated with gonad differentiation suggest that several of the examined proteins, especially Ilf2 and Ilf3 (interleukin enhancer-binding factors 2 and 3), Raldh3 (retinaldehyde dehydrogenase type 3), Zgc:195027 (low density lipoprotein-related receptor protein 3) and Sept5a (septin 5a), may play a specific role in the sexual differentiation in zebrafish.

Antibodies trap tissue migrating helminth larvae and prevent tissue damage by driving IL-4Rα-independent alternative differentiation of macrophages

Approximately one-third of the world's population suffers from chronic helminth infections with no effective vaccines currently available. Antibodies and alternatively activated macrophages (AAM) form crucial components of protective immunity against challenge infections with intestinal helminths. However, the mechanisms by which antibodies target these large multi-cellular parasites remain obscure. Alternative activation of macrophages during helminth infection has been linked to signaling through the IL-4 receptor alpha chain (IL-4Rα), but the potential effects of antibodies on macrophage differentiation have not been explored. We demonstrate that helminth-specific antibodies induce the rapid trapping of tissue migrating helminth larvae and prevent tissue necrosis following challenge infection with the natural murine parasite Heligmosomoides polygyrus bakeri (Hp). Mice lacking antibodies (JH (-/-)) or activating Fc receptors (FcRγ(-/-)) harbored highly motile larvae, developed extensive tissue damage and accumulated less Arginase-1 expressing macrophages around the larvae. Moreover, Hp-specific antibodies induced FcRγ- and complement-dependent adherence of macrophages to larvae in vitro, resulting in complete larval immobilization. Antibodies together with helminth larvae reprogrammed macrophages to express wound-healing associated genes, including Arginase-1, and the Arginase-1 product L-ornithine directly impaired larval motility. Antibody-induced expression of Arginase-1 in vitro and in vivo occurred independently of IL-4Rα signaling. In summary, we present a novel IL-4Rα-independent mechanism of alternative macrophage activation that is antibody-dependent and which both mediates anti-helminth immunity and prevents tissue disruption caused by migrating larvae.

Repeated and predictable patterns of ecotypic differentiation during a biological invasion: lake-stream divergence in parapatric Swiss stickleback

The relative importance of ecological selection and geographical isolation in promoting and constraining genetic and phenotypic differentiation among populations is not always obvious. Interacting with divergent selection, restricted opportunity for gene flow may in some cases be as much a cause as a consequence of adaptation, with the latter being a hallmark of ecologi- cal speciation. Ecological speciation is well studied in parts of the native range of the three-spined stickleback. Here, we study this process in a recently invaded part of its range. Switzerland was colonized within the past 140 years from at least three different colonization events involving differ- ent stickleback lineages. They now occupy diverse habitats, ranging from small streams to the pelagic zone of large lakes. We use replicated systems of parapatric lake and stream populations, some of which trace their origins to different invasive lineages, to ask (i) whether phenotypic divergence occurred among populations inhabiting distinct habitats, (ii) whether trajec- tories of phenotypic divergence follow predictable parallel patterns and (iii) whether gene flow constrains divergent adaptation or vice versa. We find consistent phenotypic divergence between populations occupying distinct habitats. This involves parallel evolution in several traits with known eco- logical relevance in independent evolutionary lineages. Adaptive divergence supersedes homogenizing gene flow even at a small spatial scale. We find evidence that adaptive phenotypic divergence places constraints on gene flow over and above that imposed by geographical distance, signalling the early onset of ecological speciation.

Insights into the regulation of GPEET procyclin during differentiation from early to late procyclic forms of Trypanosoma brucei.

The procyclic form of Trypanosoma brucei colonises the gut of its insect vector, the tsetse fly. GPEET and EP procyclins constitute the parasite's surface coat at this stage of the life cycle, and the presence or absence of GPEET distinguishes between early and late procyclic forms, respectively. Differentiation from early to late procyclic forms in vivo occurs in the fly midgut and can be mimicked in culture. Our analysis of this transition in vitro delivered new insights into the process of GPEET repression. First, we could show that parasites followed a concrete sequence of events upon triggering differentiation: after undergoing an initial growth arrest, cells lost GPEET protein, and finally late procyclic forms resumed proliferation. Second, we determined the stability of both GPEET and EP mRNA during differentiation. GPEET mRNA is exceptionally stable in early procyclic forms, with a half-life >6h. The GPEET mRNA detected in late procyclic form cultures is a mixture of transcripts from both bona fide late procyclic forms and GPEET-positive 'laggard' parasites present in these cultures. However, its stability was clearly reduced during differentiation and in late procyclic form cultures. Alternatively processed GPEET transcripts were enriched in samples from late procyclic forms, suggesting that altered mRNA processing might contribute to repression of GPEET in this developmental stage. In addition, we detected GPEET transcripts with non-templated oligo(U) tails that were enriched in late procyclic forms. To the best of our knowledge, this is the first study reporting a uridylyl-tailed, nuclear-encoded mRNA species in trypanosomatids or any other protozoa.

RNA-dependent genome processing during nuclear differentiation: the model systems of stichotrichous ciliates

We introduce ciliated protozoa, and more specifically the stichotrichous ciliates Oxytricha and Stylonychia, as biological model systems for the analysis of programmed DNA-reorganization processes during nuclear differentiation. These include DNA excision, DNA elimination, reordering of gene segments and specific gene amplification. We show that small nuclear RNAs specify DNA sequences to be excised or retained, but also discuss the need for a RNA template molecule derived from the parental nucleus for these processes. This RNA template guides reordering of gene segments to become functional genes and determines gene copy number in the differentiated nucleus. Since the template is derived from the parental macronucleus, gene reordering and DNA amplification are inherited in a non-Mendelian epigenetic manner.

Magma differentiation fractionates Mo isotope ratios: Evidence from the Kos Plateau Tuff (Aegean Arc)

We investigated high temperature Mo isotope fractionation in a hydrous supra-subduction volcano-plutonic system (Kos, Aegean Arc, Greece) in order to address the debate on the δ98/95Mo variability of the continental crust. In this igneous system, where differentiation is interpreted to be dominated by fractional crystallization, bulk rock data from olivine basalt to dacite show δ98/95Mo ratios increasing from +0.3 to +0.6‰ along with Mo concentrations increasing from 0.8 to 4.1 μg g−1. Data for hornblende and biotite mineral separates reveal the extraction of light Mo into crystallizing silicates, with minimum partition coefficients between hornblende- silicate melt and biotite-silicate melt of 0.6 and 0.4 δ98/95Mo, respectively. Our data document significant Mo isotope fractionation at magmatic temperatures, hence, the igneous contribution to continental runoff is variable, besides probable source-related variability. Based on these results and published data an average continental δ98/95Mo of +0.3 to +0.4‰ can be derived. This signature corresponds more closely to the average of published data of dissolved Mo loads of large rivers than previous estimates and is consistent with an upper limit of δ98/95Mo = 0.4‰ of the Earth's upper crust as derived from the analysis of molybdenites.

Directed differentiation and functional maturation of cortical interneurons from human embryonic stem cells

Human pluripotent stem cells are a powerful tool for modeling brain development and disease. The human cortex is composed of two major neuronal populations: projection neurons and local interneurons. Cortical interneurons comprise a diverse class of cell types expressing the neurotransmitter GABA. Dysfunction of cortical interneurons has been implicated in neuropsychiatric diseases, including schizophrenia, autism, and epilepsy. Here, we demonstrate the highly efficient derivation of human cortical interneurons in an NKX2.1::GFP human embryonic stem cell reporter line. Manipulating the timing of SHH activation yields three distinct GFP+ populations with specific transcriptional profiles, neurotransmitter phenotypes, and migratory behaviors. Further differentiation in a murine cortical environment yields parvalbumin- and somatostatin-expressing neurons that exhibit synaptic inputs and electrophysiological properties of cortical interneurons. Our study defines the signals sufficient for modeling human ventral forebrain development in vitro and lays the foundation for studying cortical interneuron involvement in human disease pathology.

Core formation and mantle differentiation on Mars

Geochemical investigation of Martian meteorites (SNC meteorites) yields important constraints on the chemical and geodynamical evolution of Mars. These samples may not be representative of the whole of Mars; however, they provide constraints on the early differentiation processes on Mars. The bulk composition of Martian samples implies the presence of a metallic core that formed concurrently as the planet accreted. The strong depletion of highly siderophile elements in the Martian mantle is only possible if Mars had a large scale magma ocean early in its history allowing efficient separation of a metallic melt from molten silicate. The solidification of the magma ocean created chemical heterogeneities whose ancient origin is manifested in the heterogeneous 142Nd and 182W abundances observed in different meteorite groups derived from Mars. The isotope anomalies measured in SNC meteorites imply major chemical fractionation within the Martian mantle during the life time of the short-lived isotopes 146Sm and 182Hf. The Hf-W data are consistent with very rapid accretion of Mars within a few million years or, alternatively, a more protracted accretion history involving several large impacts and incomplete metal-silicate equilibration during core formation. In contrast to Earth early-formed chemical heterogeneities are still preserved on Mars, albeit slightly modified by mixing processes. The preservation of such ancient chemical differences is only possible if Mars did not undergo efficient whole mantle convection or vigorous plate tectonic style processes after the first few tens of millions of years of its history.