Neurogenesis in the water flea Daphnia magna (Crustacea, Branchiopoda) suggests different mechanisms of neuroblast formation in insects and crustaceans

Within euarthropods, the morphological and molecular mechanisms of early nervous system development have been analysed in insects and several representatives of chelicerates and myriapods, while data on crustaceans are fragmentary. Neural stem cells (neuroblasts) generate the nervous system in insects and in higher crustaceans (malacostracans); in the remaining euarthropod groups, the chelicerates (e.g. spiders) and myriapods (e.g. millipedes), neuroblasts are missing. In the latter taxa, groups of neural precursors segregate from the neuroectoderm and directly differentiate into neurons and glial cells. In all euarthropod groups, achaete–scute homologues are required for neuroblast/neural precursor group formation. In the insects Drosophila melanogaster and Tribolium castaneum achaete–scute homologues are initially expressed in clusters of cells (proneural clusters) in the neuroepithelium but expression becomes restricted to the future neuroblast. Subsequently genes such as snail and prospero are expressed in the neuroblasts which are required for asymmetric division and differentiation. In contrast to insects, malacostracan neuroblasts do not segregate into the embryo but remain in the outer neuroepithelium, similar to vertebrate neural stem cells. It has been suggested that neuroblasts are present in another crustacean group, the branchiopods, and that they also remain in the neuroepithelium. This raises the questions how the molecular mechanisms of neuroblast selection have been modified during crustacean and insect evolution and if the segregation or the maintenance of neuroblasts in the neuroepithelium represents the ancestral state. Here we take advantage of the recently published Daphnia pulex (branchiopod) genome and identify genes in Daphnia magna that are known to be required for the selection and asymmetric division of neuroblasts in the fruit fly D. melanogaster. We unambiguously identify neuroblasts in D. magna by molecular marker gene expression and division pattern. We show for the first time that branchiopod neuroblasts divide in the same pattern as insect and malacostracan neuroblasts. Furthermore, in contrast to D. melanogaster, neuroblasts are not selected from proneural clusters in the branchiopod. Snail rather than ASH is the first gene to be expressed in the nascent neuroblasts suggesting that ASH is not required for the selection of neuroblasts as in D. melanogaster. The prolonged expression of ASH in D. magna furthermore suggests that it is involved in the maintenance of the neuroblasts in the neuroepithelium. Based on these and additional data from various representatives of arthropods we conclude that the selection of neural precursors from proneural clusters as well as the segregation of neural precursors represents the ancestral state of neurogenesis in arthropods. We discuss that the derived characters of malacostracans and branchiopods – the absence of neuroblast segregation and proneural clusters – might be used to support or reject the possible groupings of paraphyletic crustaceans.     

The tumor suppressor protein DLC1 is regulated by PKD-mediated GAP domain phosphorylation

Deleted in liver cancer 1 (DLC1) is a tumor suppressor protein that is frequently downregulated in various tumor types. DLC1 contains a Rho GTPase activating protein (GAP) domain that appears to be required for its tumor suppressive functions. Little is known about the molecular mechanisms that regulate DLC1. By mass spectrometry we have mapped a novel phosphorylation site within the DLC1 GAP domain on serine 807. Using a phospho-S807-specific antibody, our results identify protein kinase D (PKD) to phosphorylate this site in DLC1 in intact cells. Although phosphorylation on serine 807 did not directly impact on in vitro GAP activity, a DLC1 serine-to-alanine exchange mutant inhibited colony formation more potently than the wild type protein. Our results thus show that PKD-mediated phosphorylation of DLC1 on serine 807 negatively regulates DLC1 cellular function.     

Impaired nutrient signaling and body weight control in a Na+ neutral amino acid cotransporter (Slc6a19)-deficient mouse

Amino acid uptake in the intestine and kidney is mediated by a variety of amino acid transporters. To understand the role of epithelial neutral amino acid uptake in whole body homeostasis, we analyzed mice lacking the apical broad-spectrum neutral (0) amino acid transporter B(0)AT1 (Slc6a19). A general neutral aminoaciduria was observed similar to human Hartnup disorder which is caused by mutations in SLC6A19. Na(+)-dependent uptake of neutral amino acids into the intestine and renal brush-border membrane vesicles was abolished. No compensatory increase of peptide transport or other neutral amino acid transporters was detected. Mice lacking B(0)AT1 showed a reduced body weight. When adapted to a standard 20% protein diet, B(0)AT1-deficient mice lost body weight rapidly on diets containing 6 or 40% protein. Secretion of insulin in response to food ingestion after fasting was blunted. In the intestine, amino acid signaling to the mammalian target of rapamycin (mTOR) pathway was reduced, whereas the GCN2/ATF4 stress response pathway was activated, indicating amino acid deprivation in epithelial cells. The results demonstrate that epithelial amino acid uptake is essential for optimal growth and body weight regulation.     

Proteolytic cleavage inactivates the Staphylococcus aureus lipoteichoic acid synthase

Lipoteichoic acid (LTA) is a crucial cell envelope component in Gram-positive bacteria. In Staphylococcus aureus, the polyglycerolphosphate LTA molecule is synthesized by LtaS, a membrane-embedded enzyme with five N-terminal transmembrane helices (5TM domain) that are connected via a linker region to the C-terminal extracellular enzymatic domain (eLtaS). The LtaS enzyme is processed during bacterial growth, and the eLtaS domain is released from the bacterial membrane. Here we provide experimental evidence that the proteolytic cleavage following residues 215Ala-Leu-Ala217 is performed by the essential S. aureus signal peptidase SpsB, as depletion of spsB results in reduced LtaS processing. In addition, the introduction of a proline residue at the +1 position with respect to the cleavage site, a substitution known to inhibit signal peptidase-dependent cleavage, abolished LtaS processing at this site. It was further shown that the 5TM domain is crucial for enzyme function. The observation that the construction of hybrid proteins between two functional LtaS-type enzymes resulted in the production of proteins unable to synthesize LTA suggests that specific interactions between the 5TM and eLtaS domains are required for function. No enzyme activity was detected upon expression of the 5TM and eLtaS domains as separate fragments, indicating that the two domains cannot assemble postsynthesis to form a functional enzyme. Taken together, our data suggest that only the full-length LtaS enzyme is active in the LTA synthesis pathway and that the proteolytic cleavage step is used as a mechanism to irreversibly inactivate the enzyme.     

Distribution of soil organic matter between fractions and aggregate size classes in grazed semiarid steppe soil profiles

Grazed steppe ecosystems are discussed as one of the big global carbon sinks that may have the potential to sequester large amounts of atmospheric CO₂ and mitigate the effects of global change if grazing is abandoned or management improved. But until today, little is known about sequestration potentials and stabilisation mechanisms in complete soil profiles of semiarid grasslands and how these systems react to grazing cessation. We applied a combined aggregate size, density and particle size fractionation procedure to sandy steppe soils under different grazing intensities (continuously grazed = Cg, winter grazing = Wg, ungrazed since 1999 = Ug99, ungrazed since 1979 = Ug79). Higher inputs of organic matter in ungrazed treatments led to higher amounts of OC in coarse aggregate size classes (ASC) and especially in particulate organic matter (POM) fractions across all depth. These processes started in the topsoil and took more than 5 years to reach deeper soil horizons (>10 cm). After 25 years of grazing cessation, subsoils showed clearly higher POM amounts. We found no grazing-induced changes of soil organic matter (SOM) quantity in fine ASC and particle size fractions. Current C-loading of fine particle size fractions was similar between differently grazed plots and decreased with depth, pointing towards free sequestration capacities in deeper horizons. Despite these free capacities, we found no increase in current C-loading on fine mineral soil fractions after 25 years of grazing exclusion. Silt and clay fractions appeared to be saturated. We suppose empirical estimations to overestimate sequestration potentials of particle size fractions or climatic conditions to delay the decomposition and incorporation of OM into these particle size fractions. POM quality was analysed using solid-state ¹³C NMR spectroscopy to clarify if grazing cessation changed chemical composition of POM in different ASC and soil depths via changing litter quality or changing decomposition dynamics. We found comparable POM compositions between different grazing intensities. POM is decomposed hierarchically from coarse to fine particles in all soil depths and grazing cessation has not affected the OM decomposition processes. The surplus of OM due to grazing cessation was predominately sequestered in readily decomposable POM fractions across all affected horizons. We question the long-term stabilisation of OM in these steppe soils during the first 25 years after grazing cessation and request more studies in the field of long-term OM stabilisation processes and assessment of carbon sequestration capacities to consider deeper soil horizons.     

Grazing changes topography-controlled topsoil properties and their interaction on different spatial scales in a semi-arid grassland of Inner Mongolia, P.R. China

Semiarid steppe ecosystems account for large terrestrial areas and are considered as large carbon (C) sinks. However, fundamental information on topsoil sensitivity to grazing is lacking across different spatial scales including the effects of topography. Our interdisciplinary approach considering soil chemical, physical, and vegetation properties included investigations on pit scale (square-metre scale), plot scale (hectare scale), and the scale of a landscape section (several hectares). Five different sites, representing a grazing intensity gradient, ranging from a long-term grazing exclosure to a heavily grazed site were used. On the pit scale, data about aggregate size distribution, quantity of different soil organic carbon (SOC) pools, SOC mineralisation, hydraulic conductivity and shear strength was available for topsoil samples from representative soil profiles. Spatial variability of topographical parameters, topsoil texture, bulk density, SOC, water repellency, and vegetation cover was analysed on the basis of regular, orthogonal grids in differently grazed treatments by using two different grid sizes on the plot scale and landscape section. On the pit scale, intensive grazing clearly decreased soil aggregation and the amount of fresh, litter-like particulate organic matter (POM). The weak aggregation in combination with animal trampling led to an enhanced mineralisation of SOC, higher topsoil bulk densities, lower infiltration rates, and subsequently to a higher risk of soil erosion. On the plot scale, the effects of soil structure disruption due to grazing are enhanced by the degradation of vegetation patches and resulted in a texture-controlled wettability of the soil surface. In contrast, topsoils of grazing exclosures were characterised by advantageous mechanical topsoil characteristics and SOC-controlled wettability due to higher POM contents. A combined geostatistical and General Linear Model approach identified topography as the fundamental factor creating the spatial distribution of texture fractions and related soil parameters on the scale of a landscape section. Grazing strongly interfered with the topography-controlled particle relocation processes in the landscape and showed strongest effects on the aboveground biomass production and biomass-related soil properties like SOC stocks. We conclude that interdisciplinary multi-scale analyses are essential (i) to differentiate between topography- and grazing-controlled spatial patterns of topsoil and vegetation properties, and (ii) to identify the main grazing-sensitive processes on small scales that are interacting with the spatial distribution and relocation processes on larger scales.     

Diphosphoinositol polyphosphates: what are the mechanisms?

In countries where adulthood is considered to be attained at age eighteen, 2011 can be the point at which the diphosphoinositol polyphosphates might formally be described as "coming of age", since these molecules were first fully defined in 1993 (Menniti et al., 1993; Stephens et al., 1993b). But from a biological perspective, these polyphosphates cannot quite be considered to have matured into the status of being independently-acting intracellular signals. This review has discussed several of the published proposals for mechanisms by which the diphosphoinositol polyphosphates might act. We have argued that all of these hypotheses need further development.We also still do not know a single molecular mechanism by which a change in the levels of a particular diphosphoinositol polyphosphate can be controlled. Yet, despite all these gaps in our understanding, there is an enduring anticipation that these molecules have great potential in the signaling field. Reflecting our expectations of all teenagers, it should be our earnest hope that in the near future the diphosphoinositol polyphosphates will finally grow up.     

Non-CpG hypermethylation in placenta of mutation-induced intrauterine growth restricted bovine foetuses

The existence of non-CpG methylation in mammalian DNA has mainly been observed in embryonic stem cells, but its functional significance is uncertain. We found an age-dependent non-CpG hypermethylation in DMR at the 3′ end of the MIMT1 in the placenta of intrauterine growth restricted foetuses in cattle. Data suggest that this DMR play a role in epigenetic regulation of the PEG3 domain.     

Unanticipated Success Stories: An Interview with Angelika Amon

THE Genetics Society of America Medal is awarded to an individual for outstanding contributions to the field of genetics in the past 15 years. Recipients of the GSA Medal are recognized for elegant and highly meaningful contributions to modern genetics. The 2014 recipient, Angelika B. Amon, has uncovered key principles governing the cell cycle and was the first to demonstrate a connection between the physical completion of anaphase and the initiation of mitotic exit. More recently, her research has focused on the consequences of aneuploidy. GENETICS spoke with Dr. Amon about her approach to science and what is next on the horizon.Open in a separate windowAngelika B. Amon