Summary

Sequential addition of segments in the posteriorly growing end of the embryo is a developmental mechanism common to many bilaterians. However, posterior growth and patterning in most animals also entails the establishment of a ‘posterior organiser’ that expresses the Caudal and Wnt proteins and has been proposed to be an ancestral feature of animal development. We have studied the functional relationships between the Wnt-driven organiser and the segmentation mechanisms in a basal insect, the cockroach Periplaneta americana. Here, posteriorly-expressed Wnt1 promotes caudal and Delta expression early in development to generate a growth zone from which segments will later bud off. caudal maintains the undifferentiated growth zone by dampening Delta expression, and hence Notch-mediated segmentation occurs just outside the caudal domain. In turn, Delta expression maintains Wnt1, maintaining this posterior gene network until all segments have formed. This feedback between caudal, Wnt and Notch-signalling in regulating growth and segmentation seems conserved in other arthropods, with some aspects found even in vertebrates. Thus our findings not only support an ancestral Wnt posterior organiser, but also impinge on the proposals for a common origin of segmentation in arthropods, annelids and vertebrates.

Summary

The generation of cellular microtubules is initiated at specific sites such as the centrosome and the Golgi apparatus that contain nucleation complexes rich in γ-tubulin. The microtubule growing plus-ends are stabilized by plus-end tracking proteins (+TIPs), mainly EB1 and associated proteins. Myomegalin was identified as a centrosome/Golgi protein associated with cyclic nucleotide phosphodiesterase. We show here that Myomegalin exists as several isoforms. We characterize two of them. One isoform, CM-MMG, harbors a conserved domain (CM1), recently described as a nucleation activator, and is related to a family of γ-tubulin binding proteins, which includes Drosophila centrosomin. It localizes at the centrosome and at the cis-Golgi in an AKAP450-dependent manner. It recruits γ-tubulin nucleating complexes and promotes microtubule nucleation. The second isoform, EB-MMG, is devoid of CM1 domain and has a unique N-terminus with potential EB1-binding sites. It localizes at the cis-Golgi and can localize to microtubule plus-ends. EB-MMG binds EB1 and affects its loading on microtubules and microtubule growth. Depletion of Myomegalin by small interfering RNA delays microtubule growth from the centrosome and Golgi apparatus, and decreases directional migration of RPE1 cells. In conclusion, the Myomegalin gene encodes different isoforms that regulate microtubules. At least two of these have different roles, demonstrating a previously unknown mechanism to control microtubules in vertebrate cells.

Summary

Thrombospondins are multimeric extracellular matrix glycoproteins that play important roles in development, synaptogenesis and wound healing in mammals. We previously identified four putative thrombospondins in the genome of the starlet sea anemone Nematostella vectensis. This study presents the first analysis of these thrombospondins, with the goals of understanding fundamental roles of thrombospondins in the Eumetazoa. Reverse transcriptase PCR showed that each of the N. vectensis thrombospondins (Nv85341, Nv22035, Nv168100 and Nv30790) is transcribed. Three of the four thrombospondins include an RGD or KGD motif in their thrombospondin type 3 repeats at sites equivalent to mammalian thrombospondins, suggesting ancient roles as RGD integrin ligands. Phylogenetic analysis based on the C-terminal regions demonstrated a high level of sequence diversity between N. vectensis thrombospondins. A full-length cDNA sequence was obtained for Nv168100 (NvTSP168100), which has an unusual domain organization. Immunohistochemistry with an antibody to NvTSP168100 revealed labeling of neuron-like cells in the mesoglea of the retractor muscles and the pharynx. In situ hybridization and quantitative PCR showed that NvTSP168100 is upregulated during regeneration. Immunohistochemistry of the area of regeneration identified strong immunostaining of the glycocalyx, the carbohydrate-rich matrix coating the epidermis, and electron microscopy identified changes in glycocalyx organization during regeneration. Thus, N. vectensis thrombospondins share structural features with thrombospondins from mammals and may have roles in the nervous system and in matrix reorganization during regeneration.

Summary

Rnd proteins are atypical Rho family proteins that do not hydrolyse GTP and are instead regulated by expression levels and post-translational modifications. Rnd1 and Rnd3/RhoE induce loss of actin stress fibres and cell rounding in multiple cell types, whereas responses to Rnd2 are more variable. Here we report the responses of endothelial cells to Rnd proteins. Rnd3 induces a very transient decrease in stress fibres but subsequently stimulates a strong increase in stress fibres, in contrast to the reduction observed in other cell types. Rnd2 also increases stress fibres whereas Rnd1 induces a loss of stress fibres and weakening of cell–cell junctions. Rnd3 does not act through any of its known signalling partners and does not need to associate with membranes to increase stress fibres. Instead, it acts by increasing RhoB expression, which is then required for Rnd3-induced stress fibre assembly. Rnd2 also increases RhoB levels. These data indicate that the cytoskeletal response to Rnd3 expression is dependent on cell type and context, and identify regulation of RhoB as a new mechanism for Rnd proteins to affect the actin cytoskeleton.

Summary

Brain structures such as the outer subventricular zone (OSVZ) and the inner fiber layer (IFL) in the developing cerebral cortex are especially prominent in higher mammals. However, the molecular mechanisms underlying the formation of the OSVZ are still largely unknown, mainly because genetic manipulations that can be applied to the OSVZ in higher mammals had been poorly available. Here we developed and validated a rapid and efficient genetic manipulation technique for germinal zones including the OSVZ using in utero electroporation in developing gyrencephalic carnivore ferrets. We also determined the optimal conditions for using in utero electroporation to express transgenes in germinal zones. Using our electroporation procedure, the morphology of GFP-positive cells in the OSVZ was clearly visible even without immunostaining, and multiple genes were efficiently co-expressed in the same cells. Furthermore, we uncovered that fibers, which seemed to correspond to those in the IFL of monkeys, also existed in ferrets, and were derived from newly generated cortical neurons. Our technique promises to be a powerful tool for investigating the fundamental mechanisms underlying the formation and abnormalities of the cerebral cortex in higher mammals.

Summary

DEADSouth mRNA is a component of germ plasm in Xenopus laevis and encodes a DDX25 DEAD-box RNA helicase. To determine the intracellular localization of DEADSouth protein, we injected mRNA encoding DEADSouth tagged with mCherry fluorescent protein into fertilized eggs from transgenic Xenopus expressing EGFP fused with a mitochondrial targeting signal. The DEADSouth-mCherry fusion protein was localized to the germ plasm, a mitochondria-rich region in primordial germ cells (PGCs). DEADSouth overexpression resulted in a reduction of PGC numbers after stage 20. Conversely, DEADSouth knockdown using an antisense locked nucleic acid gapmer inhibited movement of the germ plasm from the cortex to the perinuclear region, resulting in inhibition of PGC division at stage 12 and a decrease in PGC numbers at later stages. The knockdown phenotype was rescued by intact DEADSouth mRNA, but not mutant mRNA encoding inactive DEADSouth helicase. Surprisingly, it was also rescued by mouse vasa homolog and Xenopus vasa-like gene 1 mRNAs that encode DDX4 RNA helicases. The rescue was dependent on the 3′ untranslated region (3′UTR) of DEADSouth mRNA, which was used for PGC-specific expression. The 3′UTR contributed to localization of the injected mRNA to the germ plasm, resulting in effective localization of DEADSouth protein. These results demonstrate that localization of DEADSouth helicase to the germ plasm is required for proper PGC development in Xenopus laevis.

Summary

The mechanisms underlying the regenerative abilities of certain model species are of central importance to the basic understanding of pattern formation. Complex organisms such as planaria and salamanders exhibit an exceptional capacity to regenerate complete body regions and organs from amputated pieces. However, despite the outstanding bottom-up efforts of molecular biologists and bioinformatics focused at the level of gene sequence, no comprehensive mechanistic model exists that can account for more than one or two aspects of regeneration. The development of computational approaches that help scientists identify constructive models of pattern regulation is held back by the lack of both flexible morphological representations and a repository for the experimental procedures and their results (altered pattern formation). No formal representation or computational tools exist to efficiently store, search, or mine the available knowledge from regenerative experiments, inhibiting fundamental insights from this huge dataset. To overcome these problems, we present here a new class of ontology to encode formally and unambiguously a very wide range of possible morphologies, manipulations, and experiments. This formalism will pave the way for top-down approaches for the discovery of comprehensive models of regeneration. We chose the planarian regeneration dataset to illustrate a proof-of-principle of this novel bioinformatics of shape; we developed a software tool to facilitate the formalization and mining of the planarian experimental knowledge, and cured a database containing all of the experiments from the principal publications on planarian regeneration. These resources are freely available for the regeneration community and will readily assist researchers in identifying specific functional data in planarian experiments. More importantly, these applications illustrate the presented framework for formalizing knowledge about functional perturbations of morphogenesis, which is widely applicable to numerous model systems beyond regenerating planaria, and can be extended to many aspects of functional developmental, regenerative, and evolutionary biology.

Summary

During the maternal-to-zygotic transition (MZT), maternal proteins in oocytes are degraded by the ubiquitin–proteasome system (UPS), and new proteins are synthesized from the zygotic genome. However, the specific mechanisms underlying the UPS at the MZT are not well understood. We identified a molecule named zygote-specific proteasome assembly chaperone (ZPAC) that is specifically expressed in mouse gonads, and expression of ZPAC was transiently increased at the mouse MZT. ZPAC formed a complex with Ump1 and associated with precursor forms of 20S proteasomes. Transcription of ZPAC genes was also under the control of an autoregulatory feedback mechanism for the compensation of reduced proteasome activity similar to Ump1 and 20S proteasome subunit gene expression. Knockdown of ZPAC in early embryos caused a significant reduction of proteasome activity and decrease in Ump1 and mature proteasomes, leading to accumulation of proteins that need to be degraded at the MZT and early developmental arrest. Therefore, a unique proteasome assembly pathway mediated by ZPAC is important for progression of the mouse MZT.

Summary

Mutations in rbf1, the Drosophila homologue of the RB tumour suppressor gene, generate defects in cell cycle control, cell death, and differentiation during development. Previous studies have established that EGFR/Ras activity is an important determinant of proliferation and survival in rbf1 mutant cells. Here, we report that Capicua (Cic), an HMG box transcription factor whose activity is regulated by the EGFR/Ras pathway, regulates both proliferation and survival of RB-deficient cells in Drosophila. We demonstrate that cic mutations allow rbf1 mutant cells to bypass developmentally controlled cell cycle arrest and apoptotic pressure. The cooperative effect between Cic and RBF1 in promoting G1 arrest is mediated, at least in part, by limiting Cyclin E expression. Surprisingly, we also found evidence to suggest that cic mutant cells have decreased levels of reactive oxygen species (ROS), and that the survival of rbf1 mutant cells is affected by changes in ROS levels. Collectively, our results elucidate the importance of the crosstalk between EGFR/Ras and RBF1 in coordinating cell cycle progression and survival.

Summary

Actin and myosin II play major roles in cell migration. Whereas pseudopod extension by actin polymerization has been intensively researched, less attention has been paid to how the rest of the actin cytoskeleton such as the actin cortex contributes to cell migration. In this study, cortical actin and myosin II filaments were simultaneously observed in migrating Dictyostelium cells under total internal reflection fluorescence microscopy. The cortical actin and myosin II filaments remained stationary with respect to the substratum as the cells advanced. However, fluorescence recovery after photobleaching experiments and direct observation of filaments showed that they rapidly turned over. When the cells were detached from the substratum, the actin and myosin filaments displayed a vigorous retrograde flow. Thus, when the cells migrate on the substratum, the cortical cytoskeleton firmly holds the substratum to generate the motive force instead. The present studies also demonstrate how myosin II localizes to the rear region of the migrating cells. The observed dynamic turnover of actin and myosin II filaments contributes to the recycling of their subunits across the whole cell and enables rapid reorganization of the cytoskeleton.

Summary

Understanding locomotor energetics is imperative, because energy expended during locomotion, a requisite feature of primate subsistence, is lost to reproduction. Although metabolic energy expenditure can only be measured in extant species, using the equations of motion to calculate mechanical energy expenditure offers unlimited opportunities to explore energy expenditure, particularly in extinct species on which empirical experimentation is impossible. Variability, either within or between groups, can manifest as changes in size and/or shape. Isometric scaling (or geometric similarity) requires that all dimensions change equally among all individuals, a condition that will not be met in naturally developing populations. The Froude number (Fr), with lower limb (or hindlimb) length as the characteristic length, has been used to compensate for differences in size, but does not account for differences in shape.

To determine whether or not shape matters at the intraspecific level, we used a mechanical model that had properties that mimic human variation in shape. We varied crural index and limb segment circumferences (and consequently, mass and inertial parameters) among nine populations that included 19 individuals that were of different size. Our goal in the current work is to understand whether shape variation changes mechanical energy sufficiently enough to make shape a critical factor in mechanical and metabolic energy assessments.

Our results reaffirm that size does not affect mass-specific mechanical cost of transport (Alexander and Jayes, 1983) among geometrically similar individuals walking at equal Fr. The known shape differences among modern humans, however, produce sufficiently large differences in internal and external work to account for much of the observed variation in metabolic energy expenditure, if mechanical energy is correlated with metabolic energy. Any species or other group that exhibits shape differences should be affected similarly to that which we establish for humans. Unfortunately, we currently do not have a simple method to control or adjust for size–shape differences in individuals that are not geometrically similar, although musculoskeletal modeling is a viable, and promising, alternative. In mouse-to-elephant comparisons, size differences could represent the largest source of morphological variation, and isometric scaling factors such as Fr can compensate for much of the variability. Within species, however, shape differences may dominate morphological variation and Fr is not designed to compensate for shape differences. In other words, those shape differences that are “reasonably close” at the mouse-to-elephant level may become grossly different for within-species energetic comparisons.

Summary

Although tight junctions (TJ) have been extensively studied in simple epithelial cells, it is still unknown whether their organization is coupled to cell differentiation in stratified epithelia. We studied the expression of TJ in RCE1(5T5) cells, an in vitro model which mimics the sequential steps of rabbit corneal epithelial differentiation. RCE1(5T5) cells expressed TJ components which were assembled once cells constituted differentiated epithelia, as suggested by the increase of transepithelial electrical resistance (TER) which followed a similar kinetic to the expression of the early differentiation marker Pax-6. TJ were functional as indicated by the establishment of an epithelial barrier nonpermeable to ruthenium red or a biotin tracer. In immunostaining experiments, TJ were located at the superficial cells from the suprabasal layers; Western blot and RT-PCR suggested that TJ were composed of claudins (cldn) -1, -2, -4, cingulin (cgn), occludin (ocln) and ZO-1. Semi-quantitative RT-PCR and TER measurements showed that TJ became organized when cells began to form a 3–5 layers stratified epithelium; TER increased once cells reached confluence, with a time course comparable to the raise in the expression of cgn, cldn-2 and -4. Nevertheless, cldn-1, -2, ZO-1 and ocln were present in the cells from the beginning of cultivation, suggesting that TER increases mainly depend on TJ assembly. While EGF increased epithelial barrier strength, retinoic acid disrupted it, increasing paracellular flux about 2-fold; this effect was concentration dependent and completely reversible. Our results suggest that TJ assembly is tightly linked to the expression of corneal epithelial terminal phenotype.

Summary

The gastrointestinal (GI) tract defines the digestive system and is composed of the stomach, intestine and colon. Among the major cell types lining radially along the GI tract are the epithelium, mucosa, smooth muscles and enteric neurons. The Hedgehog (Hh) pathway has been implicated in directing various aspects of the developing GI tract, notably the mucosa and smooth muscle growth, and enteric neuron patterning, while the Ret signaling pathway is selectively required for enteric neuron migration, proliferation, and differentiation. The growth arrest specific gene 1 (Gas1) encodes a GPI-anchored membrane protein known to bind to Sonic Hh (Shh), Indian Hh (Ihh), and Ret. However, its role in the GI tract has not been examined. Here we show that the Gas1 mutant GI tract, compared to the control, is shorter, has thinner smooth muscles, and contains more enteric progenitors that are abnormally distributed. These phenotypes are similar to those of the Shh mutant, supporting that Gas1 mediates most of the Shh activity in the GI tract. Because Gas1 has been shown to inhibit Ret signaling elicited by Glial cell line-derived neurotrophic factor (Gdnf), we explored whether Gas1 mutant enteric neurons displayed any alteration of Ret signaling levels. Indeed, isolated mutant enteric progenitors not only showed increased levels of phospho-Ret and its downstream effectors, phospho-Akt and phospho-Erk, but also displayed altered responses to Gdnf and Shh. We therefore conclude that phenotypes observed in the Gas1 mutant are due to a combination of reduced Hh signaling and increased Ret signaling.

Summary

During preimplantation development, the embryo must establish totipotency and enact the earliest differentiation choices, processes that involve extensive chromatin modification. To identify novel developmental regulators, we screened for genes that are preferentially transcribed in the pluripotent inner cell mass (ICM) of the mouse blastocyst. Genes that encode chromatin remodeling factors were prominently represented in the ICM, including Chd1l, a member of the Snf2 gene family. Chd1l is developmentally regulated and expressed in embryonic stem (ES) cells, but its role in development has not been investigated. Here we show that inhibiting Chd1l protein production by microinjection of antisense morpholinos causes arrest prior to the blastocyst stage. Despite this important function in vivo, Chd1l is non-essential for cultured ES cell survival, pluripotency, or differentiation, suggesting that Chd1l is vital for events in embryos that are distinct from events in ES cells. Our data reveal a novel role for the chromatin remodeling factor Chd1l in the earliest cell divisions of mammalian development.

Summary

In insect groups with the ability of endothermy, the thermoregulatory capacity has a direct relation to body mass. To verify this relationship in vespine wasps, we compared the thermoregulation of hornets (Vespa crabro), the largest species of wasps in Central Europe, with two smaller wasps (Vespula vulgaris and Vespula germanica) in the entire range of ambient temperature (Ta: ~0–40°C) where the insects exhibited foraging flights.

Despite the great difference in body weight of Vespula (V. vulgaris: 84.1±19.0 mg, V. germanica: 74.1±9.6 mg) and Vespa (477.5±59.9 mg), they exhibited similarities in the dependence of thorax temperature on Ta on their arrival (mean Tth = 30–40°C) and departure (mean Tth = 33–40°C) at the nest entrance. However, the hornets’ thorax temperature was up to 2.5°C higher upon arrival and up to 3°C lower at departure. The thorax temperature excess (Tth−Ta) above ambient air of about 5–18°C indicates a high endothermic capacity in both hornets and wasps. Heat gain from solar radiation elevated the temperature excess by up to 1°C. Results show that hornets and wasps are able to regulate their body temperature quite well, even during flight. A comparison of flight temperature with literature reports on other vespine wasps revealed a dependence of the Tth on the body mass in species weighing less than about 200 mg.

Summary

Goldfish have been used for cold acclimation studies, which have focused on changes in glycolytic and oxidative enzymes or alterations in lipid composition in skeletal muscle. Here we examine the effects of cold acclimation on the functional properties of isolated mitochondria and permeabilized fibers from goldfish white skeletal muscle, focusing on understanding the types of changes that occur in the mitochondrial respiratory states. We observed that cold acclimation promoted a significant increase in the mitochondrial oxygen consumption rates. Western blot analysis showed that UCP3 was raised by ∼1.5-fold in cold-acclimated muscle mitochondria. Similarly, we also evidenced a rise in the adenine nucleotide translocase content in cold-acclimated muscle mitochondria compared to warm-acclimated mitochondria (0.96±0.05 vs 0.68±0.02 nmol carboxyatractyloside mg−1 protein). This was followed by a 2-fold increment in the citrate synthase activity, which suggests a higher mitochondrial content in cold-acclimated goldfish. Even with higher levels of UCP3 and ANT, the effects of activator (palmitate) and inhibitors (carboxyatractyloside and GDP) on mitochondrial parameters were similar in both warm- and cold-acclimated goldfish. Thus, we propose that cold acclimation in goldfish promotes an increase in functional oxidative capacity, with higher mitochondrial content without changes in the mitochondrial uncoupling pathways.

Summary

Protein phosphorylation and dephosphorylation is a key mechanism for the spatial and temporal regulation of many essential developmental processes and is especially prominent during mitosis. The multi-subunit protein phosphatase 2A (PP2A) enzyme plays an important, yet poorly characterized role in dephosphorylating proteins during mitosis. PP2As are heterotrimeric complexes comprising a catalytic, structural, and regulatory subunit. Regulatory subunits are mutually exclusive and determine subcellular localization and substrate specificity of PP2A. At least 3 different classes of regulatory subunits exist (termed B, B′, B″) but there is no obvious similarity in primary sequence between these classes. Therefore, it is not known how these diverse regulatory subunits interact with the same holoenzyme to facilitate specific PP2A functions in vivo. The B″ family of regulatory subunits is the least understood because these proteins lack conserved structural domains. RSA-1 (regulator of spindle assembly) is a regulatory B″ subunit required for mitotic spindle assembly in Caenorhabditis elegans. In order to address how B″ subunits interact with the PP2A core enzyme, we focused on a conditional allele, rsa-1(or598ts), and determined that this mutation specifically disrupts the protein interaction between RSA-1 and the PP2A structural subunit, PAA-1. Through genetic screening, we identified a putative interface on the PAA-1 structural subunit that interacts with a defined region of RSA-1/B″. In the context of previously published results, these data propose a mechanism of how different PP2A B-regulatory subunit families can bind the same holoenzyme in a mutually exclusive manner, to perform specific tasks in vivo.

Summary

Natal dispersal of sea turtles is an energetically demanding activity that is fuelled primarily by aerobic metabolism. However, during intense exercise reptiles can use anaerobic metabolism to supplement their energy requirements. We assessed anaerobic metabolism in dispersing hatchling loggerhead and flatback turtles by measuring the concentrations of blood lactate during crawling and at different times during the first four hours of their frenzy swim. We also measured concentrations of blood glucose and corticosterone. Blood lactate (12.13 to 2.03 mmol/L), glucose (6.25 to 3.8 mmol/L) and corticosterone (8.13 to 2.01 ng/mL) concentrations decreased significantly over time in both loggerhead and flatback hatchlings and no significant differences were found between the species. These results indicate that anaerobic metabolism makes a significant contribution to the dispersal phase of hatchling sea turtles during the beach crawl and the first few hours of the frenzy swim.

Summary

Gene duplication is an important means of generating new genes. The major mechanisms by which duplicated genes are preserved in the face of purifying selection are thought to be neofunctionalization, subfunctionalization, and increased gene dosage. However, very few duplicated gene families in vertebrate species have been analyzed by functional tests in vivo. We have therefore examined the three vertebrate Myb genes (c-Myb, A-Myb, and B-Myb) by cytogenetic map analysis, by sequence analysis, and by ectopic expression in Drosophila. We provide evidence that the vertebrate Myb genes arose by two rounds of regional genomic duplication. We found that ubiquitous expression of c-Myb and A-Myb, but not of B-Myb or Drosophila Myb, was lethal in Drosophila. Expression of any of these genes during early larval eye development was well tolerated. However, expression of c-Myb and A-Myb, but not of B-Myb or Drosophila Myb, during late larval eye development caused drastic alterations in adult eye morphology. Mosaic analysis implied that this eye phenotype was cell-autonomous. Interestingly, some of the eye phenotypes caused by the retroviral v-Myb oncogene and the normal c-Myb proto-oncogene from which v-Myb arose were quite distinct. Finally, we found that post-translational modifications of c-Myb by the GSK-3 protein kinase and by the Ubc9 SUMO-conjugating enzyme that normally occur in vertebrate cells can modify the eye phenotype caused by c-Myb in Drosophila. These results support a model in which the three Myb genes of vertebrates arose by two sequential duplications. The first duplication was followed by a subfunctionalization of gene expression, then neofunctionalization of protein function to yield a c/A-Myb progenitor. The duplication of this progenitor was followed by subfunctionalization of gene expression to give rise to tissue-specific c-Myb and A-Myb genes.

Summary

Most clinically distinguishable malignant tumors are characterized by specific mutations, specific patterns of chromosomal rearrangements and a predominant mechanism of genetic instability but it remains unsolved whether modifications of cancer genomes can be explained solely by mutations and selection through the cancer microenvironment.

It has been suggested that internal dynamics of genomic modifications as opposed to the external evolutionary forces have a significant and complex impact on Darwinian species evolution. A similar situation can be expected for somatic cancer evolution as molecular key mechanisms encountered in species evolution also constitute prevalent mutation mechanisms in human cancers. This assumption is developed into a systems approach of carcinogenesis which focuses on possible inner constraints of the genome architecture on lineage selection during somatic cancer evolution. The proposed systems approach can be considered an analogy to the concept of evolvability in species evolution.

The principal hypothesis is that permissive or restrictive effects of the genome architecture on lineage selection during somatic cancer evolution exist and have a measurable impact. The systems approach postulates three classes of lineage selection effects of the genome architecture on somatic cancer evolution: i) effects mediated by changes of fitness of cells of cancer lineage, ii) effects mediated by changes of mutation probabilities and iii) effects mediated by changes of gene designation and physical and functional genome redundancy. Physical genome redundancy is the copy number of identical genetic sequences. Functional genome redundancy of a gene or a regulatory element is defined as the number of different genetic elements, regardless of copy number, coding for the same specific biological function within a cancer cell. Complex interactions of the genome architecture on lineage selection may be expected when modifications of the genome architecture have multiple and possibly opposed effects which manifest themselves at disparate times and progression stages.

Dissection of putative mechanisms mediating constraints exerted by the genome architecture on somatic cancer evolution may provide an algorithm for understanding and predicting as well as modifying somatic cancer evolution in individual patients.

Summary

Paired-like homeodomain transcription factor 1 (PITX1) was specifically up-regulated in patients with facioscapulohumeral muscular dystrophy (FSHD) by comparing the genome-wide mRNA expression profiles of 12 neuromuscular disorders. In addition, it is the only known direct transcriptional target of the double homeobox protein 4 (DUX4) of which aberrant expression has been shown to be the cause of FSHD. To test the hypothesis that up-regulation of PITX1 contributes to the skeletal muscle atrophy seen in patients with FSHD, we generated a tet-repressible muscle-specific Pitx1 transgenic mouse model in which expression of PITX1 in skeletal muscle can be controlled by oral administration of doxycycline. After PITX1 was over-expressed in the skeletal muscle for 5 weeks, the mice exhibited significant loss of body weight and muscle mass, decreased muscle strength, and reduction of muscle fiber diameters. Among the muscles examined, the tibialis anterior, gastrocnemius, quadricep, bicep, tricep and deltoid showed significant reduction of muscle mass, while the soleus, masseter and diaphragm muscles were not affected. The most prominent pathological change was the development of atrophic muscle fibers with mild necrosis and inflammatory infiltration. The affected myofibers stained heavily with NADH-TR with the strongest staining in angular-shaped atrophic fibers. Some of the atrophic fibers were also positive for embryonic myosin heavy chain using immunohistochemistry. Immunoblotting showed that the p53 was up-regulated in the muscles over-expressing PITX1. The results suggest that the up-regulation of PITX1 followed by activation of p53-dependent pathways may play a major role in the muscle atrophy developed in the mouse model.

Summary

FMRP is an evolutionarily conserved protein that is highly expressed in neurons and its deficiency causes fragile X mental retardation syndrome. FMRP controls the translation of target mRNAs in part by promoting their dynamic transport in neuronal RNA granules. We have previously shown that high expression of mammalian FMRP induces formation of granules termed FMRP granules. These RNA granules are reminiscent of neuronal granules, of stress granules, as well as of the recently described in vitro-assembled granules. In contrast with mammalian FMRP, which has two paralog proteins, Drosophila FMRP (dFMRP) is encoded by a single gene that has no paralog. Using this genetically simple organism, we investigated formation and dynamics of FMRP granules. We found that increased expression of dFMRP in Drosophila cells induces the formation of dynamic dFMRP RNA granules. Mutagenesis studies identified the N-terminal protein–protein domain of dFMRP as a key determinant for FMRP granules formation. The RGG RNA binding motif of dFMRP is dispensable for dFMRP granules formation since its deletion does not prevent formation of those granules. Deletion of the RGG motif reduced, however, dFMRP trafficking between FMRP granules and the cytosol. Similarly, deletion of a large part of the KH RNA binding motif of dFMRP had no effect on formation of dFMRP-granules, but diminished the shuttling activity of dFMRP. Our results thus suggest that the mechanisms controlling formation of RNA granules and those promoting their dynamics are uncoupled. This study opens new avenues to further elucidate the molecular mechanisms controlling FMRP trafficking with its associated mRNAs in and out of RNA granules.

Summary

Many scleractinian coral species host epizoic acoelomorph flatworms, both in aquaculture and in situ. These symbiotic flatworms may impair coral growth and health through light-shading, mucus removal and disruption of heterotrophic feeding. To quantify the effect of epizoic flatworms on zooplankton feeding, we conducted video analyses of single polyps of Galaxea fascicularis (Linnaeus 1767) grazing on Artemia nauplii in the presence and absence of symbiotic flatworms. 18S DNA analysis revealed that flatworms inhabiting G. fascicularis belonged to the genus Waminoa (Convolutidae), which were hosted at a density of 3.6±0.4 individuals polyp−1. Polyps hosting flatworms exhibited prey capture rates of 2.2±2.5, 3.4±4.5 and 2.7±3.4 nauplii polyp−1 30 min−1 at prey concentrations of 250, 500 and 1,000 nauplii L−1, respectively. Polyps that had their flatworms removed displayed prey capture rates of 2.7±1.6, 4.8±4.1 and 16.9±10.3 nauplii polyp−1 30 min−1. Significant main and interactive effects of flatworm presence and ambient prey concentration were found, reflected by the fact that flatworms significantly impaired host feeding rates at the highest prey density of 1,000 nauplii L−1. In addition, flatworms displayed kleptoparasitism, removing between 0.1±0.3 and 0.6±1.1 nauplii 30 min−1 from the oral disc of their host, or 5.3±3.3 to 50.0±2.1% of prey acquired by the coral. We suggest classifying the coral-associated Waminoa sp. as an epizoic parasite, as its presence may negatively affect growth and health of the host.

Summary

The expression of the spalt genes is regulated by the Decapentaplegic signalling pathway in the Drosophila wing. These genes participate in the patterning of the longitudinal wing veins by regulating the expression of vein-specific genes, and in the establishment of cellular affinities in the central region of the wing blade epithelium. The Spalt proteins act as transcription factors, most likely regulating gene expression by repression, but the identity of their target genes in the wing is still unknown. As a preliminary step to unravel the genetic hierarchy controlled by the Spalt proteins, we have analysed their requirements during wing development, and addressed to what extent they mediate all the functions of the Decapentaplegic pathway in this developmental system. We identify additional functions for Spalt in cell division, survival, and maintenance of epithelial integrity. Thus, Spalt activity is required to promote cell proliferation, acting in the G2/M transition of the cell cycle. The contribution of Spalt to cell division is limited to the central region of the wing blade, as they do not mediate the extra growth triggered by Decapentaplegic signalling in the peripheral regions of the wing disc. In addition, Spalt function is required to maintain cell viability in cells exposed to high levels of Decapentaplegic signalling. This aspect of Spalt function is related to the repression of JNK signalling in the spalt domain of expression. Finally, we further characterise the requirements of Spalt to maintain epithelial integrity by regulating cellular affinities between cells located in the central wing region. Our results indicate that Spalt function mediates most of the requirements identified for Decapentaplegic signalling, contributing to establish the cellular qualities that differentiate central versus peripheral territories in the wing blade.

Summary

During Drosophila development region-specific regulation of target genes by Hox proteins is modulated by genetic interactions with various cofactors and genetic collaborators. During embryogenesis one such modulator of Hox target specificity is the zinc-finger transcription factor Teashirt (Tsh) that is expressed in the developing trunk and cooperatively functions with trunk-specific Hox proteins to promote appropriate segment fate. This embryonic function of Tsh is characterized as homeotic since loss of embryonic Tsh activity leads to transformation of trunk segments toward head identity. In addition to this embryonic homeotic role, Tsh also performs vital Hox-independent functions through patterning numerous embryonic, larval and adult structures. Here we address whether the homeotic function of Tsh is maintained throughout development by investigating its contribution to patterning the adult abdomen. We show that Tsh is expressed throughout the developing abdomen and that this expression is dependent on the three Bithorax Hox proteins Ultrabithorax, Abdominal-A and Abdominal-B. Conditional reduction of Tsh activity during pupation reveals broad homeotic roles for this transcription factor throughout the adult abdomen. Additionally we show that, as during embryogenesis, the tsh paralog tiptop (tio) plays a partially redundant role in this homeotic activity.