Identification of a critical control element directing expression of the muscle-specific transcription factor MRF4 in the mouse embryo

AbstractSkeletal muscle development in the vertebrate embryo critically depends on the myogenic regulatory factors (MRFs) including MRF4 and Myf5. Both genes exhibit distinct expression patterns during mouse embryogenesis, although they are genetically closely linked with multiple regulatory elements dispersed throughout the common gene locus. MRF4 has a biphasic expression profile, first in somites and later in foetal skeletal muscles. Here, we demonstrate by transgenic analysis that elements within a 7.5-kb promoter fragment of the MRF4 gene are sufficient to drive the embryonic wave of expression very similar to the endogenous gene in somites of mouse embryos. In contrast, a 3-kb fragment of the proximal promoter fails to support expression in the myotome, suggesting that essential cis-acting elements are located between −7.5 and −3 kb upstream of MRF4. Further analysis of this sequence delimits an essential region between −6.6 and −5.6 kb that together with the 3-kb promoter fragment directs transgene expression in the epaxial myotome of all somites during the appropriate developmental period. These data provide evidence that the partly overlapping expression patterns of Mrf4 and Myf5 in somites are controlled by distinct regulatory elements. We also show that 11.4 kb sequence upstream of MRF4, including the promoter and the somitic control region identified in this study, is not sufficient to elicit target specificity towards the strong Myf5 (−58/−48 kb) enhancer, suggesting that additional yet unidentified elements are necessary to convey promoter selectivity and protect the MRF4 gene from this enhancer

DifferentMRF4Knockout Alleles Differentially Disrupt Myf-5 Expression:cis-Regulatory Interactions at theMRF4/Myf-5Locus

AbstractThree different null alleles of the myogenic bHLH geneMRF4/herculin/Myf-6were created recently. The three alleles were similar in design but were surprisingly different in the intensity of their phenotypes, which ranged from complete viability of homozygotes to complete lethality. One possible explanation for these differences is that each mutation altered expression from the nearbyMyf-5gene to a different extent. This possibility was first raised by the observation that the most severeMRF4knockout allele expresses no Myf-5 RNA and is a developmental phenocopy of theMyf-5null mutation. Furthermore, initial studies of the two weaker alleles had shown that their differences in viability correlate with the intensity of rib skeletal defects, and the most extreme version of this rib defect is the hallmark phenotype ofMyf-5null animals. In the present study we tested this hypothesis for the two milderMRF4alleles. By analyzing compound heterozygous animals carrying either the intermediate or the weakestMRF4knockout allele on one chromosome 10 and aMyf-5knockout allele on the other chromosome, we found that both of theseMRF4alleles apparently downregulate Myf-5 expression by acis-acting mechanism. Compound heterozygotes showed increased mortality of the normally viableMRF4allele, together with intensified rib defects for bothMRF4alleles and increased deficits in myotomal Myf-5 expression. The allele-specific gradation in phenotypes also suggested that rib morphogenesis is profoundly sensitive to quantitative differences in Myf-5 function if Myf-5 products drop below hemizygous levels. The mechanistic basis forcisinteractions at theMRF4/Myf-5locus was further examined by fusing a DNA segment containing the entireMRF4structural gene, including all sequences deleted in the threeMRFknockout alleles, with a basal promoter and alacZreporter. Transgenic embryos showed specific LacZ expression in myotomes in a pattern that closely resembles the expression of Myf-5 RNA.cis-acting interactions betweenMyf-5andMRF4may therefore play a significant role in regulating expression of these genes in the early myotomes of wildtype embryos

Chick CFC Controls Lefty1 Expression in the Embryonic Midline and Nodal Expression in the Lateral Plate

AbstractMembers of the EGF-CFC family of proteins have recently been implicated as essential cofactors for Nodal signaling. Here we report the isolation of chick CFC and describe its expression pattern, which appears to be similar to Cfc1 in mouse. During early gastrulation, chick CFC was asymmetrically expressed on the left side of Hensen's node as well as in the emerging notochord, prechordal plate, and lateral plate mesoderm. Subsequently, its expression became confined to the heart fields, notochord, and posterior mesoderm. Implantation experiments suggest that chick CFC expression in the lateral plate mesoderm is dependent on BMP signaling, while in the midline its expression depends on an Activin-like signal. The asymmetric expression domain within Hensen's node was not affected by application of FGF8, Noggin, or Shh antibody. Implantation of cells expressing human or mouse CFC2, or chick CFC on the right side of Hensen's node randomized heart looping without affecting expression of genes involved in left–right axis formation, including SnR, Nodal, Car, or Pitx2. Application of antisense oligodeoxynucleotides to the midline of Hamburger–Hamilton stage 4-5 embryos also randomized heart looping, but in contrast to the overexpression experiments, antisense oligodeoxynucleotide treatment resulted in bilateral expression of Nodal, Car, Pitx2, and NKX3.2, whereas Lefty1 expression in the midline was transiently lost. Application of the antisense oligodeoxynucleotides to the lateral plate mesoderm abolished Nodal expression. Thus, chick CFC seems to have a dual function in left–right axis formation by maintaining Nodal expression in the lateral plate mesoderm and controlling expression of Lefty1 expression in the midline territory

Raver2, a new member of the hnRNP family

AbstractRaver2 was identified as a novel member of the hnRNP family based on sequence homology within three RNA recognition motifs and its general domain organization reminiscent of the previously described raver1 protein. Like raver1, raver2 contains two putative nuclear localization signals and a potential nuclear export sequence, and also displays nucleo-cytoplasmic shuttling in a heterokaryon assay. In glia cells and neurons, raver2 localizes to the nucleus. Moreover, the protein interacts with polypyrimidine tract binding protein (PTB) suggesting that it may participate in PTB-mediated nuclear functions. In contrast to ubiquitously expressed raver1, raver2 exerts a distinct spatio-temporal expression pattern during embryogenesis and is essentially restricted to brain, lung, and kidney in the adult mouse

Induced Arp2/3 Complex Depletion Increases FMNL2/3 Formin Expression and Filopodia Formation.

The Arp2/3 complex generates branched actin filament networks operating in cell edge protrusion and vesicle trafficking. Here we employ a conditional knockout mouse model permitting tissue- or cell-type specific deletion of the murine Actr3 gene (encoding Arp3). A functional Actr3 gene appeared essential for fibroblast viability and growth. Thus, we developed cell lines for exploring the consequences of acute, tamoxifen-induced Actr3 deletion causing near-complete loss of functional Arp2/3 complex expression as well as abolished lamellipodia formation and membrane ruffling, as expected. Interestingly, Arp3-depleted cells displayed enhanced rather than reduced cell spreading, employing numerous filopodia, and showed little defects in the rates of random cell migration. However, both exploration of new space by individual cells and collective migration were clearly compromised by the incapability to efficiently maintain directionality of migration, while the principal ability to chemotax was only moderately affected. Examination of actin remodeling at the cell periphery revealed reduced actin turnover rates in Arp2/3-deficient cells, clearly deviating from previous sequestration approaches. Most surprisingly, induced removal of Arp2/3 complexes reproducibly increased FMNL formin expression, which correlated with the explosive induction of filopodia formation. Our results thus highlight both direct and indirect effects of acute Arp2/3 complex removal on actin cytoskeleton regulation

The Arp2/3 complex is crucial for colonisation of the mouse skin by melanoblasts

The Arp2/3 complex is essential for the assembly of branched filamentous actin, but its role in physiology and development is surprisingly little understood. Melanoblasts deriving from the neural crest migrate along the developing embryo and traverse the dermis to reach the epidermis, colonising the skin and eventually homing within the hair follicles. We have previously established that Rac1 and Cdc42 direct melanoblast migration in vivo. We hypothesised that the Arp2/3 complex might be the main downstream effector of these small GTPases. Arp3 depletion in the melanocyte lineage results in severe pigmentation defects in dorsal and ventral regions of the mouse skin. Arp3 null melanoblasts demonstrate proliferation and migration defects and fail to elongate as their wild-type counterparts. Conditional deletion of Arp3 in primary melanocytes causes improper proliferation, spreading, migration and adhesion to extracellular matrix. Collectively, our results suggest that the Arp2/3 complex is absolutely indispensable in the melanocyte lineage in mouse development, and indicate a significant role in developmental processes that require tight regulation of actin-mediated motility

Planbare Schwangerschaft - perfektes Kind?: Wechselwirkungen von Medizin und Gesellschaft

Die Nationale Akademie der Wissenschaften Leopoldina und die Konrad-Adenauer-Stiftung widmeten sich in zwei gemeinsamen Veranstaltungen im Jahr 2017 der Frage nach der Beeinflussung von Fortpflanzung und den gesellschaftlichen Rahmenbedingungen, die den Wunsch danach befördern. Das Aufschieben der Fortpflanzung in ein höheres Lebensalter ist ein zentraler Trend des Demografischen Wandels. Medizinische Angebote reagieren hierbei auf die Nachfrage, offerieren aber gleichzeitig Angebote, die die reproduktiven Möglichkeiten für Frauen noch einmal deutlich zu erweitern versprechen: das Social Freezing. Die Pränatalmedizin wiederum lässt mit immer weiteren Möglichkeiten der Testung und Diagnose den Wunsch nach einem möglichst gesunden, perfekten Kind in scheinbar greifbare Nähe rücken. Aktuell wird diskutiert, ob die Kosten der nicht-invasiven Pränataltests (NIPT) von den gesetzlichen Krankenkassen übernommen werden sollen. Diese Entwicklungen halten oftmals schleichend in unserem Alltag Einzug – ohne, dass ihre ganze gesellschaftliche Tragweite immer die notwendige Berücksichtigung erfährt. Die Autorinnen und Autoren möchten mit dem vorliegenden Papier einen Anstoß zur Diskussion geben, indem einige dieser Entwicklungen und die Frage nach dem gesellschaftlichen Hintergrund skizziert werden

Arp3 controls the podocyte architecture at the kidney filtration barrier

Podocytes, highly specialized epithelial cells, build the outer part of the kidney filtration barrier and withstand high mechanical forces through a complex network of cellular protrusions. Here, we show that Arp2/3-dependent actin polymerization controls actomyosin contractility and focal adhesion maturation of podocyte protrusions and thereby regulates formation, maintenance, and capacity to adapt to mechanical requirements of the filtration barrier. We find that N-WASP-Arp2/3 define the development of complex arborized podocyte protrusions in vitro and in vivo. Loss of dendritic actin networks results in a pronounced activation of the actomyosin cytoskeleton and the generation of over-maturated but less efficient adhesion, leading to detachment of podocytes. Our data provide a model to explain podocyte protrusion morphology and their mechanical stability based on a tripartite relationship between actin polymerization, contractility, and adhesion

The Chlamydia psittaci Genome: A Comparative Analysis of Intracellular Pathogens

Chlamydiaceae are a family of obligate intracellular pathogens causing a wide range of diseases in animals and humans, and facing unique evolutionary constraints not encountered by free-living prokaryotes. To investigate genomic aspects of infection, virulence and host preference we have sequenced Chlamydia psittaci, the pathogenic agent of ornithosis.A comparison of the genome of the avian Chlamydia psittaci isolate 6BC with the genomes of other chlamydial species, C. trachomatis, C. muridarum, C. pneumoniae, C. abortus, C. felis and C. caviae, revealed a high level of sequence conservation and synteny across taxa, with the major exception of the human pathogen C. trachomatis. Important differences manifest in the polymorphic membrane protein family specific for the Chlamydiae and in the highly variable chlamydial plasticity zone. We identified a number of psittaci-specific polymorphic membrane proteins of the G family that may be related to differences in host-range and/or virulence as compared to closely related Chlamydiaceae. We calculated non-synonymous to synonymous substitution rate ratios for pairs of orthologous genes to identify putative targets of adaptive evolution and predicted type III secreted effector proteins.This study is the first detailed analysis of the Chlamydia psittaci genome sequence. It provides insights in the genome architecture of C. psittaci and proposes a number of novel candidate genes mostly of yet unknown function that may be important for pathogen-host interactions

Differential cross section measurements for the production of a W boson in association with jets in proton–proton collisions at √s = 7 TeV

Measurements are reported of differential cross sections for the production of a W boson, which decays into a muon and a neutrino, in association with jets, as a function of several variables, including the transverse momenta (pT) and pseudorapidities of the four leading jets, the scalar sum of jet transverse momenta (HT), and the difference in azimuthal angle between the directions of each jet and the muon. The data sample of pp collisions at a centre-of-mass energy of 7 TeV was collected with the CMS detector at the LHC and corresponds to an integrated luminosity of 5.0 fb[superscript −1]. The measured cross sections are compared to predictions from Monte Carlo generators, MadGraph + pythia and sherpa, and to next-to-leading-order calculations from BlackHat + sherpa. The differential cross sections are found to be in agreement with the predictions, apart from the pT distributions of the leading jets at high pT values, the distributions of the HT at high-HT and low jet multiplicity, and the distribution of the difference in azimuthal angle between the leading jet and the muon at low values.United States. Dept. of EnergyNational Science Foundation (U.S.)Alfred P. Sloan Foundatio