In vivo importance of homologous recombination DNA repair for mouse neural stem and progenitor cells

We characterized the in vivo importance of the homologous recombination factor RAD54 for the developing mouse brain cortex in normal conditions or after ionizing radiation exposure. Contrary to numerous homologous recombination genes, Rad54 disruption did not impact the cortical development without exogenous stress, but it dramatically enhanced the radiation sensitivity of neural stem and progenitor cells. This resulted in the death of all cells irradiated during S or G2, whereas the viability of cells irradiated in G1 or G0 was not affected by Rad54 disruption. Apoptosis occurred after long arrests at intra-S and G2/M checkpoints. This concerned every type of neural stem and progenitor cells, showing that the importance of Rad54 for radiation response was linked to the cell cycle phase at the time of irradiation and not to the differentiation state. In the developing brain, RAD54-dependent homologous recombination appeared absolutely required for the repair of damages induced by ionizing radiation during S and G2 phases, but not for the repair of endogenous damages in normal conditions. Altogether our data support the existence of RAD54-dependent and -independent homologous recombination pathways.     

CRIM1 complexes with ß-catenin and cadherins, stabilizes cell-cell junctions and is critical for neural morphogenesis

In multicellular organisms, morphogenesis is a highly coordinated process that requires dynamically regulated adhesion between cells. An excellent example of cellular morphogenesis is the formation of the neural tube from the flattened epithelium of the neural plate. Cysteine-rich motor neuron protein 1 (CRIM1) is a single-pass (type 1) transmembrane protein that is expressed in neural structures beginning at the neural plate stage. In the frog Xenopus laevis, loss of function studies using CRIM1 antisense morpholino oligonucleotides resulted in a failure of neural development. The CRIM1 knockdown phenotype was, in some cases, mild and resulted in perturbed neural fold morphogenesis. In severely affected embryos there was a dramatic failure of cell adhesion in the neural plate and complete absence of neural structures subsequently. Investigation of the mechanism of CRIM1 function revealed that it can form complexes with ?-catenin and cadherins, albeit indirectly, via the cytosolic domain. Consistent with this, CRIM1 knockdown resulted in diminished levels of cadherins and ?-catenin in junctional complexes in the neural plate. We conclude that CRIM1 is critical for cell-cell adhesion during neural development because it is required for the function of cadherin-dependent junctions.     

Sizzled-Tolloid Interactions Maintain Foregut Progenitors by Regulating Fibronectin-Dependent BMP Signaling

The liver, pancreas, and lungs are induced from endoderm progenitors by a series of dynamic growth factor signals from the mesoderm, but how the temporal-spatial activity of these signals is controlled is poorly understood. We have identified an extracellular regulatory loop required for robust bone morphogenetic protein (BMP) signaling in the Xenopus foregut. We show that BMP signaling is required to maintain foregut progenitors and induce expression of the secreted frizzled related protein Sizzled (Szl) and the extracellular metalloprotease Tolloid-like 1 (Tll1). Szl negatively regulates Tll activity to control deposition of a fibronectin (FN) matrix between the mesoderm and endoderm, which is required to maintain BMP signaling. Foregut-specific Szl depletion results in a loss of the FN matrix and failure to maintain robust pSmad1 levels, causing a loss of foregut gene expression and organ agenesis. These results have implications for BMP signaling in diverse contexts and the differentiation of foregut tissue from stem cells.     

The core endodermal gene network of vertebrates: combining developmental precision with evolutionary flexibility

Embryonic development combines paradoxical properties: it has great precision, it is usually conducted at breakneck speed and it is flexible on relatively short evolutionary time scales, particularly at early stages. While these features appear mutually exclusive, we consider how they may be reconciled by the properties of key early regulatory networks. We illustrate these ideas with the network that controls development of endoderm progenitors. We argue that this network enables precision because of its intrinsic stability, self propagation and dependence on signalling. The network enables high developmental speed because it is rapidly established by maternal inputs at multiple points. In turn these properties confer flexibility on an evolutionary time scale because they can be initiated in many ways, while buffering essential progenitor cell populations against changes in their embryonic environment on both evolutionary and developmental time scales. Although stable, these networks must be capable of rapid dissolution as cell differentiation progresses. While we focus on the core early endodermal network of vertebrates, we argue that these properties are likely to be general in early embryonic stem cell populations, such as mammalian ES cells.     

Body protein does not vary despite seasonal changes in fat in the White Stork Ciconia ciconia

To understand how a large soaring bird, the White Stork Ciconia ciconia , copes with energy constraints, we compared changes in body mass in 14 captive adult storks with the body composition of 12 free-ranging adult storks found dead from accidents. The captive storks, already in an enclosure for several years, were fed ad libitum . They were weighed daily for 1.53.5 years using an automatic device. The bodies of the accidentally killed storks were analysed to determine total water, lipid, protein and ash contents, and to assess the biochemical composition of certain organs. Females were on average 20% lighter and 24% smaller than males, but the body mass of the sexes varied in parallel throughout the year. Body mass peaked in December and January (2530% above minimal body mass), due essentially to large fat stores in subcutaneous and abdominal adipose tissues. Body mass and body lipid rapidly decreased from February to June, whether the storks reared chicks successfully or not, and remained minimal for a few days into July. In contrast to birds using flapping flight, no variation in body protein or pectoral muscle protein was observed while breeding, even though the moult occurred then, nor in August, before the time when wild storks migrate. An endogenous regulation of body fuels is discussed.     

Large-scale production of a therapeutic protein in transgenic tobacco plants: effect of subcellular targeting on quality of a recombinant dog gastric lipase

A recombinant dog gastric lipase with therapeutic potential for the treatment of exocrine pancreatic insufficiency was expressed in transgenic tobacco plants. We targeted the protein using two different signal sequences for either vacuolar retention or secretion. In both cases, an active glycosylated recombinant protein was obtained. The recombinant enzymes and the native enzyme displayed similar properties including acid resistance and acidic optimum pH. The proteolytic maturation and the specific activity of the recombinant proteins, however, were found to be dependent on subcellular compartmentalization. Expression levels of recombinant dog gastric lipase were about 5% and 7% of acid extractable plant proteins for vacuolar retention and secretion respectively. This expression system already has allowed the production of tens of grams of purified lipase through open-field culture of transgenic tobacco plants.     

Facteurs de stress psychologique et qualité du sperme dans une population de 450 hommes infertiles slovènes

The purpose of this prospective study was to examine the relationship between psychological stress and male reproductive function. The study population consisted of 450 men attending the infertility outpatient clinic and 45 men participating in an IVF-ICSI programme. Psychological stress was measured in four ways. Firstly, the “WHO (five) Well-Being Index” (1995 version) assessed the risk of depression in the previous two-week period. Secondly, Zung’s Anxiety Scale Inventory was used to assess the features and severity of anxiety. Thirdly, the global reaction to various types of chronic life stress events related to family, partner, sexual behaviour, job strain and stress related to infertility treatment, with a more intense reaction corresponding to a higher score. Fourthly, the participant indicated by no (=1) or yes (=2) whether he had been exposed to the following acute stress situations: unemployment, moving house, personal illness or illness of a close relative, road accident. All questionnaires were completed after collection of the semen sample. The incidence of depression and anxiety symptoms in the two study populations (6.5% and 5.3%, and 20.3% and 30.8%, respectively) was twice as high as that observed in the general population. Anxiety was more pronounced (50%) in ICSI patients than in conventional IVF patients. To assess a relationship between psychological stress factors and male reproductive function, we used multiple regression analysis after adjustment of sperm parameters for age, sexual abstinence, type of infertility (primary or secondary), elevated body temperature, varicocele, and history of cryptorchidism and genital infection. Depression scoring was associated with increased sperm concentration (a 7.3% increase by score unit), an intense reaction to chronic stress was associated with a decreased sperm count (3.1% by score unit), and decreased motility “a”, motility “a+b”, and vitality (0.5, 0.5, and 0.6% by score unit, respectively). Exposure to acute stress was associated with increased motility “a+b” (3.5% when the man was exposed to acute stress). Analysis using dichotomous division of fertile and infertile men did not reveal any significant correlation between any stress factor and infertility. These findings indicate that depression and anxiety are frequent in a population of infertile men attending the outpatient clinic or during IVF-ICSI attempts, while significant changes of sperm characteristics are related to depression (decreased sperm concentration), intense reaction to chronic stress (decreased sperm count, motility “a” and “a+b”, and vitality) and exposure to acute stress (better motility “a+b”). This study confirms that stress factors must be taken into account in the management of infertile couples with particular attention to depression and reaction to stress.