A short upstream promoter region mediates transcriptional regulation of the mouse doublecortin gene in differentiating neurons.

peer reviewedABSTRACT: BACKGROUND: Doublecortin (Dcx), a MAP (Microtubule-Associated Protein), is transiently expressed in migrating and differentiating neurons and thereby characterizes neuronal precursors and neurogenesis in developing and adult neurogenesis. In addition, reduced Dcx expression during development has been related to appearance of brain pathologies. Here, we attempt to unveil the molecular mechanisms controlling Dcx gene expression by studying its transcriptional regulation during neuronal differentiation. RESULTS: To determine and analyze important regulatory sequences of the Dcx promoter, we studied a putative regulatory region upstream from the mouse Dcx coding region (pdcx2kb) and several deletions thereof. These different fragments were used in vitro and in vivo to drive reporter gene expression. We demonstrated, using transient expression experiments, that pdcx2kb is sufficient to control specific reporter gene expression in cerebellar cells and in the developing (E14.5) brain. We determined the temporal profile of Dcx promoter activity during neuronal differentiation of mouse embryonic stem cells (mESC) and found that transcriptional activation of the Dcx gene varies along with neuronal differentiation of mESC. Deletion experiments and sequence comparison of Dcx promoters across rodents, human and chicken revealed the importance of a highly conserved sequence in the proximal region of the promoter required for specific and strong expression in neuronal precursors and young neuronal cells. Further analyses revealed the presence in this short sequence of several conserved, putative transcription factor binding sites: LEF/TCF (Lymphoid Enhancer Factor/T-Cell Factor) which are effectors of the canonical Wnt pathway; HNF6/OC2 (Hepatocyte Nuclear Factor-6/Oncecut-2) members of the ONECUT family; and NF-Y/CAAT (Nuclear Factor-Y). CONCLUSIONS: Studies of Dcx gene regulatory sequences using native, deleted and mutated constructs suggest that fragments located upstream of the Dcx coding sequence are sufficient to induce specific Dcx expression in vitro: in heterogeneous differentiated neurons from mESC, in primary mouse cerebellar neurons (PND3) and in organotypic slices cultures. Furthermore, a region in the 3'-end region of the Dcx promoter is highly conserved across several species and exerts positive control on Dcx transcriptional activation. Together, these results indicate that the proximal 3'-end region of the mouse Dcx regulatory sequence is essential for Dcx gene expression during differentiation of neuronal precursors

Nitrate enrichment & heat stress impacts the physiology of the coral A. kenti and the composition of its associated microbiome

Coral reefs are unarguably under increasing pressures arising from various environmental stressors. Coral survival in the face of environmental change relies heavily on nutrient exchanges between the host and the photosynthetic endosymbionts. While the functional contribution of the coral microbiome remains poorly understood, increasing evidence suggests that associated microorganisms are essential for coral resilience as they are intricately linked to nutrient cycling and energy flows in the ecosystem. Nitrogen underpins many aspects of coral holobiont functioning but the effect of its availability in its most abundant environmental form, nitrate, on the coral response to stress is equivocal: while nitrate sustains symbiont communities, it has also been reported to have adverse effects on the response to oxidative stress and to accentuate bleaching. In this study, using a crossed treatment experimental design in a mesocosm setup, we investigated the responses of the coral Acropora kenti to a nitrate enrichment of 5 µM in combination with a heat stress of 4 DHW over a period of 3 weeks. Corals’ health was monitored throughout the experiment and corals’ physiological response to the different treatments was assessed at the start of the stress and at the end of the experiment by measuring respiration rates, photosynthetic capacity, growth rates, symbiont densities, pigment and protein contents. In addition, corals were sampled to identify the composition of the associated symbiont and microbial communities using high-throughput sequencing of the genes ITS2 and 16S respectively. The heat stress treatment induced moderate to severe bleaching that was not alleviated by the increased nitrate supply. Nuances in the physiological data and the integration with the sequencing data give valuable inputs into the holobiont’s functioning by disentangling the effect of nitrate availability and heat stress on the resilience of the coral and the stability of its associated symbiotic and microbial communities

Nitrate in the coral symbiosis: from the regulation of its assimilation to its impact on the physiology of the holobiont

In oligotrophic reef systems, coral holobionts are remarkably efficient at assimilating nitrogen through heterotrophic feeding or the uptake of dissolved inorganic nitrogen. Symbiodiniaceae are vital partners of the symbiosis for nutrient assimilation. In addition to providing translocated photosynthates, they account for most of the uptake of dissolved inorganic nitrogen. Although NO3- is the most abundant source of nitrogen in the ocean, little is known about the mechanisms regulating its assimilation by the holobiont. Coral hosts are unable to reduce nitrate as they lack the necessary enzymes, whereas Symbiodiniaceae have been shown to express the enzyme nitrate reductase (NR). However, the evidence supporting the active reduction of nitrate by the symbiotic algae during symbiosis is scarce and equivocal. This research aimed at deciphering the pathways of NO3- assimilation in both free-living Symbiodiniaceae and in hospite symbionts while also investigating the relevance of inorganic nitrogen source in physiological responses to stress. We investigated the expression and regulation of NR both in free-living Symbiodiniaceae and in in hospite symbionts using a combined western blot and qRT-PCR approach. We showed that the expression and regulation of NR in free-living Symbiodiniaceae is a dynamic and reversible process impacted by NO3- and NH4+ concentrations. Symbionts from N-depleted corals incubated with NO3- enriched seawater showed an increase in NR synthesis over time. Interestingly, NR protein synthesis did not correlate with NR gene expression, hinting towards a potential post-transcriptional regulation of the enzyme. Additionally, we investigated the impacts of inorganic N source (NO3- vs NH4+ vs N depletion) in combination with stress on the physiology of Symbiodiniaceae (photosynthetic responses, ROS and NO production). The availability of inorganic nitrogen improved photosynthetic capacity while reducing ROS production. Moreover, preliminary experiments showed that NO3- and NH4+ had differential effects on the physiological responses of Symbiodiniaceae subjected to stress

Functionally Fractal Urban Networks: Geospatial Co-location and Homogeneity of Infrastructure

Just as natural river networks are known to be globally self-similar, recent research has shown that human-built urban networks, such as road networks, are also functionally self-similar, and have fractal topology with power-law node-degree distributions (p(k) = a k). Here we show, for the first time, that other urban infrastructure networks (sanitary and storm-water sewers), which sustain flows of critical services for urban citizens, also show scale-free functional topologies. For roads and drainage networks, we compared functional topological metrics, derived from high-resolution data (70,000 nodes) for a large US city providing services to about 900,000 citizens over an area of about 1,000 km2. For the whole city and for different sized subnets, we also examined these networks in terms of geospatial co-location (roads and sewers). Our analyses reveal functional topological homogeneity among all the subnets within the city, in spite of differences in several urban attributes. The functional topologies of all subnets of both infrastructure types resemble power-law distributions, with tails becoming increasingly power-law as the subnet area increases. Our findings hold implications for assessing the vulnerability of these critical infrastructure networks to cascading shocks based on spatial interdependency, and for improved design and maintenance of urban infrastructure networks

Endothelium-Dependent Effects of Heat Shock on Vasomotor Tone: Role of eNOS and HSP70

peer reviewe