Mosaic analysis of stem cell function and wound healing in the mouse corneal epithelium

<p>Abstract</p> <p>Background</p> <p>The mouse corneal epithelium is a continuously renewing 5–6 cell thick protective layer covering the corneal surface, which regenerates rapidly when injured. It is maintained by peripherally located limbal stem cells (LSCs) that produce transient amplifying cells (TACs) which proliferate, migrate centripetally, differentiate and are eventually shed from the epithelial surface. LSC activity is required both for normal tissue maintenance and wound healing. Mosaic analysis can provide insights into LSC function, cell movement and cell mixing during tissue maintenance and repair. The present study investigates cell streaming during corneal maintenance and repair and changes in LSC function with age.</p> <p>Results</p> <p>The initial pattern of corneal epithelial patches in <it>XLacZ</it>^+/-X-inactivation mosaics was replaced after birth by radial stripes, indicating activation of LSCs. Stripe patterns (clockwise, anticlockwise or midline) were independent between paired eyes. Wound healing in organ culture was analysed by mosaic analysis of <it>XLacZ</it>^+/-eyes or time-lapse imaging of GFP mosaics. Both central and peripheral wounds healed clonally, with cells moving in from all around the wound circumference without significant cell mixing, to reconstitute striping patterns. Mosaic analysis revealed that wounds can heal asymmetrically. Healing of peripheral wounds produced stripe patterns that mimicked some aberrant striping patterns observed in unwounded corneas. Quantitative analysis provided no evidence for an uneven distribution of LSC clones but showed that corrected corneal epithelial stripe numbers declined with age (implying declining LSC function) but stabilised after 39 weeks.</p> <p>Conclusion</p> <p>Striping patterns, produced by centripetal movement, are defined independently and stochastically in individual eyes. Little cell mixing occurs during the initial phase of wound healing and the direction of cell movement is determined by the position of the wound and not by population pressure from the limbus. LSC function declines with age and this may reflect reduced LSCs numbers, more quiescent LSCs or a reduced ability of older stem cells to maintain tissue homeostasis. The later plateau of LSC function might indicate the minimum LSC function that is sufficient for corneal epithelial maintenance. Quantitative and temporal mosaic analyses provide new possibilities for studying stem cell function, tissue maintenance and repair.</p

Growth and Fecundity of Several Weed Species in Corn and Soybean

Do weeds that emerge later in the season justify additional control costs\u27? If crop yield is not reduced or few or no seeds arc added to the soil seed hank, then no control may he needed. Eight weed species were sown in corn (Zea mays L.) and soybean I Glycine max (L.) Mcrr.l (i) before crop emergence, (ii) at crop emergence, (iii) at V-1, and (iv) at V-2 stages of crop growth in 2002 and 2003. Weed seed was sown close to the crop row and thinned to 1.3 plants m 2• Weed growth and fecundity were influenced by species, time of planting, and year. Only barnyarclgrass (Echinochloa crus-galli L.), rcclroot pigwced (Amaranthus retniflexus L.), and vclvetlcaf (Abutilon theophrasti L.) survived to produce seed. Plants from the pre-emergence seeding had the largest canopy and produced the most seeds. Harnyardgrass had maximum canopy cover in early .July in corn and late .Inly in soybean hut only produced seed in corn. Rcclroot pigweecl and vclvctleaf had maximum canopy cover in late August or midSeptember, and some plants from most seeding elates survived and produced seed in both corn and soybean. However, plants that grew from seed sown at V-1 and V-2 crnp grnwth stages did not reduce yield or biomass of adjacent crop plants, had low fecundity, and may not warrant treatment. Control may be necessary, however, to prevent yield losses if weeds arc present at high densities or to prevent establishment of uncommon species

DNaseI Hypersensitivity and Ultraconservation Reveal Novel, Interdependent Long-Range Enhancers at the Complex Pax6 Cis-Regulatory Region

The PAX6 gene plays a crucial role in development of the eye, brain, olfactory system and endocrine pancreas. Consistent with its pleiotropic role the gene exhibits a complex developmental expression pattern which is subject to strict spatial, temporal and quantitative regulation. Control of expression depends on a large array of cis-elements residing in an extended genomic domain around the coding region of the gene. The minimal essential region required for proper regulation of this complex locus has been defined through analysis of human aniridia-associated breakpoints and YAC transgenic rescue studies of the mouse smalleye mutant. We have carried out a systematic DNase I hypersensitive site (HS) analysis across 200 kb of this critical region of mouse chromosome 2E3 to identify putative regulatory elements. Mapping the identified HSs onto a percent identity plot (PIP) shows many HSs correspond to recognisable genomic features such as evolutionarily conserved sequences, CpG islands and retrotransposon derived repeats. We then focussed on a region previously shown to contain essential long range cis-regulatory information, the Pax6 downstream regulatory region (DRR), allowing comparison of mouse HS data with previous human HS data for this region. Reporter transgenic mice for two of the HS sites, HS5 and HS6, show that they function as tissue specific regulatory elements. In addition we have characterised enhancer activity of an ultra-conserved cis-regulatory region located near Pax6, termed E60. All three cis-elements exhibit multiple spatio-temporal activities in the embryo that overlap between themselves and other elements in the locus. Using a deletion set of YAC reporter transgenic mice we demonstrate functional interdependence of the elements. Finally, we use the HS6 enhancer as a marker for the migration of precerebellar neuro-epithelium cells to the hindbrain precerebellar nuclei along the posterior and anterior extramural streams allowing visualisation of migratory defects in both pathways in Pax6(Sey/Sey) mice

Winter Wheat Grain Yield Response to Fungicide Application is Influenced by Cultivar and Rainfall

Winter wheat is susceptible to several fungal pathogens throughout the growing season and foliar fungicide application is one of the strategies used in the management of fungal diseases in winter wheat. However, for fungicides to be profitable, weather conditions conducive to fungal disease development should be present. To determine if winter wheat yield response to fungicide application at the flowering growth stage (Feekes 10.5.1) was related to the growing season precipitation, grain yield from fungicide treated plots was compared to non-treated plots for 19 to 30 hard red winter wheat cultivars planted at 8 site years from 2011 through 2015. At all locations, Prothioconazole + Tebuconazole or Tebuconazole alone was applied at flowering timing for the fungicide treated plots. Grain yield response (difference between treated and non-treated) ranged from 66-696 kg/ha across years and locations. Grain yield response had a positive and significant linear relationship with cumulative rainfall in May through June for the mid and top grain yield ranked cultivars (R2=54%, 78%, respectively) indicating that a higher amount of accumulated rainfall in this period increased chances of getting a higher yield response from fungicide application. Cultivars treated with a fungicide had slightly higher protein content (up to 0.5%) compared to non-treated. These results indicate that application of fungicides when there is sufficient moisture in May and June may increase chances of profitability from fungicide application

Notch signaling in T cells is essential for allergic airway inflammation, but expression of the Notch ligands Jagged 1 and Jagged 2 on dendritic cells is dispensable

__Background:__ Allergic asthma is characterized by a TH2 response induced by dendritic cells (DCs) that present inhaled allergen. Although the mechanisms by which they instruct TH2 differentiation are still poorly understood, expression of the Notch ligand Jagged on DCs has been implicated in this process. __Objective:__ We sought to establish whether Notch signaling induced by DCs is critical for house dust mite (HDM)-driven allergic airway inflammation (AAI) in vivo. __Methods:__ The induction of Notch ligand expression on DC subsets by HDM was quantified by using quantitative real-time PCR. We used an HDM-driven asthma mouse model to compare the capacity of Jagged 1 and Jagged 2 single- and double-deficient DCs to induce AAI. In addition, we studied AAI in mice with a T cell-specific deletion of recombination signal-binding protein for immunoglobulin Jκ region (RBPJκ), a downstream effector of Notch signaling. __Results:__ HDM exposure promoted expression of Jagged 1, but not Jagged 2, on DCs. In agreement with published findings, in vitro-differentiated and HDM-pulsed Jagged 1 and Jagged 2 double-deficient DCs lacked the capacity to induce AAI. However, after in vivo intranasal sensitization and challenge with HDM, DC-specific Jagged 1 or Jagged 2 single- or double-deficient mice had eosinophilic airway inflammation and a TH2 cell activation phenotype that was not different from that in control littermates. In contrast, RBPJκ-def

Identification of Novel Craniofacial Regulatory Domains Located far Upstream of SOX9 and Disrupted in Pierre Robin Sequence.

Mutations in the coding sequence of SOX9 cause campomelic dysplasia (CD), a disorder of skeletal development associated with 46,XY disorders of sex development (DSDs). Translocations, deletions, and duplications within a ∼2 Mb region upstream of SOX9 can recapitulate the CD-DSD phenotype fully or partially, suggesting the existence of an unusually large cis-regulatory control region. Pierre Robin sequence (PRS) is a craniofacial disorder that is frequently an endophenotype of CD and a locus for isolated PRS at ∼1.2-1.5 Mb upstream of SOX9 has been previously reported. The craniofacial regulatory potential within this locus, and within the greater genomic domain surrounding SOX9, remains poorly defined. We report two novel deletions upstream of SOX9 in families with PRS, allowing refinement of the regions harboring candidate craniofacial regulatory elements. In parallel, ChIP-Seq for p300 binding sites in mouse craniofacial tissue led to the identification of several novel craniofacial enhancers at the SOX9 locus, which were validated in transgenic reporter mice and zebrafish. Notably, some of the functionally validated elements fall within the PRS deletions. These studies suggest that multiple noncoding elements contribute to the craniofacial regulation of SOX9 expression, and that their disruption results in PRS

Dielectric quantification of conductivity limitations due to nanofiller size in conductive powders and nanocomposites

Conducting submicron particles are well-suited as filler particles in non-conducting polymer matrices to obtain a conducting composite with a low percolation threshold. Going to nanometer-sized filler particles imposes a restriction to the conductivity of the composite, due to the reduction of the density of states involved in the hopping process between the particles, compared to its value within the crystallites. We show how those microscopic parameters that govern the charge-transport processes across many decades of length scales, can accurately and consistently be determined by a range of dielectric-spectroscopy techniques from a few Hz to infrared frequencies. The method, which is suited for a variety of systems with restricted geometries, is applied to densely packed 7-nm-sized tin-oxide crystalline particles with various degree of antimony doping and the quantitative results unambiguously show the role of the nanocrystal charging energy in limiting the hopping process.Comment: 6 pages, 4 figure

Controlled overexpression of Pax6 in vivo negatively autoregulates the Pax6 locus, causing cell-autonomous defects of late cortical progenitor proliferation with little effect on cortical arealization

Levels of expression of the transcription factor Pax6 vary throughout corticogenesis in a rostro-lateral(high) to caudo-medial(low) gradient across the cortical proliferative zone. Previous loss-of-function studies have indicated that Pax6 is required for normal cortical progenitor proliferation, neuronal differentiation, cortical lamination and cortical arealization, but whether and how its level of expression affects its function is unclear. We studied the developing cortex of PAX77 YAC transgenic mice carrying several copies of the human PAX6 locus with its full complement of regulatory regions. We found that PAX77 embryos express Pax6 in a normal spatial pattern, with levels up to three times higher than wild type. By crossing PAX77 mice with a new YAC transgenic line that reports Pax6 expression (DTy54), we showed that increased expression is limited by negative autoregulation. Increased expression reduces proliferation of late cortical progenitors specifically, and analysis of PAX77↔wild-type chimeras indicates that the defect is cell autonomous. We analyzed cortical arealization in PAX77 mice and found that, whereas the loss of Pax6 shifts caudal cortical areas rostrally, Pax6 overexpression at levels predicted to shift rostral areas caudally has very little effect. These findings indicate that Pax6 levels are stabilized by autoregulation, that the proliferation of cortical progenitors is sensitive to altered Pax6 levels and that cortical arealization is not