A new transgenic reporter line reveals Wnt-dependent Snai2 re-expression and cranial neural crest differentiation in Xenopus

During vertebrate embryogenesis, the cranial neural crest (CNC) forms at the neural plate border and subsequently migrates and differentiates into many types of cells. The transcription factor Snai2, which is induced by canonical Wnt signaling to be expressed in the early CNC, is pivotal for CNC induction and migration in Xenopus. However, snai2 expression is silenced during CNC migration, and its roles at later developmental stages remain unclear. We generated a transgenic X. tropicalis line that expresses enhanced green fluorescent protein (eGFP) driven by the snai2 promoter/enhancer, and observed eGFP expression not only in the pre-migratory and migrating CNC, but also the differentiating CNC. This transgenic line can be used directly to detect deficiencies in CNC development at various stages, including subtle perturbation of CNC differentiation. In situ hybridization and immunohistochemistry confirm that Snai2 is re-expressed in the differentiating CNC. Using a separate transgenic Wnt reporter line, we show that canonical Wnt signaling is also active in the differentiating CNC. Blocking Wnt signaling shortly after CNC migration causes reduced snai2 expression and impaired differentiation of CNC-derived head cartilage structures. These results suggest that Wnt signaling is required for snai2 re-expression and CNC differentiation

A new Transgenic Reporter Line Reveals Wnt-dependent Snai2 Re-expression and Cranial Neural Crest Differentiation in Xenopus

During vertebrate embryogenesis, the cranial neural crest (CNC) forms at the neural plate border and subsequently migrates and diferentiates into many types of cells. The transcription factor Snai2, which is induced by canonical Wnt signaling to be expressed in the early CNC, is pivotal for CNC induction and migration in Xenopus. However, snai2 expression is silenced during CNC migration, and its roles at later developmental stages remain unclear. We generated a transgenic X. tropicalis line that expresses enhanced green fuorescent protein (eGFP) driven by the snai2 promoter/enhancer, and observed eGFP expression not only in the pre-migratory and migrating CNC, but also the diferentiating CNC. This transgenic line can be used directly to detect defciencies in CNC development at various stages, including subtle perturbation of CNC diferentiation. In situ hybridization and immunohistochemistry confrm that Snai2 is re-expressed in the diferentiating CNC. Using a separate transgenic Wnt reporter line, we show that canonical Wnt signaling is also active in the diferentiating CNC. Blocking Wnt signaling shortly after CNC migration causes reduced snai2 expression and impaired diferentiation of CNC-derived head cartilage structures. These results suggest that Wnt signaling is required for snai2 reexpression and CNC diferentiation

Genetic Drivers of Kidney Defects in the DiGeorge Syndrome

Background The DiGeorge syndrome, the most common of the microdeletion syndromes, affects multiple organs, including the heart, the nervous system, and the kidney. It is caused by deletions on chromosome 22q11.2; the genetic driver of the kidney defects is unknown. Methods We conducted a genomewide search for structural variants in two cohorts: 2080 patients with congenital kidney and urinary tract anomalies and 22,094 controls. We performed exome and targeted resequencing in samples obtained from 586 additional patients with congenital kidney anomalies. We also carried out functional studies using zebrafish and mice. Results We identified heterozygous deletions of 22q11.2 in 1.1% of the patients with congenital kidney anomalies and in 0.01% of population controls (odds ratio, 81.5; P=4.5×10(-14)). We localized the main drivers of renal disease in the DiGeorge syndrome to a 370-kb region containing nine genes. In zebrafish embryos, an induced loss of function in snap29, aifm3, and crkl resulted in renal defects; the loss of crkl alone was sufficient to induce defects. Five of 586 patients with congenital urinary anomalies had newly identified, heterozygous protein-altering variants, including a premature termination codon, in CRKL. The inactivation of Crkl in the mouse model induced developmental defects similar to those observed in patients with congenital urinary anomalies. Conclusions We identified a recurrent 370-kb deletion at the 22q11.2 locus as a driver of kidney defects in the DiGeorge syndrome and in sporadic congenital kidney and urinary tract anomalies. Of the nine genes at this locus, SNAP29, AIFM3, and CRKL appear to be critical to the phenotype, with haploinsufficiency of CRKL emerging as the main genetic driver. (Funded by the National Institutes of Health and others.)

Alignment and theory in Corporate Real Estate alignment models

This paper deepens the understanding of Corporate Real Estate (CRE) alignment through a meta-study of twenty existing alignment models. A qualitative hermeneutic method interpreted the models and their articles. This holistic analysis found alignment to be more complex and pluralistic than the individual models assumed. Four dimensions operating simultaneously were evident – a multi-valent relationship, multiple alignment forms, multiple cognitive objects to align and alignment in multiple directions. Alignment theorisation had positive and negative aspects. Positive is that good science was evident and had improved over time. Negative is that model theorisation had occurred mostly in isolation and was constrained by simplifications required to make modelling tractable. The research makes a meta-theoretical contribution through a more complete theorisation of CRE alignment as a phenomenon. This addresses a disordered sense to prior theory thereby representing a major conceptual improvement. A new alignment model is not proposed; rather through developed understanding a basis is provided to examine alignment in both theorisation and practice

Mechanisms regulating lens fiber cell elongation

Duncan, Melinda K.How changes in cell shape are driven by events at the molecular and cellular levels is an ongoing area of investigation for developmental biologists. Lens epithelial cells differentiate into lens fiber cells and undergo a significant cell shape change during this process. Understanding how these cell shape changes occur could allow for a deeper understanding of similar processes in other tissues. Further, disorders of eye and lens development such as congenital cataracts and microphthalmia, which are the major causes of visual disability in children, can in some cases be caused by misregulation of cell shape. The cell fate decision between lens epithelial and fiber cell requires the transcription factor, Prox1, a global regulator of lens fiber preferred gene expression. RNAseq identified numerous cytoskeletal associated proteins mediating fiber cell elongation. Kinesin 1a (Kif1a), microtubule associated end binding protein RP/EB (Mapre3) and tubulin-β6 (Tubb6) are three genes differentially expressed in Prox1 null lenses that were prioritized for further study as possible regulators of lens fiber cell elongation based on their evolutionary conservation, lens preferred expression, and the availability of antibodies. While the expression of Kif1a, Mapre3, and Tubb6 have been characterized in mice, this thesis sought to use an alternative model to study their function due to the high cost of using mice. Xenopus tropicalis is a low-cost amphibian system for genetic and developmental biology research. Immunofluorescence for the established lens fiber marker, aquaporin0 (Aqp0) and histological staining methods confirmed lens fiber elongation occurs between stages 32/34 and 35/36. Therefore, the function of these three cytoskeletal associated genes were characterized using X. tropicalis. Morpholino-driven knockdowns of these genes offers insight into these genes’ role in the elongation process. Preliminarily, Kif1a knockdown produced an embryonic lethal phenotype before lens development occurred. Mapre3 knockdown resulted in an ocular pigmentation defect. Lastly, Tubb6 knockdown resulted in microphthalmia and reduced expression of the fiber cell marker, Aquaporin 0. Therefore, Tubb6 and Mapre3 may be essential for lens and/or eye morphogenesis. The future of this project will expand the preliminary data provided at the conclusion of this study to elucidate the function of these cytoskeletal associated genes in the regulation of lens fiber elongation.University of Delaware, Department of Biological SciencesM.S

Exploratory Evaluation of Nutrient Enrichment and Frog Response at Conboy Lake National Wildlife Refuge

53 pp. Bookmarks supplied by UO. References, map, tables, figures, appendices. Captured August 17, 2007.Reports on the results of a study on the effects of nutrient enrichment on the Oregon spotted frog in the Conboy Lake National Wildlife Refuge. Makes recommendations for management based on the results

Surface-Induced Deprotection of THP-Protected Hydroxamic Acids on Titanium Dioxide

Hydroxamic acids chelate metals with high affinity and form hydrolytically stable complexes with metal oxides such as TiO₂. However, these appealing binding properties can cause problems during the preparation and application of metallocatalysts with appended hydroxamate anchoring groups. Here we show that the tetrahydropyran (THP) O-protected hydroxamate group can be cleaved in situ on a TiO₂ surface at room temperature, leading to the surface-bound species. Surface-mediated deprotection has several advantages over direct surface functionalization including increased hydrolytic stability of the covalent interaction with the metal oxide surface and decreased aggregation of the surface species. Application of the surface-mediated chelation method for dye-sensitized photoelectrochemical cells (DSPC) was examined using the organic dye MK-2. Results show that the surface-mediated deprotection led to improved DSPC performance attributed to a decrease in dye aggregation relative to a DSPC prepared using standard methods. This simplified approach using THP-protected hydroxamates provides a convenient new method for functionalizing metal oxides

Unfolded Protein Response in Cancer: IRE1α Inhibition by Selective Kinase Ligands Does Not Impair Tumor Cell Viability

The kinase/endonuclease inositol requiring enzyme 1 (IRE1α), one of the sensors of unfolded protein accumulation in the endoplasmic reticulum that triggers the unfolded protein response (UPR), has been investigated as an anticancer target. We identified potent allosteric inhibitors of IRE1α endonuclease activity that bound to the kinase site on the enzyme. Structure–activity relationship (SAR) studies led to <b>16</b> and <b>18</b>, which were selective in kinase screens and were potent against recombinant IRE1α endonuclease as well as cellular IRE1α. The first X-ray crystal structure of a kinase inhibitor (<b>16</b>) bound to hIRE1α was obtained. Screening of native tumor cell lines (>300) against selective IRE1α inhibitors failed to demonstrate any effect on cellular viability. These results suggest that IRE1α activity is not essential for viability in most tumor cell lines, in vitro, and that interfering with the survival functions of the UPR may not be an effective strategy to block tumorigenesis