Characterization of miRNA processing machinery in the embryonic chick lung

Lung development is a very complex process that relies on the interaction of several signaling pathways that are controlled by precise regulatory mechanisms. Recently, microRNAs (miRNAs), small non-coding regulatory RNAs, have emerged as new players involved in gene expression regulation controlling several biological processes, such as cellular differentiation, apoptosis and organogenesis, in both developmental and disease processes. Failure to correctly express some specific miRNAs or a component of their biosynthetic machinery during embryonic development is disastrous, resulting in severe abnormalities. Several miRNAs have already been identified as modulators of lung development. Regarding the spatial distribution of the processing machinery of miRNAs, only two of its members (dicer1 and argonaute) have been characterized. The present work characterizes the expression pattern of drosha, dgcr8, exportin-5 and dicer1 in early stages of the embryonic chick lung by whole mount in situ hybridization and cross-section analysis. Overall, these genes are co-expressed in dorsal and distal mesenchyme and also in growing epithelial regions. The expression pattern of miRNA processing machinery supports the previously recognized regulatory role of this mechanism in epithelial and mesenchymal morphogenesis.QRE

Genetic and Evolutionary Analyses of the Human Bone Morphogenetic Protein Receptor 2 (BMPR2) in the Pathophysiology of Obesity

Human bone morphogenetic protein receptor 2 (BMPR2) is essential for BMP signalling and may be involved in the regulation of adipogenesis. The BMPR2 locus has been suggested as target of recent selection in human populations. We hypothesized that BMPR2 might have a role in the pathophysiology of obesity.Evolutionary analyses (dN/dS, Fst, iHS) were conducted in vertebrates and human populations. BMPR2 mRNA expression was measured in 190 paired samples of visceral and subcutaneous adipose tissue. The gene was sequenced in 48 DNA samples. Nine representative single nucleotide polymorphisms (SNPs) were genotyped for subsequent association studies on quantitative traits related to obesity in 1830 German Caucasians. An independent cohort of 925 Sorbs was used for replication. Finally, relation of genotypes to mRNA in fat was examined.The evolutionary analyses indicated signatures of selection on the BMPR2 locus. BMPR2 mRNA expression was significantly increased both in visceral and subcutaneous adipose tissue of 37 overweight (BMI>25 and <30 kg/m²) and 80 obese (BMI>30 kg/m²) compared with 44 lean subjects (BMI< 25 kg/m²) (P<0.001). In a case-control study including lean and obese subjects, two intronic SNPs (rs6717924, rs13426118) were associated with obesity (adjusted P<0.05). Combined analyses including the initial cohort and the Sorbs confirmed a consistent effect for rs6717924 (combined P = 0.01) on obesity. Moreover, rs6717924 was associated with higher BMPR2 mRNA expression in visceral adipose tissue.Combined BMPR2 genotype-phenotype-mRNA expression data as well as evolutionary aspects suggest a role of BMPR2 in the pathophysiology of obesity

Whole-Mount Skeletal Staining

The first step in almost every investigation of skeletal phenotypes is analysis of whole-mount skeletal preparations. Whole-mount skeletal staining permits evaluation of the shapes and sizes of skeletal elements in their appropriate locations. The technique is thus the major method for detecting changes in skeletal patterning. Because cartilage and bone can be distinguished by differential staining, this technique is also a powerful means to assess the pace of skeletal maturation. This protocol covers staining of the pre- and postnatal mouse skeleton using Alcian blue and Alizarin red to identify cartilage and bone, respectively