Follistatin antagonizes transforming growth factor-beta3-induced epithelial-mesenchymal transition in vitro: implications for murine palatal development supported by microarray analysis.

Epithelial-mesenchymal transition (EMT) is involved in normal embryonic development as well as in tumor progression and invasiveness. This process is also known to be a crucial step in palatogenesis during fusion of the bi-lateral palatal processes. Disruption of this step results in a cleft palate, which is among the most frequent birth defects in humans. A number of genes and encoded proteins have been shown to play a role in this developmental stage. The central role is attributed to the cytokine transforming growth factor-beta3 (TGF-beta3), which is expressed in the medial edge epithelium (MEE) already before the fusion process. The MEE covers the tips of the growing palatal shelves and eventually undergoes EMT or programmed cell death (apoptosis). TGF-beta3 is described to induce EMT in embryonic palates. With regard to the early expression of this molecule before the fusion process, it is not well understood which mechanisms prevent the TGF-beta3 producing epithelial cells from undergoing differentiation precociously. We used the murine palatal fusion to study the regulation of EMT. Specifically, we analyzed the MEE for the expression of known antagonists of TGF-beta molecules using in situ hybridization and detected the gene coding for Follistatin to be co-expressed with TGF-beta3. Further, we could show that Follistatin directly binds to TGF-beta3 and that it completely blocks TGF-beta3-induced EMT of the normal murine mammary gland (NMuMG) epithelial cell line in vitro. In addition, we analyzed the gene expression profile of NMuMG cells during TGF-beta3-induced EMT by microarray hybridization, detecting strong changes in the expression of apoptosis-regulating genes.

LNA-modified oligodeoxynucleotide hybridization with DNA microarrays printed on nanoporous membrane slides.

We report a robust method for the detection of hybridization events using a microarray-based assay on a nanoporous membrane platform. The technique is characterized by a hybridization time of only 1 hour and uses Cy5-labeled, 7-mer oligodeoxynucleotide probes modified with locked nucleic acid (LNA) nucleotides. We show that the volume of the DNA spotted onto a nanomembrane can be reduced to approximately 4 nL with detectable signal intensity. Moreover, the amount of the DNA target could be reduced to 4 fmol. The described approach could dramatically increase the throughput of techniques based on sequencing by hybridization, such as oligofingerprinting, by decreasing the total number of probes that are needed for analysis of large clone sets and reduction of the sample/reagent consumption. The method is particularly advantageous when numerous hybridization-based assays must be performed for characterization of sample sets of 100,000 or more.