The Hedgehog (Hh) signaling pathway has critical functions during embryogenesis of both invertebrate and vertebrate species [1]; defects in this pathway in humans can cause developmental disorders as well as neoplasia [2]. Although the Gli1, Gli2, and Gli3 zinc finger proteins are known to be effectors of Hh signaling in vertebrates, the mechanisms regulating activity of these transcription factors remain poorly understood [3] [4]. In Drosophila, activity of the Gli homolog Cubitus interruptus (Ci) is likely to be modulated by its interaction with a cytoplasmic complex containing several other proteins [5] [6], including Costal2, Fused (Fu), and Suppressor of fused (Su(fu)), the last of which has been shown to interact directly with Ci [7]. We have cloned mouse Suppressor of fused (mSu(fu)) and detected its 4.5 kb transcript throughout embryogenesis and in several adult tissues. In cultured cells, mSu(fu) overexpression inhibited transcriptional activation mediated by Sonic hedgehog (Shh), Gli1 and Gli2. Co-immunoprecipitation of epitope-tagged proteins indicated that mSu(fu) interacts with Gli1, Gli2, and Gli3, and that the inhibitory effects of mSu(fu) on Gli1's transcriptional activity were mediated through interactions with both amino- and carboxy-terminal regions of Gli1. Gli1 was localized primarily to the nucleus of both HeLa cells and the Shh-responsive cell line MNS-70; co-expression with mSu(fu) resulted in a striking increase in cytoplasmic Gli1 immunostaining. Our findings indicate that mSu(fu) can function as a negative regulator of Shh signaling and suggest that this effect is mediated by interaction with Gli transcription factors.
Recent advances in high-resolution imaging have provided valuable novel insights into structural relationships within cells and tissues both in vitro and in vivo. An analysis of this kind is regularly done by optical sectioning using either confocal or deconvolution microscopy. However, the reconstruction of 3D images suffers from light scattering and absorption with increasing depth by finite transparency of the used media. Photobleaching of fluorochromes has been especially troublesome and often the only remedy for loss of signal during optical sectioning is to reduce the number of sections. This causes disparities in the x-y and z dimensions of voxels, which lead to vertical distortion of the original stack of images and necessitates interpolation. Interpolation is necessary to fill up the gaps between consecutive sections in the original image stack to obtain cubic voxels. The present manuscript describes a novel method for adaptive compensation of attenuation of light intensity in stacks of fluorescence microscopy images that is based on a physical model of light attenuation. First, we use a fast interpolation technique to generate a cubic voxel-based volume stack with the aid of a contribution look up table. With the contribution look up table, multiple calculations are avoided, which substantially reduces the computational time without compromising the accuracy of the restoration procedure. Second, each section within the resulting volume is processed to rectify its intensity values that have been altered due to photobleaching and scattering and absorption. The method allows to define the last good section in the stack and the correction is then done automatically.
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