Highly efficient 3D fluorescence microscopy with a scanning laser optical tomograph.

Optical Projection Tomography (OPT) proved to be useful for the three-dimensional tracking of fluorescence signals in biological model organisms with sizes up to several millimeters. This tomographic technique detects absorption as well as fluorescence to create multimodal three-dimensional data. While the absorption of a specimen is detected very fast usually less than 0.1% of the fluorescence photons are collected. The low efficiency can result in radiation dose dependent artifacts such as photobleaching and phototoxicity. To minimize these effects as well as artifacts introduced due to the use of a CCD- or CMOS- camera-chip, we constructed a Scanning Laser Optical Tomograph (SLOT). Compared to conventional fluorescence OPT our first SLOT enhanced the photon collection efficiency a hundredfold.

Development of nitrergic neurons in the nervous system of the locust embryo.

We followed the development of the nitric oxide-cyclic guanosine monophosphate (NO-cGMP) system during locust embryogenesis in whole mount nervous systems and brain sections by using various cytochemical techniques. We visualized NO-sensitive neurons by cGMP immunofluorescence after incubation with an NO donor in the presence of the soluble guanylyl cyclase (sGC) activator YC-1 and the phosphodiesterase-inhibitor isobutyl-methyl-xanthine (IBMX). Central nervous system (CNS) cells respond to NO as early as 38% embryogenesis. By using the NADPH-diaphorase technique, we identified somata and neurites of possible NO-synthesizing cells in the CNS. The first NADPH-diaphorase-positive cell bodies appear around 40% embryogenesis in the brain and at 47% in the ventral nerve cord. The number of positive cells reaches the full complement of adult cells at 80%. In the brain, some structures, e.g., the mushroom bodies acquire NADPH-diaphorase staining only postembryonically. Immunolocalization of L-citrulline confirmed the presence of NOS in NADPH-diaphorase-stained neurons and, in addition, indicated enzymatic activity in vivo. In whole mount ventral nerve cords, citrulline immunolabeling was present in varying subsets of NADPH-diaphorase-positive cells, but staining was very variable and often weak. However, in a regeneration paradigm in which one of the two connectives between ganglia had been crushed, strong, reliable staining was observed as early as 60% embryogenesis. Thus, citrulline immunolabeling appears to reflect specific activity of NOS. However, in younger embryos, NOS may not always be constitutively active or may be so at a very low level, below the citrulline antibody detection threshold. For the CNS, histochemical markers for NOS do not provide conclusive evidence for a developmental role of this enzyme.

Fibroblast growth factor-2 regulates the stability of nuclear bodies.

Nuclear bodies are distinct subnuclear structures. The survival of motoneuron (SMN) gene is mutated or deleted in patients with the neurodegenerative disease spinal muscular atrophy (SMA). The gene product SMN is a marker protein for one class of nuclear bodies denoted as nuclear gems. SMN has also been found in Cajal bodies, which co-localize with gems in many cell types. Interestingly, SMA patients display a reduced number of gems. Little is known about the regulation of nuclear body formation and stabilization. We have previously shown that a nuclear isoform of the fibroblast growth factor-2 (FGF-2(23)) binds directly to SMN. In this study, we analyzed the consequences of FGF-2(23) binding to SMN with regard to nuclear body formation. On a molecular level, we showed that FGF-2(23) competed with Gemin2 (a component of the SMN complex that is necessary for gem stabilization) for binding to SMN. Down-regulation of Gemin2 by siRNA caused destabilization of SMN-positive nuclear bodies. This process is reflected in both cellular and in vivo systems by a negative regulatory function of FGF-2 in nuclear body formation: in HEK293 cells, FGF-2(23) decreased the number of SMN-positive nuclear bodies. The same effect could be observed in motoneurons of FGF-2 transgenic mice. This study demonstrates the functional role of a growth factor in the regulation of structural entities of the nucleus.