Cord Of Transgenic Mice Research Articles

Intravital multiphoton fluorescence imaging and optical manipulation of spinal cord in mice, using a compact fiber laser system.

Near-infrared ultrafast lasers are widely used for multiphoton excited fluorescence microscopy in living animals. Ti:Sapphire lasers are typically used for multiphoton excitation, but their emission wavelength is restricted below 1,000 nm. The aim of this study is to evaluate the performance of a compact Ytterbium-(Yb-) fiber laser at 1,045 nm for multiphoton excited fluorescence microscopy in spinal cord injury. In this study, we employed a custom-designed microscopy system with a compact Yb-fiber laser and evaluated the performance of this system in in vivo imaging of brain cortex and spinal cord in YFP-H transgenic mice. For in vivo imaging of brain cortex, sharp images of basal dendrites, and pyramidal cells expressing EYFP were successfully captured using the Yb-fiber laser in our microscopy system. We also performed in vivo imaging of axon fibers of spinal cord in the transgenic mice. The obtained images were almost as sharp as those obtained using a conventional ultrafast laser system. In addition, laser ablation and multi-color imaging could be performed simultaneously using the Yb-fiber laser. The high-peak pulse Yb-fiber laser is potentially useful for multimodal bioimaging methods based on a multiphoton excited fluorescence microscopy system that incorporates laser ablation techniques. Our results suggest that microscopy systems of this type could be utilized in studies of neuroscience and clinical use in diagnostics and therapeutic tool for spinal cord injury in the future.

The mouse mafB 5'-upstream fragment directs gene expression in myelomonocytic cells, differentiated macrophages and the ventral spinal cord in transgenic mice.

The b-Zip transcription factor MafB is an essential determinant of neural development and an inducer of monocytic differentiation. The MafB protein is expressed in a variety of tissues including the developing spinal cord, retina, myelomonocytic lineages of hematopoietic cells, and peritoneal macrophages. However, the tissue-specific regulatory mechanism of mafB gene expression and its biological relevance have not been examined in detail. Here, we report, for the first time, analysis of the regulatory mechanism and tissue-specific expression of the mafB gene in vivo using transgenic mice. A transgene, containing the 8.2-kb sequence flanking the 5' end of the mafB exon, directed the expression of a GFP reporter gene specifically in the retina, myelomonocytic lineages of hematopoietic cells, peritoneal macrophages and the ventral spinal cord. In situ hybridization analysis showed that the reporter gene expression specifically recapitulates the endogenous expression profile of mafB in the retina and spinal cord. FACS analysis revealed that the Gr-1, Mac-1 and F4/80 antigens were present on most of the GFP-positive hematopoietic cells from transgenic adult bone marrow and spleen. On the other hand, B220 CD4, 8, and Ter119 cells were almost absent from among the GFP-positive cells examined. These observations suggest that gene regulatory regions located in the 8.2-kb upstream region of mafB are responsible for directing mafB expression in the retina, myelomonocytic lineages, peritoneal macrophages and the ventral spinal cord.

Mutant SOD1 linked to familial amyotrophic lateral sclerosis, but not wild-type SOD1, induces ER stress in COS7 cells and transgenic mice

Mutations in a Cu, Zn-superoxide dismutase (SOD1) cause motor neuron death in human familial amyotrophic lateral sclerosis (FALS) and its mouse model, suggesting that mutant SOD1 has a toxic effect on motor neurons. However, the question of how the toxic function is gained has not been answered. Here, we report that the mutant SOD1s linked to FALS, but not wild-type SOD1, aggregated in association with the endoplasmic reticulum (ER) and induced ER stress in the cDNA-transfected COS7 cells. These cells showed an aberrant intracellular localization of mitochondria and microtubules, which might lead to a functional disturbance of the cells. Motor neurons of the spinal cord in transgenic mice with a FALS-linked mutant SOD1 also showed a marked increase of GRP78/BiP, an ER-resident chaperone, just before the onset of motor symptoms. These data suggest that ER stress is involved in the pathogenesis of FALS with an SOD1 mutation.

Zic1 Promotes the Expansion of Dorsal Neural Progenitors in Spinal Cord by Inhibiting Neuronal Differentiation

The role of Zic1 was investigated by altering its expression status in developing spinal cords. Zic genes encode zinc finger proteins homologous to Drosophila Odd-paired. In vertebrate neural development, they are generally expressed in the dorsal neural tube. Chick Zic1 was initially expressed evenly along the dorsoventral axis and its expression became increasingly restricted dorsally during the course of neurulation. The dorsal expression of Zic1 was regulated by Sonic hedgehog, BMP4, and BMP7, as revealed by their overexpressions in the spinal cord. When Zic1 was misexpressed on the ventral side of the chick spinal cord, neuronal differentiation was inhibited irrespective of the dorsoventral position. In addition, dorsoventral properties were not grossly affected as revealed by molecular markers. Concordantly, when Zic1 was overexpressed in the dorsal spinal cord in transgenic mice, we observed hypercellularity in the dorsal spinal cord. The transgene-expressing cells were increased in comparison to those of truncated mutant Zic1-bearing mice. Conversely, we observed a significant cell number reduction without loss of dorsal properties in the dorsal spinal cords of Zic1-deficient mice. Taken together, these findings suggest that Zic1 controls the expansion of neuronal precursors by inhibiting the progression of neuronal differentiation. Notch-mediated inhibition of neuronal differentiation is likely to act downstream of Zic genes since Notch1 is upregulated in Zic1-overexpressing spinal cords in both the mouse and the chick.

A core region of the mafK gene IN promoter directs neurone-specific transcription in vivo.

MafK serves as a required subunit of erythroid transcription factor NF-E2 and also functions with various heterodimeric CNC family proteins. MafK expression begins in early mesoderm and is observed in mesenchymal and haematopoietic cells, as well as in neurones during mouse development. In mesodermal descendants, MafK mRNA begins with a distal first exon (called IM), whereas the mRNA in neurones begins with a proximal first exon (IN). To elucidate the mechanisms that underlie the tissue-specific transcription of the mafK gene, and to gain insights into the functions of MafK during neural development, we analysed the activity of the mafK IN promoter. A detailed investigation of mafK expression in the embryonic spinal cord revealed that IN-initiated mRNA is expressed in the ventral side of the spinal cord. Transient transfection analysis of reporter plasmids bearing the IN promoter and upstream regions revealed that the 'core' region of this promoter (nt -67 to -9) is active and that its integrity is crucial for this activity. The core region was also capable of directing the tissue-specific transcription of a reporter gene in neural cells of the spinal cord in transgenic mice in vivo. These results demonstrate that the specific expression of mafK in neural cells is determined, at least in part, by the core region of the IN promoter.

Reactive astrocytes express nitric oxide synthase in the spinal cord of transgenic mice expressing a human Cu/Zn SOD mutation.

The distribution of the neuronal isoform of nitric oxide synthase (nNOS) in the spinal cord of transgenic mice expressing a mutated human copper/zinc superoxide dismutase gene was enhanced when investigated by immunocytochemistry. Immunocytochemistry showed intensely stained NOS-immunoreactive (IR) glial cells with the appearance of astrocytes in the spinal cord and brain stem of transgenic mice, but none were observed at these sites in control mice. Using antisera directed against GFAP, the specific marker for astrocyte, the glial cells were confirmed by immunocytochemistry to be astrocytes. This immunocytochemical evidence suggests that nitric oxide may mediate glutamate neurotoxicity, and this study provides the first in vivo evidence that nitric oxide may be implicated in the pathologic process of human familial amyotrophic lateral sclerosis.