Expression of Drosophila omb-related T-box genes in the developing human and mouse neural retina.

PURPOSE: To examine the role of Drosophila optomotor blind (omb)-related T-box genes in development of human and mouse retina. METHODS: Mouse Tbx2, Tbx3, and Tbx5 and human TBX2 cDNAs were isolated from retinal cDNA libraries by hybridization to the Drosophila omb gene. Gene expression patterns in developing retina were analyzed by in situ hybridization. RESULTS: TBX2/Tbx2, TBX3/Tbx3, and TBX5/Tbx5 were expressed asymmetrically across the embryonic neural retina with highest levels of mRNA within dorsal and peripheral retina. The dorsoventral gradient of TBX2 expression disappeared before the ganglion cell layer (GCL) formed. Its expression then became restricted to the inner neuroblastic retina and later to the GCL and inner nuclear layer (INL). The dorsal expression domains of TBX5/Tbx5 and TBX3/Tbx3 were maintained during formation of the GCL. As the retina matured, TBX3/Tbx3 expression was restricted to the INL, and TBX5/Tbx5 was expressed within the GCL. CONCLUSIONS: The expression pattern of TBX2, TBX3, and TBX5 within the developing retina supports the idea that the encoded transcription factors play a role in providing positional information important for topographic mapping and in differentiation of distinct cell types across the laminar axis of the retina.

Gene delivery from the E3 region of replicating human adenovirus: evaluation of the 6.7 K/gp19 K region.

The use of genetically engineered, replication-selective viruses to treat cancer is being realized with viruses such as ONYX-015, a human adenovirus that selectively destroys p53 mutant cancer cells. To enhance further the clinical efficacy of ONYX-015 and viruses like it, we have developed a novel gene delivery system for replicating adenoviruses. This system has two unique features. First, it uses the endogenous adenoviral gene expression machinery (promoter, splicing, polyadenylation) to drive transgene expression. Second, a single region or gene in the multi-gene E3 transcription unit is selectively substituted for by the therapeutic transgene(s). Analyzing various transgene substitutions for the 6.7 K/gp19 K region of E3, we demonstrate the following: (1) transgene expression in this system is predictable and mimics the substituted endogenous gene expression pattern, (2) expression of surrounding E3 genes can be retained, (3) the insertion site choice can effect both the transgene expression level and the viral life cycle, and, (4) expression levels from this system are superior to those generated from a replication-defective virus using the HCMV enhancer-promoter and this is dependent on viral DNA replication. This unique methodology has broad application to the rapidly evolving field of replicating virus-based therapies.

Temporal and spatial expression patterns of the CRX transcription factor and its downstream targets. Critical differences during human and mouse eye development.

Cone--rod homeobox (CRX), a paired-like homeobox transcription factor, plays a major role in photoreceptor development and maintenance of the retina. Fifteen different mutations in the CRX gene have been identified as a cause of blinding retinal dystrophy. As a step towards characterizing the underlying pathophysiology of disease, temporal and spatial gene expression patterns during human and mouse eye development were investigated for CRX and for downstream retinally expressed genes, postulated to be transactivated by CRX. We found that human CRX was expressed at 10.5 weeks post-conception (p.c.). This was significantly later than observed in mouse development. Immunocytochemistry in human retina showed that CRX protein was not detected until >4 weeks later at 15 weeks p.c., implying that it would be unable to transactivate PDEB, IRBP and arrestin, which were all expressed before 15 weeks. These data therefore eliminate CRX as the major transcriptional activator of these three genes from a wide group of retinal genes that can be transactivated by CRX in vitro. Additionally, PDEB was expressed 2 weeks before CRX whereas murine Pdeb was expressed after Crx, highlighting a potential difference for the role of PDEB in human eye development. Previous data had shown CRX expression in the adult human retina to be photoreceptor-specific; however, we demonstrate that this gene is also expressed in the inner nuclear layer (INL) of the human and mouse retina by in situ hybridization and immunocytochemistry. INL localization of murine Crx was confirmed in rd/rd,cl mice, as in this mouse model the photoreceptors are absent. We have found important differences in the temporal expression of this gene in human and mouse retina, although spatial expression of the CRX gene appears to be conserved. In addition, downstream targets of CRX in vitro might not represent in vivo function during development. These data support concerns about the extent to which we can extrapolate from rodent models regarding embryonic development and disease pathophysiology.

Method for characterization of adhesion properties of trace explosives in fingerprints and fingerprint simulations.

The near inevitable transfer of explosive particulate matter through fingerprints makes it possible to detect concealed explosives through surface sampling. Repeatable and well-characterized fingerprint simulation facilitates quantitative comparison between particulate sampling methods for subsequent detection of trace explosive residues. This study employs a simple, but reproducible sampling system to determine the accuracy of a fingerprint simulation. The sampling system uses a gas jet to entrain particles from a substrate and the resulting airborne particles are then aspirated onto a Teflon filter. A calibrated Barringer IonScan 400 ion mobility spectrometer was used to determine the mass of explosive material collected on the filter. The IonScan 400 was calibrated with known masses of 2,4,6-trinitrotoluene (TNT). The resulting calibration curve is in good agreement with that obtained by Garofolo et al. (1994) for an earlier model of the instrument. The collection efficiency of the sampling system was measured for three particle sizes (8.0. 10.0, and 13.0 microm) using spherical polystyrene particles laced with known quantities of TNT. Collection efficiency ranged from less than 1% for the larger particles to 5% for the smaller particles. Particle entrainment from the surface was monitored with dark field imaging of the remaining particles. The sampling system was then applied to two C4 test samples--a fingerprint transfer and a dry Teflon transfer. Over 100 ng of RDX was collected from the dry transfer sample, while less than 1 ng was collected from the fingerprint transfer. Possible explanations for this large difference are presented based on the system calibration.

Spontaneous axonal regeneration after optic nerve injury in adult rat.

Optic nerves of adult rats were crushed 2 mm behind the eye to examine the ability of retinal ganglion cells (RGCs) to regenerate their axons. Some animals were treated with the immunophilin ligands FK 506 or GPI 1046 for up to 4 weeks. After 10 days to 16 months, regenerating RGC axons were visualized using anterograde tracing and/or electron microscopy. A small proportion of RGC axons regenerated across the lesion site and grew very slowly along the entire optic nerve. Immunophilin ligands had no obvious effect. The regenerating axons were about 0.2 microm in diameter, and usually in clusters surrounded by astrocyte processes. Thus, some CNS axons can spontaneously regenerate long distances within degenerate white matter and this slow regeneration is not accelerated by immunophilin ligands.