AB986 Sigma-AldrichAnti-β Galactosidase Antibody, bacterial

Sonic Hedgehog (SHH) and WNT proteins are key regulators in many developmental processes, like embryonic patterning and brain development. In the brain, SHH is expressed in a gradient starting in the floor plate (FP) progressing ventrally in the midbrain, where it is thought to be involved in the development and specification of mesodiencephalic dopaminergic (mdDA) neurons. GLI2A-mediated SHH-signaling induces the expression of Gli1, which is inhibited when cells start expressing SHH themselves. To determine whether mdDA neurons receive GLI2A-mediated SHH-signaling during differentiation, we used a BAC-transgenic mouse model expressing eGFP under the control of the Gli1 promoter. This mouse-model allowed for mapping of GLI2A-mediated SHH-signaling temporal and spatial in the mouse midbrain. Since mdDA neurons are born from E10.5, peaking at E11.0-E12.0, we examined Gli1-eGFP embryos at E11.5, E12.5, and E13.5, indicating whether Gli1 was induced before or during mdDA development and differentiation. Our data indicate that GLI2A-mediated SHH-signaling is not involved in mdDA neuronal differentiation. However, it appears to be involved in the differentiation of neurons which make up a subset of the red nucleus (RN). In order to detect whether mdDA neuronal differentiation may be under the control of canonical WNT-signaling, we used a transgenic mouse-line expressing LacZ under the influence of stable β-catenin. Here, we show that TH+ neurons of the midbrain receive canonical WNT-signaling during differentiation. Therefore, we suggest that early SHH-signaling is indirectly involved in mdDA development through early patterning of the midbrain area, whereas canonical WNT-signaling is directly involved in the differentiation of the mdDA neuronal population.

Recent studies have shown that ephrin-B2 on sensory afferent fibers from the dorsal root ganglia (DRG) controls transmission of pain sensation to the spinal cord. We examined ephrin-B2 expression in mouse DRG and spinal cord using an ephrin-B2/ß-galactosidase chimeric allele. We found that ephrin-B2 is expressed exclusively in proprioceptive neurons and fibers in neonates, while expression in lamina III and IV of the adult spinal cord was observed in addition to that in the deeper laminae. We confirmed that ephrin-B2 protein causes co-clustering of EphB2 and glutamate receptors in spinal cord neurons. Our data are consistent with a role for ephrin-B2 in transmission of positional information to the CNS, and thus suggest a role in synaptic plasticity of spinal cord locomotor circuits that are known to be sensitive to proprioceptive sensory input after spinal cord injury.

Recent studies have demonstrated that RNAi is a promising approach for treating autosomal dominant disorders. However, discrimination between wild-type and mutant transcripts is essential, to preserve wild-type expression and function. A single nucleotide polymorphism (SNP) is present in more than 70% of patients with Machado-Joseph disease (MJD). We investigated whether this SNP could be used to inactivate mutant ataxin-3 selectively. Lentiviral-mediated silencing of mutant human ataxin-3 was demonstrated in vitro and in a rat model of MJD in vivo. The allele-specific silencing of ataxin-3 significantly decreased the severity of the neuropathological abnormalities associated with MJD. These data demonstrate that RNAi has potential for use in MJD treatment and constitute the first proof-of-principle for allele-specific silencing in the central nervous system.

Whereas the mammalian retina possesses a repertoire of factors known to establish general retinal cell types, these factors alone cannot explain the vast diversity of neuronal subtypes. In other CNS regions, the differentiation of diverse neuronal pools is governed by coordinately acting LIM-homeodomain proteins including the Islet-class factor Islet-1 (Isl1). We report that deletion of Isl1 profoundly disrupts retinal function as assessed by electroretinograms and vision as assessed by optomotor behavior. These deficits are coupled with marked reductions in mature ON- and OFF-bipolar (greater than 76%), cholinergic amacrine (93%), and ganglion (71%) cells. Mosaic deletion of Isl1 permitted a chimeric analysis of "wild-type" cells in a predominantly Isl1-null environment, demonstrating a cell-autonomous role for Isl1 in rod bipolar and cholinergic amacrine development. Furthermore, the effects on bipolar cell development appear to be dissociable from the preceding retinal ganglion cell loss, because Pou4f2-null mice are devoid of similar defects in bipolar cell marker expression. Expression of the ON- and OFF-bipolar cell differentiation factors Bhlhb4 and Vsx1, respectively, requires the presence of Isl1, whereas the early bipolar cell marker Prox1 initially did not. Thus, Isl1 is required for engaging bipolar differentiation pathways but not for general bipolar cell specification. Spatiotemporal expression analysis of additional LIM-homeobox genes identifies a LIM-homeobox gene network during bipolar cell development that includes Lhx3 and Lhx4. We conclude that Isl1 has an indispensable role in retinal neuron differentiation within restricted cell populations and this function may reflect a broader role for other LIM-homeobox genes in retinal development, and perhaps in establishing neuronal subtypes.

The mammalian retina is comprised of six major neuronal cell types and is subdivided into more morphological and physiological subtypes. The transcriptional machinery underlying these subtype fate choices is largely unknown. The LIM-homeodomain protein, Isl1, plays an essential role in central nervous system (CNS) differentiation but its relationship to retinal neurogenesis remains unknown. We report here its dynamic spatiotemporal expression in the mouse retina. Among bipolar interneurons, Isl1 expression commences at postnatal day (P)5 and is later restricted to ON-bipolar cells. The intensity of Isl1 expression is found to segregate the pool of ON-bipolar cells into rod and ON-cone bipolar cells with higher expression in rod bipolar cells. As bipolar cell development proceeds from P5-10 the colocalization of Isl1 and the pan-bipolar cell marker Chx10 reveals the organization of ON-center bipolar cell nuclei to the upper portion of the inner nuclear layer. Further, whereas Isl1 is predominantly a ganglion cell marker prior to embryonic day (E)15.5, at E15.5 and later its expression in nonganglion cells expands. We demonstrate that these Isl1-positive, nonganglion cells acquire the expression of amacrine cell markers embryonically, likely representing nascent cholinergic amacrine cells. Taken together, Isl1 is expressed during the maturation of and is later maintained in retinal ganglion cells and subtypes of amacrine and bipolar cells where it may function in the maintenance of these cells into adulthood.

Correct cell cycle regulation and terminal mitosis are critical for nervous system development. The retinoblastoma (Rb) protein is a key regulator of these processes, as Rb-/- embryos die by E15.5, exhibiting gross hematopoietic and neurological defects. The extensive apoptosis in Rb-/- embryos has been attributed to aberrant S phase entry resulting in conflicting growth control signals in differentiating cells. To assess the role of Rb in cortical development in the absence of other embryonic defects, we examined mice with telencephalon-specific Rb deletions. Animals carrying a floxed Rb allele were interbred with mice in which cre was knocked into the Foxg1 locus. Unlike germline knockouts, mice specifically deleted for Rb in the developing telencephalon survived until birth. In these mutants, Rb-/- progenitor cells divided ectopically, but were able to survive and differentiate. Mutant brains exhibited enhanced cellularity due to increased proliferation of neuroblasts. These studies demonstrate that: (i) cell cycle deregulation during differentiation does not necessitate apoptosis; (ii) Rb-deficient mutants exhibit enhanced neuroblast proliferation; and (iii) terminal mitosis may not be required to initiate differentiation.

Development of a multicellular organism requires precise coordination of cell division and cell type determination. The selector homeoprotein Even skipped (Eve) plays a very specific role in determining cell identity in the Drosophila embryo, both during segmentation and in neuronal development. However, studies of gene expression in eve mutant embryos suggest that eve regulates the embryonic expression of the vast majority of genes. We present here genetic interaction and phenotypic analysis showing that eve functions in the trol pathway to regulate the onset of neuroblast division in the larval CNS. Surprisingly, Eve is not detected in the regulated neuroblasts, and culture experiments reveal that Eve is required in the body, not the CNS. Furthermore, the effect of an eve mutation can be rescued both in vivo and in culture by the hormone ecdysone. These results suggest that eve is required to produce a trans-acting factor that stimulates cell division in the larval brain.

Focal demyelination models provide powerful tools to study demyelination and remyelination in the central nervous system. In this report, we present a novel technique, which selectively targets oligodendrocytes within the spinal cord of transgenic mice to produce focal demyelination. Transgenic mice expressing the E. coli LacZ (beta-galactosidase) gene from the myelin basic protein promotor allowed for oligodendrocyte-specific cleavage of topically applied fluorescein-di-beta-galactopyranoside liberating photoactivatable fluorescein. Subsequent fluorescence illumination generated oxygen radicals that oxidized a second exogenous substrate, 3-amino-9-ethyl carbazole, to form a toxic precipitate within oligodendrocytes. Histochemical staining of the spinal cord dorsal columns 8 days following phototargeting revealed that the treated region no longer contained beta-galactosidase-positive cells. Focal demyelination of the dorsal columns was observed to a depth of 150 microm in transverse semithin plastic sections. Numerous bundles of naked axons interspersed with myelin, debris-laden macrophages, and reactive astrocytes were evident by electron microscopy. Remyelination of axons by both oligodendrocytes and invading Schwann cells was observed within the treated region 14 days after phototargeting. Newly generated oligodendrocytes were identified within the demyelinated region by their incorporation of bromodeoxyuridine. Thus, this novel focal demyelination protocol provides: (1) a method for selective targeted ablation of oligodendrocytes in vivo, (2) control over the extent of the demyelinated region, with (3) an environment that maintains its remyelination capacity. Phototargeted ablation of oligodendrocytes may therefore be a useful model for studying axon-glia interactions, axon regeneration within a demyelinated zone, and remyelination of axons.

Analysis of LacZ gene expression is conventionally inferred from blue staining that results from exposure of the transfected cells or tissue to the substrate 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal). Such histochemical staining reports not whether the gene product is present or absent, but where it is active. We investigated the hypothesis that identification of activity, as opposed to presence, of the enzyme underestimates gene expression following LacZ gene transfer. Under conditions optimized for in vitro histochemistry, up to 20% of cells stably transfected with nls-LacZ remained unstained by X-Gal. In contrast, immunostaining with a monoclonal or a polyclonal anti-beta-galactosidase (beta-Gal) antibody positively stained 99% of the cell nuclei. Following in vivo transfection of naked DNA encoding for nls-LacZ, X-Gal staining disclosed 2.7 +/- 1.7 positive nuclei per LacZ-transfected animal, or a transfection efficiency of 0.015%. In contrast, immunohistochemical staining disclosed 118 +/- 32.7 positive nuclei per transfected animal, yielding a transfection efficiency of 0.64% (p < 0.0001 versus X-Gal staining). Thus, 42.9 times more positive cells were detected by antibody than X-Gal staining. Finally, LacZ gene expression following intramuscular gene transfer with an adenoviral vector was observed in 7.6% of skeletal muscle cells assessed with X-Gal; anti-beta-Gal antibody identified 21.8% of cells as being successfully transfected (p < 0.0001). Thus, X-Gal histochemistry following gene transfer of constructs encoding LacZ may underestimate the anatomic extent of gene expression. The superior sensitivity of immunostaining suggests that anti-beta-Gal antibody represents the preferred analytical tool for light microscopic evaluation of LacZ gene transfer.