The roles of Pax6 in the cornea,retina, and olfactory epithelium of the developing mouse embryo

The roles of Pax6 were investigated in the murine eye and the olfactory epithelium by analysing gene expression and distribution of Pax6(-/-) cells in Pax6(+/+) <--> Pax6(-/-) chimeras. It was found that between embryonic days E10.5 and E16.5 Pax6 is autonomously required for cells to contribute fully not only to the corneal epithelium, where Pax6 is expressed at high levels, but also to the to the corneal stroma and endothelium, where the protein is detected at very low levels. Pax6(-/-) cells contributed only poorly to the neural retina, forming small clumps of cells that were normally restricted to the ganglion cell layer at E16.5. Pax6(-/-) cells in the retinal pigment epithelium could express Trp2, a component of the pigmentation pathway, at E14.5 and a small number went on to differentiate and produce pigment at E16.5. The segregation and near-exclusion of mutant cells from the nasal epithelium mirrored the behaviour of mutant cells in other developmental contexts, particularly the lens, suggesting that common primary defects may be responsible for diverse Pax6-related phenotypes.     

Dynamic expression of Pax6 in the shark olfactory system: evidence for the presence of Pax6 cells along the olfactory nerve pathway

Pax6 is involved in the control of neuronal specification, migration, and differentiation in the olfactory epithelium and in the generation of different interneuron subtypes in the olfactory bulb. Whether these roles are conserved during evolution is not known. Cartilaginous fish are extremely useful models for assessing the ancestral condition of brain organization because of their phylogenetic position. To shed light on the evolution of development of the olfactory system in vertebrates and on the involvement of Pax6 in this process, we analyzed by in situ hybridization and immunohistochemistry the expression pattern of Pax6 in the developing olfactory system in a basal vertebrate, the lesser spotted dogfish Scyliorhinus canicula. This small shark is becoming an important fish model in studies of vertebrate development. We report Pax6 expression in cells of the olfactory epithelium and olfactory bulb, and present the first evidence in vertebrates of strings of Pax6-expressing cells extending along the developing olfactory nerve. The results indicate the olfactory epithelium as the origin of these cells. These data are compatible with a role for Pax6 in the development of the olfactory epithelium and fibers, and provide a basis for future investigations into the mechanisms that regulate development of the olfactory system throughout evolution.     

Pax2 expression patterns in the developing chick inner ear

The fate specification of the developing vertebrate inner ear could be determined by complex regulatory genetic pathways involving the Pax2/5/8 genes. Pax2 expression has been reported in the otic placode and vesicle of all vertebrates that have been studied. Loss-of-function experiments suggest that the Pax2 gene plays a key role in the development of the cochlear duct and acoustic ganglion. Despite all these data, the role of Pax2 gene in the specification of the otic epithelium is still only poorly defined. In the present work, we report a detailed study of the spatial and temporal Pax2 expression patterns during the development of the chick inner ear. In the period analysed, Pax2 is expressed only in some presumptive sensory patches, but not all, even though all sensory patches show the scattered Pax2 expression pattern later on. We also show that Pax2 is also expressed in several non-sensory structures.     

Multiple elements in human beta-globin locus control region 5' HS 2 are involved in enhancer activity and position-independent, transgene expression.

The human beta-globin Locus Control Region (LCR) has two important activities. First, the LCR opens a 200 kb chromosomal domain containing the human epsilon-, gamma- and beta-globin genes and, secondly, these sequences function as a powerful enhancer of epsilon-, gamma- and beta-globin gene expression. Erythroid-specific, DNase I hypersensitive sites (HS) mark sequences that are critical for LCR activity. Previous experiments demonstrated that a 1.9 kb fragment containing the 5' HS 2 site confers position-independent expression in transgenic mice and enhances human beta-globin gene expression 100-fold. Further analysis of this region demonstrates that multiple sequences are required for maximal enhancer activity; deletion of SP1, NF-E2, GATA-1 or USF binding sites significantly decrease beta-globin gene expression. In contrast, no single site is required for position-independent transgene expression; all mice with site-specific mutations in 5' HS 2 express human beta-globin mRNA regardless of the site of transgene integration. Apparently, multiple combinations of protein binding sites in 5' HS 2 are sufficient to prevent chromosomal position effects that inhibit transgene expression.     

Essential and nonessential elements in the 3' nontranslated region of Bovine viral diarrhea virus

The 3' nontranslated region (NTR) of the pestivirus Bovine viral diarrhea virus (BVDV), a close relative of human Hepatitis C virus, consists of three stem-loops which are separated by two single-stranded regions. As in other positive-stranded RNA viruses, the 3' NTR of pestiviruses is involved in crucial processes of the viral life cycle. While several studies characterized cis-acting elements within the 3' NTR of a BVDV replicon, there are no studies addressing the significance of these elements in the context of a replicating virus. To examine the functional importance of 3' NTR elements, a set of 4-base deletions and deletions of each of the three stem-loops were introduced into an infectious BVDV cDNA clone. Emerging mutant viruses were characterized with regard to plaque phenotype, growth kinetics, and synthesis of viral RNA. The results indicated that presence of stem-loop (SL) I and the 3'-terminal part of the single-stranded region between stem-loops I and II are indispensable for pestiviral replication. In contrast, deletions within SL II and SL III as well as absence of either SL II or SL III still allowed efficient viral replication; however, a mutant RNA lacking both SL II and SL III was not infectious. The results of this study provide a detailed map of the essential and nonessential elements within the 3' NTR of BVDV and contribute to our understanding of sequence and structural elements important for efficient viral replication of pestiviruses in natural host cells.