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To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2008.12.008 Byline: Jane C. Quinn (a)(b), Michael Molinek (a), John O. Mason (a), David J. Price (a) Keywords: Gli3; Telencephalon; Mouse; Chimera; Forebrain Abstract: The Gli3 zinc finger transcription factor is expressed in developing forebrain, with the highest levels of expression in dorsal telencephalon. In Gli3.sup.-/- embryos the dorsal telencephalon is abnormally small and fails to develop dorsomedial telencephalic structures, including hippocampus and cortical hem, while the ventral telencephalon appears to expand. A hurdle to understanding the underlying mechanisms is that abnormalities of developing Gli3.sup.-/- telencephalic cells in Gli3.sup.-/- mutants result from a combination of their own cell autonomous defects and defects in the Gli3.sup.-/- cells that surround them. Here we used chimeras to identify some of the defects of Gli3.sup.-/- telencephalic cells that are likely to be autonomous by studying how Gli3.sup.-/- cells develop when surrounded by a majority of wild-type cells. We found that Gli3.sup.-/- cells are present in all components of the Gli3.sup.-/- a Gli3.sup.+/+ chimeric forebrain, including dorsomedial structures, in proportions that either equal or exceed proportions found elsewhere in the embryo. Gli3.sup.-/- cells segregate from Gli3.sup.+/+ cells to form many abnormal structures particularly in dorsal telencephalon. Gli3.sup.-/- cells in some locations are misspecified: in those parts of the dorsal telencephalon near to its boundaries with the diencephalon and the ventral telencephalon, mutant cells express sets of transcription factors expressed by wild-type cells on the other side of the boundary. Elsewhere in the dorsal telencephalon, in the diencephalon and in the ventral telencephalon, mutant cells express sets of transcription factors similar to those expressed by their immediately surrounding wild-type cells. We propose that an important cell autonomous action of Gli3 is to regulate the competence of dorsal telencephalic cells, preventing cells near to its boundaries expressing regulatory factors normally restricted to adjacent tissues. Author Affiliation: (a) Centre for Integrative Physiology, The University of Edinburgh, Hugh Robson Building, George Square, Edinburgh EH8 9XD, UK (b) School of Veterinary and Animal Sciences, Charles Sturt University, Wagga Wagga, NSW, Australia Article History: Received 17 September 2008; Revised 9 December 2008; Accepted 10 December 2008

The roles of Pax6 were investigated in the murine eye and the olfactory epithelium by analysing gene expression and distribution of [Pax6.sup.-/-] cells in [Pax6.sup.+/+] [left and right arrow] [Pax6.sup.-/-] 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.sup.-/-] 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.sup.-/-] 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. Keywords: Chimera; Chimaera; Pax6; Eye development; Olfactory epithelium; Cornea; Retina; Lens

Mutations in the Pax6 gene disrupt telencephalic development, resulting in a thin cortical plate, expansion of proliferative layers, and the absence of the olfactory bulb. The primary defect in the neuronal cell population of the developing cerebral cortex was analysed by using mouse chimeras containing a mixture of wild-type and Pax6-deficient cells. The chimeric analysis shows that Pax6 influences cellular activity throughout corticogenesis. At early stages, Pax6-deficient and wildtype cells segregate into exclusive patches, indicating an inability of different cell genotypes to interact. At later stages, cells are sorted further based on telencephalic domains. Pax6-deficient cells are specifically reduced in the mediocaudal domain of the dorsal telencephalon, indicating a role in regionalization. In addition, Pax6 regulates the process of radial migration of neuronal precursors. Loss of Pax6 particularly affects movement of neuronal precursors at the subventricular zone/intermediate zone boundary at a transitional migratory phase essential for entry into the intermediate zone. We suggest that the primary role of Pax6 is the continual regulation of cell surface properties responsible for both cellular identity and radial migration, defects of which cause regional cell sorting and abnormalities of migration in chimeras. Keywords: Pax6; Small eye; Cerebral cortex; Neuronal migration; Cell autonomous

Abstract Background The transcription factor Foxg1 is an important regulator of telencephalic cell cycles. Its inactivation causes premature lengthening of telencephalic progenitor cell cycles and increased neurogenic divisions, leading to severe hypoplasia of the telencephalon. These proliferation defects could be a secondary consequence of the loss of Foxg1 caused by the abnormal expression of several morphogens (Fibroblast growth factor 8, bone morphogenetic proteins) in the telencephalon of Foxg1 null mutants. Here we investigated whether Foxg1 has a cell autonomous role in the regulation of telencephalic progenitor proliferation. We analysed Foxg1+/+↔Foxg1-/- chimeras, in which mutant telencephalic cells have the potential to interact with, and to have any cell non-autonomous defects rescued by, normal wild-type cells. Results Our analysis showed that the Foxg1-/- cells are under-represented in the chimeric telencephalon and the proportion of them in S-phase is significantly smaller than that of their wild-type neighbours, indicating that their under-representation is caused by a cell autonomous reduction in their proliferation. We then analysed the expression of the cell-cycle regulator Pax6 and found that it is cell-autonomously downregulated in Foxg1-/- dorsal telencephalic cells. We went on to show that the introduction into Foxg1-/- embryos of a transgene designed to reverse Pax6 expression defects resulted in a partial rescue of the telencephalic progenitor proliferation defects. Conclusions We conclude that Foxg1 exerts control over telencephalic progenitor proliferation by cell autonomous mechanisms that include the regulation of Pax6, which itself is known to regulate proliferation cell autonomously in a regional manner.

Abstract Background Embryonic stem (ES) cells can differentiate into all cell types and have been used extensively to study factors affecting neuronal differentiation. ES cells containing mutations in known genes have the potential to provide useful in vitro models for the study of gene function during neuronal differentiation. Recently, mouse ES cell lines lacking the neurogenic transcription factor Pax6 were reported; neurons derived from these Pax6-/- ES cells died rapidly after neuronal differentiation in vitro. Results Here we report the derivation of new lines of Pax6-/- ES cells and the assessment of their ability to survive and differentiate both in vitro and in vivo. Neurons derived from our new Pax6-/- lines were viable and continued to elaborate processes in culture under conditions that resulted in the death of neurons derived from previously reported Pax6-/- ES cell lines. The new lines of Pax6-/-ES cells showed reduced neurogenic potential, mimicking the effects of loss of Pax6 in vivo. We used our new lines to generate Pax6-/- ↔ Pax6+/+ chimeras in which the mutant cells survived and displayed the same phenotypes as Pax6-/- cells in Pax6-/- ↔ Pax6+/+ chimeras made by embryo aggregation. Conclusions We suggest that loss of Pax6 from ES cells reduces their neurogenic capacity but does not necessarily result in the death of derived neurons. We offer these new lines as additional tools for those interested in the generation of chimeras and the analysis of in vitro ES cell models of Pax6 function during neuronal differentiation, embryonic and postnatal development.

Gene-directed tissue repair offers the clinician, human or veterinary, the chance to enhance cartilage regeneration and repair at a molecular level. Non-viral plasmid vectors have key biosafety advantages over viral vector systems for regenerative therapies due to their episomal integration however, conventional non-viral vectors can suffer from low transfection efficiency. Our objective was to identify and validate in vitro a novel non-viral gene expression vector that could be utilized for ex vivo and in vivo delivery to stromal-derived mesenchymal stem cells (MSCs). Minicircle plasmid DNA vector containing green fluorescent protein (GFP) was generated and transfected into adipose-derived MSCs from three species: canine, equine and rodent and transfection efficiency was determined. Both canine and rat cells showed transfection efficiencies of approximately 40% using minicircle vectors with equine cells exhibiting lower transfection efficiency. A Sox9-expressing minicircle vector was generated and transfected into canine MSCs. Successful transfection of the minicircle-Sox9 vector was confirmed in canine cells by Sox9 immunostaining. This study demonstrate the application and efficacy of a novel non-viral expression vector in canine and equine MSCs. Minicircle vectors have potential use in gene-directed regenerative therapies in non-rodent animal models for treatment of cartilage injury and repair. [ABSTRACT FROM AUTHOR]

Photosensitivity in animals is defined as a severe dermatitis that results from a heightened reactivity of skin cells and associated dermal tissues upon their exposure to sunlight, following ingestion or contact with UV reactive secondary plant products. Photosensitivity occurs in animal cells as a reaction that is mediated by a light absorbing molecule, specifically in this case a plant-produced metabolite that is heterocyclic or polyphenolic. In sensitive animals, this reaction is most severe in non-pigmented skin which has the least protection from UV or visible light exposure. Photosensitization in a biological system such as the epidermis is an oxidative or other chemical change in a molecule in response to light-induced excitation of endogenous or exogenously-delivered molecules within the tissue. Photo-oxidation can also occur in the plant itself, resulting in the generation of reactive oxygen species, free radical damage and eventual DNA degradation. Similar cellular changes occur in affected herbivores and are associated with an accumulation of photodynamic molecules in the affected dermal tissues or circulatory system of the herbivore. Recent advances in our ability to identify and detect secondary products at trace levels in the plant and surrounding environment, or in organisms that ingest plants, have provided additional evidence for the role of secondary metabolites in photosensitization of grazing herbivores. This review outlines the role of unique secondary products produced by higher plants in the animal photosensitization process, describes their chemistry and localization in the plant as well as impacts of the environment upon their production, discusses their direct and indirect effects on associated animal systems and presents several examples of well-characterized plant photosensitization in animal systems. [ABSTRACT FROM AUTHOR]

Background: Embryonic stem (ES) cells can differentiate into all cell types and have been used extensively to study factors affecting neuronal differentiation. ES cells containing mutations in known genes have the potential to provide useful in vitro models for the study of gene function during neuronal differentiation. Recently, mouse ES cell lines lacking the neurogenic transcription factor Pax6 were reported; neurons derived from these Pax6-/- ES cells died rapidly after neuronal differentiation in vitro. Results: Here we report the derivation of new lines of Pax6-/- ES cells and the assessment of their ability to survive and differentiate both in vitro and in vivo. Neurons derived from our new Pax6-/- lines were viable and continued to elaborate processes in culture under conditions that resulted in the death of neurons derived from previously reported Pax6-/- ES cell lines. The new lines of Pax6-/-ES cells showed reduced neurogenic potential, mimicking the effects of loss of Pax6 in vivo. We used our new lines to generate Pax6-/- ↔ Pax6+/+ chimeras in which the mutant cells survived and displayed the same phenotypes as Pax6-/- cells in Pax6-/- ↔ Pax6+/+ chimeras made by embryo aggregation. Conclusions: We suggest that loss of Pax6 from ES cells reduces their neurogenic capacity but does not necessarily result in the death of derived neurons. We offer these new lines as additional tools for those interested in the generation of chimeras and the analysis of in vitro ES cell models of Pax6 function during neuronal differentiation, embryonic and postnatal development. [ABSTRACT FROM AUTHOR]

As the telencephalon first emerges from anterior neuroectoderm, signalling molecules and transcription factors combine to specify the identity and fate of cells in each of its regions. Studies of both naturally occurring and transgenic mutant mice have identified many genes that contribute to this process. The development of telencephalon and its regions is specified by signalling molecules produced at sites both surrounding and within the telencephalon. Different parts of the telencephalon express different combinations of transcription factors that control processes including proliferation, cell fate determination and migration in order to create the unique phenotype of each region. [Copyright &y& Elsevier]

Small eye (Pax6 Sey ) is a semi-dominant mutation affecting development of the eyes, brain and nasal structures. The mutant phenotype arises from defects within the Pax6 gene and several mutant alleles have been identified. A previous study reported that Pax6 Sey /Pax6 Sey homozygotes, in a random-bred stock, had a median cartilaginous rod-like structure in the nasal region and 80% had supernumerary upper incisor teeth. In this study we show that supernumerary upper incisor teeth and a previously unreported nasal capsule-derived cartilaginous ’spur’ occur in compound heterozygous Pax6 Sey-Neu /Pax6 Sey and homozygous Pax6 Sey /Pax6 Sey fetuses from several strains of mice. The frequencies of the abnormal phenotypes were not related to allele type but showed variable penetrance, which was dependent on genetic background. The median nasal cartilaginous rod-like structure was present in all homozygous small eye fetuses. The Pax6 Sey /Pax6 Sey homozygote may provide insight into the complex gene interactions involved in eye, nasal and craniofacial morphogenesis. [ABSTRACT FROM AUTHOR]