Long-range downstream enhancers are essential for Pax6 expression

Pax6 is a developmental control gene with an essential role in development of the eye, brain and pancreas. Pax6, as many other developmental regulators, depends on a substantial number of cis-regulatory elements in addition to its promoters for correct spatiotemporal and quantitative expression. Here we report on our analysis of a set of mice transgenic for a modified yeast artificial chromosome carrying the human PAX6 locus. In this 420 kb YAC a tauGFP-IRES-Neomycin reporter cassette has been inserted into the PAX6 translational start site in exon 4. The YAC has been further engineered to insert LoxP sites flanking a 35 kb long, distant downstream regulatory region (DRR) containing previously described DNaseI hypersensitive sites, to allow direct comparison between the presence or absence of this region in the same genomic context. Five independent transgenic lines were obtained that vary in the extent of downstream PAX6 locus that has integrated. Analysis of transgenic embryos carrying full-length and truncated versions of the YAC indicates the location and putative function of several novel tissue-specific enhancers. Absence of these distal regulatory elements abolishes expression in specific tissues despite the presence of more proximal enhancers with overlapping specificity, strongly suggesting interaction between these control elements. Using plasmid-based reporter transgenic analysis we provide detailed characterization of one of these enhancers in isolation. Furthermore, we show that overexpression of a short PAX6 isoform derived from an internal promoter in a multicopy YAC transgenic line results in a microphthalmia phenotype. Finally, direct comparison of a single-copy line with the floxed DRR before and after Cre-mediated deletion demonstrates unequivocally the essential role of these long-range control elements for PAX6 expression.     

Conditional activation of Pax6 in the developing cortex of transgenic mice causes progenitor apoptosis

During development, Pax6 is expressed in a rostrolateral-high to caudomedial-low gradient in the majority of the cortical radial glial progenitors and endows them with neurogenic properties. Using a Cre/loxP-based approach, we studied the effect of conditional activation of two Pax6 isoforms, Pax6 and Pax6-5a, on the corticogenesis of transgenic mice. We found that activation of either Pax6 or Pax6-5a inhibits progenitor proliferation in the developing cortex. Upon activation of transgenic Pax6, specific progenitor pools with distinct endogenous Pax6 expression levels at different developmental stages show defects in cell cycle progression and in the acquisition of apoptotic or neuronal cell fate. The results provide new evidence for the complex role of Pax6 in mammalian corticogenesis.     

DNaseI hypersensitivity and ultraconservation reveal novel, interdependent long-range enhancers at the complex Pax6 cis-regulatory region

The PAX6 gene plays a crucial role in development of the eye, brain, olfactory system and endocrine pancreas. Consistent with its pleiotropic role the gene exhibits a complex developmental expression pattern which is subject to strict spatial, temporal and quantitative regulation. Control of expression depends on a large array of cis-elements residing in an extended genomic domain around the coding region of the gene. The minimal essential region required for proper regulation of this complex locus has been defined through analysis of human aniridia-associated breakpoints and YAC transgenic rescue studies of the mouse smalleye mutant. We have carried out a systematic DNase I hypersensitive site (HS) analysis across 200 kb of this critical region of mouse chromosome 2E3 to identify putative regulatory elements. Mapping the identified HSs onto a percent identity plot (PIP) shows many HSs correspond to recognisable genomic features such as evolutionarily conserved sequences, CpG islands and retrotransposon derived repeats. We then focussed on a region previously shown to contain essential long range cis-regulatory information, the Pax6 downstream regulatory region (DRR), allowing comparison of mouse HS data with previous human HS data for this region. Reporter transgenic mice for two of the HS sites, HS5 and HS6, show that they function as tissue specific regulatory elements. In addition we have characterised enhancer activity of an ultra-conserved cis-regulatory region located near Pax6, termed E60. All three cis-elements exhibit multiple spatio-temporal activities in the embryo that overlap between themselves and other elements in the locus. Using a deletion set of YAC reporter transgenic mice we demonstrate functional interdependence of the elements. Finally, we use the HS6 enhancer as a marker for the migration of precerebellar neuro-epithelium cells to the hindbrain precerebellar nuclei along the posterior and anterior extramural streams allowing visualisation of migratory defects in both pathways in Pax6(Sey/Sey) mice.     

Genetic control of dorsal-ventral identity in the telencephalon: opposing roles for Pax6 and Gsh2

We have examined the genetic mechanisms that regulate dorsal-ventral identity in the embryonic mouse telencephalon and, in particular, the specification of progenitors in the cerebral cortex and striatum. The respective roles of Pax6 and Gsh2 in cortical and striatal development were studied in single and double loss-of-function mouse mutants. Gsh2 gene function was found to be essential to maintain the molecular identity of early striatal progenitors and in its absence the ventral telencephalic regulatory genes Mash1 and Dlx are lost from most of the striatal germinal zone. In their place, the dorsal regulators, Pax6, neurogenin 1 and neurogenin 2 are found ectopically. Conversely, Pax6 is required to maintain the correct molecular identity of cortical progenitors. In its absence, neurogenins are lost from the cortical germinal zone and Gsh2, Mash1 and Dlx genes are found ectopically. These reciprocal alterations in cortical and striatal progenitor specification lead to the abnormal development of the cortex and striatum observed in Pax6 (small eye) and Gsh2 mutants, respectively. In support of this, double homozygous mutants for Pax6 and Gsh2 exhibit significant improvements in both cortical and striatal development compared with their respective single mutants. Taken together, these results demonstrate that Pax6 and Gsh2 govern cortical and striatal development by regulating genetically opposing programs that control the expression of each other as well as the regionally expressed developmental regulators Mash1, the neurogenins and Dlx genes in telencephalic progenitors.