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

Circulatory Effects of Inhaled Iloprost in the Newborn Preterm Lamb.

Inhaled nitric oxide (iNO) has an established role in the treatment of pulmonary hypertension in the newborn. However, costs and potential toxicity associated with iNO have generated interest in alternative inhaled selective pulmonary vasodilators such as iloprost. In a preterm lamb model of respiratory distress syndrome, we studied effects of increasing doses of iloprost followed by iNO on right ventricular pressure (RVP) and circulation including cerebral oxygenation. Fetal sheep were randomized to three doses (0.2 - 4 mg/kg) of iloprost (n=9) or saline (n=10), administered as 15 min inhalations with 15 min intervals after a 60-min postnatal stabilisation. No differences were found in RVP, arterial pO2, or cardiac index according to treatment. The cerebral oxygenation, measured with near-infrared spectroscopy, deteriorated in control lambs, but not in iloprost lambs. Iloprost treatment followed by iNO resulted in a larger decrease (p=0.007) in RVP than saline treatment followed by iNO. In conclusion, iloprost stabilised cerebral oxygenation and when followed by iNO had a larger effect on RVP than iNO alone. Although species differences may be relevant, these results suggest that iloprost should be studied in newborn infants for the treatment of pulmonary hypertension