DNaseI Hypersensitivity and Ultraconservation Reveal Novel, Interdependent Long-Range Enhancers at the Complex Pax6 Cis-Regulatory Region
Figure 8
Examination of GFP fluorescence patterns in YAC transgenic mice at E15.5, in comparison with 64YFP, a YFP expressing targeted gene-trap allele for Pax6.
(A) Schematic diagram of the Pax6 genomic locus. Pax6 exons are shown as black rectangles, exons of the adjacent Elp4 gene, transcribed from the opposite strand, are shown below the line. Location of the 64YFP knock-in cassette is shown, as well as the E60 elements (orange ellipses), the HS sites of the DRR and the HS5+ and HS6 elements (orange ellipses). The composition of the YAC reporter lines is shown below, including the multicopy full length Y223, single-copy full length DTy54, the single-copy line Y001 lacking the distal-most 20kb including the RB enhancer, and the DRR (downstream regulatory region) deletion line ΔDRR, which is directly derived from Y001 by Cre-mediated deletion of the 25 kb DRR region. The positions of the LoxP sites are indicated by red triangles. The DRR contains a number of hypersensitive sites (HS sites) including the HS5 and HS6 enhancers [33]. (B1–5) In the 64YFP knock-in allele and the YAC lines Y223, DTy54 and Y001 fluorescence signal is seen in the pre-cerebellar neuroepithelium (pcn). The signal in the pcn is absent when the DRR is deleted (B5). (C1–5 ) Lateral views of dissected brains at E15.5 with anterior side to the right. GFP signal is present in the cortex (c), cerebellum (cb), optic chiasm (oc), anterior extramural streams (aes) and pontine gray (pn) nuclei, and weakly visible in the posterior extramural stream (pes) and lateral reticulate nuclei (lrn). While the cortex remains positive in the ΔDRR line, no signal is seen in the eas or pn (C5). (D1–5) Ventral views. Expression is conspicuous in the cortex (c), and olfactory bulbs (ob) of all transgenic lines. It is also clearly seen in the aes, pn and optic nerve/chiasm (oc) of the 64YFP, Y223, DTy54 and Y001 lines, but absent from the aes and pn, as well as from the optic nerve/chiasm (oc) in the ΔDRR line (E1–3) Dorsolateral views of the hindbrain regions with long exposure, showing signal in the precerebellar neuro-epithelium (pcn) and migratory streams (pes, aes) of the 64YFP, Y223, and DTy54 lines. Signal in the cerebella (cb) of the YAC transgenic lines is weak compared to the YFP signal of the 64YFP knock-in line. (F1–2) Fluorescence signal at the lateral sides of the diencephalon is seen in a transverse section through the region of an Y001 brain (F1), but is missing in an equivalent section through a ΔDRR brain. (G1–5) Skulls of E15.5 embryos after the brains have been removed. Fluorescence in a 64YFP embryo highlights Pax6 expression in the eyes and olfactory epithelium. None of the YAC transgenic lines show fluorescent signal in the olfactory epithelium (oe). Expression is present in the eyes (e) of the Y223, DTy54 and Y001 lines, but missing in the ΔDRR line, apart from expression in the cornea (white arrow in G5).