Figure 4

Caudalized gene expression and thalamic innervation in the brain of Sp8 mutants. (a, a', b, b') WMISH on dissected E12.5 forebrains, using Emx2 and Pax6 riboprobes. The Emx2 gradient is up-regulated in cKO (arrows in (a, a')). The expression level of Pax6 is diminished (arrowheads in (b, b')) in Sp8 mutants. Analysis of the area specific marker genes (c, c') EphrinA5, (d, d') EphA7, (e, e') Coup-TF1 and (f, f') ID-2 on E18.5 sagittal sections using ISH (rostral is to the left). The visual cortex area in cKO cortices appears expanded towards the rostral brain, as demonstrated by EphA7, ID-2 and Coup-TF1 expression (strong rostral domain of EphA7 in (d'), strong ID-2 domain in (f'), strong domain of Coup-TF1 in (e')). The somatosensory cortex (area between the red arrows in (c, d') and between the black and red arrows in (f)) shifts rostrally in Sp8 conditional mutants (compare (c, d) with (c', d'), and (f) with the area indicated by the asterisk in (f')). The motor cortex expression area appears condensed (compare rostral to left arrow in (c, c', d, d')) in Sp8 deficient specimens. (g, g') Coronal sections of E18.5 brains labeled with DiI and DiO and counterstained with DAPI. In controls, DiI, placed in the visual cortex (inset in (g)), retrogradelly labels only cells in the dLGN (g). Placing DiO in the somatosensory cortex (inset in (g)) marks only cells in the VP (g). In the mutants the green dye, placed into somatosensory cortex (inset in (g')) labels cells in the VP and the dLGN (g'). (h) GST-pull down reveals that Emx2 lacking the homeobox (Emx2ΔHox) does not bind GST, GST-Sp8 (GST-Sp8FL) or GST-Sp8 lacking zinc fingers (GST-Sp8ΔZn) (lanes 2–4). Full-length Emx2 protein (Emx2FL) does not bind GST, but interacts with GST-Sp8 (GST-Sp8FL) and GST-SP8 lacking zinc fingers (GST-Sp8ΔZn) (lanes 6–8). Lanes 1 and 5 show 10% of the radiolabeled Emx2 isoforms, used as input for the binding assays in lanes 2–4 and 6–8.