Fig. 6. Expression of activated Notch1 results in overproduction of hindbrain choroid plexus epithelium but not of other rhombic lip lineages. (A) Schematic of a transverse section through the hindbrain (hb), with the boxed area demarcating the region of the rhombic lip (RL) shown in B-D. (B-D) Detection of Notch1, Gdf7 and Math1 mRNA on E11.5 hindbrain serial sections. Notch1 is expressed in the rhombic lip, but not in the hindbrain roof plate epithelium (hRPe); Gdf7 is expressed in the distal tip of the rhombic lip and into the hRPe; and Math1 is expressed in the rhombic lip ventral to the Gdf7 expression territory. (E-J,L) Comparison of singly transgenic R26::stop-Notch1-ICD::IRES-nGFP (E,G,I) and doubly transgenic Gdf7::cre; R26::stop-Notch1-ICD::IRES-nGFP (F,H,J,L) brains. (E,F) Posterior (pos) whole-mount (wm) view of P7 brains processed for Ttr mRNA detection [hindbrain choroid plexus epithelium (hCPe) marker] showing enlarged hCPe in doubly transgenic tissue (F,H,J,L). (G,H) Sagittal sections showing enlarged postnatal day 0 (P0) hCPe (red boxes) and abnormal cerebellar architecture in doubly but not singly transgenic animals. Inset in G shows Ttr+ hCPe on a serial section to that shown in G. (I,J) Hematoxylin and Eosin staining at P7 shows abnormal cytoarchitecture of hCP in doubly transgenic tissues. (K) P0 doubly transgenic Wnt1::Flpe; Flpe-responsive nßgal indicator distinguishing hCPe (green by immunodetection of ßgal) from underlying mesenchyme and vasculature. BrdU was injected into animals at P0, and tissue was harvested 2 hours later. BrdU immunodetection is shown in red. No co-labeled cells were found in wild-type hCPe, as shown. (L) Similar BrdU injection into doubly transgenic Gdf7::cre; R26::stop-Notch1-ICD::IRES-nGFP animals showed numerous co-labeled cells (arrowheads; GFP positivity identifying hCPe cells and expression of Notch1-ICD, and red indicating BrdU incorporation as a measure of proliferation). (M-V) Comparison of singly transgenic R26::stop-Notch1-ICD::IRES-nGFP and doubly transgenic Math1::cre; R26::stop-Notch1-ICD::IRES-nGFP brains. Coronal sections show no differences in size, relative cell density (Barhl1 expression at P0) or proliferation (Ki-67 immunodetection at P5 shown in red; U,V) of Math1-derived rhombic lip lineages, including mossy fiber neurons of the pontine gray nucleus (PGN, M-P), cerebellar granule cell precursors (CGC, white arrowheads, Q-V) and granule cells of the cochlear nucleus (CNGC, red arrowheads, Q,R). GFP positivity (immunodetection shown in green) reflects expression of the Notch1-ICD transgene in PGN (O,P) and CGC (S,T). White boxes in M-N demarcate regions of the PGN shown in O,P. DAPI is shown in blue. 4v, fourth ventricle; bs, brainstem; cb, cerebellum; CP, choroid plexus; hb, hindbrain; mz, post-mitotic mantle zone; RPe, roof plate epithelium; vz, ventricular zone.