Figure 2.

Brn3a and Islet1/2 co-localize with Npr2/cGKI in midbrain and rhombomere 1 and the ligand CNP is localized in the hindbrain but absent from the midbrain and forebrain. (A₁) Percentage of cGKI-positive cells of Npr2-expressing cells in the mesencephalon and in rhombomere 1 at E12.5. (A₂,₃) Percentage of Npr2(β-Gal)/cGKI-positive or Npr2(β-Gal)/cGKI/neurofilament-positive cells of total midbrain or rhombomere 1 cells. Total cells were identified by DAPI. Anti-β-galactosidase staining was performed using the Npr2-LacZ reporter mouse line to monitor Npr2-expressing cells. (A₄,B) Co-localization of Brn3a with Npr2(β-Gal)/cGKI-positive cells in the midbrain or rhombomere 1 at E12.5. (A₅,C) Co-localization of Islet1 with Npr2(β-Gal)/cGKI-positive cells in the midbrain or rhombomere 1 at E12.5. The Npr2-LacZ reporter line was used to monitor expression of Npr2 in (B, C) by using an antibody to β-galactosidase. Scale bar for (B₁-C₆), 50 μm. (D) Localization of CNP at E8.5, lateral (D₁) and dorsal (D₂) view of whole mounts. Expression analysis was done with the CNP-LacZ reporter mouse line using X-Gal staining. Localization of CNP at E9.5, lateral (D₃) and dorsal (D₄) view. (D₅) Localization of CNP at E10.5 lateral view. Scale bar for (D₁–D₅) 30 μm. (E) Cross section of the hindbrain at the level of rhombomere 2 at E11.5 indicating that CNP is localized throughout all layers of the hindbrain. Anti-β-galactosidase staining of the CNP-lacZ reporter mouse line was used to monitor CNP. Scale bar, 250 μm. Hb, hindbrain; Mb, midbrain; Pros, prosencephalon; r2, rhombomere 2; r4, rhombomere 4; Sc, spinal cord. (F) Western blot of extracts of hindbrain, midbrain, and forebrain from CNP-LacZ reporter mice at E13.5 using anti-β-galactosidase to monitor expression of CNP. Equal loading was demonstrated by anti-GAPDH. Molecular mass markers are indicated at the right of the panel in kD.