Neural tube closure defects in m-numb homozygous mutant embryos. (A) Immunoblot analysis of m-Numb protein expression in wild-type (+/+), heterozygous (Δ5,6/+), and homozygous mutant (Δ5,6/Δ5,6) mice using an antibody against the C-terminal m-Numb (19). Anti-Actin antibody was used as a control. (B–E) Scanning electron micrograph of wild-type (B, E10.5, lateral view) and m-numb homozygous mutant (C, E10.5, frontal view; D, E11.5, lateral view; E, E10.5, dorsal view) embryos. (Scale bar is 200 μm.) Arrow marks the midbrain–hindbrain junction; black arrowhead marks the hindbrain–spinal cord junction; white arrowhead marks the eye; and ∗ marks the first branchial arch.

Precocious production of neurons in the m-numb mutant forebrain at E10.5. (A–H) Heterozygous control (m-numb^Δ5,6/+). (I–S) Homozygous mutant (m-numb^Δ5,6/Δ5,6). Because of the defects in cranial neural tube closure, molecular markers (BF-1 and Pax6) are used to identify the mutant telencephalon. I–L, M and N, and O–Q are adjacent sections, respectively. C and D, E and F, G and H, K and L, O and P, and R and S are double-staining of the same section, respectively. Notice that sections from the wild-type forebrain (telencephalon and diencephalon) have very few cells that show immunostaining for Neurofilament (C, E, and G) or MAP2 (F and H), both of which are neuronal markers, whereas those from the mutant forebrain have Neurofilament (K, N, O, and R) and MAP2 (P and S)-positive cells throughout the forebrain. Both in the wild-type (D) and the mutant (L and Q) forebrain, most of the cells in the neuroepithelium are positive for Nestin, a neural progenitor cell marker. Insets in H (wild type) and S (mutant) show BrdUrd incorporation in the forebrain after a 2-h pulse. The arrow in B, E, and F marks the junction between mesencephalon and diencephalon, whereas the arrowhead in O, P, and Q marks the border of Pax-6 expression domain (O, Inset). TE, telencephalon; DE, diencephalon; ME, mesencephalon; HB, hindbrain; OS, optic stalk; VZ, ventricular zone.

Targeted disruption of the m-numb gene. (A) Schematic drawings of the targeting vector, the wild-type, the floxed, and the mutant m-numb alleles. Exons 5 and 6 encode amino acids 68–139 of the m-Numb protein. All EcoRI (E) and HindIII (H) sites are shown, whereas only the relevant BamHI (B), XhoI (O), and XbaI (X) sites are shown. Open triangles represent PCR primers as described in Materials and Methods. (B) PCR analysis of the wild-type (+), floxed (flox), and mutant (Δ5,6) m-numb alleles.

Neural tube patterning in wild-type and m-numb mutant embryos. (A and D) E11.5 m-numb^Δ5,6 heterozygous (+/−) and homozygous (−/−) embryos. Notice the extensive bleeding in the homozygous mutant embryo. (B and E) Fgf17 expression at the midbrain–hindbrain junction (arrow). (C and F) Otx2 expression in the midbrain and forebrain. Arrow marks the midbrain–hindbrain junction.