Olfactory structures develop normally in Ebf2-null mice. Histochemical staining conducted on vibratome sections of E12.5 (A) and E13.5 (B)mice. lacZ expression can be observed in the olfactory epithelium (oe), in the vomeronasal organ (vno) and alongside olfactory/vomeronasal fibers (arrows). (C,D) Peripherin-positive fibers connecting the olfactory epithelium to the hypothalamus (arrows) are normally distributed in wild-type (C) and mutant (D) embryonic brains alike (arrowhead indicates fibers in the nasal mesenchyme). (E,F) Nissl staining of P30 paraffin coronal sections. The mutant olfactory bulb (F) is histologically indistinguishable from the wild-type one (E). cp, cribriform plate; epl, external plexiform layer; gl, glomerular layer; gr, granule cell layer; hy, hypothalamus; ipl, internal plexiform layer; lot, lateral olfactory tract; m, mitral cell layer; mot, medial olfactory tract; ns, nasal septum; oe, olfactory epithelium; vno, vomeronasal organ. Scale bars: 100 μm in A; 200 μm in B; 150 μm in C,D; 100 μm in E,F.

Migrating GnRH-neurons express Ebf2. Arrowheads in A-C indicate cell bodies of migrating neurons. (A) GnRH-like immunostaining in Ebf2^-/- neurons migrating dorsally through the nasal mesenchyme at embryonic day 15. (B) Anti β-galactosidase staining in the same neurons. (C) Superimposed signals from A and B revealed co-expression of Ebf2 and GnRH in migrating neurons. (D) Hoechst nuclear counterstaining of same field. As described (Wray, 2001), GnRH-neurons migrate through cell-poor, nerve fiber-rich regions. Scale bar: 20 μm.

Defective migration of GnRH-neurons from the olfactory epithelium to the hypothalamus. (A,C,E) Normal controls; (B,D,F,G) Ebf2^-/- coisogenic mutants; (A,B) E15; (C,D,G) P0; (E,F) P30. (A) In E15 wild-type embryos, migrating GnRH-neurons (arrows, brown staining, nuclear counterstaining in cyan) are mostly located dorsal to the cribriform plate (cp) and ventral to the olfactory bulb (ob). (B) Mutant neurons (arrows) migrate slowly out of the vomeronasal organ (vno). (Inset) Mutant neurons form dense clusters, often devoid of leading or trailing processes. (C) GnRH-positive fibers (arrows) reach the median eminence (ME) of the hypothalamus in wild-type newborn brains. (D) No GnRH immunostaining in the ME of mutant P0 brains. (E) GnRH-positive neurons (brown) in the preoptic region of the wild-type hypothalamus. (F) No GnRH immunostaining in the corresponding region of mutant brains. (G) At birth, mutant GnRH-positive neuronal cell bodies (black arrowheads) and fibers (white arrowheads) are ectopically located in the forebrain close to the midline at the interface between olfactory bulb (ob) and rostral telencephalic cortex (cx), dorsal to the nasal septum (ns) and cribriform plate. See empty box in inset for localization. Arrow in inset indicates physiological migration route. cp, cribriform plate; cx, telencephalic cortex; ME, median eminence; ns, nasal septum; ob, olfactory bulb; vno, vomeronasal organ. Scale bars: 100 μm in A-F; 50 μm in G.

Hypoplasia of seminipherous tubules and reduced spermatogenesis in Ebf2-null mice. (A,C,E) Wild type; (B,D,F) Ebf2^-/-. (A,B) Hematoxylin and Eosin (HE) staining of seminipherous tubules. In B, the luminal side of the tubule contains very few aggregations of spermatids compared with wild-type tubule. (C,D) Anti BrdU immunostaining reveals a sharply reduced number of proliferating spermatogonia in mutant tubules (arrows). (E,F) HE-stained epidydimes. The lumen of a mutant epidydimis (F) contains cell debris and hardly any sperm cells. The latter are recognizable as Hematoxylin-stained (dark purple) dots in the normal control (E). Scale bar: 40 μm.

Ebf2 is expressed in embryonic, postnatal glia and in postnatal motoneurons. (A) E12.5 DRG neurons positive for the heavy chain neurofilament (NF-H) are negative for lacZ staining (Ebf2); however, lacZ staining is observed in presumptive satellite cells adjacent to NF-H positive neurons. (B) In E12.5 dorsal roots, NF-H-like staining corresponding to DRG neuron axons does not colocalize with lacZ staining. (C) In E16.5, peripheral glial cell precursors are positive for p75 NGFR and lacZ. (D) In semi-thin transverse sections of P15 null mutant sciatic nerve, Ebf2 (lacZ staining) is expressed in both myelin forming (msc, arrow) and non-myelin forming (nmsc, arrowhead) Schwann cells. (E) Electron microscopy (EM) image of a heterozygous P15 sciatic nerve stained with bluo-gal, revealing Ebf2 expression (arrows) in nmsc. (F) EM image of a P15-null mutant nerve stained with bluo-gal, revealing persistent expression of Ebf2 in mutant msc (arrows). (G) Low-magnification image of a vibratome transverse section of an heterozygous P30 spinal cord, at the level of the lumbar swelling, revealing Ebf2 expression in dorsal (2,3), commissural (4,10) and ventral (9) laminae of the gray matter. H corresponds to the boxed area in G and reveals Ebf2 colocalization (arrows) with the motoneuron marker choline acetyltransferase (ChAT). 2-9, spinal cord layers 2-9; cs, corticospinal tract; gr, fasciculus gracilis. Scale bars: 10 μm in A,C; 5 μm in B; 10 μ m in D; 2 μm in E; 1 μm in F; 80 μm in H.

Large axons are hypomyelinated in the Ebf2^-/- sciatic nerve. (A) Toluidine Blue-stained transverse semi-thin section of a wild-type sciatic nerve at postnatal day 30. (B) Section of a P30 Ebf2-null sciatic nerve that corresponds to that in A. Note reduced myelin thickness of sample large caliber Ebf2^-/- axons indicated by arrowheads in B, in comparison with Ebf2^+/+ fibers of similar sizes in A. Scale bar: 10 μm.

Defective axon sorting, dysmyelination and axonal degeneration in the mutant sciatic nerve. (A) Normal control nerve at postnatal day 30 (P30); note orderly arrangement of uniformly sized axons sorted within the cytoplasm of a non-myelin forming Schwann cell (nmsc), and, in parallel, a myelin forming Schwann cell (msc) surrounding a single large diameter axon (ax) with a myelin cuff (m). (B-F) sciatic nerve sections from Ebf2^-/- mice. (B) At P30, two 4 μm axons (arrows) are abnormally fasciculated within the cytoplasm of a nmsc. Likewise, in C (P60), a single Schwann cell ensheathes an unmyelinated axon and a myelinated one; arrow indicates SC basal lamina joining myelinated and unmyelinated axons; note adjacent unmyelinated 3 μm axon (arrowhead). (D) A longitudinal section from a P45 nerve reveals segmental dysmyelination: an internode is myelinated (arrowheads) proximal to a node (n), while the subsequent internode remains unmyelinated (arrows). (E) A hypomyelinated axon at P60, a stage at which myelination is complete in controls; arrow indicates an abnormally thin myelin sheath. (F) A P30 axon shows cytoplasmic vacuoles (arrow), indicative of axonal degeneration. ax, axon; m, myelin; msc, myelin-forming Schwann cell; n, node of Ranvier; nmsc, nonmyelin-forming Schwann cell. Scale bars: 1 μm in A-E; 0.5 μm in F.