Fezl regulates the differentiation and axon targeting of layer 5 subcortical projection neurons in cerebral cortex

Abstract

During the development of the cerebral cortex, progenitor cells produce neurons that migrate to laminar positions appropriate for their birth dates, adopt specific neuronal identities, and form appropriate local and long-distance axonal connections. Here, we report that forebrain embryonic zinc-finger-like protein (Fezl), a putative zinc-finger transcriptional repressor, is required for the differentiation of projection neurons in cortical layer 5. In Fezl-deficient mice, these neurons display molecular, morphological, and axonal targeting defects. The corticospinal tract was absent in Fezl(-/-) mice, corticotectal and pontine projections were severely reduced, and Fezl-expressing neurons formed aberrant axonal projections. The expression of many molecular markers for deep-layer neurons was reduced or absent in the Fezl(-/-) cerebral cortex. Most strikingly, Ctip2, a transcription factor required for the formation of the corticospinal tract, was not expressed in the Fezl-deficient cortex. These results suggest that Fezl regulates the differentiation of layer 5 subcortical projection neurons.

Fig. 1.

In situ hybridization for genes normally expressed by deep layer neurons at P15. (A and B) ER81 is normally expressed in layer 5 and hippocampus, but expression in layer 5 is severely reduced in Fezl mutants. (C and D) Expression of Foxo1 in layer 5 is reduced in Fezl^-/- mice. (E and F) Foxp2, which is normally expressed in layer 6 and striatum, is lost from layer 6 in Fezl^-/- brains. (G and H) The expression of Grg4 in layers 5 and 6 is severely reduced in mutants. (Scale bar, 0.5 mm.)

Fig. 2.

PLAP staining reveals defective axonal projections in Fezl^-/- brains at P0. (A-F) Sagittal sections showing PLAP staining in Fezl^+/- brains (A-C) and Fezl^-/- brains (D and E). (A and D) PLAP stains axons in the internal capsule and cerebral peduncle of control and mutant brains. PLAP activity is strong in the anterior commissure in Fezl^-/- brains (D) but much weaker in heterozygotes (A). (B and E) A subset of axons in both controls and mutants leaves the internal capsule and enters the thalamus (th). (C and F) PLAP prominently marks the pyramidal tract in heterozygotes (arrowhead), but this labeling is absent in Fezl mutants. (G-P) Coronal sections showing PLAP staining in Fezl^+/- (G, I, K, M, O, and Q) and Fezl^-/- brains (H, J, L, N, P, and R). (G-J) The corpus callosum is apparent in heterozygotes (G and I) but absent in Fezl mutants (H and J). Some axons in Fezl^-/- mice take an aberrant pathway into the septum (arrowhead in H). Mutants show increased PLAP in the external capsule and posterior limb of the anterior commissure (J, arrowhead) compared with controls (I). (K and L) PLAP marks axons in the internal capsule (arrowhead), but these axons appear disorganized in mutants. (M and N) More PLAP positive axons in the cerebral peduncle turn dorsally into the thalamus (arrowhead) in Fezl^-/- brains than in controls. (O and P) PLAP activity in the cerebral peduncle (arrowhead) is dramatically reduced in Fezl^-/- brains. (Q and R) As axons approach the pyramidal decussation, PLAP marks the pyramidal tract (arrowhead) in controls, but little or no staining is visible in mutants. ic, internal capsule; cp, cerebral peduncle; pt, pyramidal tract; th, thalamus; cc, corpus callosum; ac, anterior commissure; ec, external capsule.

Fig. 3.

Defects in layer 5 projections to the pyramidal decussion in Fezl^-/- mutants. Sagittal sections showing retrograde labeling from the pyramidal decussation. The extent of retrograde labeling is indicated by arrowheads. Fluorescence images have been inverted in A and C. (A) Retrograde labeling normally marks layer 5 neurons throughout the motor and somatosensory areas. (B) High-power view of the region delineated by the box in A. (C) In Fezl^-/- mutants, only layer 5 neurons within somatosensory cortex (arrowheads) retain projections to the spinal cord. (D) High-power view of the boxed region in C, showing sparse labeling in the mutant.

Fig. 4.

Abnormal axon projections from the somatosensory and motor cortices of Fezl^-/- mice. Sagittal sections showing BDA-labeled axons traced from somatosensory (A-F) or motor cortex (G-J) of adult mice. (A and B) BDA injections into somatosensory cortex normally label axons that innervate neighboring cortical areas. Axons in Fezl^-/- mice fail to cross the corpus callosum and instead form Probst bundles (asterisk), and some labeled axon bundles enter rostral motor areas (arrowhead). (C and D) Control axons from somatosensory cortex project to the superior colliculus, which contains many fewer labeled axons in Fezl mutants. (E) Somatosensory axons innervate the reticular nucleus, the lateral part of the ventral posterior nucleus and the posterior nucleus of the thalamus. (F) Thalamic projections in Fezl^-/- mice show a grossly normal pattern, but more axons appear to innervate the medial ventral posterior nucleus, pretectum, and zona inserta. (G and H) BDA-labeled axons from motor cortex normally branch in the central pons, which contains many fewer axons in mutants. (I) Axons from motor cortex extend through the pyramidal tract, cross in the pyramidal decussation, then descend in the CST. (J) No axonal labeling was visible in the pyramidal decussation or CST of Fezl^-/- mutants. mot, motor cortex; wm, white matter; som, somatosensory cortex; hip, hippocampus; sc, superior colliculus; ic, inferior colliculus; rt, reticular thalamic nucleus; vp, ventral posterior nucleus; pt, pretectum; and zi, zona inserta. (Scale bar, 0.5 mm.)

Fig. 5.

Reduced expression of CSMN genes in Fezl mutant mice. Sagittal sections from P15 (A-L) or P0 (M-P) brains. (A and B) Ctip2 is normally expressed in layers 5 and 6 of cerebral cortex, hippocampus (hip) and striatum (str), but cortical expression is lost in Fezl^-/- mice. (C and D) Crim1 is expressed in layers 2, 3, and 5 in control brains, but layer 5 expression is lost in mutants. (E and F) Neurofilament heavy chain (NFH) in layer 5 is severely reduced in mutants. (G and H) Expression of Igfbp4 expands into layer 6 in Fezl mutants. (I and J) The expression of Lix1 in layer 5 is severely reduced in mutants. (K and L) Layer 5 cells express Synaptotagmin 9 in controls but not in Fezl mutants. (M and N) Expression of Riken 2010001O09 in layer 5 at P0 is lost in the Fezl mutants. (O and P) The CSMN general identity gene Contactin 6 is expressed normally in mutants. (Scale bar, 0.5 mm.)