Zfp312 is required for subcortical axonal projections and dendritic morphology of deep-layer pyramidal neurons of the cerebral cortex

Abstract

Pyramidal neurons of the cerebral cortex display marked layer- and subtype-specific differences in their axonal projections and dendritic morphologies. Here we show that transcription factor Zfp312 is selectively expressed by layer V and VI subcortical projection pyramidal neurons and their progenitor cells. Knocking down Zfp312 with small interfering RNAs dramatically reduced the number of subcortical axonal projections from deep-layer pyramidal neurons and altered their dendritic morphology. In contrast, misexpression of Zfp312 in cortically projecting pyramidal neurons of layers II and III induced the expression of Tbr1, a transcription factor enriched in deep-layer neurons, and the formation of ectopic subcortical axonal projections. Thus, our results indicate that transcription factor Zfp312 plays a critical role in layer- and neuronal subtype-specific patterning of cortical axonal projections and dendritic morphologies.

Fig. 1.

Zfp312 expression in early VZ progenitors and deep-layer pyramidal neurons. (A) Quantitative real-time RT-PCR analysis of Zfp312 expression. (B) Sagittal section of adult brain hybridized with Zfp312 riboprobe. (C and D) Nissl staining and Zfp312 ISH and immunohistochemistry in neocortex and CA1 hippocampus. (Insets) Nuclear localization of Zfp312 in neocortical layer V (C′) and hippocampal (D′) pyramidal neurons. (E) Zfp312 ISH in whole-mount embryos and sagittal sections of embryonic neocortical wall and P7 neocortex. Zfp312 is expressed by early ventricular zone (VZ) progenitors (E10.5 and E12.5). From E14.5, Zfp312 is down-regulated by VZ progenitors, and Zfp312-expressing neurons are localized solely in the subplate (SP), deep-layer cortical plate (DL CP), and layers V and VI and absent in the upper-layer CP (UL CP) and layers II to IV. (Scale bar, 275 μm in C, 140 μm in D, and 200 μm in E.)

Fig. 2.

Zfp312 is exclusively expressed by subcortically projecting neurons. (Left) Schematic depictions of stereotaxical injection sites of retrograde tracer BDA-3k and expected projection routes (red). (Right) Representative images of neurons in the adult primary motor neocortex retrogradely labeled with BDA-3k (red) and colabeled by Zfp312 ISH (green). Zfp312⁺ neurons project to the striatum, thalamus, and brainstem (arrowheads). (Scale bar, 50 μm.)

Fig. 3.

Zfp312 is required for subcortical projections and axonal fasciculation. (A and B) Zfp312 siRNAs dramatically decreased the expression and nuclear localization of Zfp312-GFP in N2a cells. N2a cells were transfected with Zfp312-GFP in the presence of 10-fold excess of pCRLH (A) or pLVTH (B) expressing either scr siRNAs or Zfp312 siRNAs. GFP from pLVTH was used as an internal control (B). pLVTH expressing either scr siRNA (C) or Zfp312 siRNAs (D) was delivered by in utero electroporation to cortical VZ progenitors at E12.5 and analyzed at P14. GFP⁺ apical dendrites (blue arrowheads) and axons (red arrowheads) were present in the control cortex. In control brains, numerous GFP⁺ axons were present in the white matter (WM), striatum (st) internal capsule (IC), and thalamus (Th) and formed axonal fascicles (red arrowheads). GFP⁺ subcortical axons were dramatically reduced in brains expressing Zfp312 siRNAs and do not form fasciculated bundles (open arrowheads). (Scale bar, 300 μm in C and D.)

Fig. 4.

Zfp312 regulates pyramidal morphology, dendritic patterning, and spine number. (A and B) Neurolucida 3D reconstructions of layer V neurons in the primary motor cortex electroporated at E12.5 with pLVTH expressing either control (scr) siRNAs or Zfp312 siRNAs and analyzed at P14. Zfp312 siRNAs significantly reduced basal dendritic tree complexity (D; P = 0.001), spine number (arrowheads in A′ and B′, quantification in E; P < 0.001), and soma size (F; P < 0.001) and affected the vertical orientation of apical dendrites (C; P = 0.027).

Fig. 5.

Zfp312 siRNAs do not alter the generation, migration, or development of upper-layer pyramidal neurons in the primary somatosensory cortex electroporated at E12.5 with pLVTH. (A) Quantification of laminar distribution of GFP⁺ neurons at P14 showing no differences between neurons expressing scr siRNA and Zfp312 siRNA (P > 0.05 for each bin). Error bars indicate SD. (B) Zfp312 siRNAs alter morphology of layer V (arrows) but not layer III pyramidal neurons (arrowhead) at P14. (C) Zfp312 siRNAs do not affect the expression of upper-layer marker Pou3f3 (red) at P0 (arrowheads). (Scale bar, 200 μm in B and 400 μm in C.)

Fig. 6.

Zfp312 misexpression in layer II/III callosal neurons induces Tbr1 and ectopic subcortical axonal projections. Neocortical VZ progenitors were electroporation in utero at E17 with pCLEG or pCLEG-Zfp312 and analyzed by GFP immunohistochemistry at P14. (A) Control GFP⁺ neurons in the uppermost layer (green arrowheads) send axons through the white matter and corpus callosum (blue arrowheads). Zfp312-misexpressing but not control GFP⁺ neurons have ectopic subcortical axons (A and B; red arrows) and are retrogradely labeled by BDA-3k injected into the pons (C and D; arrowheads). (E and F)A small number of neurons electroporated with control pCLEG expressed Tbr1 (E), whereas the vast majority of neurons misexpressing Zfp312 coexpressed Tbr1 (F) (arrowheads). (Scale bar, 500 μm in A and B,50 μm in C and D, and 90 μm in E and F.)