Lmo4 synergizes with Fezf2 to promote direct in vivo reprogramming of upper layer cortical neurons and cortical glia towards deep-layer neuron identities
Fig 1
Synergistic effect of Fezf2 and Lmo4 in upper- to deep-layer neuron reprogramming.
(A) Schematic representation of the experimental procedure and vectors. cGFP, cLmo4 (cL), cFezf2 (cF), or cFezf2 and cLmo4 (cF+cL) plasmids were electroporated into E14.5 somatosensory (S1) embryonic cortices. Brains were collected at P7. (B) IF of GFP, UL marker Cux1, and DL V marker Ctip2 on a coronal slice of a cFezf2 and cLmo4-electroporated brain. The white box indicates the magnification image on the right side. (C) Percentage of S1 electroporated UL neurons expressing upper vs. DL markers. (D) Representative images of Cux1, Ctip2, Fog2, Pcp4, and Darpp32 IF staining in electroporated brains. Full and empty arrowheads indicate whether GFP+ cells co-express or not, respectively, the marker. To the right, confocal images of high-magnification panels showing 3D reconstructions of double staining. Sidebars represent projections along the x–z axes (right) and the y–z axes (below). (E) Tract tracing of UL GFP+ axons upon electroporation of cGFP, cFezf2 or cFezf2 and cLmo4 in P7 brains. In control cases, GFP+ axons cross the CC to reach the contralateral hemisphere. In cFezf2-electroporated brains, fewer GFP+ axons cross the CC, and many are found in the striatum (Str) and subcerebral targets, such as IC, thalamus (Th), CP, and SC. In cFezf2 and cLmo4-electroporated brains, no projections are observed along the CC, and almost all GFP+ axons project through the Str to subcerebral targets. White boxes indicate the regions magnified in the panels below. Full and empty arrows indicate the presence or absence of axons, respectively. Scale bars: C = 1,000 μm (left, macro image) and 200 μm (right, magnified image); D = 20 μm; E = 1,000 μm (top row, macro images), 200 μm (magnified images). Results are expressed as mean ± SEM. Two-way ANOVA with Tukey’s post hoc correction was used for statistical analysis, *p < 0.05, **p < 0.01, ***p < 0.001. n = 3 brains for each plasmid. Extended data and statistics are listed in S1 Data. CC, corpus callosum; CP, cerebral peduncle; DL, deep layers; GFP, green fluorescent protein; IC, internal capsule; IF, immunofluorescence; UL, upper layers.