OTX2 Transcription Factor Controls Regional Patterning within the Medial Ganglionic Eminence and Regional Identity of the Septum

Abstract

The Otx2 homeodomain transcription factor is essential for gastrulation and early neural development. We generated Otx2 conditional knockout (cKO) mice to investigate its roles in telencephalon development after neurulation (approximately embryonic day 9.0). We conducted transcriptional profiling and in situ hybridization to identify genes de-regulated in Otx2 cKO ventral forebrain. In parallel, we used chromatin immunoprecipitation sequencing to identify enhancer elements, the OTX2 binding motif, and de-regulated genes that are likely direct targets of OTX2 transcriptional regulation. We found that Otx2 was essential in septum specification, regulation of Fgf signaling in the rostral telencephalon, and medial ganglionic eminence (MGE) patterning, neurogenesis, and oligodendrogenesis. Within the MGE, Otx2 was required for ventral, but not dorsal, identity, thus controlling the production of specific MGE derivatives.

Figure 1. Otx2 expression in conditional knock outs (cKOs)

(A-B”) ISH on E11.5 coronal sections from (A–A”) Otx2f/+ and (B–B”) Otx2f/−; RxCre embryos using a full length Otx2 riboprobe. Otx2 transcription appears upregulated in the MGE of RxCre cKOs (arrowheads and arrows in A’, A”, B’, B”), and that the MGE SVZ and MZ are hypoplastic (asterisks in A’, B’). (C–H) Anti-OTX2 IHC: in RxCre cKOs (C–F), OTX2 protein expression is absent in cKO forebrains except in the dorsomedial caudal cortex, hippocampal anlage, and choroid plexus (arrows, C’-C”, D’–D”). E and F show higher magnification views of the boxed regions in C’, D’. (G–H) In Nkx2.1Cre cKOs, OTX2 expression was reduced in the MGE. A–D”: rostrocaudal series of coronal sections. Abbreviations: Se, septum; MGE, medial ganglionic eminence; LGE, lateral ganglionic eminence; dCx, dorsomedial cortex; Hp, hippocampal anlage; POA, preoptic area; Di, diencephalon; CP, choroid plexus; OE, olfactory epithelium. Scale bars: A, E = 0.25mm, G = 0.4mm.

Figure 2. Anti-OTX2 ChIP-seq results

“Called” peak locations relative to genomic loci are shown for genes (alphabetically organized) that are deregulated in Otx2 cKOs, and for which ChIP-seq peaks were identified within ~1 megabase (MB) of the gene body. Note the different scale bars for individual panels. Black arrows and text identify the Otx2-regulated gene of interest, grey arrows and text designate nearby genes. For each panel, italicized “up” or “down” indicates whether the gene was upregulated or downregulated in RxCre cKO forebrains. The yellow stars indicate that the OTX2-ChIP-seq peak had an OTX2 binding motif. Abbreviations: chr: chromosome; kb: kilobase

Figure 3. Otx2 restricts the domain of Fgf expression and controls regional specification of the RPC

(A–D, F–Q’) ISH comparing gene expression in (A, C, F–Q) Otx2f/+ and (B, D, F’–Q’) Otx2f/-; RxCre embryos at E11.5. (A–B) Fgf8, (C–D) Fgf17, (F–G’) Sprouty1, (H–I’) Sprouty2, (J–J’, N-N’) Mkp3, (K–M’) En2, (O–Q’) Pax3. (E) Quantification of the rostral expansion of Fgf expression in cKO and control embryos at E11.5 (mean +/− st. dev.). For each embryo, we calculated the distance from the caudal septum (where Fgf8 and Fgf17 are expressed) to the rostral limit of Fgf expression, and expressed this as a percentage of the total (rostral telencephalic pole > caudal septum) distance. Rostral expansion was statistically significant for Fgf8 (p < 0.001) but not Fgf17 (p = 0.17; 2-tailed t tests, unequal variance). Abbreviation: mPFC, medial prefrontal cortex. Scale bars: A–D = 0.5mm, F–G’ = 0.2mm, H–Q’ = 0.25mm.

Figure 4. Otx2 cKOs exhibit deficits in molecular markers of oligodendrogenesis

ISH on coronal hemisections from (A–I) Otx2f/+, (A’–I’) Otx2f/−; RxCre, embryos: (A–C’) Olig1 at E11.5, (D–F’) Olig2 at E11.5, (G–I’) Olig1 at E13.5. Three planes of section are shown along the rostral-caudal axis. Abbreviations: vMGE, ventral MGE; dMGE, dorsal MGE; vSe, ventral septum. Scale bars: A–F’ = 0.5mm, G–I’ = 0.4mm.

Figure 5. Reduced neurogenesis and proliferation in the basal ganglia of E11.5 Otx2 cKOs

ISH and IHC on coronal hemisections (control: left, mutant: right). (A–H’) ISH: (A–H) Otx2f/+, (A’, C’, D’–F’) Otx2f/−; RxCre, and (B’, G’–H’) Otx2f/−; Nkx2.1Cre embryos using probes to (A–B’) Dlx1, (C–C’) Arx, (E–H’) Robo2. Anti-Tuj1 IHC (D) Otx2f/+ and (D’) Otx2f/−; RxCre. (I–J’) Anti- pH3 IHC: (I’, I) Otx2f/+ and (J, J’) Otx2f/−; RxCre embryos. I’–J’: high magnification of I, J. Red lines: neural/mesenchymal boundary; purple arrowheads: pH3+ SVZ cells; yellow rectangles highlight similar VZ regions of the vMGE in K–L’ showing upregulation of Hes1 and Id4, respectively. (K, L) Otx2f/+ and (K’, L’) Otx2f/-; RxCre embryos. (M–N’) Shh reduction in MZ and increase in VZ. Abbreviations: mes: mesenchyme; SVZ: subventricular zone; VZ ventricular zone. Scale bars: 0.5mm.

Figure 6. Re-specification of the vMGE towards the POA fate in Otx2 RxCre cKOs

ISH on E11.5 coronal hemisections from (A–S) Otx2f/+, (A’–O’) Otx2f/−; RxCre, and (P’S’) Otx2f/−; Nkx2.1Cre embryos showing expanded expression of POA genes (A–M’, U–W’) and diminished expression of vMGE genes (N–S’, T–T’) in cKOs. For each experiment (except Tal2 and Slit2 in Nkx2.1Cre cKOs), two or three sections are shown to demonstrate effects at different rostral-caudal planes. (A–C’) Hmx2 (Nkx5.2), (D–F’) Dbx1, (G–G’) Tgfb3, (H–I’, U–U’) Slit2, (J–K’) Arhgap22, (L–M’, V–W’) Sox14, (N–O’, T–T’) Tal2, (P–Q’) Tll2, (R–S’) Sall3. Scale bars: 0.5mm.

Figure 7. E13.5-E15.5 MGE and POA development in Otx2 cKOs. ISH on (A–O)

Otx2f/+ and (A’–O’) Otx2f/−; RxCre coronal hemisections, using the following probes: (AC’) Nkx5.1, (D–E’) COUP-TF1, (F–F’) ER81, (G–I’) Zic1, (J–L’) Nkx2.1, (M–M’) Ikaros, (NO’) Sizn1. Arrowheads in G indicate three streams of neurons or progenitors that appear to emanate from the POA or vMGE and migrate toward the MGE, LGE, and ventral cortex; these streams are not apparent in cKOs. Arrowheads in N–O’ point to the dMGE: Sizn1 expression appears to shift ventrally from its vLGE domain into the dMGE in cKOs. All are E13.5 except for Ikaros, which is E15.5. Note that in L’, the dark region in the CGE (*) is a tissue fold, not ISH signal. Abbreviations: GP, globus pallidus; Str, striatum. Scale bars: 0.5mm.