Ventralized dorsal telencephalic progenitors in Pax6 mutant mice generate GABA interneurons of a lateral ganglionic eminence fate

Abstract

The transcription factor Pax6 is expressed by progenitors in the ventricular zone (VZ) of dorsal telencephalon (dTel), which generate all cortical glutamatergic neurons, but not by progenitors in the medial ganglionic eminence (MGE), which generate cortical GABAergic interneurons (GABA INs), or the lateral ganglionic eminence (LGE), which generate GABA INs that normally migrate to the olfactory bulb. We show that perinatally, Pax6(sey/sey) mice, which lack functional Pax6 protein, have large subpial ectopias in dTel and ventral telencephalon connected by cell streams arising from an aberrant paraventricular ectopia found throughout dTel. The subpial and paraventricular ectopias and connecting cell streams are comprised of postmitotic neurons expressing markers for GABA INs characteristic of a LGE fate. Marker analyses show that dTel VZ progenitors in Pax6 mutants are progressively ventralized, acquiring expression of regulatory genes normally limited to GE progenitors; by midneurogenesis, the entire dTel VZ exhibits ventralization. This ventralization of the dTel VZ is paralleled by the expression of markers for GABA INs superficial to it, suggesting that it ectopically produces GABA INs, leading to their ectopias and a thinner cortical plate due to diminished production of glutamatergic neurons. Genetic lineage tracing demonstrates that the GABA INs comprising the ectopias are from a cortical Emx1 lineage generated in the dTel VZ, definitively showing that dTel progenitors and progeny acquire a ventral, GE, fate in Pax6 mutants. Thus, Pax6 delimits the appropriate proliferative zone for GABA INs and regulates their numbers and distributions by repressing the ventral fates of dTel progenitors and progeny.

Fig. 1.

Markers specific for GABA INs reveal ectopias on the dorsal and ventral telencephalic surface in E18.5 Pax6^sey/sey mice. Whole-mount E18.5 WT and Pax6^sey/sey brains were processed for ISH by using digoxigenin-labeled riboprobes for GAD67 or Dlx-1/2. Dorsal (A) and ventral (A′) views of WT brains show GAD67 expression over the neocortex and OBs (black arrowheads). Dorsal (B and C) and ventral (B′ and C′) views of Pax6 mutants show ectopias containing GABA INs marked with GAD67 (B and B′) or Dlx-1/2 (C and C′). Dorsomedial ectopias (red arrowheads), ventrolateral ectopias (white arrowheads), and dorsal neocortical ectopias (red dashed circles) are marked. Black arrowheads mark INs attempting to migrate to the absent OBs.

Fig. 2.

Surface ectopias in E18.5 Pax6 mutants are subpial and connected to a paraventricular ectopia of GABA INs. Sections from E18.5 Pax6 mutants were processed for ISH by using digoxigenin-labeled riboprobes for ErbB4. (A) Coronal sections show the dorsomedial (red arrowheads) and ventrolateral (black arrowheads) ectopias. Streams of INs (red arrows) connect the paraventricular (black arrows) and dorsomedial ectopias, shown at higher magnification (Inset). (B) Sagittal sections (rostral faces left) show the dorsomedial ectopias. INs positioned between the paraventricular and dorsomedial ectopias are shown at higher magnification (Inset). (C) Higher-power view of cortical wall in an E18.5 Pax6 mutant shows ErbB4-expressing cells (arrowhead) in the CP. The paraventricular ectopia is marked with an asterisk. ErbB4-expressing cells in the IZ have a morphology resembling migrating INs (arrow). Str, striatum; Ctx, cortex; Cg, cingulate cortex; lv, lateral ventricle.

Fig. 3.

Neurons that comprise the ectopias in Pax6 mutants selectively express a marker for GABA INs of a LGE fate but not markers for a MGE fate or for cortical glutamatergic neurons. Coronal sections from E18.5 WT and Pax6 mutants were processed for ISH by using S35-labeled riboprobes, using the indicated markers and were DAPI-stained. Ectopias containing ErbB4-expressing cells (A′ and A″) are seen in Pax6 mutants but not in WT (A). Emx1 labels CP glutamatergic neurons in WT (B) and Pax6 mutants (B′ and B″) but does not mark these ectopias (B′ and B″). Sp8 marks the ectopias (C and C′), whereas Lhx6 is dramatically excluded from the ectopias (D) but is present in the CP and MZ. Boxed regions in the Pax6 mutants are shown at higher magnification (A″, B″, and C′). Streams of GABA INs (yellow arrows) connect the paraventricular ectopia to the dorsomedial ectopias (yellow arrowheads). Streams (white arrows) from the deep paraventricular ectopia (asterisk) also are continuous with the ventrolateral ectopias (white arrowheads). Ncx, neocortex; Pcx, piriform cortex; Hyp, hypothalamus.

Fig. 4.

Ectopias of GABA INs develop in mid- to late cortical neurogenesis in Pax6 mutants. Coronal sections from E15.5, E16.5 Pax6^sey/sey, and E16.5 WT brains were processed for ISH by using S35-labeled Dlx-1/2 riboprobes and were DAPI-stained. Only the paraventricular ectopia of Dlx-1/2-expressing cells is evident at E15.5 (A). By E16.5, Pax6 mutants (B) show large streams (yellow arrows) of Dlx-1/2-positive cells between the paraventricular ectopia and their entry point into the MZ (yellow arrowheads), which are not seen in WT (B′). Tangentially migrating INs in the MZ of the Hp (white arrowheads) are seen in both E16.5 Pax6 mutant (B) and WT (C). Serial coronal sections through an E18.5 Pax6 mutant (C, C′, and C″) show streams of INs that appear to migrate from the neocortex to the pial surface. The green arrowhead marks the location of INs in the Hp MZ (C″).

Fig. 5.

Progenitors in the dTel VZ of Pax6 mutants undergo rapid ventralization between E13.5 and E14.5. Coronal sections of E13.5 and E14.5 WT and Pax6 mutant brains were processed for ISH by using S35-labeled riboprobes for the indicated markers and were DAPI-stained. A, B, D, and E and A′, B′, D′, and E′ are from E13.5 WT and Pax6 mutants, respectively; C and C′ are from E12.5 WT and Pax6 mutants; and F-J and F′-J′ are from E14.5 WT and Pax6 mutants, respectively. White arrowheads mark the expression boundary between WT vTel and dTel, whereas red arrowheads mark the shifted marker expression boundary in Pax6 mutants. Yellow arrow in I′ marks aberrant superficial expression of Ngn2. The two normal migratory streams of ErbB4-expressing cells, through the MZ (red arrow) and IZ (white arrow), are seen at E14.5 in both WT and Pax6 mutants (H′).

Fig. 6.

GABA INs that form ectopias in Pax6 mutants are of an Emx1 lineage and generated by dTel progenitors. Coronal sections from an E18.5 Pax6^sey/sey;Emx1-Cre;R26R brain processed by X-Gal histochemistry to reveal β-gal-labeled cells of an Emx1 lineage and DAPI-stained. DAPI reveals the paraventricular heterotopia (pvh), dorsal ectopia (arrowheads, A-C), and cell stream migrating toward the ventrolateral ectopia (D). β-gal reporter densely labels cells in the CP and in each of the ectopias (A′-D′). The LGE, which never expresses Emx1, does not contain an appreciable number of β-gal cells (A′ and D′). (A-C and A′-C′) Arrows mark cell streams connecting the pvh to the dorsal subpial ectopia. (D and D′) Arrowheads mark the cell streams extending from the pvh to the ventrolateral ectopias. Red arrowheads (B and B′) denote the MZ of the Hp.