GABAergic interneuron lineages selectively sort into specific cortical layers during early postnatal development

Abstract

It is of considerable interest to determine how diverse subtypes of γ-aminobutyric acidergic (GABAergic) interneurons integrate into the functional network of the cerebral cortex. Using inducible in vivo genetic fate mapping approaches, we found that interneuron precursors arising from the medial ganglionic eminence (MGE) and caudal ganglionic eminence (CGE) at E12.5, respectively, populate deep and superficial cortical layers in a complementary manner in the mature cortex. These age-matched populations initiate tangential migration into the cortex simultaneously, migrate above and below the cortical plate in a similar ratio, and complete their entrance into the cortical plate by P1. Surprisingly, while these 2 interneuron populations show a comparable layer distribution at P1, they subsequently segregate into distinct cortical layers. In addition, the initiation of the radial sorting within each lineage coincided well with the upregulation of the potassium/chloride cotransporter KCC2. Moreover, layer sorting of a later born (E16.5) CGE-derived population occurred with a similar time course to the earlier born E12.5 cohorts, further suggesting that this segregation step is controlled in a subtype specific manner. We conclude that radial sorting within the early postnatal cortex is a key mechanism by which the layer-specific integration of GABAergic interneurons into the emerging cortical network is achieved.

Figure 1.

The MGE and CGE provide interneurons into cortical layers through a distinct temporal methodology. A schematic drawing comparing the contribution of MGE- versus CGE-derived interneurons with different cortical layers based on birthdate. While MGE-derived interneurons exhibit inside-out layering, interneurons derived from the CGE preferentially occupy superficial layers irrespective of their birthdate. Importantly, MGE- and CGE-derived interneurons generated at E12.5 (red bracket) show a complementary bias in their contribution to deep versus superficial cortical layers, respectively.

Figure 2.

Age-matched E12.5 MGE- and CGE-derived interneurons are indistinguishable during tangential migration period. (A) The migration pathways of E12.5 MGE- (Olig2-CreER; Z/EG, Miyoshi et al. 2007) and CGE- (Mash1BAC-CreER; RCE:loxP, Miyoshi et al. 2010) derived populations were compared at E14.5. After 2 days, both interneuron populations transited from their site of origin in the ventral telencephalon to the cortex. The MGE domain is delineated by Nkx2-1 expression (left), and the dorsolateral limit of the CGE is indicated by Mash1 expression (right). Note that interneurons first appear in the intermediate zone in both cases (inset). (B) An E14.5 coronal section from the telencephalon carrying GAD1-EGFP, Nkx2-1BAC-Cre, and R26R-stop-LacZ (left). The entire GABAergic population is labeled by EGFP (brown), while the majority of MGE-derived cells are fate mapped as indicated by their beta-galactosidase enzymatic reactivity (blue) (right). A high magnification of cortex showing both MGE- (blue) and CGE- (brown without blue) derived cells migrating through the intermediate, marginal, and subventricular zones of the cortex. (C) Tangential migration of E12.5 MGE- (left) or CGE- (right) derived interneurons at E16.5. Fate-mapped cells express EGFP, and cortical histology is revealed by 4′,6-diamidino-2-phenylindole (DAPI) nuclear counter staining (pseudocolored in red). Fate-mapped cells from both the E12.5 MGE and CGE take similar tangential migration paths with migrating above (MZ, around 25%) and below (IZ and SVZ, around 60%) the cortical plate. CP, cortical plate; IZ, intermediate zone; MZ, marginal zone; SVZ, subventricular zone; VZ, ventricular zone. Scale bars: (A) 200 μm, (B, C) 100 μm.

Figure 3.

MGE- and CGE-derived interneurons differentially sort into distinct cortical layers after P1. (A) Representative examples of E12.5 MGE-derived interneurons in P1, P3, and P5 cortices. Triple-labeled sections for EGFP, RORß (marks layers IV and V), and 4′,6-diamidino-2-phenylindole (DAPI) nuclear counter stain (pseudocolored in red) are each shown with fate-mapped cells labeled in green. (B, C) Representative examples of E12.5 (B) and E16.5 (C) CGE-derived interneurons in P1, P3, P5, and P7 cortices. (D) The layer distribution of E12.5 MGE- and E12.5 or E16.5 CGE-derived cortical interneurons at P1, P3, P5, P7, and their final locations at P21. At P1, the superficial to deep layer ratio of interneurons derived from the E12.5 MGE and CGE are quite similar (compare blue and red color bar graphs). However, by P3, we observe a decrease in the numbers of layer I and an increase in the numbers of layer V interneurons in the E12.5 MGE-derived population, suggesting a superficial to deep layer migration of this population (dotted arrow). This superficial to deep migration continues till around P5, at which time these neurons have almost completed integrating into their target layers. By contrast, in the E12.5 CGE-derived interneuron population, the number of fate-mapped cells decreased in layers I and VI and increased in layers II/III. This suggests a net cell migration of CGE-derived cells toward layer II/III from both deeper and more superficial layers (dotted arrows). This migration seems to finalize at around P7, which is few days later than the ones derived from the age-matched MGE. Interestingly, although they are born 4 days later, E16.5 CGE-derived interneurons at P1 occupy the cortical layers in a similar proportion to the 2 lineages originated from E12.5. After this period, E16.5 CGE-derived interneurons are sorted into target layers in a very similar manner to the ones from earlier at E12.5. WM, white matter; IZ, intermediate zone. Scale bars 50 μm.

Figure 4.

KCC2 upregulation occurs during the radial sorting of interneurons. (A) Representative examples of KCC2-positive (arrowhead) and KCC2-negative (open arrowhead) cells at P3 derived from the E12.5 MGE and CGE, respectively. (B) A graph showing the proportion of KCC2-positive interneurons fate mapped from the E12.5 MGE and CGE. An increase in KCC2 expression occurs at the time interneurons are actively sorted (E12.5 MGE-derived: around P3, E12.5 CGE-derived: around P5). Scale bar 50 μm.

Figure 5.

Postnatal radial sorting of E12.5 MGE- and CGE-derived interneurons upon entering the cortical plate. A summary diagram illustrating that the layer-specific integration of E12.5-derived cortical interneurons only occurs after entering the cortical plate. During embryogenesis, interneurons tangentially migrate through the MZ (marginal zone), IZ (intermediate zone), and SVZ (subventricular zone). E12.5-generated interneurons switch their mode of migration from tangential to radial as they invade the cortical plate. At P1, both MGE- and CGE-derived interneurons are relatively uniformly distributed across all cortical layers. After P1, E12.5 MGE- and CGE-derived interneurons become preferentially distributed into deep and superficial cortical layers, respectively. This sorting step takes place at P1 onward until interneurons reach their final locations.