Gene expression in cortical interneuron precursors is prescient of their mature function

Abstract

At present little is known about the developmental mechanisms that give rise to inhibitory gamma-aminobutyric acidergic interneurons of the neocortex or the timing of their subtype specification. As such, we performed a gene expression microarray analysis on cortical interneuron precursors isolated through their expression of a Dlx5/6(Cre-IRES-EGFP) transgene. We purified these precursors from the embryonic mouse neocortex at E13.5 and E15.5 by sorting of enhanced green fluorescent protein-expressing cells. We identified novel transcription factors, neuropeptides, and cell surface genes whose expression is highly enriched in embryonic cortical interneuron precursors. Our identification of many of the genes known to be selectively enriched within cortical interneurons validated the efficacy of our approach. Surprisingly, we find that subpopulations of migrating cortical interneurons express genes encoding for proteins characteristic of mature interneuron subtypes as early as E13.5. These results provide support for the idea that many of the genes characteristic of specific cortical interneuron subtypes are evident prior to their functional integration into cortical microcircuitry. They suggest interneurons are already relegated to specific genetic subtypes shortly after they become postmitotic. Moreover, our work has revealed that many of the genes expressed in cortical interneuron precursors have been independently linked to neurological disorders in both mice and humans.

Figure 1.

Genetic fate mapping of Dlx5/6 expressing cells. Representative examples of sections for Dlx5/6 fate mapping. EGFP, DAPI, and various immunohistochemical markers on coronal sections of P30 brains. (A, B) show colocalization of GABA and EGFP in the vast majority of fate mapped cells. (C–G) Partial colocalization of EGFP and the interneuron subtype markers PV, SST, NPY, CR, and VIP signals. (H) No colocalization of GFP and PDGFRα signals was never observed. The frequency of cortical interneuron markers at P30 in mouse somatosensory barrel cortex PV, SST, NPY, CR, and VIP are calculated by comparing the number of cells that are double positive for EGFP and the markers examined as a ratio of the total number of EGFP cells (I). The histogram (I) summarizes the cell fate mapping in the Dlx5/6^{Cre-IRES-EGFP} mice. Virtually all cells seen in the cortex are GABAergic. Notably the frequency and the distribution of various cell expressing immunomarkers shown is precisely consistent with that reported previously. Scale bars: (A) 35 μm, (B) 5 μm, (C–H) 35 μm.

Figure 2.

Isolation of cortical interneuron precursors using the transgenic Dlx5/6^{Cre-IRES-EGFP} mouse. EGFP immunohistochemistry on coronal sections of E13.5 and E15.5 brains. (A, B) The cortical region indicated was dissected, dissociated and FACS sorted at both ages (A) E13.5 and (B) E15.5. (C, D) In situ hybridization on similar cryosections using the Gad1 antisense probe. Note that within the cortex the distribution of the EGFP precursors shown in (A) and (B) closely resembles the pattern of Gad1 expression seen in (C) and (D). Scale bars: (A–D) 250 μm.

Figure 3.

Classes of genes enriched in the interneuron population. The proportion of different classes of genes with assigned function, which are enriched in the interneuron population by at least 2-fold at E13.5 (total number genes: 416) (A) and E15.5 (total number genes: 371) (B). In this study we validated the expression of some interneuron enriched genes encoding transcription factors, neuropeptides, channels, and ion transport, and finally genes encoding cellular interaction molecules.

Figure 4.

Expression of genes encoding transcription factors. (A–F) Expression patterns of genes encoding transcription factors enriched in interneuron precursors at E13.5 and E15.5 revealed by in situ hybridization. Satb1, Neud4, and Ssbp2 were broadly expressed in postmitotic interneurons at both ages. (G–L) Carhsp1, Cux2, and Zfxh1 were strongly expressed in migrating interneurons and detected within the ganglionic eminences. (M–P) Pbx3 and Pou3f4 were more strongly expressed in the ganglionic eminences and were downregulated in interneurons as they migrated toward the cortex. Scale bars: (A–F) 250 μm.

Figure 5.

Expression of neuropeptides. (A–F) In situ hybridization for Sst, Npy, and Cck and double immunostaining for (G) EGFP/ SST, and (H) EGFP/ NPY of Dlx5/6^{Cre-IRES-EGFP} mouse shows that subpopulations of cortical interneuron precursors express SST and NPY (arrows). Panels (G) and (H) corresponds to the location of the box in (A). (I) Microarray fold enrichment (color code) in interneuron precursors for the neuropeptides Sst, Npy, Cck, and neuropeptide receptors Npy2r, Sstr2, and Cckar2. Scale bars: (A–F) 250 μm, (G–H) 10 μm.

Figure 6.

Expression of genes encoding cellular interaction molecules. In situ hybridization for Nxph1, Nkcc1 (C, D), Viaat (E, F), Kcc2 (G, H), Gria1 (I, J), and Cacnb4. (I) Microarray fold enrichment (color code) in the interneuron precursor population for select genes involved in cell surface interactions, GABA signaling, glutamate signaling, and ionic regulation. Scale bars: (A–L) 250 μm.