In this report, we adopted a conservation-based bioinformatic approach to identify potential molecular regulators of GABAergic identity in the mammalian telencephalon. GFP-marked GABAergic neurons from the nematode, C. elegans, were isolated by FACS for microarray profiling. These data revealed enrichment (≥ 1.7×) of 17 transcripts encoding conserved proteins with potential roles in gene regulation in the nematode. BLASTP of these C. elegans proteins identified mouse homologs and 62 independent transcripts corresponding to these mammalian transcription factors were assessed for expression in E14.5 mouse brain. The data generated in our comparative strategy revealed several highly conserved players in GABAergic interneuron differentiation, including Arx, Nkx2.1 and Cux2[22–24]. The positive identification of these transcripts supports the utility of our bioinformatic approach as a productive strategy for identifying conserved determinants of neuronal fate. Of the reciprocal BLASTP top hits, 14 unique transcripts showed relevant in situ hybridization patterns for telencephalic GABAergic neurogenesis, with 3 having known roles (Arx, Cux1, Cux2). Indeed, mutations in ARX have been associated with human brain function and interneuron pathology as identified in OMIM . The 11 remaining top reciprocal hits with relevant expression patterns serve as novel candidate genes (Ip6k1, Ip6k2, Trerf1, C130039O16Rik, Ezh2, Taf11, Med6, Thoc4, Refbp2, Med8, Tcfap4). While not top reciprocal hits, based on striking expression pattern alone, Hist1h1a, Fox1, Myst3 and Suv39h1 warrant further attention. This is especially true as reciprocity is not a perfect predictor of candidacy, as two proteins with known function in GABAergic specification were not top reciprocal hits (NKX2.1 and beta-Catenin).
Mammalian GABAergic cells are generated in the preoptic area and ganglionic eminence of the ventral pallium during embryogenesis [8, 42–44]. The three main subdivisions of the ganglionic eminence-lateral (LGE), medial (MGE) and caudal (CGE)-generate a diverse portfolio of GABAergic cells. The LGE produces GABAergic projection neurons of the striatum and interneurons of the amygdala and the olfactory bulbs whereas the MGE and CGE produce the majority of cortical and striatal interneurons, although each contributes a different repertoire of cell types. Cells from the MGE (for example, Nkx2.1-expressing cells) settle in cortical layers in an inside-out fashion based on cell birth date, whereas the most ventral MGE cells generate neurons of the globus pallidus and striatal cholinergic neurons . In contrast, cells from the CGE tend to migrate to upper layers, independent of birthday, and comprise 15 to 30% of all cortical interneurons . It is curious that of all of the transcription factors that we mapped, Nkx2.1 was the only one that was limited to one of the three progenitor pools.
It is clear that the gene regulatory transcripts identified in our study, with the exception of Nkx2.1, do not delineate these well-known pools of progenitor populations. The absence of tissue specificity could mean that these transcription factors exercise general roles in neuronal differentiation as opposed to functioning as selective determinants of GABAergic fate. However, the broader expression beyond the boundaries of these defined progenitor zones does not preclude a role for the protein products of these transcripts in contributing to the development of a selective neuronal type. For example, these candidates may be permissive for a particular fate or act in combination with other gene products with more limited expression patterns.
The data generated by our comparative approach blend with and add to the existing data on mammalian transcription factors that could play a role in the full development of GABAergic fates. There have been several efforts in mouse embryogenesis to use transcription profiling of microdissected GABAergic proliferative zones or fluorescent sorting of enhanced GFP (EGFP)-positive interneurons in dissected embryonic brain. For example, Batista-Brito et al.  used FACS to isolate embryonic interneurons from presumptive neocortex of E13.5 and E15.5 Dlx5/6Cre-IRIS-EGFP mice. They contrasted the transcriptomes of EGFP-positive (interneurons) and EGFP-negative cells (all other cell types) and identified several enriched transcripts, including Arx and Cux2, as in our study. Because of the region dissected, Nkx2.1 was not enriched, as its expression wanes as interneurons leave the medial ganglionic eminence. They also identified several other candidate transcription factors, including some with association with neurological disorders. Faux et al.  performed a similar experiment contrasting the transcriptomes of interneurons in the cortex versus the ganglionic eminence using GAD67-EGFP FACS isolated cells obtained at E13.5 and E15.5. Among other transcription factors, Faux et al. also show increased expression of Cux2. Cux2 was also identified in a similar study by Marsh et al. . By changing the contrasted pools of mRNA, the Faux et al. study addressed a different question than the Batista-Brito et al. study. The purpose of the Faux et al. study was to enrich for transcripts that may play a role in the migration of interneurons, while the Batista-Brito et al. study addressed the question of what genes are differentially expressed in interneurons versus other cell types in the embryonic cortex. Clearly, the contrasted pool of mRNA makes a difference in what transcripts appear to play a role in aspects of interneuron specification , migration  and maturation . Indeed, contrasting mRNA pools from CGE, LGE and MGE can provide candidates for specifying interneuron subtype .
While the comparative approach used here has identified novel potential candidates in the specification of interneurons, there are limitations. The experimental design would not detect elements of chromatin structure or microRNAs, for example, as mechanisms of transcriptional regulation. Our analysis was limited to transcripts that encode proteins involved in gene regulation; other protein classes (for example, receptor tyrosine kinases, ion channels) could also be involved. Moreover, the results are correlational; the expression patterns of these novel candidates overlap with areas that produce GABAergic cells, but do not show that these transcripts participate in GABA fate. Functional studies will be necessary to determine a role for these potential novel players. Additionally, while the comparative data used in this study are based on protein sequence homologies, the ultimate goal is to identify functional orthologs across species. Because true functional orthology is determined over time with experimental methods outside of the scope of this manuscript, we implore the reader to view these data as a first step on the path to identifying potential functional orthologs in conserved gene regulation networks to specify a GABAergic fate.
While this comparative approach revealed several highly conserved players in GABAergic neurogenesis, including Nkx2.1, Arx and Cux2, we failed to identify some known factors in mammalian forebrain specification, including Olig-2, although we did identify other basic helix-loop-helix (bHLH) transcription factors, such as Tcfap4. Also noticeably absent from the list were Lhx6 (lim-4 in C. elegans), Mash1 and Dlx1/2, all of which have been demonstrated to play a role in GABAergic differentiation in the mammalian forebrain. We note that a related LIM homeodomain protein, LIM-6, is required for differentiation and expression of UNC-25/GAD in a subset of C. elegans GABAergic neurons .
While unc-30 is the top candidate with the highest enrichment in GABAergic cells in the worm data set, none of the mammalian homologs (Pitx1, Pitx2, Pitx3) revealed expression in known GABAergic proliferative zones of the forebrain, even though there was expression in other brain areas at E14.5. Pitx2 is highly expressed in GABA neuron progenitors in diencephalon/mesencephalon , where it is known to drive Gad67 expression . This role is also conserved in the C. elegans homolog, unc-30. In fact, both mammalian Pitx2 and C. elegans unc-30 can both be used to activate Gad67 transcription in vitro and in vivo. While Pitx2 and unc-30 clearly give rise to a GABA phenotype, based on the absence of Pitx2 expression in the forebrain, there are other mechanisms that regulate GABA phenotype in the interneurons of the telencephalon. More than one type of transcription factor or combination of transcription factors likely can drive the GABAergic fate. Indeed, GABAergic fate regulation in the worm offers a striking parallel to the mouse: unc-30 drives GABAergic fate in ventral cord motor neurons but not in GABAergic motor neurons in the head where the LIM homeodomain lim-6 is required; similarly, Pitx2 is highly expressed in diencephalon/mesencephalon GABAergic progenitors and drives Gad67 expression but is not required for differentiation of forebrain GABAergic interneurons that depend on ARX. Additionally, alr-1, the worm homolog of ARX, regulates gene expression in worm GABA motor neurons .