GABAergic Interneuron Differentiation in the Basal Forebrain Is Mediated through Direct Regulation of Glutamic Acid Decarboxylase Isoforms by Dlx Homeobox Transcription Factors

Abstract

GABA is the key inhibitory neurotransmitter in the cortex but regulation of its synthesis during forebrain development is poorly understood. In the telencephalon, members of the distal-less () homeobox gene family are expressed in, and regulate the development of, the basal ganglia primodia from which many GABAergic neurons originate and migrate to other forebrain regions. The double knock-out mice die at birth with abnormal cortical development, including loss of tangential migration of GABAergic inhibitory interneurons to the neocortex (Anderson et al., 1997a). We have discovered that specific promoter regulatory elements of glutamic acid decarboxylase isoforms (1 and 2), which regulate GABA synthesis from the excitatory neurotransmitter glutamate, are direct transcriptional targets of both DLX1 and DLX2 homeoproteins Further gain- and loss-of-function studies and demonstrated that both DLX1 and DLX2 are necessary and sufficient for gene expression. DLX1 and/or DLX2 activated the transcription of both genes, and defects in function disrupted the differentiation of GABAergic interneurons with global reduction in GABA levels in the forebrains of the double knock-out mouse Identification of genes as direct transcriptional targets is significant; it extends our understanding of gene function in the developing forebrain beyond the regulation of tangential interneuron migration to the differentiation of GABAergic interneurons arising from the basal telencephalon, and may help to unravel the pathogenesis of several developmental brain disorders. GABA is the major inhibitory neurotransmitter in the brain. We show that homeobox genes regulate GABA synthesis during forebrain development through direct activation of glutamic acid decarboxylase enzyme isoforms that convert glutamate to GABA. This discovery helps explain how mutations result in abnormal forebrain development, due to defective differentiation, in addition to the loss of tangential migration of GABAergic inhibitory interneurons to the neocortex. Reduced numbers or function of cortical GABAergic neurons may lead to hyperactivity states such as seizures (Cobos et al., 2005) or contribute to the pathogenesis of some autism spectrum disorders. GABAergic dysfunction in the basal ganglia could disrupt the learning and development of complex motor and cognitive behaviors (Rubenstein and Merzenich, 2003).

Distal-less genesDlx1/Dlx2repress oligodendrocyte genesis through transcriptional inhibition ofOlig2expression in the developing vertebrate forebrain

“…Dlx1 is proposed to be the founding member of the vertebrate Dlx family because it is most closely related to the Dll gene of Drosophila and amphioxus (Panganiban & Rubenstein, 2002). However, DLX2 is expressed before DLX1 during forebrain development in the mouse (Eisenstat et al, 1999;Le et al, 2017), and previously we also found that DLX2 is more robust than DLX1 as a transcriptional activator (Le et al, 2017;Q. P. Zhou et al, 2004) or transcriptional repressor (Le et al, 2007), signifying a more important role for the Dlx2 gene in forebrain development.…”

“…We have optimized our ChIP protocol to preferentially obtain homeoproteingenomic DNA complexes from embryonic tissues in situ (Q. P. Zhou et al, 2004). With the sensitive and highly specific antisera we possess (Eisenstat et al, 1999), several direct transcriptional targets of DLX1/2 proteins during forebrain development have been determined including the Dlx5/6 intergenic enhancer, neuropilin-2 and the glutamic acid decarboxylase genes Gad1/Gad2 (Le et al, 2007;Le et al, 2017;Q. P. Zhou et al, 2004).…”

GABAergic Neuron-Specific Whole-Brain Transduction by AAV-PHP.eB Incorporated with a New GAD65 Promoter

“…A previous study revealed pivotal roles of Dlx1 and Dlx2, members of the Dlx gene, in GABA synthesis [24]. Recently, Le et al demonstrated that Dlx1 and Dlx2 directly bind to and activate the transcription of the Gad promoters [25]. Moreover, using chromatin immunoprecipitation (ChIP) of the embryonic forebrain, they identi ed two Dlx target sequences within the Gad2 promoter regions.…”

“…The Dlx target regions were at nucleotide positions of -2,294 --2,088 and − 958 --598 (Region i and Region ii, respectively) ( Fig. 1A), both of which contained speci c homeodomain DNA-binding tetranucleotide TAAT/ATTA motifs [25]. Thus, these regions were postulated to be critical for the Gad2 promoter in terms of inhibitory neuron speci city as well as promoter activity.…”

Transcriptomic changes due to early, chronic alcohol exposure during cortical development implicate regionalization, cell-type specification, synaptogenesis and WNT signaling as primary determinants of fetal alcohol Spectrum Disorders

“…ASCL1, GSX2, DLX1/2/5/6), studies with DLX1/2 knockout mice, while leading to an embryonic lethal phenotype, surprisingly still demonstrate the generation of GABAergic neurons(Le 2007). Interestingly, one study additionally found an accumulation of INs in the ganglionic eminences, though this may be due to impaired migration to the cortex rather than a proliferative effect per se(Le 2007(Le , 2017. Strengthening this hypothesis, a recent reportfrom Tasic et al utilized single-cell RNA sequencing and clustering algorithms to assess cellular diversity in the mouse visual cortex and identified 49 distinct cell types, including two novel NDNF + IN subtypes (2.61-fold upregulated with EtOH) (Tasic 2016).…”