During vertebrate forebrain formation, Dlx homeobox genes play essential roles in the differentiation, migration and survival of subpallial precursor cells that will later give rise to diverse subtypes of γ-aminobutyric acid (GABA)-expressing neurons, including inhibitory cortical interneurons in mammals. They also participate in the regulation of the Gad genes encoding the enzymes necessary for GABA synthesis. In mice, at least four cis-regulatory elements (CREs) control Dlx expression in the telencephalon and diencephalon: URE2 and I12b in the Dlx1/Dlx2 bigene cluster, and I56i and I56ii in the Dlx5/Dlx6 bigene cluster. However, little is known so far with respect to the function of orthologous dlx genes and their regulatory elements during zebrafish GABAergic neuron development. To investigate whether similar dlx-mediated pathways exist in the early developing zebrafish forebrain, we generated independent lines of transgenic zebrafish carrying two distinct GFP reporter constructs driven by a β-globin minimal promoter: one containing a ∼1.4kb dlx5a/dlx6a intergenic sequence (encompassing I56i and I56ii) and one with a ∼1.1kb fragment containing only the I56i CRE, respectively. The expression patterns of these two transgenes were compared with that obtained with another construct containing the ∼1.4kb dlx5a/dlx6a intergenic sequence and driven by a ∼3.5kb dlx6a 5'-flanking fragment. Our comparative analysis showed that GFP expression of the three transgene is largely overlapping throughout the ventral forebrain. Intriguingly, the dlx6a 5'-flanking fragment has a major impact on transgene expression in the mesencephalic tectum. Furthermore, comparison of transgene expression between the ∼1.4kb and ∼1.1kb intergenic fragments did not show any specific spatial expression conferred by I56ii. Almost all GFP-expressing cells in the transgenic zebrafish are GABA-positive and also express various GABAergic interneuron markers. Together, our data suggest that zebrafish dlx5a/dlx6a intergenic CREs may be involved in a conserved genetic pathway necessary for proper dlx expression during zebrafish GABAergic neuron development.
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