The Distal-less homeobox (Dlx) genes encode homeodomain transcription factors found in all animals of the phylum Chordata. These genes are involved in early vertebrate development of limbs, sensory organs, branchial arches and the forebrain (telencephalon and diencephalon). The mouse and human genomes each have six Dlx genes organized into convergently transcribed bigene clusters (Dlx1/2, Dlx3/4 and Dlx5/6). In the forebrain, Dlx1/2 and Dlx5/6 genes play essential roles in GABAergic neuron proliferation, migration and survival. Each bigene cluster includes a short intergenic region (~3.5-16kb) harboring cis-regulatory elements (CREs) that control expression of the Dlx genes. The Dlx1/2 intergenic region harbors the I12b/I12a CREs, while Dlx5/6 includes I56i/I56ii. In determining the regulatory roles of the CREs on Dlx activity and forebrain development, I have characterized the phenotypic changes that occur in mice that have an I56i enhancer deletion. I have also characterized mice with double deletions of I56i and I12b as well as mice that harbored an I12b deletion and have a SNP in the I56i enhancer (vI56i). Mutant mice with a single targeted deletion of I56i are viable, fertile and do not show obvious developmental defects. These mice have significant decreases in Dlx5/6, Gad1/Gad2 and Evf-2 expression in the forebrain and have defects related to GABAergic neuron development. The ΔI56i mutants demonstrate a behavioral phenotype related to anxiety and learning deficits. Mice that lack the I12b enhancer and have the vI56i do not show morphological abnormalities but have severely disrupted Dlx expression. When mice are homozygous for the I56i and I12b enhancer deletion, they do not survive past post natal day 5 and exhibit a dwarfed body size. These mice look weak and seem to have limited motor ability. In characterizing mice with targeted deletions of highly conserved Dlx enhancers, we will have a better understanding of forebrain development.

The temporal and spatial patterning that underlies morphogenetic events is controlled by gene regulatory networks (GRNs). These operate through a combinatorial code of DNA – binding transcription factor proteins, and non – coding DNA sequences (cis-regulatory elements, or CREs), that specifically bind transcription factors and regulate nearby genes. By comparatively studying the development of different species, we can illuminate lineage – specific changes in gene regulation that account for morphological evolution. The central nervous system of vertebrates is composed of diverse neural cells that undergo highly coordinated programs of specialization, migration and differentiation during development. Approximately 20% of neurons in the cerebral cortex are GABAergic inhibitory interneurons, which release the neurotransmitter gamma-aminobutyric acid (GABA). Diseases such as autism, schizophrenia and epilepsy are associated with defects in GABAergic interneuron function. Several members of the distal-less homeobox (Dlx) transcription factor family are implicated in a GRN underlying early GABAergic interneuron development in the forebrain. I examined the role played by orthologous dlx genes in the development of GABAergic interneurons in the zebrafish forebrain. I found that when ascl1a transcription factor is down-regulated through the micro-injection of translation – blocking morpholino oligonucleotides, Dlx gene transcription is decreased in the diencephalon, but not the telencephalon. Similarly, gad1a transcription is also decreased in this region for these morphants. As gad1a encodes an enzyme necessary for the production of GABA, these genes are implicated in a cascade underlying GABAergic interneuron development in the diencephalon.

The Distal-less homeobox (Dlx) genes encode homeodomain transcription factors found in all animals of the phylum Chordata. These genes are involved in early vertebrate development of limbs, sensory organs, branchial arches and the forebrain (telencephalon and diencephalon). The mouse and human genomes each have six Dlx genes organized into convergently transcribed bigene clusters (Dlx1/2, Dlx3/4 and Dlx5/6). In the forebrain, Dlx1/2 and Dlx5/6 genes play essential roles in GABAergic neuron proliferation, migration and survival. Each bigene cluster includes a short intergenic region (~3.5-16kb) harboring cis-regulatory elements (CREs) that control expression of the Dlx genes. The Dlx1/2 intergenic region harbors the I12b/I12a CREs, while Dlx5/6 includes I56i/I56ii. In determining the regulatory roles of the CREs on Dlx activity and forebrain development, I have characterized the phenotypic changes that occur in mice that have an I56i enhancer deletion. I have also characterized mice with double deletions of I56i and I12b as well as mice that harbored an I12b deletion and have a SNP in the I56i enhancer (vI56i). Mutant mice with a single targeted deletion of I56i are viable, fertile and do not show obvious developmental defects. These mice have significant decreases in Dlx5/6, Gad1/Gad2 and Evf-2 expression in the forebrain and have defects related to GABAergic neuron development. The ΔI56i mutants demonstrate a behavioral phenotype related to anxiety and learning deficits. Mice that lack the I12b enhancer and have the vI56i do not show morphological abnormalities but have severely disrupted Dlx expression. When mice are homozygous for the I56i and I12b enhancer deletion, they do not survive past post natal day 5 and exhibit a dwarfed body size. These mice look weak and seem to have limited motor ability. In characterizing mice with targeted deletions of highly conserved Dlx enhancers, we will have a better understanding of forebrain development.

Abstract The Dlx genes play an important role in the differentiation and migration of gamma-aminobutyric acid (GABA) interneurons of mice. GABAergic interneurons are born in the proliferative zones of the ventral telencephalon and migrate to the cortex early during mouse development. Single Dlx mutant mice show only subtle phenotypes. However, the migration of immature interneurons is blocked in the ventral telencephalon of Dlx1/Dlx2 double mutant mice leading to reduction of GABAergic interneurons in the cortex. Also, Dlx5/Dlx6 expression is almost entirely absent in the forebrain, most probably due to cross-regulatory mechanisms. In zebrafish, the role of dlx genes in GABAergic interneuron development is unknown. By injecting Morpholino, we double knocked down dlx1 and dlx2 genes in wildtype zebrafish to investigate the function of the two genes in zebrafish GABAergic interneuron development. By comparing different subsets of GABAergic interneuron development in wildtype and dlx1/2 morphant zebrafish forebrain, we found out that at 3dpf, 4dpf and 7dpf, double knockdown of dlx1 and dlx2 genes in zebrafish remarkably reduced the number of Calbindin-, Somatostatin- and Parvalbumin-positive GABAergic neurons, whereas the development of Calretinin-positive neurons is slightly affected. These results suggest that in zebrafish, dlx1a and dlx2a genes are important for the development of certain subtypes of GABAergic interneurons (Calbindin-, Somatostatin- and Parvalbumin-positive neurons) and may have minor influence on Calretinin-positive neuron development. This also suggests that different regulatory mechanisms are involved in the development of the different subtypes of GABAergic interneurons.

Abstract The Dlx genes play an important role in the differentiation and migration of gamma-aminobutyric acid (GABA) interneurons of mice. GABAergic interneurons are born in the proliferative zones of the ventral telencephalon and migrate to the cortex early during mouse development. Single Dlx mutant mice show only subtle phenotypes. However, the migration of immature interneurons is blocked in the ventral telencephalon of Dlx1/Dlx2 double mutant mice leading to reduction of GABAergic interneurons in the cortex. Also, Dlx5/Dlx6 expression is almost entirely absent in the forebrain, most probably due to cross-regulatory mechanisms. In zebrafish, the role of dlx genes in GABAergic interneuron development is unknown. By injecting Morpholino, we double knocked down dlx1 and dlx2 genes in wildtype zebrafish to investigate the function of the two genes in zebrafish GABAergic interneuron development. By comparing different subsets of GABAergic interneuron development in wildtype and dlx1/2 morphant zebrafish forebrain, we found out that at 3dpf, 4dpf and 7dpf, double knockdown of dlx1 and dlx2 genes in zebrafish remarkably reduced the number of Calbindin-, Somatostatin- and Parvalbumin-positive GABAergic neurons, whereas the development of Calretinin-positive neurons is slightly affected. These results suggest that in zebrafish, dlx1a and dlx2a genes are important for the development of certain subtypes of GABAergic interneurons (Calbindin-, Somatostatin- and Parvalbumin-positive neurons) and may have minor influence on Calretinin-positive neuron development. This also suggests that different regulatory mechanisms are involved in the development of the different subtypes of GABAergic interneurons.

Abstract The Dlx genes play an important role in the differentiation and migration of gamma-aminobutyric acid (GABA) interneurons of mice. GABAergic interneurons are born in the proliferative zones of the ventral telencephalon and migrate to the cortex early during mouse development. Single Dlx mutant mice show only subtle phenotypes. However, the migration of immature interneurons is blocked in the ventral telencephalon of Dlx1/Dlx2 double mutant mice leading to reduction of GABAergic interneurons in the cortex. Also, Dlx5/Dlx6 expression is almost entirely absent in the forebrain, most probably due to cross-regulatory mechanisms. In zebrafish, the role of dlx genes in GABAergic interneuron development is unknown. By injecting Morpholino, we double knocked down dlx1 and dlx2 genes in wildtype zebrafish to investigate the function of the two genes in zebrafish GABAergic interneuron development. By comparing different subsets of GABAergic interneuron development in wildtype and dlx1/2 morphant zebrafish forebrain, we found out that at 3dpf, 4dpf and 7dpf, double knockdown of dlx1 and dlx2 genes in zebrafish remarkably reduced the number of Calbindin-, Somatostatin- and Parvalbumin-positive GABAergic neurons, whereas the development of Calretinin-positive neurons is slightly affected. These results suggest that in zebrafish, dlx1a and dlx2a genes are important for the development of certain subtypes of GABAergic interneurons (Calbindin-, Somatostatin- and Parvalbumin-positive neurons) and may have minor influence on Calretinin-positive neuron development. This also suggests that different regulatory mechanisms are involved in the development of the different subtypes of GABAergic interneurons.

Abstract The Dlx genes play an important role in the differentiation and migration of gamma-aminobutyric acid (GABA) interneurons of mice. GABAergic interneurons are born in the proliferative zones of the ventral telencephalon and migrate to the cortex early during mouse development. Single Dlx mutant mice show only subtle phenotypes. However, the migration of immature interneurons is blocked in the ventral telencephalon of Dlx1/Dlx2 double mutant mice leading to reduction of GABAergic interneurons in the cortex. Also, Dlx5/Dlx6 expression is almost entirely absent in the forebrain, most probably due to cross-regulatory mechanisms. In zebrafish, the role of dlx genes in GABAergic interneuron development is unknown. By injecting Morpholino, we double knocked down dlx1 and dlx2 genes in wildtype zebrafish to investigate the function of the two genes in zebrafish GABAergic interneuron development. By comparing different subsets of GABAergic interneuron development in wildtype and dlx1/2 morphant zebrafish forebrain, we found out that at 3dpf, 4dpf and 7dpf, double knockdown of dlx1 and dlx2 genes in zebrafish remarkably reduced the number of Calbindin-, Somatostatin- and Parvalbumin-positive GABAergic neurons, whereas the development of Calretinin-positive neurons is slightly affected. These results suggest that in zebrafish, dlx1a and dlx2a genes are important for the development of certain subtypes of GABAergic interneurons (Calbindin-, Somatostatin- and Parvalbumin-positive neurons) and may have minor influence on Calretinin-positive neuron development. This also suggests that different regulatory mechanisms are involved in the development of the different subtypes of GABAergic interneurons.

The Dlx homeobox genes encode homeodomain transcription factors that are involved in multiple developmental aspects. In the brain, these genes take part in neuronal migration and differentiation, more precisely in the migration and differentiation of GABAergic neurons. Dysfunctions in the GABAergic system can lead to various pathological conditions, where impaired inhibitory function is one of the main causes of several neuropathies characterized by neuronal hyperexcitability. The Dlx genes are organized as bi-gene clusters and highly conserved cis-regulatory elements have been previously characterized to be fundamental for the regulation of Dlx expression in developing embryos of different vertebrates. The activity of these regulatory elements and the Dlx genes has been well studied in developmental stages of mice and zebrafish, but little is known about their activity in the adult brain. The extensive neurogenesis that takes place in the adult zebrafish brain provides an ideal platform for the visualization of mechanisms involving dlx genes during adulthood and their possible involvement in adult neurogenesis. Here we show novel information concerning the expression of dlx1a, dlx2a, dlx5a and dlx6a in the adult zebrafish brain and provide insight into the identity of cells that express dlx. We also demonstrate the involvement of dlx genes in brain regeneration and through lineage tracing, their fate determination in the adult zebrafish brain. Analyses in the adult zebrafish has revealed that all four dlx paralogs are expressed in the forebrain and midbrain throughout adulthood and expression is found in almost all areas presenting continuous proliferation. Most dlx-expressing cells present GABAergic neuronal identity in the adult forebrain where, in some areas they were identified as the Calbindin subtype. In some areas of the midbrain, especially within the hypothalamus, many dlxexpressing cell co-localized with a marker for neural stem cells. However, cells expressing dlx iii genes did not co-localize with markers for proliferating cells or for glia. Investigations during brain regeneration in response to injury in the adult zebrafish brain has revealed that dlx5a expression decreases shortly after lesion and that the dlx5a/6a bi-gene cluster, more specifically, dlx5a, is up regulated during the peak of regeneration response proposing a possible role for dlx during regeneration in adults. Studies of lineage tracing have shown the progeny of dlx1a/2a-expressing cells in adults are located within small clusters in different areas of the adult brain where they seem to become mature neurons. Our observations provide a better understanding about the role of dlx genes during adulthood, further contributing to the general knowledge of the molecular pathways involved in adult neurogenesis and regeneration in the zebrafish adult brain.

The vertebrate Dlx gene family consists of multiple convergently transcribed bigene clusters and encodes a group of homeodomain-containing transcription factors crucial for the development of forebrain, branchial arches, sensory organs and limbs. At least four cis-regulatory elements (CREs) are responsible for Dlx expression in the forebrain: URE2 and I12b in the Dlx1/Dlx2 (zebrafish dlx1a/dlx2a) locus, and, I56i and I56ii in the Dlx5/Dlx6 (zebrafish dlx5a/dlx6a) locus. Here, we first show that unlike the other three enhancers, mouse I56ii CRE targets a group of GABAergic projection neurons expressing striatal markers Meis2 and Islet1. Meis2 and Islet1 proteins can activate reporter gene transcription via the I56ii CRE, suggesting that they may be potential upstream regulators of Dlx genes in vivo. To determine whether there exists a dlx-mediated regulatory pathway during zebrafish GABAergic neuron formation, we establish two independent lines of transgenic fish in which the GFP reporter gene is controlled by a 1.4kb dlx5a/dlx6a intergenic sequence (encompassing zebrafish I56i and I56ii) and a 1.1kb fragment containing only I56i CRE, respectively. Our observations reveal that dlx5a/dlx6a regulatory elements exhibit a fairly specific activity in the zebrafish forebrain and may be essential for GABAergic neuron generation, while I56i and I56ii are likely to play distinct roles in modulating this process in different subpopulations of cells. Disruption of dlx1a/dlx2a or dlx5a/dlx6a function leads to a marked decrease of enhancer activity in the diencephalon and midbrain as well as a comparatively lesser extent of reduction in the telencephalon. In order to define the specific contribution of various individual CREs to overall Dlx regulation, we also generate a mutant mouse model in which I12b CRE is selectively deleted. Despite that mice homozygous for I12b loss develop normally and harbor no overt morphological defects in the forebrain, targeted deletion of this enhancer results in a significant reduction of Dlx1/Dlx2 transcript levels and seemingly perturbs cell proliferation in the subpallial telencephalon, particularly in the ventricular and subventricular zones of ganglionic eminences. Taken together, these data illustrate a complex and dynamic Dlx regulation in the early developing forebrain through the implications of multiple Dlx CREs with overlapping and diverse functions.

The vertebrate Dlx gene family consists of multiple convergently transcribed bigene clusters and encodes a group of homeodomain-containing transcription factors crucial for the development of forebrain, branchial arches, sensory organs and limbs. At least four cis-regulatory elements (CREs) are responsible for Dlx expression in the forebrain: URE2 and I12b in the Dlx1/Dlx2 (zebrafish dlx1a/dlx2a) locus, and, I56i and I56ii in the Dlx5/Dlx6 (zebrafish dlx5a/dlx6a) locus. Here, we first show that unlike the other three enhancers, mouse I56ii CRE targets a group of GABAergic projection neurons expressing striatal markers Meis2 and Islet1. Meis2 and Islet1 proteins can activate reporter gene transcription via the I56ii CRE, suggesting that they may be potential upstream regulators of Dlx genes in vivo. To determine whether there exists a dlx-mediated regulatory pathway during zebrafish GABAergic neuron formation, we establish two independent lines of transgenic fish in which the GFP reporter gene is controlled by a 1.4kb dlx5a/dlx6a intergenic sequence (encompassing zebrafish I56i and I56ii) and a 1.1kb fragment containing only I56i CRE, respectively. Our observations reveal that dlx5a/dlx6a regulatory elements exhibit a fairly specific activity in the zebrafish forebrain and may be essential for GABAergic neuron generation, while I56i and I56ii are likely to play distinct roles in modulating this process in different subpopulations of cells. Disruption of dlx1a/dlx2a or dlx5a/dlx6a function leads to a marked decrease of enhancer activity in the diencephalon and midbrain as well as a comparatively lesser extent of reduction in the telencephalon. In order to define the specific contribution of various individual CREs to overall Dlx regulation, we also generate a mutant mouse model in which I12b CRE is selectively deleted. Despite that mice homozygous for I12b loss develop normally and harbor no overt morphological defects in the forebrain, targeted deletion of this enhancer results in a significant reduction of Dlx1/Dlx2 transcript levels and seemingly perturbs cell proliferation in the subpallial telencephalon, particularly in the ventricular and subventricular zones of ganglionic eminences. Taken together, these data illustrate a complex and dynamic Dlx regulation in the early developing forebrain through the implications of multiple Dlx CREs with overlapping and diverse functions.