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, 239 (8), 2298-306

The Relationship Between Dlx and gad1 Expression Indicates Highly Conserved Genetic Pathways in the Zebrafish Forebrain

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The Relationship Between Dlx and gad1 Expression Indicates Highly Conserved Genetic Pathways in the Zebrafish Forebrain

Ryan B MacDonald et al. Dev Dyn.

Abstract

The Dlx genes encode a family of transcription factors important for the development of the vertebrate forebrain. These genes have very similar expression domains during the development of the telencephalon in mice and play a role in gamma-aminobutyric acid (GABAergic) interneuron differentiation. We have used triple fluorescent in situ hybridization to study the relative expression domains of the dlx and gad1 genes in the zebrafish telencephalon and diencephalon. We also generated transgenic zebrafish with regulatory elements from the zebrafish dlx1a/2a locus. The zebrafish dlx regulatory elements recapitulated dlx expression in the forebrain and mimicked the relationship between the expression of the dlx genes and gad1. Finally, we show that a putative enhancer located downstream of dlx2b can also activate reporter gene expression in a tissue-specific manner similar to endogenous dlx2b expression. Our results indicate the dlx genes are regulated by an evolutionarily conserved genetic pathway and may play a role in GABAergic interneuron differentiation in the zebrafish forebrain.

Figures

Figure 1
Figure 1. Similar forebrain expression of the two genes from a dlx cluster
Single z transversal sections of triple fluorescent in situ hybridization shows dlx1a, dlx2a, and dlx2b expression domains at 48hpf in the telencephalon (A-A’”) and diencephalon (B-B’”), as well as dlx5a and dlx6a (C-C’” and D-D’”). Schematic summaries representing the overlaps in dlx expression are shown in A””, B””, C”” and D””: the solid lines define the boundaries of detectable expression; the zones of relatively higher expression are colored when applicable. The boundaries of expression domains are comparable for dlx1a, dlx2a and dlx2b but dlx2b specifically displays a more intense zone of expression in a dorsal-lateral domain (arrow in A””). Boundaries of expression domains for dlx5a and dlx6a are overall similar although dlx6a was hardly detected in the ventral domain of the prethalamus (arrowhead on D””). Levels of section are shown on the schematic in (E). Shared expression domains of clustered genes (and their paralogs) are mapped on a schematic representation of a brain section at the level of the telencephalon (F) and of the diencephalon (G): dlx1a/2a/2b in blue and dlx5a/6a in red. Scale bar: 100 μm.
Figure 2
Figure 2. Expression domains of dlx genes with respect to gad1 expression
Single z sections of triple fluorescent in situ hybridization for dlx1a, dlx5a, and gad1 in the telencephalon at 24hpf (A-A”, para-sagital section, anterior is on the left), 36hpf (B-B”, transverse section in the telencephalon, dorsal is to the top), and 48hpf (C-C”, transversal section in the telencephalon; D-D”, transversal section in the diencephalon; dorsal is to the top) with a colored merge of all three channels on A’”, B’”, C’” and D’”. For each section level, a schematic representation is given to localize the boundaries of each expression domain (solid line) and a domain of higher level of expression is represented as a colored surface. Panel E gives a schematic representation of the partially nested expression domains in the telencephalon (left half) and diencephalon (right half) at 48hpf, based on the data shown in panels C-C’” and D-D’”: the proximal (ventricular) domain where only dlx1a is detected is shown in green and the lateral domain where gad1 only is detected is colored red. Scale bar: 100 μm.
Figure 3
Figure 3. Regulatory elements of dlx1a/2a and dlx2b drive reporter gene expression in the zebrafish forebrain and branchial arches
Genomic organization of the dlx1a/dlx2a and of the dlx2b loci (A). Exons are represented in white and UTRs in black. Intergenic regulatory elements are shown as blue (URE2 and I12b) and red boxes (I56i and I56ii). The dlx2bDRE is shown as a purple box. Schematic representation of the transgene constructs (containing the β-globin minimal promoter (grey) linked to GFP (green)) used to generate transgenic zebrafish (B). The Tg(dlx1a/2aIG:GFP) construct contains a 6kb of the intergenic fragment from the dlx1a/dlx2a intergenic region, the Tg(URE2dlx1a/2a:GFP) contains a 900 bp fragment encompassing the URE2 enhancer and the Tg(dlx2bDRE:GFP) contains a 200 base pair fragment downstream of dlx2b. Activity of the transgene constructs in the forebrain at 24hpf and 48hpf (C-E) Tg(URE2dlx1a/2a:GFP) transgene (F-H) Tg(dlx1a/2aIG:GFP) transgene (I-K) Tg(dlx2bDRE:GFP). tel, telencephalon; di, diencephalon; md, mandibular portion of branchial arches. Scale bar: 100 μm.
Figure 4
Figure 4. The dlx regulatory elements recapitulate endogenous dlx expression in the forebrain of the zebrafish at 48hpf
Whole mount in situ with probes against dlx1a (A,B), dlx2a (C,D) and gfp (E, F) Tg(dlx1a/2aIG:GFP) is expressed in the telencephalon, ventral thalamus, and hypothalamus. There is also expression in the pharyngeal arches (arrowhead), which recapitulates a small portion of the endogenous dlx arch expression domain. G,H) URE2 activity closely resembles the expression of Tg(dlx1a/2aIG:GFP) in the telencephalon, ventral thalamus, and hypothalamus and mimics dlx expression in these tissues. tel, telencephalon; pTh, prethalamus; hyp, hypothalamus; di, diencephalon; md, mandible; ba, branchial arches. Scale bar: 100 μm.
Figure 5
Figure 5. Immunolocalization of GFP and Gad proteins on transverse cryosections of the telencephalon at 48hpf
Expression of GFP (A, B, C) and Gad proteins (A’, B’, C’) is detected in the three transgenic lines, a merged image is presented in panels A”, B” and C” to show overlap (yellow arrows). An interpretative schematic representation of a hemi-section is presented in each case in panels A’”, B’” and C’”. Scale bars: 100 μm.
Figure 6
Figure 6
Schematic representation of the overlaps in dlx and gad expression (left) and of the activity of dlx regulatory elements (right) in the zebrafish forebrain.

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