Expression of dlx genes in the normal and regenerating brain of adult zebrafish

Dysfunctions in the GABAergic system lead to various pathological conditions and impaired inhibitory function is one of the causes behind neuropathies characterized by neuronal hyper excitability. The Dlx homeobox genes are involved in the development of nervous system, neural crest, branchial arches and developing appendages. Dlx genes also take part in neuronal migration and differentiation during development, more precisely, in the migration and differentiation of GABAergic neurons. Functional analysis of dlx genes has mainly been carried out in developing zebrafish embryos and larvae, however information regarding the expression and roles of these genes in the adult zebrafish brain is still lacking. The extensive neurogenesis that takes place in the adult zebrafish brain, makes them a good model for the visualization of mechanisms involving dlx genes during adulthood in physiological conditions and during regeneration of the nervous system. We have identified the adult brain regions where transcripts of dlx1a, dlx2a, dlx5a and dlx6a genes are normally found and have confirmed that within telencephalic domains, there is high overlapping expression of the four dlx paralogs with a marker for GABAergic neurons. Co-localization analyses carried with the Tg(dlx6a-1.4kbdlx5a/dlx6a:GFP) reporter line have also shown that in some areas of the diencephalon, cells expressing the dlx5a/6a bigene may have a neural stem cell identity. Furthermore, investigations in a response to stab wound lesions, have demonstrated a possible participation of the dlx5a/6a bigene, most likely of dlx5a, during regeneration of the adult zebrafish brain. These observations suggest a possible participation of dlx-expressing cells during brain regeneration in adult zebrafish and also provide information on the role of dlx genes under normal physiological conditions in adults.

under normal physiological conditions in adults.

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The transcription factors encoded by Dlx genes play key roles in the 50 patterning of the vertebrate limb and the central nervous system (CNS) (1), more 51 specifically, Dlx genes are required in the development of the mammalian brain (2,3).

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These genes are required for correct migration and differentiation of progenitors that 53 will later give rise to GABAergic interneurons (4). Dlx1 -/-/Dlx2 -/mutant mice show a 54 loss of GABAergic interneuron differentiation in the ventral telencephalon, supporting 55 the notion of this requirement for Dlx genes in the differentiation to GABAergic 56 interneurons (5). In the case of zebrafish, dlx genes are also expressed in the 57 developing brain (6) (7). At 24-48 hours-post-fertilization (hpf), there is partial 58 overlapping expression of dlx and gad1 genes in the zebrafish forebrain (8).

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Nevertheless, information regarding the activity and functions of Dlx genes in the 60 adult brain is still scarce and non-existent in the zebrafish.

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The majority of vertebrates have six Dlx genes which are organized in 62 convergently transcribed bigene pairs, namely Dlx1/Dlx2, Dlx3/Dlx4 and Dlx5/Dlx6 63 (9) (10) (11). In zebrafish, the dlx1a/dlx2a and dlx5a/dlx6a (orthologs of mouse 64 Dlx1/Dlx2 and Dlx5/Dlx6) are expressed in the developing brain. Previously 65 described cis-regulatory regions are essential for driving the expression of these 66 bigenes in the brain. The deletion of one of these regions in mice, I56ii, has been 67 shown to impair the expression of Dlx genes and of potential targets including Gad2 68 and other striatal markers (12   these specific subtype markers (Fig. 16). Co-labeling with calretinin has shown very 312 little if any co-localization with GFP. Only a few dlx5a/6a-expressing cells appear to 313 be calretinin neurons in the periventricular gray zone (PGZ) (Fig 2M-N). In fact, within 314 the PGZ area, a few dlx5a/6a-expressing cells have also shown a calbindin identity.

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Interestingly and in contrast to observations at developmental stages, in the 316 adult zebrafish brain, we observed many dlx5a/6a-expressing cells co-localizing with telencephalic area (Vs), we observed a higher percentage of co-localization than in 335 the domains of the telencephalon as mentioned before (Fig 3).

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We did not observe apparent changes in the expression of dlx1a, dlx2a or 437 dlx6a at 7dpl (Fig 5J), nor in the expression patterns of dlx1a, dlx2a, dlx5a or dlx6a at 438 the site of injury where the needle was inserted in the ventricule of telencephalon at 439 7dpl.

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Possible changes in expression levels of dlx1a, dlx2a, dlx5a and dlx6a were 441 further quantified by qRT-PCR at 7dpl. Slight increases in dlx1a, dlx2a and dlx6a 442 transcripts were seen but did not reach statistical significance at 7dpl. However, there was a significant increase in dlx5a expression in the telencephalon of lesioned 444 adult zebrafish at 7dpl (Fig.5 J).

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Changes in dlx expression during brain regeneration was further examined 446 using the Tg(dlx6a-1.4kbdlx5a/6a:GFP) reporter line and direct counting of GFP 447 positive cells. The dorsal and ventral areas of the ventral telencephalon is where 448 constitutive proliferation takes place and are also the regions were an increase 449 seemed more visible, therefore this area was selected for quantification (Fig.6)