Neurogenin1 is a determinant of zebrafish basal forebrain dopaminergic neurons and is regulated by the conserved zinc finger protein Tof/Fezl

Abstract

The development of vertebrate basal forebrain dopaminergic (DA) neurons requires the conserved zinc finger protein Too Few (Tof͞ Fezl) in zebrafish. However, how Tof͞Fezl regulates the commitment and differentiation of these DA neurons is not known. Proneural genes encoding basic helix-loop-helix transcription factors regulate the development of multiple neuronal lineages, but their involvement in vertebrate DA neuron determination is unclear. Here we show that neurogenin 1 (ngn1), a vertebrate proneural gene related to the Drosophila atonal, is expressed in and required for specification of DA progenitor cells, and when overexpressed leads to supernumerary DA neurons in the forebrain of zebrafish. Overexpression of ngn1 is also sufficient to induce tyrosine hydroxylase expression in addition to the panneuronal marker Hu in nonneural ectoderm. We further show that Tof͞Fezl is required to establish basal forebrain ngn1-expressing DA progenitor domains. These findings identify Ngn1 as a determinant of brain DA neurons and provide insights into how Tof͞Fezl regulates the development of these clinically important neuronal types.neurogenin 1 ͉ pluripotent neural stem cell ͉ neurotransmitter phenotype ͉ commitment and differentiation T he determination of neurotransmitter phenotype is an important aspect of neuronal differentiation, and in this regard, dopaminergic (DA) neurons have attracted considerable attention because of their functional and medical importance (1). Degeneration of substantia nigra DA neurons in humans is a hallmark of Parkinson's disease, and the malfunction of DA neurons in other brain regions is implicated in psychiatric disorders and neuroendocrine dysregulation. Therefore, understanding the determination of DA phenotype and the specification of DA neuronal circuitry may provide mechanistic and therapeutic insights into these disorders. To date, only limited number of known or putative transcriptional regulators, including Pax6, Dlx, Nurr1, Lmx1a, Lmx1b, Msx1, Foggy, and Too Few (Tof͞Fez1), have been implicated in the specification of DA phenotype in vertebrates (2-7, 35). Despite this knowledge, the mechanisms leading to the early commitment of pluripotent neural stem cells to DA lineage remain elusive.The earliest DA neurons in zebrafish are detected at Ϸ24 h postfertilization (hpf) in the basal forebrain (8). They express tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine synthesis, and dopamine transporter (DAT), a protein involved in dopamine reuptake (9). Later during development, these DA neurons have both ascending and descending projections, and are believed to be homologous to mammalian DA neurons of both the basal forebrain and midbrain (10,11).Through forward genetic analysis, an adult viable zebrafish mutant named too few (tof m808 ) has been isolated that displays selective deficits of basal forebrain DA as well as adjacent serotonergic (5HT) neurons (8). Molecular characterizations have revealed that the too few mutant carries a point mutation that changes Cys-287 t...

Loss of PINK1 Function Affects Development and Results in Neurodegeneration in Zebrafish

“…This sized PINK1 band has been described in the human brain Consistent with loss of PINK1 causing increased GSK3␤ activity, we observed an increase in the expression of fezl (a gene regulated by the wnt/GSK3␤ pathway) (Hashimoto et al, 2000b) and its downstream target neurogenin-1 in our PINK1 morphants. Our PINK1 morphants had decreased numbers of dopaminergic neurons, whereas neurogenin-1 overexpression in otherwise normal zebrafish leads to an increase in this neuronal population (Jeong et al, 2006). Thus, it is likely that the phenotype mediated by GSK3␤ activity in the brain is independent of neurogenin-1 and is mediated by other pathways regulated by this broad-acting enzyme.…”

“…However, fezl mRNA, one of the factors required for the development of the dopaminergic system in zebrafish, was selectively increased in the posterior diencephalon of PINK1 morphants. Neurogenin-1, a downstream target of fezl (Jeong et al, 2006), was similarly upregulated in PINK1 morphants (Fig. 2 E).…”

Neural protein Olig2 acts upstream of the transcriptional regulator sim1 to specify diencephalic dopaminergic neurons

“…We used Neurog1 as a genetic neuroanatomical landmark as this is the first gene product known to appear in PT progenitors destined towards the DA neuronal fate (Jeong et al, 2006;Russek-Blum et al, 2008). To allow high-resolution expression analysis of sim1 and olig2, we performed fluorescent in situ hybridization of sim1 mRNA in transgenic embryos expressing either the olig2::gfp (Shin et al, 2003) or the neurog1::gfp (Blader et al, 2003) fluorescent reporters ( Fig.…”

Expression of ventral diencephalon‐enriched genes in zebrafish

“…Currently, in addition to those found in mice or human beings, several factors, including Ngn1, that play important roles in the development of zebrafish DA neurons, have been discovered. Ngn1 was reported to be expressed in DA progenitors and essential for the specification of DA progenitors (Jeong et al, 2006). Fezl and Otp were also reported to play important roles in the development of a certain subgroup of DA neurons (Levkowitz et al, 2003;Ryu et al, 2007).…”