A mutation in cnot8, component of the Ccr4-not complex regulating transcript stability, affects expression levels of developmental regulators and reveals a role of Fgf3 in development of caudal hypothalamic dopaminergic neurons.

While regulation of the activity of developmental control genes at the transcriptional level as well as by specific miRNA-based degradation are intensively studied, little is known whether general cellular mechanisms controlling mRNA decay may contribute to differential stability of mRNAs of developmental control genes. Here, we investigate whether a mutation in the deadenylation dependent mRNA decay pathway may reveal differential effects on developmental mechanisms, using dopaminergic differentiation in the zebrafish brain as model system. In a zebrafish genetic screen aimed at identifying genes controlling dopaminergic neuron development we isolated the m1061 mutation that selectively caused increased dopaminergic differentiation in the caudal hypothalamus, while other dopaminergic groups were not affected. Positional cloning revealed that m1061 causes a premature stop codon in the cnot8 open reading frame. Cnot8 is a component of the Ccr4-Not complex and displays deadenylase activity, which is required for removal of the poly (A) tail in bulk mRNA turnover. Analyses of expression of developmental regulators indicate that loss of Cnot8 activity results in increased mRNA in situ hybridization signal levels for a subset of developmental control genes. We show that in the area of caudal hypothalamic dopaminergic differentiation, mRNA levels for several components of the FGF signaling pathway, including Fgf3, FGF receptors, and FGF target genes, are increased. Pharmacological inhibition of FGF signaling or a mutation in the fgf3 gene can compensate the gain of caudal hypothalamic dopaminergic neurons in cnot8m1061 mutants, indicating a role for Fgf3 in control of development of this dopaminergic population. The cnot8m1061 mutant phenotype provides an in vivo system to study roles of the Cnot8 deadenylase component of the mRNA decay pathway in vertebrate development. Our data indicate that attenuation of Cnot8 activity differentially affects mRNA levels of developmental control genes.

Sim1a and Arnt2 contribute to hypothalamo-spinal axon guidance by regulating Robo2 activity via a Robo3-dependent mechanism.

Precise spatiotemporal control of axon guidance factor expression is a prerequisite for formation of functional neuronal connections. Although Netrin/Dcc- and Robo/Slit-mediated attractive and repulsive guidance of commissural axons have been extensively studied, little is known about mechanisms controlling mediolateral positioning of longitudinal axons in vertebrates. Here, we use a genetic approach in zebrafish embryos to study pathfinding mechanisms of dopaminergic and neuroendocrine longitudinal axons projecting from the hypothalamus into hindbrain and spinal cord. The transcription factors Sim1a and Arnt2 contribute to differentiation of a defined population of dopaminergic and neuroendocrine neurons. We show that both factors also control aspects of axon guidance: Sim1a or Arnt2 depletion results in displacement of hypothalamo-spinal longitudinal axons towards the midline. This phenotype is suppressed in robo3 guidance receptor mutant embryos. In the absence of Sim1a and Arnt2, expression of the robo3 splice isoform robo3a.1 is increased in the hypothalamus, indicating negative control of robo3a.1 transcription by these factors. We further provide evidence that increased Robo3a.1 levels interfere with Robo2-mediated repulsive axon guidance. Finally, we show that the N-terminal domain unique to Robo3a.1 mediates the block of Robo2 repulsive activity. Therefore, Sim1a and Arnt2 contribute to control of lateral positioning of longitudinal hypothalamic-spinal axons by negative regulation of robo3a.1 expression, which in turn attenuates the repulsive activity of Robo2.

Analysis of transcriptional codes for zebrafish dopaminergic neurons reveals essential functions of Arx and Isl1 in prethalamic dopaminergic neuron development.

Distinct groups of dopaminergic neurons develop at defined anatomical sites in the brain to modulate function of a large diversity of local and far-ranging circuits. However, the molecular identity as judged from transcription factor expression has not been determined for all dopaminergic groups. Here, we analyze regional expression of transcription factors in the larval zebrafish brain to determine co-expression with the Tyrosine hydroxylase marker in dopaminergic neurons. We define sets of transcription factors that clearly identify each dopaminergic group. These data confirm postulated relations to dopaminergic groups defined for mammalian systems. We focus our functional analysis on prethalamic dopaminergic neurons, which co-express the transcription factors Arx and Isl1. Morpholino-based knockdown reveals that both Arx and Isl1 are strictly required for prethalamic dopaminergic neuron development and appear to act in parallel. We further show that Arx contributes to patterning in the prethalamic region, while Isl1 is required for differentiation of prethalamic dopaminergic neurons.

Expression of the paralogous tyrosine hydroxylase encoding genes th1 and th2 reveals the full complement of dopaminergic and noradrenergic neurons in zebrafish larval and juvenile brain.

The development of dopaminergic and noradrenergic neurons has received much attention based on their modulatory effect on many behavioral circuits and their involvement in neurodegenerative diseases. The zebrafish (Danio rerio) has emerged as a new model organism with which to study development and function of catecholaminergic systems. Tyrosine hydroxylase is the entry enzyme into catecholamine biosynthesis and is frequently used as a marker for catecholaminergic neurons. A genome duplication at the base of teleost evolution resulted in two paralogous zebrafish tyrosine hydroxylase-encoding genes, th1 and th2, the expression of which has been described previously only for th1. Here we investigate the expression of th2 in the brain of embryonic and juvenile zebrafish. We optimized whole-mount in situ hybridization protocols to detect gene expression in the anatomical three-dimensional context of whole juvenile brains. To confirm whether th2-expressing cells may indeed use dopamine as a neurotransmitter, we also included expression of dopamine beta hydroxylase, dopa decarboxylase, and dopamine transporter in our analysis. Our data provide the first complete account of catecholaminergic neurons in the zebrafish embryonic and juvenile brain. We identified four major th2-expressing neuronal groups that likely use dopamine as transmitter in the zebrafish diencephalon, including neurons of the posterior preoptic nucleus, the paraventricular organ, and the nuclei of the lateral and posterior recesses in the caudal hypothalamus. th2 expression in the latter two groups resolves a previously reported discrepancy, in which strong dopamine but little tyrosine hydroxylase immunoreactivity had been detected in the caudal hypothalamus. Our data also confirm that there are no mesencephalic DA neurons in zebrafish.