The zebrafish mutation m865 affects formation of dopaminergic neurons and neuronal survival, and maps to a genetic interval containing the sepiapterin reductase locus.

The zebrafish mutation m865 was isolated during a large-scale mutagenesis screen aimed at identifying genes involved in the development and maintenance of subgroups of neurons in the zebrafish central nervous system. The phenotype of m865 mutant embryos shows defects in the development of dopaminergic neurons in the pretectum and of retinal amacrine cells, as well as abnormal caudal dopaminergic cluster in the diencephalon. The effects of the mutation appear not to be restricted to dopaminergic neurons, as development of other neurotransmitter systems (serotonergic and cholinergic) is impaired as well. Furthermore, increased apoptosis is localized to the m865 mutant retina and in the optic tectum starting at 24hpf, and may lead to the observed reduced size of the mutant head and eye. Early patterning is not affected in m865 mutant embryos, and expression of genes known to play a role in dopaminergic cell differentiation is normal except for reduced expression of nurr1 in the mutant retina. Thus the m865 mutation does not specifically affect dopaminergic neuron development. m865 was genetically mapped to linkage group 5, and the critical genomic interval could be narrowed down to a region of 110 kb, containing four candidate genes. For one of these candidate genes, sepiapterin reductase (spr), a requirement for neuronal survival has previously been implicated, including dopaminergic neurons. Identification of the mutated gene should lead to a more detailed understanding of the defects observed in m865 mutant embryos, and potentially could enhance the understanding of the development and maintenance of specific dopaminergic neuronal populations.

Differential roles of transcriptional mediator complex subunits Crsp34/Med27, Crsp150/Med14 and Trap100/Med24 during zebrafish retinal development.

The transcriptional mediator complex has emerged as an important component of transcriptional regulation, yet it is largely unknown whether its subunits have differential functions in development. We demonstrate that the zebrafish mutation m885 disrupts a subunit of the mediator complex, Crsp34/Med27. To explore the role of the mediator in the control of retinal differentiation, we employed two additional mutations disrupting the mediator subunits Trap100/Med24 and Crsp150/Med14. Our analysis shows that loss of Crsp34/Med27 decreases amacrine cell number, but increases the number of rod photoreceptor cells. In contrast, loss of Trap100/Med24 decreases rod photoreceptor cells. Loss of Crsp150/Med14, on the other hand, only slightly reduces dopaminergic amacrine cells, which are absent from both crsp34(m885) and trap100(lessen) mutant embryos. Our data provide evidence for differential requirements for Crsp34/Med27 in developmental processes. In addition, our data point to divergent functions of the mediator subunits Crsp34/Med27, Trap100/Med24, and Crsp150/Med14 and, thus, suggest that subunit composition of the mediator contributes to the control of differentiation in the vertebrate CNS.

Depletion of minichromosome maintenance protein 5 in the zebrafish retina causes cell-cycle defect and apoptosis.

In multicellular organisms, the control of genome duplication and cell division must be tightly coordinated. Essential roles of the minichromosome maintenance (MCM) proteins for genome duplication have been well established. However, no genetic model has been available to address the function of MCM proteins in the context of vertebrate organogenesis. Here, we present positional cloning of a zebrafish mcm5 mutation and characterization of its retina phenotype. In the retina, mcm5 expression correlates closely with the pattern of cell proliferation. By the third day of development, mcm5 is down-regulated in differentiated cells but is maintained in regions containing retinal stem cells. We demonstrate that a gradual depletion of maternally derived MCM5 protein leads to a prolonged S phase, cell-cycle-exit failure, apoptosis, and reduction in cell number in mcm5(m850) mutant embryos. Interestingly, by the third day of development, increased apoptosis is detectable only in the retina, tectum, and hindbrain but not in other late-proliferating tissues, suggesting that different tissues may employ distinct cellular programs in responding to the depletion of MCM5.

Noradrenergic neurons in the zebrafish hindbrain are induced by retinoic acid and require tfap2a for expression of the neurotransmitter phenotype.

Tfap2a is a transcriptional activator expressed in many different cell types, including neurons, neural crest derivatives and epidermis. We show that mutations at the zebrafish locus previously called mont blanc (mob) or lockjaw (low) encode tfap2a. The mutant phenotype reveals that tfap2a is essential for the development of hindbrain noradrenergic (NA) neurons of the locus coeruleus, medulla and area postrema, as well as for sympathetic NA neurons, epibranchial placode derived visceral sensory ganglia, and craniofacial and trunk crest derivatives. We focus our analysis on the role of tfap2a NA differentiation in the CNS. In the locus coeruleus, Phox2a and Tfap2a are co-expressed and are both required for NA development. By contrast, in the medulla Phox2a and Tfap2a are expressed in adjacent overlapping domains, but only tfap2a activity is required for NA differentiation, as NA neurons develop normally in soulless/phox2a mutant medulla. phox2a and tfap2a do not appear to affect each others expression. Our studies show that two distinct inductive mechanisms control NA development in the zebrafish hindbrain. For the posterior hindbrain, we identify retinoic acid as an important signal to induce NA differentiation in the medulla oblongata and area postrema, where it expands the tfap2a expression domain and thus acts upstream of tfap2a. By contrast, previous work revealed Fgf8 to be involved in specification of NA neurons in the locus coeruleus. Thus, although the inductive signals may be distinct, hindbrain NA neurons of the locus coeruleus and the posterior groups both require Tfap2a to establish their noradrenergic identity.