Signaling requirements

The availability of many mutations affecting components of signaling pathways involved in brain patterning and cell differentiation allowed us to address signaling requirements for DA neuronal specification in zebrafish. In mammalian embryos, many mutations affecting major signaling pathways are lethal prior to DA system development. In contrast, most mutant zebrafish embryos survive for at least two days since a functional cardiovascular system is not required for early development and store of maternal proteins enables cells to survive. As experiments in mammalian systems indicated a requirement of Shh and FGF8 signaling for dopaminergic neuron development (Ye et al., 1998), we tested contribution of these signaling pathways (summarized in Table 1; (Holzschuh et al., 2003b). For the Shh pathway, both syu (shh) and smu (Shh co-receptor Smoothened) mutant embryos have been analyzed. In Shh signaling deficient embryos, the early ventral dience-phalic and the olfactory bulb DA groups form, but pretectal and retinal amacrine DA cells are reduced or absent. Thus the floorplate-derived Shh signal may not contribute to specification of early ventral diencephalic DA

Table 1. Impact of signaling pathways on specification and differentiation of zebrafish catecholaminergic neurons

Signal Catecholaminergic groups

Affected Non affected Not determined*

Shh (smu, syu) PT(-0), vDC(-), AC(-) OB, LC, MO PO, SP, HY

FGF8 (ace) LC(0), vDCa(-) OB, PT, vDCp, AC, MO PO, HY

Nodal/TGF (MZsur) vDC(-0) LC, MO, AC PT, OB, PO, SP, HY

Nodal/TGF (oep or cyc) PT(0), vDC(0), HY(0), LC, MO PO, SP

Retinoic acid (>24hpf) MO (0) PT, OB, vDC, PO, SP, LC HY, AC

Abbreviations for DA groups: PT pretectal DA neurons, OB olfactory bulb DA neurons, vDCa anterior DA group ventral diencephalon (group 1 according to Rink and Wullimann), vDCp posterior DA groups in the ventral diencephalon (groups 2-6 according to Rink and Wullimann), PO preoptic group, SP DA group of subpallium, HY hypothalamic DA groups, AC amacrine, DA cells of retina, LC locus coeruleus NA neurons, MO medulla oblongata area postrema NA neurons, (-) - reduced number of cells, (0) - absent, (-0) - reduced or absent, *not determined because embryos show global defects neurons, but Shh derived from the zona limit-ans intrathalamica at the border of prosomers 2 and 3 may be involved in specification of precursors of DA neurons in the pretectum.

Analysis of ace mutant zebrafish, which are devoid of FGF8, revealed that FGF8 contributes both to specification of DA and NA neurons. Locus coeruleus NA neurons are completely absent in ace mutant embryos. Within the ventral diencephalon, the caudal DA groups form, but the anteriormost cluster of DA cells, corresponding to group 1 neurons in Rink and Wullimann (2002), appears to be absent in mutant embryos. An expression domain for FGF8 has been reported to exist in the posterior tuberculum, and may be the source of FGF signaling required for the development of these neurons. The pretectal DA group appears to develop independently of FGF8, even though an FGF8 expression domain has been reported in close proximity of this DA group in the dorsal thalamus. The fact that the majority of early differentiating ventral diencephalic DA neurons develop even in ace and smu double mutant embryos, which should be devoid of both FGF8 and Shh signaling, led us to consider additional pathways that may contribute to DA development. Nodal signals of the TGFbeta family play an important role in development of the ventral diencephalon. However, a complete depletion of Nodal signals in cyc mutants (affecting Nodal related protein 2), or in oep mutant embryos (affecting the Oep Nodal co-receptor) also deletes a significant portion of the ventral diencephalon. This makes it difficult to distinguish whether the absence of ventral diencephalic DA neurons in these mutants is caused by early patterning defects or defects in specification of DA neurons. The analysis of the zebrafish sur mutation, which affects Fast1/FoxH1, a transcription factor and transducer of Nodal signals, was more informative. In MZsur mutant embryos, which lack both maternal and zygotic expression of Fast1/FoxH1, DA neurons are often completely absent from the ventral dience-phalon. Analysis of the expression pattern of dbx1a, a genes expressed in the basal plate of prosomere 3, indicates that the posterior tuberculum still forms in MZsur embryos. This argues for the role of Nodal/TGFbeta family signals in specification of the ventral DA groups. Whether this involves direct induction of ventral diencephalon (DC) DA cells, or regulation of the prepattern of this region remains to be addressed.

Retinoic acid is directly involved in neuronal differentiation as well as in hindbrain patterning. When zebrafish embryos older

than 24 hpf are exposed to retinoic acid, segment identity, as judged by establishment of the Hox gene code is already complete and a shift in segment identities can not be induced. However, exogenous retinoic acid induces an expansion of the medullary NA group often well into rhombomere 3 (Holzschuh et al., 2003a). These findings argue that within a dorsoventral domain competent to form NA neurons, the rostro-caudal retinoic acid gradient determines the anterior limit of NA differentiation. Other CNS DA or NA groups are not affected by RA under these conditions.

Delta/Notch signaling is also involved in DA specification, but its role has not been analyzed in detail. Mutations in mib, a ubi-quitin ligase required for proper Delta/Notch signaling, generate supernummary DA neurons in all clusters, indicating that this neuro-genic switch is broadly involved in restricting precursors from differentiating into DA neurons (Holzschuh and Driever, unpublished).

Several other signaling systems play important roles in neural differentiation. However, for some, including the Wnt signaling pathway, manipulation of the signaling pathway in the whole embryo causes severe early anterioposterior patterning defects in the neural plate which makes it impossible to analyze its specific contributions to DA or NA specification. For some other signaling pathways, mutations are currently not available.

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