The hindbrain (brainstem) of all vertebrates follows a segmental developmental strategy and has been the focus of intense study not only for its intrinsic interest but also as a model for how more complex regions of the brain are patterned. Segmentation ultimately serves to organize the development of neuronal populations and their projections, and regional diversity is achieved through each segment having its own identity. The latter being established through differential expression of a hierarchy of transcription factors, including Hox genes, Krox20, and Kreisler/Valentino. Here we identify a novel signaling center in the zebrafish embryo that arises prior to establishment of segmental patterning and which is located centrally within the hindbrain territory in a region that corresponds to the presumptive rhombomere 4. We show that signaling from this region by two members of the FGF family of secreted proteins, FGF3 and FGF8, is required to establish correct segmental identity throughout the hindbrain and for subsequent neuronal development. Spatiotemporal studies of Fgf expression suggest that this patterning mechanism is conserved during hindbrain development in other vertebrate classes.

Characterisation of human craniofacial syndromes and studies in transgenic mice have demonstrated the requirement for Fgf signalling during morphogenesis of membrane bone of the cranium. Here, we report that Fgf activity is also required for development of the oro-pharyngeal skeleton, which develops first as cartilage with some elements subsequently becoming ossified. We show that inhibition of FGF receptor activity in the zebrafish embryo following neural crest emigration from the neural tube results in complete absence of neurocranial and pharyngeal cartilages. Moreover, this Fgf signal is required during a 6-h period soon after initiation of neural crest migration. The spatial and temporal expression of Fgf3 and Fgf8 in pharyngeal endoderm and ventral forebrain and its correlation with patterns of Fgf signalling activity in migrating neural crest makes them candidate regulators of cartilage development. Inhibition of Fgf3 results in the complete absence of cartilage elements that normally form in the third, fourth, fifth, and sixth pharyngeal arches, while those of the first, second, and seventh arches are largely unaffected. Inhibition of Fgf8 alone has variable, but mild, effects. However, inhibition of both Fgf3 and Fgf8 together causes a complete absence of pharyngeal cartilages and the near-complete loss of the neurocranial cartilage. These data implicate Fgf3 and Fgf8 as key regulators of cartilage formation in the vertebrate head.

Complex spatiotemporal expression patterns of fgf3 and fgf8 within the developing zebrafish forebrain suggest their involvement in its regionalisation and early development. These factors have unique and combinatorial roles during development of more posterior brain regions, and here we report similar findings for the developing forebrain. We show that Fgf8 and Fgf3 regulate different aspects of telencephalic development, and that Fgf3 alone is required for the expression of several telencephalic markers. Within the diencephalon, Fgf3 and Fgf8 act synergistically to pattern the ventral thalamus, and are implicated in the regulation of optic stalk formation, whereas loss of Fgf3 alone results in defects in ZLI development. Forebrain commissure formation was abnormal in the absence of either Fgf3 or Fgf8; however, most severe defects were observed in the absence of both. Defects were observed in patterning of both the midline territory, within which the commissures normally form, and neuronal populations, whose axons comprise the commissures. Analysis of embryos treated with an FGFR inhibitor suggests that continuous FGF signalling is required from gastrulation stages for normal forebrain patterning, and identifies additional requirements for FGFR activity. (Fukuchi-Shimogori and Grove, 2001), and by analysis of the zebrafish acerebellar (ace) mutant (Shanmugalingam et al., 2000). Although detailed analyses are lacking, fgf3 is also expressed in the forebrain, (Mahmood et al., 1996;Raible and Brand, 2001;Walshe et al., 2002) and its ectopic expression affects the expression of certain forebrain markers .We report a complex and dynamic expression pattern for fgf3 in the zebrafish forebrain, which partially overlaps with that of fgf8. Using morpholino oligonucleotides to inhibit Fgf3 and Fgf8, both individually and together, we identify unique functions for Fgf3 in both telencephalon and several regions of the diencephalon, and in combinatorial actions with Fgf8. In addition, we report further roles for Fgf8 in forebrain development. MATERIALS AND METHODS Fish stocksZebrafish, Danio rerio, of the King's wild-type (kwt) strain were used throughout these studies. They were maintained at 28°C, and embryos were staged according to Kimmel et al. (Kimmel et al., 1995). Morpholino oligonucleotide injectionsFgf8, Fgf3 and Fgf control morpholino oligonucleotides (Gene Tools), at a concentration of 6 µg/µl, were injected into zebrafish embryos as previously described . Embryos were dechorionated and incubated with the FGFR inhibitor SU5402, as previously described , except that SU5402 stock solutions were prepared at 10 mM and diluted to 0.1 mM for use. In situ hybridisationIn situ hybridisation reactions were essentially performed as described previously (Shamim et al., 1999;Maroon et al., 2002), except that embryos younger than 24 hours post fertilisation (hpf) were not treated with proteinase K, and the hydrogen peroxide treatment was omitted. Cell death and divisionDividing cells were detected using an anti-phosphorylat...

Studies involving chick embryos have implicated FGFs in neural induction and patterning as well as in other developmental events. Detailed analyses of FGF receptor expression at early stages of neural development have not been reported for the chick embryo and are incomplete for other vertebrate classes. Here we show the expression patterns of three FGF receptors, (FGFR1, FGFR2 and FGFR3) in embryonic stages between gastrulation and limb bud formation, focussing particularly on neural tissues. Between neural induction and neurulation, all three receptors are expressed in the neural plate albeit with distinct and overlapping distributions. During early neuromere formation FGFR1 transcripts are present throughout the neural tube, while transcripts for FGFR2 and FGFR3 become restricted to regions of the diencephalon and spinal cord. A little later, FGFR2 and FGFR3 are additionally expressed in the anterior midbrain and within the hindbrain. During later neuromere development, FGFR1 transcripts become localised to the telencephalon, anterior dorsal diencephalon and throughout the midbrain and hindbrain, whereas FGFR2 mRNA is restricted to dorsal telencephalon, dorsoanterior midbrain and hindbrain. FGFR3 is also expressed in anterior midbrain and hindbrain during this developmental period, and is additionally expressed in the posterior telencephalon, in the pretectum, and at the zona limitans intrathalamica. The observed expression patterns of all three receptors within the hindbrain, including rhombomere boundaries, are complex and dynamic. Expression patterns within the somites, eye, head mesenchyme, branchial arches, limb buds, nephric kidney and pharynx are also described.

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