FGF-8 stimulates neuronal differentiation through FGFR-4a and interferes with mesoderm induction in Xenopus embryos

The role of fibroblast growth factors (FGFs) in neural induction is controversial [1,2]. Although FGF signalling has been implicated in early neural induction [3-5], a late role for FGFs in neural development is not well established. Indeed, it is thought that FGFs induce a precursor cell fate but are not able to induce neuronal differentiation or late neural markers [6-8]. It is also not known whether the same or distinct FGFs and FGF receptors (FGFRs) mediate the effects on mesoderm and neural development. We report that Xenopus embryos expressing ectopic FGF-8 develop an abundance of ectopic neurons that extend to the ventral, non-neural, ectoderm, but show no ectopic or enhanced notochord or somitic markers. FGF-8 inhibited the expression of an early mesoderm marker, Xbra, in contrast to eFGF, which induced ectopic Xbra robustly and neuronal differentiation weakly. The effect of FGF-8 on neurogenesis was blocked by dominant-negative FGFR-4a (DeltaXFGFR-4a). Endogenous neurogenesis was also blocked by DeltaXFGFR-4a and less efficiently by dominant-negative FGFR-1 (XFD), suggesting that it depends preferentially on signalling through FGFR-4a. The results suggest that FGF-8 and FGFR-4a signalling promotes neurogenesis and, unlike other FGFs, FGF-8 interferes with mesoderm induction. Thus, different FGFs show specificity for mesoderm induction versus neurogenesis and this may be mediated, at least in part, by the use of distinct receptors.     

FGF8 spliceforms mediate early mesoderm and posterior neural tissue formation in Xenopus

The relative contributions of different FGF ligands and spliceforms to mesodermal and neural patterning in Xenopus have not been determined, and alternative splicing, though common, is a relatively unexplored area in development. We present evidence that FGF8 performs a dual role in X. laevis and X. tropicalis early development. There are two FGF8 spliceforms, FGF8a and FGF8b, which have very different activities. FGF8b is a potent mesoderm inducer, while FGF8a has little effect on the development of mesoderm. When mammalian FGF8 spliceforms are analyzed in X. laevis, the contrast in activity is conserved. Using a loss-of-function approach, we demonstrate that FGF8 is necessary for proper gastrulation and formation of mesoderm and that FGF8b is the predominant FGF8 spliceform involved in early mesoderm development in Xenopus. Furthermore, FGF8 signaling is necessary for proper posterior neural formation; loss of either FGF8a or a reduction in both FGF8a and FGF8b causes a reduction in the hindbrain and spinal cord domains.     

Cloning and developmental expression of Sna, a murine homologue of the Drosophila snail gene.

The genetic analysis of dorsoventral patterning in Drosophila has identified a zinc-finger gene, snail, that is required for mesoderm formation. The cloning and nuclease protection analysis of a Xenopus homologue of this gene has suggested a possible role in the mesoderm of vertebrates. Here, we describe the cloning of a murine homologue of snail, Sna, and in situ hybridisation studies of its developmental expression. Sequence analysis reveals substantial conservation of the second to fifth zinc fingers, but not of the first zinc finger in the Sna gene. Expression occurs in the ectoplacental cone, parietal endoderm, embryonic and extraembryonic mesoderm, in neural crest and in condensing precartilage. Based on the timing and spatial restriction of expression in embryonic mesoderm, we suggest that Sna might be required for the early development of this tissue, as is the case for its Drosophila counterpart. In addition, we propose that Sna might have an analogous role in the development of neural crest. The expression in condensing precartilage indicates that this gene also has a later function in chondrogenesis.     

Neural crest determination by co-activation of Pax3 and Zic1 genes in Xenopus ectoderm

A number of regulatory genes have been implicated in neural crest development. However, the molecular mechanism of how neural crest determination is initiated in the exact ectodermal location still remains elusive. Here, we show that the cooperative function of Pax3 and Zic1 determines the neural crest fate in the amphibian ectoderm. Pax3 and Zic1 are expressed in an overlapping manner in the presumptive neural crest area of the Xenopus gastrula, even prior to the onset of the expression of the early bona fide neural crest marker genes Foxd3 and Slug. Misexpression of both Pax3 and Zic1 together efficiently induces ectopic neural crest differentiation in the ventral ectoderm, whereas overexpression of either one of them only expands the expression of neural crest markers within the dorsolateral ectoderm. The induction of neural crest differentiation by Pax3 and Zic1 requires Wnt signaling. Loss-of-function studies in vivo and in the animal cap show that co-presence of Pax3 and Zic1 is essential for the initiation of neural crest differentiation. Thus, co-activation of Pax3 and Zic1, in concert with Wnt, plays a decisive role for early neural crest determination in the correct place of the Xenopus ectoderm.     

Interleukin-1 regulates FGF-2 mRNA and localization of FGF-2 protein in human osteoblasts

Interleukin-1 (IL-1) and basic fibroblast growth factor (FGF-2) are potent stimulators of osteoclast formation. However, the role of FGF-2 in the responses to IL-1 in bone has not been reported. We examined the effect of IL-1 on FGF-2 mRNA and protein expression in human osteosarcoma MG-63 osteoblasts, normal human osteoblasts (NHOB), and osteoblasts from osteoarthritic patients (F2 and F13). IL-1 increased FGF-2 mRNA expression in osteoblasts within 1.5 to 3 h. Multiple FGF-2 protein isoforms were expressed in human osteoblasts. Twenty-four hours of treatment of MG-63 and NHOB cells with IL-1 increased the high-molecular-weight(HMW, 22/24 kDa) and low-molecular-weight (LMW, 18 kDa) FGF-2 proteins intracellularly. In contrast, IL-1 preferentially increased the LMW protein signal intracellularly as well as on the cell surface of F2 and F13 osteoblasts. We conclude that IL-1 is a major stimulator of FGF-2 expression in human osteoblasts. Furthermore, selective increases in the exportable LMW protein in osteoblasts from osteoarthritic patients may be of clinical relevance.     

FGF-10 stimulates limb regeneration ability in Xenopus laevis

By reciprocal transplantation experiments with regenerative and nonregenerative Xenopus limbs, we recently demonstrated that the regenerative capacity of a Xenopus limb depends on mesenchymal tissue and we suggested that fgf-10 is likely to be involved in this capacity (Yokoyama et al., 2000, Dev. Biol. 219, 18-29). However, the data obtained in that study are not conclusive evidence that FGF-10 is responsible for the regenerative capacity. We therefore investigated the role of FGF-10 in regenerative capacity by directly introducing FGF-10 protein into nonregenerative Xenopus limb stumps. Exogenously applied FGF-10 successfully stimulated the regenerative capacity, resulting in the reinduction of all gene expressions (including shh, msx-1, and fgf-10) that we examined and the regeneration of well-patterned limb structures. We report here for the first time that a certain molecule activates the regenerative capacity of Xenopus limb, and this finding suggests that FGF-10 could be a key molecule in possible regeneration of nonregenerative limbs in higher vertebrates.     

Developmental expression of the Xenopus int-2 (FGF-3) gene: activation by mesodermal and neural induction.

We have used a probe specific for the Xenopus homologue of the mammalian proto-oncogene int-2 (FGF-3) to examine the temporal and spatial expression pattern of the gene during Xenopus development. int-2 is expressed from just before the onset of gastrulation through to prelarval stages. In the early gastrula, it is expressed around the blastopore lip. This is maintained in the posterior third of the prospective mesoderm and neuroectoderm in the neurula. A second expression domain in the anterior third of the neuroectoderm alone appears in the late gastrula, which later resolves into the optic vesicles, hypothalamus and midbrain-hindbrain junction region. Further domains of expression arise in tailbud to prelarval embryos, including the stomodeal mesenchyme, the endoderm of the pharyngeal pouches and the cranial ganglia flanking the otocyst. It is shown, by treatment of blastula ectoderm with bFGF and activin, that int-2 can be expressed in response to mesoderm induction. By heterotypic grafting of gastrula ectoderm into axolotl neural plate, we have also demonstrated that int-2 can be expressed in response to neural induction. These results suggest that int-2 has multiple functions in development, including an early role in patterning of the anteroposterior body axis and a later role in the development of the tail, brain-derived structures and other epithelia.     

Tra2betal regulates P19 neuronal differentiation and the splicing of FGF-2R and GluR-B minigenes

The present study demonstrates that the expression of Tra2beta1 (Transformer 2-beta1) proteins, an SR (serine/arginine rich) protein, is developmentally up-regulated in a neural-specific pattern. The up-regulation is also observed in RA (retinoic acid) induced neural differentiation of P19 cells. Tra2betal proteins are located in the nuclei of P19 cells, which are consistent with its functional site as an SR protein. The over-expression of Tra2betal proteins promotes RA induced neuronal differentiation of P19 cells. In P19 cells, the splicing of FGF-2R (fibroblast growth factor receptor 2) minigene produces the BEK form, while the alternative splicing of GluR-B (glutamate receptor subunit B) minigene generates two products, the Flop and the Truncated isoforms. Tra2betal inhibits the BEK splicing, but it promotes the Flop splicing. The results therefore suggest that Tra2betal is involved in the regulation of alternative splicing processes during neural development, peculiarly the splicing of FGF-2R and GluR-B genes. Both FGF-2R and GluR-B genes are known to play important roles in neural differentiation.     

altFGF-2, A Novel ER-Associated FGF-2 Protein Isoform: Its Embryonic Distribution and Functional Analysis during Neural Tube Development

A novel fibroblast growth factor-2 (FGF-2) protein isoform, calledaltFGF-2, is expressed abundantly during chicken embryogenesis. The amino-terminal domain of the 21.5-kDaaltFGF-2 protein diverges completely from the other three FGF-2 proteins due to alternative splicing of their first coding exons. Furthermore, thealtFGF-2 protein, in contrast to FGF-2 proteins, is targeted predominantly to the endoplasmic reticulum. In chicken embryos,altFGF-2 and FGF-2 proteins are differentially distributed in several mesodermal structures including developing limbs and kidneys. All four FGF-2 protein isoforms are also expressed in the developing neural tube from early neural plate stages onward. In contrast to FGF-2 proteins, thealtFGF-2 isoform is distributed in a dynamic, spatially restricted pattern in notochord and ventral neural tube (floor plate and motor neurons) during specification of neuronal populations. To study the possible shared or differential signaling functions of chickenaltFGF-2 and FGF-2 gene products, they were ectopically expressed in the dorsal neural tube aspect of transgenic mouse embryos. Dorsal expression ofaltFGF-2, but not FGF-2 gene products, induced alteration of neural tube morphology in a significant fraction of mouse embryos (25%). However, no alterations of dorsoventral (d/v) neural tube polarity were detected, indicating thataltFGF-2 and FGF-2 gene products either function as permissive cofactors or regulate neural tube growth without affecting establishment of its primary d/v polarity.     

Molecular cloning and characterization of human FGF-20 on chromosome 8p21.3-p22

The fibroblast growth factors (FGFs) play important roles in morphogenesis, angiogenesis, tissue remodeling, and carcinogenesis. Human FGF-20 has been cloned and characterized in this study. FGF-20 encodes a 211-amino-acid polypeptide with the FGF-core domain. A strong hydrophobic region was found in the FGF-core domain of FGF-20; however, no typical N-terminal signal sequence was found in FGF-20, just as in FGF-9 and FGF-16. Total amino acid identities are as follows: FGF-20 vs FGF-9, 71.6%; FGF-20 vs FGF-16, 66.2%; FGF-9 vs FGF-16, 72.4%. Phylogenic analysis indicated that FGF-20, FGF-9, and FGF-16 constitute a subfamily among the FGF family. FGF-20 mRNA of 2.4 kb in size was detected in colon cancer cell line SW480 by Northern blot analysis. Lower levels of FGF-20 mRNA were detected in human fetal tissues and primary cancers by cDNA-PCR. The nucleotide sequence of FGF-20 cDNA is split into three parts in the human genome sequence of the chromosome 8p21.3-p22 region (Accession No. AB020858). These results indicate that the FGF-20 gene, located on human chromosome 8p21.3-p22, consists of three exons. Compared with the nucleotide sequence of FGF-20 cDNA determined in this study, one nucleotide deletion and one nucleotide substitution in the putative coding region were identified in human genome sequence AB020858.     

Neural crest induction by the canonical Wnt pathway can be dissociated from anterior-posterior neural patterning in Xenopus

While Wnt signaling is known to be involved in early steps of neural crest development, the mechanism remains unclear. Because Wnt signaling is able to posteriorize anterior neural tissues, neural crest induction by Wnts has been proposed to be an indirect consequence of posteriorization of neural tissues rather than a direct effect of Wnt signaling. To address the relationship between posteriorization and neural crest induction by Wnt signaling, we have used gain of function and loss of function approaches in Xenopus to modulate the level of Wnt signaling at multiple points in the pathway. We find that modulating the level of Wnt signaling allows separation of neural crest induction from the effects of Wnts on anterior-posterior neural patterning. We also find that activation of Wnt signaling induces ectopic neural crest in the anterior region without posteriorizing anterior neural tissues. In addition, Wnt signaling induces neural crest when its posteriorizing activity is blocked by inhibition of FGF signaling in neuralized explants. Finally, depletion of beta-catenin confirms that the canonical Wnt pathway is required for initial neural crest induction. While these observations do not exclude a role for posteriorizing signals in neural crest induction, our data, together with previous observations, strongly suggest that canonical Wnt signaling plays an essential and direct role in neural crest induction.