Ivor Mason

A role for FGF-8 in the initiation and maintenance of vertebrate limb bud outgrowth

BACKGROUND The outgrowth of the vertebrate limb bud is the result of a reciprocal interaction between the mesenchyme and a specialized region of the ectoderm, the apical ectodermal ridge (AER), which overlies it. Signals emanating from the AER act to maintain the underlying mesenchyme, called the progress zone, in a highly proliferative and undifferentiated state. Removal of the AER results in the cessation of limb bud growth, thus causing limb truncation. The best candidates for this AER-derived signal are members of the fibroblast growth factor (FGF) family, in particular FGF-4, which can maintain limb bud outgrowth following removal of the AER. However, FGF-4 is only expressed after considerable outgrowth has occurred and a well-developed limb bud has formed, and then only in the posterior part of the AER. Likewise, the other FGFs studied to date are not candidates for this activity. RESULTS We report evidence that a recently identified member of this family, FGF-8, is expressed in the ectoderm of the prospective limb territory prior to morphological outgrowth of the limb bud in both mouse and chick. Thereafter, expression is maintained throughout the AER during limb development. We have produced and purified the FGF-8 protein, and shown that it will substitute for the AER in maintaining limb bud outgrowth in mouse embryos from which the AER has been surgically removed. FGF-8 does not, however, maintain expression of the sonic hedgehog gene. CONCLUSIONS These results indicate that FGF-8 is an AER-derived mitogen that stimulates limb bud outgrowth. Moreover, our data suggest that FGF-8 may also be an ectodermally derived mitogen that stimulates the onset of limb bud outgrowth (budding) in the absence of a morphological AER, and indicate the possible involvement of FGF-8 in the establishment of the limb field.

FGF-7 (keratinocyte growth factor) expression during mouse development suggests roles in myogenesis, forebrain regionalisation and epithelial-mesenchymal interactions

We have isolated cDNA and genomic clones for the murine FGF-7 gene and examined its expression throughout development. Transcripts were transiently detected in the developing myocardium, differentially regulated between the atrium and ventricle. The gene was also expressed in the myotomes of the somites, coincident with FGF-4 and FGF-5 transcripts, and was detected transiently in cleaved muscles. Regional expression was detected in the ventricular zone of the developing forebrain at 14.5 d.p.c. Later in development, FGF-7 RNA was detected in mesenchymal tissues suggesting a role in epithelial-mesenchymal interactions and in the dermis consistent with its proposed role as a keratinocyte mitogen. Our results suggest that FGF-7 is likely to have diverse roles during development.

Initiation to end point: the multiple roles of fibroblast growth factors in neural development

From a wealth of experimental findings, derived from both in vitro and in vivo experiments, it is becoming clear that fibroblast growth factors regulate processes that are central to all aspects of nervous system development. Some of these functions are well known, whereas others, such as the roles of these proteins in axon guidance and synaptogenesis, have been established only recently. The emergent picture is one of remarkable economy, in which this family of ligands is deployed and redeployed at successive developmental stages to sculpt the nervous system.

FGF-1 and FGF-7 Induce Distinct Patterns of Growth and Differentiation in Embryonic Lung Epithelium

Fibroblast growth factors (FGFs) and receptors (FGFRs) are expressed in the developing lung and appear to be major regulators of lung growth and differentiation. By using mesenchyme‐free lung epithelial cultures we show that FGF‐1 (aFGF) and FGF‐7 (KGF) produce different effects in the developing lung. FGF‐1 stimulates epithelial proliferation that results in bud formation (branching), while FGF‐7 promotes epithelial proliferation that leads to formation of cyst‐like structures. In addition, FGF‐7 stimulates epithelial differentiation, stimulating expression of SP‐A and SP‐B mRNA throughout the explant, and inducing formation of focal areas of highly differentiated cells. The FGF‐1 effects on differentiation are limited to induction of surfactant protein SP‐B mRNA at the tips of the explant. The FGF‐induced patterns of growth appear to correlate with the distribution of epithelial FGFRs mRNAs; FGFR‐2 IIIb (KGFR) is diffusely expressed in the day 11 lung epithelium, while FGFR‐4 appears in distal but not in proximal sites. We propose that cyst‐like structures may result from FGF‐7 binding to the uniformly distributed FGFR‐2‐IIIb. Lung bud formation may be regulated by FGF‐1 and/or other ligands binding to FGFR‐2 and a distally located FGFR, such as FGFR‐4, leading to an increasing binding and activation of FGFRs at the tips of the explant. Thus, in the embryonic lung epithelium, growth effects of FGFs appear to be dependent on location of FGFRs, while effects on differentiation are ligand‐dependent. Dev. Dyn. 208:398–405, 1997.

Fgf8 expression defines a morphogenetic center required for olfactory neurogenesis and nasal cavity development in the mouse

In vertebrate olfactory epithelium (OE), neurogenesis proceeds continuously, suggesting that endogenous signals support survival and proliferation of stem and progenitor cells. We used a genetic approach to test the hypothesis that Fgf8 plays such a role in developing OE. In young embryos, Fgf8 RNA is expressed in the rim of the invaginating nasal pit (NP), in a small domain of cells that overlaps partially with that of putative OE neural stem cells later in gestation. In mutant mice in which the Fgf8 gene is inactivated in anterior neural structures, FGF-mediated signaling is strongly downregulated in both OE proper and underlying mesenchyme by day 10 of gestation. Mutants survive gestation but die at birth, lacking OE, vomeronasal organ (VNO), nasal cavity, forebrain, lower jaw, eyelids and pinnae. Analysis of mutants indicates that although initial NP formation is grossly normal, cells in the Fgf8-expressing domain undergo high levels of apoptosis, resulting in cessation of nasal cavity invagination and loss of virtually all OE neuronal cell types. These findings demonstrate that Fgf8 is crucial for proper development of the OE, nasal cavity and VNO, as well as maintenance of OE neurogenesis during prenatal development. The data suggest a model in which Fgf8 expression defines an anterior morphogenetic center, which is required not only for the sustenance and continued production of primary olfactory (OE and VNO) neural stem and progenitor cells, but also for proper morphogenesis of the entire nasal cavity.

Establishment of Hindbrain Segmental Identity Requires Signaling by FGF3 and FGF8

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.

Expression of sprouty2 during early development of the chick embryo is coincident with known sites of FGF signalling

The Drosophila sprouty protein is a recently-identified intracellular modulator of FGF and EGF receptor tyrosine kinase activity which antagonises ras/MAP kinase signalling. In a differential display analysis to identify genes involved in patterning the mid/hindbrain region of the chick neural tube, we have identified a sprouty orthologue, sprouty2. Here we report expression of sprouty2 transcripts in the developing chick embryo. We find a close correlation with known sites of FGF activity but little correlation with expression patterns of members of the EGF family. Initially, transcripts are associated with the primitive streak. During the period of neural tube patterning expression is detected in the anterior neuropore, in the isthmic region and in neural plate and posterior spinal cord. Transcripts are also detected in the otic placode, tail bud, mesoderm of the branchial arches, somitic myotome, retina, limb buds and gut mesenchyme; all known sites of FGF action.

Expression of FGFR1, FGFR2 and FGFR3 during early neural development in the chick embryo.

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.

Specific regions within the embryonic midbrain and cerebellum require different levels of FGF signaling during development

Prospective midbrain and cerebellum formation are coordinated by FGF ligands produced by the isthmic organizer. Previous studies have suggested that midbrain and cerebellum development require different levels of FGF signaling. However, little is known about the extent to which specific regions within these two parts of the brain differ in their requirement for FGF signaling during embryogenesis. Here, we have explored the effects of inhibiting FGF signaling within the embryonic mouse midbrain (mesencephalon) and cerebellum (rhombomere 1) by misexpressing sprouty2 (Spry2) from an early stage. We show that such Spry2 misexpression moderately reduces FGF signaling, and that this reduction causes cell death in the anterior mesencephalon, the region furthest from the source of FGF ligands. Interestingly, the remaining mesencephalon cells develop into anterior midbrain, indicating that a low level of FGF signaling is sufficient to promote only anterior midbrain development. Spry2 misexpression also affects development of the vermis, the part of the cerebellum that spans the midline. We found that, whereas misexpression of Spry2 alone caused loss of the anterior vermis, reducing FGF signaling further, by decreasing Fgf8 gene dose, resulted in loss of the entire vermis. Our data suggest that cell death is not responsible for vermis loss, but rather that it fails to develop because reducing FGF signaling perturbs the balance between vermis and roof plate development in rhombomere 1. We suggest a molecular explanation for this phenomenon by providing evidence that FGF signaling functions to inhibit the BMP signaling that promotes roof plate development.

Unique and combinatorial functions of Fgf3 and Fgf8 during zebrafish forebrain development

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.