The proper guidance of the C. elegans hermaphrodite sex myoblasts (SMs) requires the genes egl-15 and egl-17. egl-15 has been shown to encode the C. elegans orthologue of the fibroblast growth factor receptor. egl-17 was cloned and is a member of the fibroblast growth factor family, one of the first functional invertebrate FGFs known. egl-17 shares homology with other FGF members, conserving the key residues required to form the distinctive tertiary structure common to FGFs. The SM migration defect seen in egl-17 mutant animals represents complete loss of egl-17 function. While mutations in egl-17 affect only SM migrations, mutations in egl-15 can result in larval arrest, and scrawny body morphology (Burdine, 1997).
During the development of the egg-laying system in Caenorhabditis elegans hermaphrodites, central gonadal cells organize the alignment of the vulva with the sex myoblasts, the progenitors of the egg-laying muscles. A fibroblast growth factor [EGL-17(FGF)] and an FGF receptor [EGL-15(FGFR)] are involved in the gonadal signals that guide the migrations of the sex myoblasts (SMs). SMs are generated in the posterior half of the mid-body region and undergo anterior migrations to final positions that flank the center of the developing gonad. When the gonad is destroyed by laser ablation, the SMs still migrate anteriorly but take up final positions within a broad, centrally dispersed range. Thus, the gonad is responsible for the precise positioning of the SMs. This precise positioning appears to result from a gonad-dependent attractive signaling mechanism, since the SMs can migrate into novel territory in response to an altered positioning of the gonad. EGL-17(FGF) can act as an instructive guidance cue to direct the sex myoblasts to their final destinations. egl-17 reporter constructs are expressed in the primary vulval cell and EGL-17(FGF) expression in this cell correlates with the precise positioning of the sex myoblasts. It is postulated that EGL-17(FGF) helps to coordinate the development of a functional egg-laying system, linking vulval induction with proper sex myoblast migration. Vulval expression of egl-17 is dependent on vulval induction, requiring the action of a Ras-MAP kinase signal that is activated by Epidermal growth factor dependent signaling. egl-17 is expressed in vulval cells early in primary cell lineages and late in secondary cell lineages. A gonad-dependent repulsion mechanism may occur when egl-17 dependent function is eliminated (Burdine, 1998).
The tripartite organization of the central nervous system (CNS) may be an ancient character of the bilaterians. However, the elaboration of the more complex vertebrate brain depends on the midbrain-hindbrain boundary (MHB) organizer, which is absent in invertebrates such as Drosophila. The Fgf8 signaling molecule expressed in the MHB organizer plays a key role in delineating separate mesencephalon and metencephalon compartments in the vertebrate CNS. This study presents evidence that an Fgf8 ortholog establishes sequential patterns of regulatory gene expression in the developing posterior sensory vesicle, and the interleaved 'neck' region located between the sensory vesicle and visceral ganglion of the simple chordate Ciona intestinalis. The detailed characterization of gene networks in the developing CNS led to new insights into the mechanisms by which Fgf8/17/18 patterns the chordate brain. The precise positioning of this Fgf signaling activity depends on an unusual AND/OR network motif that regulates Snail, which encodes a threshold repressor of Fgf8 expression. Nodal is sufficient to activate low levels of the Snail repressor within the neural plate, while the combination of Nodal and Neurogenin produces high levels of Snail in neighboring domains of the CNS. The loss of Fgf8 patterning activity results in the transformation of hindbrain structures into an expanded mesencephalon in both ascidians and vertebrates, suggesting that the primitive MHB-like activity predates the vertebrate CNS (Imai, 2009).
This study provides a number of key insights into the compartmentalization of the chordate CNS. First, a localized Fgf8 signaling center was probably used by the last shared ancestor of ascidians and vertebrates to delineate two regions of the chordate brain (mesencephalon and metencephalon). Second, Fgf8 signaling in Ciona leads to restricted expression of Otx and FoxB in the PSV, as well as restricted expression of Pax2/5/8-A in the neck. Otx and FoxB might inhibit Hox1 expression in the forebrain via Cyp26, whereas Pax2/5/8-A might coordinate the expression of the regulatory genes required for the differentiation of metencephalon motoneurons, such as Phox2a/Arix. Finally, although the regulatory genes responsible for the compartmentalization of the vertebrate CNS (e.g. Otx, Pax2, Neurogenin, etc.) exhibit comparable patterns of expression in the Ciona CNS, there are both conserved and distinctive features of the underlying mechanism. Localized Fgf8 signaling is used to deploy these expression patterns in both systems, even though different regulatory mechanisms are used to restrict Fgf8 (Imai, 2009).
In mammals, 16 members of the Fgf family have so far been described with diverse roles in embryonic cell growth and differentiation. The expression from early streak stage to midgestation is described of two newly-identified murine genes, Fgf17 and Fgf18, that are most closely related to Fgf8 (63.7% and 56.8% identical, respectively, at the amino acid level). Fgf17 is expressed during gastrulation but at lower levels than Fgf8, while Fgf18 RNA is not expressed until later, in paraxial mesoderm. In the developing tail bud, each Fgf gene shows a different pattern of transcription. Distinct and overlapping expression patterns are also described in the developing brain and limbs (Maruoka, 1998).
It is widely accepted that tissue differentiation and morphogenesis in multicellular organisms are regulated by tightly controlled concentration gradients of morphogens. How exactly these gradients are formed, however, remains unclear. This study shows that Fgf8 morphogen gradients in living zebrafish embryos are established and maintained by two essential factors: fast, free diffusion of single molecules away from the source through extracellular space, and a sink function of the receiving cells, regulated by receptor-mediated endocytosis. Evidence is provided by directly examining single molecules of Fgf8 in living tissue by fluorescence correlation spectroscopy, quantifying their local mobility and concentration with high precision. By changing the degree of uptake of Fgf8 into its target cells, the shape of the Fgf8 gradient could be altered. These results demonstrate that a freely diffusing morphogen can set up concentration gradients in a complex multicellular tissue by a simple source-sink mechanism (Yu, 2009).
An analysis is presented of cardiovascular and pharyngeal arch development in mouse embryos hypomorphic for Fgf8. Fgf8 compound heterozygous (Fgf8neo/–) embryos have been generated. Although early analysis has demonstrated that some of these embryos have abnormal left-right (LR) axis specification and cardiac looping reversals, the number and type of cardiac defects present at term suggests an additional role for Fgf8 in cardiovascular development. Most Fgf8neo/– mutant embryos survive to term with abnormal cardiovascular patterning, including outflow tract, arch artery and intracardiac defects. In addition, these mutants have hypoplastic pharyngeal arches, small or absent thymus and abnormal craniofacial development. Neural crest cells (NCCs) populate the pharyngeal arches and contribute to many structures of the face, neck and cardiovascular system, suggesting that Fgf8 may be required for NCC development. Fgf8 is expressed within the developing pharyngeal arch ectoderm and endoderm during NCC migration through the arches. Analysis of NCC development in Fgf8neo/– mutant embryos demonstrates that NCCs are specified and migrate, but undergo cell death in areas both adjacent and distal to where Fgf8 is normally expressed. This study defines the cardiovascular defects present in Fgf8 mutants and supports a role for Fgf8 in development of all the pharyngeal arches and in NCC survival (Abu-Issa, 2002).
Deletion of chromosome 22q11, the most common microdeletion detected in humans, is associated with a life-threatening array of birth defects. Although 90% of affected individuals share the same three megabase deletion, their phenotypes are highly variable and includes craniofacial and cardiovascular anomalies, hypoplasia or aplasia of the thymus with associated deficiency of T cells, hypocalcemia with hypoplasia or aplasia of the parathyroids, and a variety of central nervous system abnormalities. Because ablation of neural crest in chicks produces many features of the deletion 22q11 syndrome, it has been proposed that haploinsufficiency in this region impacts neural crest function during cardiac and pharyngeal arch development. Few factors required for migration, survival, proliferation and subsequent differentiation of pharyngeal arch neural crest and mesoderm-derived mesenchyme into their respective cardiovascular, musculoskeletal, and glandular derivatives have been identified. However, the importance of epithelial-mesenchymal interactions and pharyngeal endoderm function is becoming increasingly clear. Fibroblast growth factor 8 is a signaling molecule expressed in the ectoderm and endoderm of the developing pharyngeal arches and known to play an important role in survival and patterning of first arch tissues. A dosage-sensitive requirement has been demonstrated for FGF8 during development of pharyngeal arch, pharyngeal pouch and neural crest-derived tissues. FGF8 deficient embryos have lethal malformations of the cardiac outflow tract, great vessels and heart due, at least in part, to failure to form the fourth pharyngeal arch arteries, altered expression of Fgf10 in the pharyngeal mesenchyme, and abnormal apoptosis in pharyngeal and cardiac neural crest. The Fgf8 mutants display the complete array of cardiovascular, glandular and craniofacial phenotypes seen in human deletion 22q11 syndromes. This represents the first single gene disruption outside the typically deleted region of human chromosome 22 to fully recapitulate the deletion 22q11 phenotype. FGF8 may operate directly in molecular pathways affected by deletions in 22q11 or function in parallel pathways required for normal development of pharyngeal arch and neural crest-derived tissues. In either case, Fgf8 may function as a modifier of the 22q11 deletion and contribute to the phenotypic variability of this syndrome (Frank, 2002).
In humans and mice, mutations in Hoxa13 cause malformation of limb and genitourinary (GU) regions. In males, one of the most common GU malformations associated with loss of Hoxa13 function is hypospadia, a condition defined by the poor growth and closure of the urethra and glans penis. By examining early signaling in the developing mouse genital tubercle, Hoxa13 has been found to be essential for normal expression of Fgf8 and Bmp7 in the urethral plate epithelium. In Hoxa13GFP-mutant mice, hypospadias occur as a result of the combined loss of Fgf8 and Bmp7 expression in the urethral plate epithelium, as well as the ectopic expression of noggin (Nog) in the flanking mesenchyme. In vitro supplementation with Fgf8 restores proliferation in homozygous mutants to wild-type levels, suggesting that Fgf8 is sufficient to direct early proliferation of the developing genital tubercle. However, the closure defects of the distal urethra and glans can be attributed to a loss of apoptosis in the urethra, which is consistent with reduced Bmp7 expression in this region. Mice mutant for Hoxa13 also exhibit changes in androgen receptor expression, providing a developmental link between Hoxa13-associated hypospadias and those produced by antagonists to androgen signaling. Finally, a novel role for Hoxa13 in the vascularization of the glans penis is also identified (Morgan, 2003).
Fibroblast growth factor 8 (Fgf8) is expressed in many domains of the developing embryo. Globally decreased FGF8 signaling during murine embryogenesis results in a hypomorphic phenotype with a constellation of heart, outflow tract, great vessel and pharyngeal gland defects that phenocopies human deletion 22q11 syndromes, such as DiGeorge. It is postulated that these Fgf8 hypomorphic phenotypes result from disruption of local FGF8 signaling from pharyngeal arch epithelia to mesenchymal cells populating and migrating through the third and fourth pharyngeal arches. To test this hypothesis, and to determine whether the pharyngeal ectoderm and endoderm Fgf8 expression domains have discrete functional roles, conditional mutagenesis of Fgf8 was performed using novel Crerecombinase drivers to achieve domain-specific ablation of Fgf8 gene function in the pharyngeal arch ectoderm and endoderm. Remarkably, ablating FGF8 protein in the pharyngeal arch ectoderm causes failure of formation of the fourth pharyngeal arch artery that results in aortic arch and subclavian artery anomalies in 95% of mutants; these defects recapitulate the spectrum and frequency of vascular defects reported in Fgf8 hypomorphs. Surprisingly, no cardiac, outflow tract or glandular defects were found in ectodermal-domain mutants, indicating that ectodermally derived FGF8 has essential roles during pharyngeal arch vascular development distinct from those in cardiac, outflow tract and pharyngeal gland morphogenesis. By contrast, ablation of FGF8 in the third and fourth pharyngeal endoderm and ectoderm causes glandular defects and bicuspid aortic valve, which indicates that the FGF8 endodermal domain has discrete roles in pharyngeal and valvar development. These results support the hypotheses that local FGF8 signaling from the pharyngeal epithelia is required for pharyngeal vascular and glandular development, and that the pharyngeal ectodermal and endodermal domains of FGF8 have separate functions (Macatee, 2003).
During kidney morphogenesis, the formation of nephrons begins when mesenchymal nephron progenitor cells aggregate and transform into epithelial vesicles that elongate and assume an S-shape. Cells in different regions of the S-shaped body subsequently differentiate into the morphologically and functionally distinct segments of the mature nephron. An allelic series of mutations was used to determine the role of the secreted signaling molecule FGF8 in nephrogenesis. In the absence of FGF8 signaling, nephron formation is initiated, but the nascent nephrons do not express Wnt4 or Lim1, and nephrogenesis does not progress to the S-shaped body stage. Furthermore, the nephron progenitor cells that reside in the peripheral zone, the outermost region of the developing kidney, are progressively lost. When FGF8 signaling is severely reduced rather than eliminated, mesenchymal cells differentiate into S-shaped bodies. However, the cells within these structures that normally differentiate into the tubular segments of the mature nephron undergo apoptosis, resulting in the formation of kidneys with severely truncated nephrons consisting of renal corpuscles connected to collecting ducts by an abnormally short tubular segment. Thus, unlike other FGF family members, which regulate growth and branching morphogenesis of the collecting duct system, Fgf8 encodes a factor essential for gene regulation and cell survival at distinct steps in nephrogenesis (Grieshammer, 2005).
In vertebrate olfactory epithelium (OE), neurogenesis proceeds continuously, suggesting that endogenous signals support survival and proliferation of stem and progenitor cells. A genetic approach was used 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 (Kawauchi, 2005).
The zinc finger transcription factor GLI3 is considered a repressor of vertebrate Hedgehog (Hh) signaling. In humans, the absence of GLI3 function causes Greig cephalopolysyndactyly syndrome, affecting the development of the brain, eye, face, and limb. Because the etiology of these malformations is not well understood, the phenotype of mouse Gli-/- mutants was examined as a model to investigate this. An up-regulation of Fgf8 is observed in the anterior neural ridge, isthmus, eye, facial primordia, and limb buds of mutant embryos, sites coinciding with the human disease. Intriguingly, endogenous apoptosis is reduced in Fgf8-positive areas in Gli-/- mutants. Since SHH is thought to be involved in Fgf8 regulation, Fgf8 expression was compared in Shh-/- and Gli-/-;Shh-/- mutant embryos. Whereas Fgf8 expression is almost absent in Shh-/- mutants, it is up-regulated in Gli-/-;Shh-/- double mutants, suggesting that SHH is not required for Fgf8 induction, and that GLI3 normally represses Fgf8 independently of SHH. In the limb bud, evidence is provided that ectopic expression of Gremlin in Gli-/- mutants might contribute to a decrease in apoptosis. Together, these data reveal that GLI3 limits Fgf8-expression domains in multiple tissues, through a mechanism that may include the induction or maintenance of apoptosis. It is concluded that Fgf8 may not be a direct target of GLI3 but that the size of the Fgf domain may be regulated by GLI3 indirectly; when GLI3 is present, it activates the expression of Bmps, which regulates cell death to alter the size of FGF8 domains (Aoto, 2002).
Initiation and maintenance of signaling centers is a key issue during embryonic development. The apical ectodermal ridge, a specialized epithelial structure and source of Fgf8, is a pivotal signaling center for limb outgrowth. Two closely related buttonhead-like zinc-finger transcription factors, Sp8 and Sp9, are expressed in the AER, and regulate Fgf8 expression and limb outgrowth. Embryological and genetic analyses have revealed that Sp8 and Sp9 are ectodermal targets of Fgf10 signaling from the mesenchyme. Wnt/ß-catenin signaling positively regulates Sp8, but not Sp9. Overexpression functional analyses in chick unveiled Sp8 and Sp9 role as positive regulators of Fgf8 expression. Moreover, a dominant-negative approach in chick and knockdown analysis with morpholinos in zebrafish revealed Sp8 and Sp9 requirement for Fgf8 expression and limb outgrowth, and further indicate that Sp8 and Sp9 have a coordinated action on Fgf8 expression. This study demonstrates that Sp8 and Sp9, via Fgf8, are involved in mediating the actions of Fgf10 and Wnt/ß-catenin signaling during vertebrate limb outgrowth (Kawakami, 2004).
Endogenous retinoids are important for patterning many aspects of the embryo including the branchial arches and frontonasal region of the embryonic face. The nasal placodes express retinaldehyde dehydrogenase-3 (RALDH3) and thus retinoids from the placode are a potential patterning influence on the developing face. Experiments have been carried out that have used Citral, a RALDH antagonist, to address the function of retinoid signaling from the nasal pit in a whole embryo model. When Citral-soaked beads are implanted into the nasal pit of stage 20 chicken embryos, the result is a specific loss of derivatives from the lateral nasal prominences. Providing exogenous retinoic acid rescues development of the beak demonstrating that most Citral-induced defects are produced by the specific blocking of RA synthesis. The mechanism of Citral effects is a specific increase in programmed cell death on the lateral (lateral nasal prominence) but not the medial side (frontonasal mass) of the nasal pit. Gene expression studies were focused on the Bone Morphogenetic Protein (BMP) pathway, which has a well-established role in programmed cell death. Unexpectedly, blocking RA synthesis decreased rather than increased Msx1, Msx2, and Bmp4 expression. Cell survival genes were examined, the most relevant of which was Fgf8, which is expressed around the nasal pit and in the frontonasal mass. Fgf8 was not initially expressed along the lateral side of the nasal pit at the start of the experiments, whereas it was expressed on the medial side. Citral prevented upregulation of Fgf8 along the lateral edge and this may have contributed to the specific increase in programmed cell death in the lateral nasal prominence. Consistent with this idea, exogenous FGF8 was able to prevent cell death, rescue most of the morphological defects and was able to prevent a decrease in retinoic acid receptorβ (Rarβ) expression caused by Citral. Together, these results demonstrate that endogenous retinoids act upstream of FGF8 and the balance of these two factors is critical for regulating programmed cell death and morphogenesis in the face. In addition, these data suggest a novel role for endogenous retinoids from the nasal pit in controlling the precise downregulation of FGF in the center of the frontonasal mass observed during normal vertebrate development (Song, 2004).
The development of the vertebrate inner ear depends on the precise expression of fibroblast growth factors. In a mutagenesis screen for zebrafish with abnormalities of inner-ear development and behavior, a mutant line, ru622, was isolated whose phenotypic characteristics resembled those of null mutants for the gene encoding fibroblast growth factor 8 (Fgf8): an inconsistent startle response, circular swimming, fused otoliths, and abnormal semicircular canals. Positional cloning disclosed that the mutant gene encodes the transcriptional corepressor Atrophin2. Both the Fgf8 protein and zebrafish 'similar expression to fgf genes' protein (Sef), an antagonist of fibroblast growth factors induced by Fgf8 itself, were found to be overexpressed in ru622 mutants. It was therefore hypothesized that an excess of Sef eliminates Fgf8 signals and produces an fgf8 null phenotype in ru622 mutants. In support of this idea, larvae whose atrophin2 expression had been diminished with morpholinos could be rescued by reducing the expression of Sef as well. It is proposed that Atrophin2 plays a role in the feedback regulation of Fgf8 signaling. When mutation of the atrophin2 gene results in the overexpression of both Fgf8 and Sef, the excessive Sef inhibits Fgf8 signaling. The resultant imbalance of Fgf8 and Sef signals then underlies the abnormal aural development observed in ru622 (Asai, 2006).
Fgf8 and Fgf4 encode FGF family members that are coexpressed in the primitive streak of the gastrulating mouse embryo. The phenotype of Fgf8-/- embryos were analyzed and it was discovered that they fail to express Fgf4 in the streak. In the absence of both FGF8 and FGF4, epiblast cells move into the streak and undergo an epithelial-to-mesenchymal transition, but most cells then fail to move away from the streak. As a consequence, no embryonic mesoderm- or endoderm-derived tissues develop, although extraembryonic tissues form. Patterning of the prospective neuroectoderm is greatly perturbed in the mutant embryos. Anterior neuroectoderm markers are widely expressed, at least in part because the anterior visceral endoderm, which provides signals that regulate marker expression, is not displaced proximally in the absence of definitive endoderm. Posterior neuroectoderm markers are not expressed, presumably because there is neither mesendoderm underlying the prospective neuroectoderm nor a morphologically normal node to provide the inductive signals necessary for their expression. This study identifies Fgf8 as a gene essential for gastrulation and shows that signaling via FGF8 and/or FGF4 is required for cell migration away from the primitive streak (Sun, 1999).
In invertebrates, FGF signaling is also necessary for cell migration. For example, in Drosophila it is required for migration and spreading of the embryonic mesoderm over the ectoderm and for branching morphogenesis of the tracheal system, and in Caenorhabditis elegans it is required for sex myoblast migration. In both organisms, ectopic expression experiments have suggested that FGFs can function as attractants for cell migration. By analogy, one might argue that FGF8 produced in the VE acts as an attractant for cell migration away from the streak. This hypothesis was tested by injecting wild-type embryonic stem cells into Fgf8 minus blastocysts, producing chimeras in which the VE presumably contained only Fgf8 minus cells, whereas the epiblast contained a mixture of wild-type and mutant cells. No defects in gastrulation were detected in four such chimeras, in which at least 25% of the embryonic cell population was derived from wild-type ES cells. In these embryos, Fgf8 minus cells contributed to all tissues, including somites, head mesenchyme, and foregut. These results indicate that lack of FGF8 in the VE is not responsible for the gastrulation defects in Fgf8 mutant embryos. Instead, FGF signaling appears to be required in the primitive streak itself, presumably to regulate the production of proteins necessary for cell migration (Sun, 1999).
Genes that encode molecules involved in adhesive interactions between cells and their surrounding extracellular matrix (ECM) are obvious candidates for the downstream targets affected by loss of Fgf8 function. Mutational analysis has shown that there is a deficit of mesoderm in embryos homozygous for null alleles of Fibronectin, Integrin alpha5, and Focal adhesion kinase, which encode a component of the ECM, part of the receptor for Fibronectin, and a nonreceptor tyrosine kinase thought to mediate Integrin signaling, respectively. This suggests that those genes might be required for cell migration away from the primitive streak. However, abnormalities in the mutant embryos are not detected until at least the late headfold stage. It therefore seems unlikely that the more severe phenotype of Fgf8 minus embryos is due to effects of FGF8/4 signaling on any one of these genes, although it remains possible that the defects are due to simultaneous effects on more than one such gene (Sun, 1999 and references).
Another type of molecule that appears to play some role in cell migration away from the primitive streak is the transcription factor T. When the behavior of T null homozygous cells is monitored in chimeras, they are found to accumulate in the mesodermal layer of the streak region, but this effect is not evident until the headfold stage. Moreover, T null mutant embryos do not show any obvious defects at the primitive streak stages. The fact that Fgf8 mutant embryos display a more severe phenotype argues against interference with T expression as the primary cause of the defect in cell migration. However, other genes related to T might be the downstream targets of FGF signaling required for cell migration away from the streak. Consistent with this hypothesis, it was found that FGF8/4 signaling regulates expression of at least some T-related genes. For example, Tbx6 is not expressed in Fgf8 minus embryos. Furthermore, although T expression is detected in epithelial cells in the mutant streak region, it is not detected in nascent mesenchymal cells that have traversed the streak and accumulated there. In contrast, T expression is detected in both the epithelial and mesenchymal portions of the streak in Fgf8+ embryos, and even in cells a short distance away from the streak. These observations are consistent with some aspects of the positive-feedback loop model proposed for regulation of the expression of Xbra and eFgf, the Xenopus orthologs of T and Fgf4, respectively and the finding that in zebrafish, expression of T and two T-related genes, spadetail and Tbx6 is regulated by FGF signaling (Sun, 1999 and references).
During gastrulation in amniotes, epiblast cells ingress through the primitive streak and migrate away to form endodermal, mesodermal, and extraembryonic structures. The detailed movement trajectories of cells emerging at different anterior-posterior positions from the primitive streak was analyzed using in vivo imaging of the movement of GFP-tagged streak cells. Cells emerging at different anterior-posterior positions from the streak show characteristic cell migration patterns, in response to guidance signals from neighboring tissues. Streak cells are attracted by sources of FGF4 and repelled by sources of FGF8. The observed movement patterns of anterior streak cells can be explained by an FGF8-mediated chemorepulsion of cells away from the streak followed by chemoattraction toward an FGF4 signal produced by the forming notochord (Yang, 2002).
Differentiation onset in the vertebrate body axis is controlled by a conserved switch from fibroblast growth factor (FGF) to retinoid signalling, which is also apparent in the extending limb and aberrant in many cancer cell lines. FGF protects tail-end stem zone cells from precocious differentiation by inhibiting retinoid synthesis, whereas later-produced retinoic acid (RA) attenuates FGF signalling and drives differentiation. The timing of RA production is therefore crucial for the preservation of stem zone cells and the continued extension of the body axis. Canonical Wnt signalling mediates the transition from FGF to retinoid signalling in the newly generated chick body axis. FGF promotes Wnt8c expression, which persists in the neuroepithelium as FGF signalling declines. Wnt signals then act here to repress neuronal differentiation. Furthermore, although FGF inhibition of neuronal differentiation involves repression of the RA-responsive gene, retinoic acid receptor β (RARβ), Wnt signals are weaker repressors of neuron production and do not interfere with RA signal transduction. Strikingly, as FGF signals decline in the extending axis, Wnt signals now elicit RA synthesis in neighbouring presomitic mesoderm. This study identifies a directional signalling relay that leads from FGF to retinoid signalling and demonstrates that Wnt signals serve, as cells leave the stem zone, to permit and promote RA activity, providing a mechanism to control the timing of the FGF-RA differentiation switch (Olivera-Martinez, 2007).
Epiblast cells adjacent to the regressing primitive streak behave as a stem zone that progressively generates the entire spinal cord and also contributes to paraxial mesoderm. Despite this fundamental task, this cell population is poorly characterised, and the tissue interactions and signalling pathways that specify this unique region are unknown. Fibroblast growth factor (FGF) is implicated but it is unclear whether it is sufficient and/or directly required for stem zone specification. It is also not understood how establishment of the stem zone relates to the acquisition of spinal cord identity as indicated by expression of caudal Hox genes (Delfino-Machin, 2005).
Many cells in the chick stem zone express both early neural and mesodermal genes; however, stem zone-specific gene expression can be induced by signals from underlying paraxial mesoderm without concomitant induction of an ambivalent neural/mesodermal cell state. The stem zone is a site of FGF/MAPK signalling, and although FGF alone does not mimic paraxial mesoderm signals, it is directly required in epiblast cells for stem zone specification and maintenance. Caudal Hox gene expression in the stem zone also depends on FGF and neither stem zone specification nor caudal Hox gene onset requires retinoid signalling. These findings thus support a two step model for spinal cord generation -- FGF-dependent establishment of the stem zone in which progressively more caudal Hox genes are expressed, followed by the retinoid-dependent assignment of spinal cord identity (Delfino-Machin, 2005).
The stem zone is a unique cell population set aside in the caudal part of the neural plate. These epiblast cells proliferate and re-arrange adjacent to the regressing primitive streak leaving behind progenitors that then generate the entire spinal cord. Cell labelling studies in the mouse suggest that self-renewing neural stem cells reside in this spinal cord primordium and divide at intervals to generate neural progenitors: such stem cells also exist in the chick embryo. There is evidence in frog, fish, chick and mouse embryos that this caudal-most part of the neural plate also contains cells that contribute to mesoderm and this mesoderm-forming potential persists in the neural/epiblast cells close to the anterior primitive streak through to tailbud stages. In higher vertebrates and in frogs, it remains unclear whether this indicates the presence of resident multipotent stem cells that can contribute to both neural and mesodermal layers, or whether different, but closely associated, cells in this region give rise to these different lineages as observed in zebrafish (Delfino-Machin, 2005).
In chick and mouse, the stem zone first becomes molecularly distinct just prior to somitogenesis, when it expresses several transcription factors that distinguish it from the rest of the neural plate. These genes include the homeodomain-containing factor, Sax1, and in the chick, the proneural gene homologue, cash4. Epiblast cells close to the primitive streak express Fgf8 and once caudal regression of the primitive streak is under way, expression of these genes spreads laterally into the morphologically defined open neural plate in both chick and mouse. This suggests that some cells in this region of the neuroepithelium co-express early mesodermal and neural genes (Delfino-Machin, 2005).
Signals from the regressing node can induce both cash4 and Sax1 in the chick; however, ablation of the node does not result in loss of Sax1 expression, suggesting that other tissues share this property. At later stages, studies show that the paraxial mesoderm beneath the established stem zone is indeed required for maintenance of cash4 and Sax1 in the embryo. There is some evidence that FGF signalling accounts for this maintenance signal from the mesoderm. The anterior primitive streak expresses Fgf2, Fgf3, Fgf4, Fgf8, Fgf12, Fgf13 and Fgf18, and most of these factors persist in the regressing streak and are present in the stem zone itself (Fgf2, Fgf3, Fgf8, Fgf18) while Fgf8, Fgf10 and Fgf18 are also expressed by paraxial mesoderm. Furthermore, FGF4 or FGF8 can locally ectopically maintain expression of cash4 and Sax1 as the spinal cord develops. However, it is not known whether FGF acts directly on epiblast cells to specify or maintain the stem zone (Delfino-Machin, 2005).
FGF signalling has long been implicated in the generation of the vertebrate body since disruption of this pathway results in failure to form this part of the embryo. The primary role of FGF signalling in mesoderm induction has made it difficult to assess its direct requirement for induction of tissue that depends on mesoderm derived signals. However, this pathway has been shown to initiate neural development in the chick embryo, in mouse ES cells and most recently in the frog embryo. MAPK activation downstream of FGF signalling is implicated in this step in the chick and in the frog acts at least in part by interfering with BMP signal transduction by inactivating the BMP intermediary protein Smad1. MAPK signalling is also required for mesoderm induction and recent data suggest that low level FGF/MAPK may initiate neural development, while higher levels promote mesoderm formation. Together, these studies indicate that serial FGF/MAPK mediated events may underpin stem zone formation and raise the possibility that prolonged exposure to such signalling is involved in specification and/or maintenance of this cell population (Delfino-Machin, 2005).
FGF signalling not only mediates cell fate specification in the early embryo but also maintains an undifferentiated cell state in many cellular contexts. During body axis extension, exposure to FGF inhibits neuronal differentiation and onset of ventral patterning genes. Furthermore, blocking FGF signalling also accelerates movement of cells out of the stem zone into the transition zone, which eventually forms the neural tube where neuronal differentiation commences. These findings indicate a role for FGF signals in keeping cells in an undifferentiated, proliferative cell state and within the stem zone. Importantly, the maintenance of this undifferentiated state may prolong the period during which cells are able to respond to caudalising signals and may thereby account for the expression of progressively more caudal Hox genes in the stem zone. These genes determine rostrocaudal character in the emerging body axis, so, for example, Hoxb8 expression identifies the spinal cord and is expressed in the neural tube caudal to somite 5. Interestingly, depending on context, many caudal Hox genes, including Hoxb8 are induced by FGF or retinoic acid (RA) signalling. However, recent work shows that FGF and retinoid signalling are mutually inhibitory in the extending body axis, and raises the possibility that initiation of caudal Hox gene expression in the stem zone under the influence of FGF switches to a dependency on somite-derived retinoids in differentiating tissues (Delfino-Machin, 2005).
This study used a panel of neural, mesodermal and stem zone-specific marker genes to characterise the stem zone region. In vitro explant assays were used to identify tissues that specify this cell group and to assess whether stem zone specific gene expression can be induced independently of mesodermal gene expression (Delfino-Machin, 2005).
Using both in vivo and in vitro assays, this study shows that signalling provided by either FGF4 or FGF8 is not sufficient to induce stem zone-specific gene expression. However, this does not rule out the possibility that FGF signalling is a necessary cofactor for stem zone specification. To distinguish between the necessity for FGF signalling for expression of early neural genes and for stem zone specification the misexpression of dnFGFR1 was induced in the established neural plate at HH4. These experiments demonstrate that onset of Sax1, but not maintenance of Sox2, depends on FGF signalling and thus identify a FGF requiring step that takes place after neural and mesodermal induction, during the generation of caudal neural tissue. It is further shown that this is a continuing requirement in the extending body axis, because maintenance of Sax1 also depends on FGF/MAPK signalling in epiblast cells (Delfino-Machin, 2005).
Since FGF signalling is not sufficient for stem zone specification, other signals provided by the caudal paraxial mesoderm must be involved. These include WNT proteins and TGFß family members. This study has focussed on the retinoid pathway, since the retinoid synthesising enzyme Raldh2 is initially expressed in the caudal paraxial mesoderm just prior to Sax1 onset. However, the onset of both Sax1 and Hoxb8 appear at the normal time in vitamin A-deficient embryos, indicating that retinoic acid is not necessary for stem zone specification. By contrast, during normal development, the Sax1 expression domain appears to expand rostrally, coincident with restriction of Raldh2 expression to rostral paraxial mesoderm. This rostral retreat of Raldh2 reflects the persisting expression of Fgf8 and bra in the stem zone and in paraxial mesoderm cells emerging from the primitive streak and is driven by the ability of FGF signalling to inhibit onset of Raldh2 (Delfino-Machin, 2005).
Interestingly, this apparent rostral expansion of Fgf8/bra/Sax1 coincides with the onset of the retinoic acid catabolising enzyme Cyp26a in the stem zone, which in the frog requires FGF signalling. This may further help to create a retinoid-free region at the caudal end of the embryo and, consistent with this, Cyp26a knockout mice exhibit a truncated phenotype also seen following exposure to retinoic acid: this suggests that excess retinoid signals drive premature differentiation of the stem zone. Stem zone specification thus depends on unknown signals from the paraxial mesoderm and FGF-mediated activity, which works at least in part by establishing a retinoid-free region (Delfino-Machin, 2005).
Left-right asymmetry in vertebrate embryos is first recognisable using molecular markers that encode secreted proteins or transcription factors. The asymmetry becomes morphologically obvious in the turning of the embryo and in the development of the heart, the gut and other visceral organs. In the chick embryo, a signaling pathway for the specification of the left body side has been demonstrated. Sonic hedgehog (Shh) protein is the first asymmetric signal identified in the node. Further downstream in this pathway are the left-specific genes nodal, lefty-1, lefty-2 and Pitx2. On the right body side, a function of the activin pathway is indicated by the right-sided expression of cActRIIa. Another key molecule in vertebrate development, fibroblast growth factor 8 (FGF8), is expressed asymmetrically on the right side of the posterior node. Transcription of FGF8 is induced by activin and the FGF8 protein inhibits the expression of nodal and Pitx2 and leads to expression of the chicken snail related gene (cSnR). Left-sided application of FGF8 randomises the direction of heart looping (Boettger, 1999).
Fibroblast growth factor (Fgf) signaling plays an important role during development of posterior mesoderm in vertebrate embryos. Blocking Fgf signaling by expressing a dominant-negative Fgf receptor inhibits posterior mesoderm development. In mice, Fgf8 appears to be the principal ligand required for mesodermal development; mouse Fgf8 mutants do not form mesoderm. In zebrafish, Fgf8 is encoded by the acerebellar locus, and, similar to its mouse otholog, is expressed in early mesodermal precursors during gastrulation. However, zebrafish fgf8 mutants have only mild defects in posterior mesodermal development, suggesting that it is not the only Fgf ligand involved in the development of this tissue. An fgf8-related gene has been identified in zebrafish, fgf24, that is co-expressed with fgf8 in mesodermal precursors during gastrulation. Using morpholino-based gene inactivation, the function of fgf24 during development was analyzed. Inhibiting fgf24 function alone has no affect on the formation of posterior mesoderm. Conversely, inhibiting fgf24 function in embryos mutant for fgf8 blocks the formation of most posterior mesoderm. Thus, fgf8 and fgf24 are together required to promote posterior mesodermal development. Phenotypic and genetic evidence is provided that both these Fgf signaling components interact with no tail and spadetail, two zebrafish T-box transcription factors that are required for the development of all posterior mesoderm. Last, fgf24 is shown to be expressed in early fin bud mesenchyme; inhibiting fgf24 function results in viable fish that lack pectoral fins (Draper, 2003).
The zebrafish T-box transcription factors spadetail (spt) and the brachyury ortholog no tail (ntl) are together essential for posterior mesoderm formation. In addition to being functionally redundant, spt and ntl also genetically interact with zygotic mutant alleles of one-eyed pinhead (Zoep), leading to synergistic mesodermal defects. Genetic and pharmacological assays have been used to address the mechanism of these interactions. Zoep and ntl are together required upstream of spt expression, accounting for the severity of the mesodermal defects in Zoep;ntl embryos. Since Xenopus brachyury is proposed to regulate fgf expression, and FGF signaling is required for spt expression, the involvement of the FGF signaling pathway in these genetic interactions was analyzed. Using a specific inhibitor of FGFR activity to indirectly assay the strength of FGF signaling in individual embryos, it was found that spt and ntl mutant embryos are both hypersensitive to the FGFR inhibitor. This hypersensitivity is consistent with the possibility that Spt and Ntl function upstream of FGF signaling. Furthermore, minor pharmacological or genetic perturbations in FGF signaling are sufficient to dramatically enhance the Zoep mutant phenotype, providing a plausible explanation for why Zoep genetically interacts with spt and ntl. Zoep and ace/fgf8 functions are essential for the formation of all posterior tissues, including spinal cord. Taken together, these data provide strong in vivo support for the regulation of FGF signaling by T-box transcription factors, and the cooperative activity of Oep and FGF signaling during the formation of posterior structures (Griffin, 2003).
Interactions between Nodal/Activin and Fibroblast growth factor (Fgf) signalling pathways have long been thought to play an important role in mesoderm formation. However, the molecular and cellular processes underlying these interactions have remained elusive. This study addresses the epistatic relationships between Nodal and Fgf pathways during early embryogenesis in zebrafish. (1) Fgf signalling is found to be required downstream of Nodal signals for inducing the Nodal co-factor One-eyed-pinhead (Oep). Thus, Fgf is likely to be involved in the amplification and propagation of Nodal signalling during early embryonic stages. This could account for the previously described ability of Fgf to render cells competent to respond to Nodal/Activin signals. In addition, overexpression data shows that Fgf8 and Fgf3 can take part in this process. (2) Combining zygotic mutations in ace/fgf8 and oep disrupts mesoderm formation, a phenotype that is not produced by either mutation alone and is consistent with this model of an interdependence of Fgf8 and Nodal pathways through the genetic regulation of the Nodal co-factor Oep and the cell propagation of Nodal signalling. Moreover, mesodermal cell populations are affected differentially by double loss-of-function of Zoep;ace. Most of the dorsal mesoderm undergoes massive cell death by the end of gastrulation, in contrast to either single-mutant phenotype. However, some mesoderm cells are still able to undergo myogenic differentiation in the anterior trunk of Zoep;ace embryos, revealing a morphological transition at the level of somites 6-8. Further decreasing Oep levels by removing maternal oep products aggravates the mesodermal defects in double mutants by disrupting the fate of the entire mesoderm. Together, these results demonstrate synergy between oep and fgf8 that operates with regional differences and is involved in the induction, maintenance, movement and survival of mesodermal cell populations (Mathieu, 2004).
During somite development, a fibroblast growth factor (FGF) signal secreted from the myotome induces formation of a scleraxis (Scx)-expressing tendon progenitor population in the sclerotome, at the juncture between the future lineages of muscle and cartilage. Scx is a tendon-specific bHLH transcription factor. While overexpression studies show that the entire sclerotome is competent to express Scx in response to FGF signaling, the normal Scx expression domain includes only the anterior and posterior dorsal sclerotome. To understand the molecular basis for this restriction, the expression of a set of genes involved in FGF signaling was examined and it was found that several members of the Fgf8 synexpression group are co-expressed with Scx in the dorsal sclerotome. Of particular interest were the Ets transcription factors Pea3 and Erm, which function as transcriptional effectors of FGF signaling. Transcriptional activation by Pea3 and Erm in response to FGF signaling is both necessary and sufficient for Scx expression in the somite, and it is proposed that the domain of the somitic tendon progenitors is regulated both by the restricted expression of Pea3 and Erm, and by the precise spatial relationship between these Ets transcription factors and the FGF signal originating in the myotome (Brent, 2004).
Fibroblast growth factors (Fgfs) have long been implicated in regulating vertebrate skeletal muscle differentiation, but their precise role(s) in vivo remain unclear. Fgf8 signalling in the somite is shown to be required for myod expression and terminal differentiation of a subset of fast muscle cells in the zebrafish lateral somite. In the absence of Fgf8, lateral somite cells transiently express myf5 but fail to make muscle and remain in a dermomyotome-like state characterised by pax3 and meox expression. Slow muscle fibres form and commence normal migration in the absence of Fgf8, but fail to traverse the expanded undifferentiated lateral somite. The Fgf8-independent residual population of medial fast muscle fibres is not Hedgehog dependent. However, Fgf8-independent medial fast muscle precursors are lacking in floatinghead mutants, suggesting that they require another ventral midline-derived signal. It is concluded that Fgf8 drives terminal differentiation of a specific population of lateral muscle precursor cells within the early somite (Groves, 2005).
Formation of the early vertebrate embryo depends on a Brachyury/Wnt autoregulatory loop within the posterior mesodermal progenitors. This study shows that exogenous retinoic acid (RA), which dramatically truncates the embryo, represses expression of the zebrafish brachyury ortholog no tail (ntl), causing a failure to sustain the loop. It was found that Ntl functions normally to protect the autoregulatory loop from endogenous RA by directly activating cyp26a1 expression. Thus, the embryonic mesodermal progenitors uniquely establish their own niche - with Brachyury being essential for creating a domain of high Wnt and low RA signaling - rather than having a niche created by separate support cells (Martin, 2010).
Common examples of stem or progenitor cell niches in both embryonic and adult organisms consist of at least two general cell types: the stem/progenitor cells and the support cells, which provide the physical and molecular environment necessary for the maintenance of the stem/progenitor cells. These data provide evidence of a unique type of progenitor cell niche consisting of only one cell type, in which mesodermal progenitor cells of the zebrafish tailbud act as their own support cells. Mesodermal progenitors express ntl, wnt3a, wnt8, and cyp26a1, all of which are required within the progenitor population as a whole, but none of which are required by individual progenitor cells in a wild-type environment. This demonstrates that the wild-type mesodermal progenitor cells act as support cells for the genetically deficient progenitors and can sustain them by creating an environment of high Wnt and low RA signaling. The primary function of Ntl, therefore, is to create the mesodermal progenitor niche through direct regulation of canonical wnt ligands and cyp26a1. While this analysis focused specifically on zebrafish, the common phenotypes of brachyury loss of function and RA treatment in different vertebrates, as well as the conservation of expression patterns of brachyury, wnts, and cyp26a1, indicates that the same mechanism is common to all vertebrates. Thus, it is suggested that expression of brachyury in the progenitor domain was a vertebrate adaptation that allowed the progenitor cells to be sustained during the long process of somitogenesis, which in some species can last for many days. This unique function of Brachyury is particularly relevant, given that recent molecular analysis of various human cancers has demonstrated that brachyury is commonly up-regulated in tumors. The up-regulation of brachyury may, in effect, be creating a cancer cell niche that maintains high Wnt signaling and low RA signaling, both of which have been extensively demonstrated to be key components of cancer growth (Martin, 2010).
The avian heart develops from paired primordia located in the anterior lateral mesoderm of the early embryo. Previous studies have found that the endoderm adjacent to the cardiac primordia plays an important role in heart specification. The current study provides evidence that fibroblast growth factor (Fgf) signaling contributes to the heart-inducing properties of the endoderm. Fgf8 is expressed in the endoderm adjacent to the precardiac mesoderm. Removal of endoderm results in a rapid downregulation of a subset of cardiac markers, including Nkx2.5 and Mef2c. Expression of these markers can be rescued by supplying exogenous Fgf8. In addition, application of ectopic Fgf8 results in ectopic expression of cardiac markers. Expression of cardiac markers is expanded only in regions where bone morphogenetic protein (Bmp) signaling is also present, suggesting that cardiogenesis occurs in regions exposed to both Fgf and Bmp signaling. Finally, evidence is presented that Fgf8 expression is regulated by particular levels of Bmp signaling. Application of low concentrations of Bmp2 results in ectopic expression of Fgf8, while application of higher concentrations of Bmp2 result in repression of Fgf8 expression. Together, these data indicate that Fgf signaling cooperates with Bmp signaling to regulate early cardiogenesis (Alsan, 2002).
Vertebrate heart development is initiated from bilateral lateral plate mesoderm that expresses the Nkx2.5 and GATA4 transcription factors, but the extracellular signals specifying heart precursor gene expression are not known. The secreted signaling factor Fgf8 is expressed in and required for development of the zebrafish heart precursors, particularly during initiation of cardiac gene expression. fgf8 is mutated in acerebellar (ace) mutants, and homozygous mutant embryos do not establish normal circulation, although vessel formation is only mildly affected. In contrast, heart development, in particular of the ventricle, is severely abnormal in acerebellar mutants. Several findings argue that Fgf8 has a direct function in development of cardiac precursor cells: fgf8 is expressed in cardiac precursors and later in the heart ventricle. Fgf8 is required for the earliest stages of nkx2.5 and gata4 expression in cardiac precursors, but not for gata6. Cardiac gene expression is restored in acerebellar mutant embryos by injecting fgf8 mRNA, or by implanting a Fgf8-coated bead into the heart primordium. Pharmacological inhibition of Fgf signaling during formation of the heart primordium phenocopies the acerebellar heart phenotype, confirming that Fgf signaling is required independent of earlier functions during gastrulation. These findings show that fgf8/acerebellar is required for induction and patterning of myocardial precursors (Reifers, 2000).
Tbx1 has been implicated as a candidate gene responsible for defective pharyngeal arch remodeling in DiGeorge/Velocardiofacial syndrome. Tbx1(+/-) mice mimic aspects of the DiGeorge phenotype with variable penetrance, and null mice display severe pharyngeal hypoplasia. Enhancer elements have been identified in the Tbx1 gene that are conserved through evolution and mediate tissue-specific expression. Transgenic mice were generated that utilize these enhancer elements to direct Cre recombinase expression in endogenous Tbx1 expression domains. Tbx1-Cre mice were used to fate map Tbx1-expressing precursors and identify broad regions of mesoderm, including early cardiac mesoderm, which is derived from Tbx1-expressing cells. The hypothesis that fibroblast growth factor 8 (Fgf8) functions downstream of Tbx1 was tested by performing tissue-specific inactivation of Fgf8 using Tbx1-Cre mice. Resulting newborn mice display DiGeorge-like congenital cardiovascular defects that involve the outflow tract of the heart. Vascular smooth muscle differentiation in the great vessels is disrupted. This data is consistent with a model in which Tbx1 induces Fgf8 expression in the pharyngeal endoderm, which is subsequently required for normal cardiovascular morphogenesis and smooth muscle differentiation in the aorta and pulmonary artery (Brown, 2004).
Birth defects, which occur in one out of 20 live births, often affect multiple organs that have common developmental origins. Human and mouse studies indicate that haploinsufficiency of the transcription factor TBX1 disrupts pharyngeal arch development, resulting in the cardiac and craniofacial features associated with microdeletion of 22q11 (del22q11), the most frequent human deletion syndrome. An allelic series of Tbx1 deficiency was generated that reveals a lower critical threshold for Tbx1 activity in the cardiac outflow tract compared with other pharyngeal arch derivatives, including the palatal bones. Mice hypomorphic for Tbx1 failed to activate expression of the forkhead transcription factor Foxa2 in the pharyngeal mesoderm, which contains cardiac outflow precursors derived from the anterior heart field. A Fox-binding site upstream of Tbx1 has been identified that interacts with Foxa2 and is necessary for pharyngeal mesoderm expression of Tbx1, revealing an autoregulatory loop that may explain the increased cardiac sensitivity to Tbx1 dose. Downstream of Tbx1, a fibroblast growth factor 8 (Fgf8) enhancer was found that is dependent on Tbx1 in vivo for regulating expression in the cardiac outflow tract, but not in pharyngeal arches. Consistent with its role in regulating cardiac outflow tract cells Tbx1 gain of function results in expansion of the cardiac outflow tract segment derived from the anterior heart field as marked by Fgf10. These findings reveal a Tbx1-dependent transcriptional and signaling network in the cardiac outflow tract that renders mouse cardiovascular development more susceptible than craniofacial development to a reduction in Tbx1 dose, similar to humans with del22q11 (Hu, 2004).
In order to understand how secreted signals regulate complex morphogenetic events, it is crucial to identify their cellular targets. By conditional inactivation of Fgfr1 and Fgfr2 and overexpression of the FGF antagonist sprouty 2 in different cell types, the role of FGF signaling was dissected during heart outflow tract development in mouse. Contrary to expectation, cardiac neural crest and endothelial cells are not primary paracrine targets. FGF signaling within second heart field mesoderm is required for remodeling of the outflow tract: when disrupted, outflow myocardium fails to produce extracellular matrix and TGFbeta and BMP signals essential for endothelial cell transformation and invasion of cardiac neural crest. It is concluded that an autocrine regulatory loop, initiated by the reception of FGF signals by the mesoderm, regulates correct morphogenesis at the arterial pole of the heart. These findings provide new insight into how FGF signaling regulates context-dependent cellular responses during development (Park, 2008).
In contrast to the paradigm of paracrine signaling established in other tissues, these data show that in pharyngeal and splanchnic mesoderm dorsal to the heart tube, called the second heart field, the cellular source of the ligand (signal) is also the target. Such an autocrine pathway can be easily understood in terms of a feedback loop that maintains FGF production within a tight range, which is crucial for FGF8 function. Secondary effects on other signaling pathways that were observed may also be integrated into this regulatory loop. Fgf8 and FGFR mutant analyses establish that the autocrine pathway not only regulates survival and proliferation of second heart field cells (a common response to FGFs), but also the secretory and signaling capacities of their derivatives in the arterial pole of the heart, called the outflow tract. The few transcriptional targets of the PEA3 family of FGF8 effector proteins thus far identified are ECM components, ECM-modifying enzymes and cell adhesion molecules, suggesting that an autocrine pathway might provide a means of regulating the ECM and microenvironment to ensure uniform signal reception and response within a specialized cell population. These findings are of biomedical importance, not only in the context of understanding the causes of congenital malformations of the outflow tract, but also because the crucial role demonstrated for an autocrine FGF signaling pathway has broad implications for understanding fundamental properties of FGF signaling in different developmental and pathological contexts (Park, 2008).
To bypass the essential gastrulation function of Fgf8 and study its role in lineages of the primitive streak, a new mouse line, T-Cre, was used to generate mouse embryos with pan-mesodermal loss of Fgf8 expression. Surprisingly, despite previous models in which Fgf8 has been assigned a pivotal role in segmentation/somite differentiation, Fgf8 is not required for these processes. However, mutant neonates display severe renal hypoplasia with deficient nephron formation. In mutant kidneys, aberrant cell death occurs within the metanephric mesenchyme (MM), particularly in the cortical nephrogenic zone, which provides the progenitors for recurring rounds of nephron formation. Prior to mutant morphological changes, Wnt4 and Lim1 expression, which is essential for nephrogenesis, is absent in MM. Furthermore, comparative analysis of Wnt4-null homozygotes reveals concomitant downregulation of Lim1 and diminished tubule formation. These data support a model whereby FGF8 and WNT4 function in concert to induce the expression of Lim1 for MM survival and tubulogenesis (Perantoni, 2005).
Fibroblast growth factor (Fgf) proteins are important regulators of pharyngeal arch development. Analyses of Fgf8 function in chick and mouse and Fgf3 function in zebrafish have demonstrated a role for Fgfs in the differentiation and survival of postmigratory neural crest cells (NCC) that give rise to the pharyngeal skeleton. An earlier, essential function for Fgf8 and Fgf3 is described in zebrafish in regulating the segmentation of the pharyngeal endoderm into pouches. Using time-lapse microscopy, it has been shown that pharyngeal pouches form by the directed lateral migration of discrete clusters of endodermal cells. In animals doubly reduced for Fgf8 and Fgf3, the migration of pharyngeal endodermal cells is disorganized and pouches fail to form. Transplantation and pharmacological experiments show that Fgf8 and Fgf3 are required in the neural keel and cranial mesoderm during early somite stages to promote first pouch formation. In addition, animals doubly reduced for Fgf8 and Fgf3 have severe reductions in hyoid cartilages and the more posterior branchial cartilages. By examining early pouch and later cartilage phenotypes in individual animals hypomorphic for Fgf function, it was found that alterations in pouch structure correlate with later cartilage defects. A model is presented in which Fgf signaling in the mesoderm and segmented hindbrain organizes the segmentation of the pharyngeal endoderm into pouches. Moreover, it is argued that the Fgf-dependent morphogenesis of the pharyngeal endoderm into pouches is critical for the later patterning of pharyngeal cartilages (Crump, 2004).
FGF, WNT, and BMP signaling promote neural crest formation at the neural plate boundary in vertebrate embryos. To understand how these signals are integrated, the role of the transcription factors Msx1 and Pax3 was analyzed. Using a combination of overexpression and morpholino-mediated knockdown strategies in Xenopus, it has been show that Msx1 and Pax3 are both required for neural crest formation, display overlapping but nonidentical activities, and that Pax3 acts downstream of Msx1. In neuralized ectoderm, Msx1 is sufficient to induce multiple early neural crest genes. Msx1 induces Pax3 and ZicR1 cell autonomously, in turn, Pax3 combined with ZicR1 activates Slug in a WNT-dependent manner. Upstream of this, WNTs initiate Slug induction through Pax3 activity, whereas FGF8 induces neural crest through both Msx1 and Pax3 activities. Thus, WNT and FGF8 signals act in parallel at the neural border and converge on Pax3 activity during neural crest induction (Monsoro-Burq, 2004).
FGF, WNT, and BMP signaling promote neural crest formation at the neural plate boundary in vertebrate embryos. To understand how these signals are integrated, the role of the transcription factors Msx1 and Pax3 was analyzed. Using a combination of overexpression and morpholino-mediated knockdown strategies in Xenopus, it was shown that Msx1 and Pax3 are both required for neural crest formation; they display overlapping but nonidentical activities, and Pax3 acts downstream of Msx1. In neuralized ectoderm, Msx1 is sufficient to induce multiple early neural crest genes. Msx1 induces Pax3 and ZicR1 cell autonomously, in turn, Pax3 combined with ZicR1 activates Slug in a WNT-dependent manner. Upstream of this, WNTs initiate Slug induction through Pax3 activity, whereas FGF8 induces neural crest through both Msx1 and Pax3 activities. Thus, WNT and FGF8 signals act in parallel at the neural border and converge on Pax3 activity during neural crest induction (Monsoro-Burq, 2005).
In all vertebrates, the neurogenic placodes are transient ectodermal thickenings that give rise to sensory neurons of the cranial ganglia. Epibranchial (EB) placodes generate neurons of the distal facial, glossopharyngeal and vagal ganglia, which convey sensation from the viscera, including pharyngeal endoderm structures, to the CNS. Recent studies have implicated signals from pharyngeal endoderm in the initiation of neurogenesis from EB placodes; however, the signals underlying the formation of placodes are unknown. Zebrafish embryos mutant for fgf3 and fgf8 do not express early EB placode markers, including foxi1 and pax2a. Mosaic analysis demonstrates that placodal cells must directly receive Fgf signals during a specific crucial period of development. Transplantation experiments and mutant analysis reveal that cephalic mesoderm is the source of Fgf signals. Finally, both Fgf3 and Fgf8 are sufficient to induce foxi1-positive placodal precursors in wild-type as well as Fgf3-plus Fgf8-depleted embryos. A model is proposed in which mesoderm-derived Fgf3 and Fgf8 signals establish both the EB placodes and the development of the pharyngeal endoderm, the subsequent interaction of which promotes neurogenesis. The coordinated interplay between craniofacial tissues would thus assure proper spatial and temporal interactions in the shaping of the vertebrate head (Nechiporu, 2007).
Gain of function mutations in fibroblast growth factor (FGF) receptors cause chondrodysplasia and craniosynostosis syndromes. Identification of the ligands interacting with FGF receptors (FGFRs) in developing bone has remained elusive, and the mechanisms by which FGF signaling regulates endochondral, periosteal, and intramembranous bone growth are as yet not known. This study shows that Fgf18 is expressed in the perichondrium and that mice homozygous for a targeted disruption of Fgf18 exhibit a growth plate phenotype similar to that observed in mice lacking Fgfr3 and an ossification defect at sites that express Fgfr2. Mice lacking either Fgf18 or Fgfr3 exhibit expanded zones of proliferating and hypertrophic chondrocytes and increased chondrocyte proliferation, differentiation, and Indian hedgehog signaling. These data suggest that FGF18 acts as a physiological ligand for FGFR3. In addition, mice lacking Fgf18 display delayed ossification and decreased expression of osteogenic markers, phenotypes not seen in mice lacking Fgfr3. These data demonstrate that FGF18 signals through another FGFR to regulate osteoblast growth. Signaling to multiple FGFRs positions FGF18 to coordinate chondrogenesis in the growth plate with osteogenesis in cortical and trabecular bone (Z. Liu, 2002).
Much of the skeleton and connective tissue of the vertebrate head is derived from cranial neural crest. During development, cranial neural crest cells migrate from the dorsal neural tube to populate the forming face and pharyngeal arches. Fgf8 and Shh, signaling molecules known to be important for craniofacial development, are expressed in distinct domains in the developing face. Specifically, in chick embryos these molecules are expressed in adjacent but non-overlapping patterns in the epithelium covering crest-derived mesenchyme that will give rise to the skeletal projections of the upper and lower beaks. It has been suggested that these molecules play important roles in patterning the developing face. The ability of FGF8 and SHH signaling, singly and in combination, to regulate cranial skeletogenesis, has been examined both in vitro and in vivo. SHH and FGF8 were found to have strong synergistic effects on chondrogenesis in vitro and are sufficient to promote outgrowth and chondrogenesis in vivo, suggesting a very specific role for these molecules in producing the elongated beak structures during chick facial development (Abzhanov, 2004).
A Japanese chick wingless mutant (Jwg) has been analyzed to elucidate the molecular mechanism underlying wing development. The expression patterns of eleven marker genes were studied to characterize the mutant. In Jwg mutants, expression of Fgf8, a marker gene for the apical ectodermal ridge (AER), is delayed and shortly disappears in the wing as the AER regresses. Likewise, Shh, which is expressed in the posterior mesoderm of the normal chick limb by late stage 18, is considerably weaker in stage 19/20 mutant wing buds; Shh is expressed normally expressed in the posterior mesenchyme of the leg bud of the same mutant embryo. Fgf4 expression, which is normally induced in the posterior domain of the AER by Shh is not detected in the Jwg mutant wing bud at stage 19 and thereafter. Expressions of limb dorsoventral (DV) patterning genes such as Wnt7a and Lmx1 and mesenchymal marker genes such as Msx2 and Lh2 (a LIM homeodomain protein) are intact in nascent Jwg limb buds. Later in development, ventral expression of dorsal marker genes Wnt7a and Lmx1 indicate that the wing bud without the AER becomes bi-dorsal. The posterior mesoderm becomes defective, as deduced from the impaired expression patterns of Sonic hedgehog, Msx1, and Prx1. Rescue of the wing was attempted by implanting Fgf8-expressing cells into the Jwg wing bud. FGF8 can rescue outgrowth of the wing bud by maintaining Shh expression. Thus, the Jwg gene seems to be involved in maintenance of the Fgf8 expression in the wing bud. Further, it is suggested that the AER is required for maintenance of the DV boundary and the polarizing activity of the established wing bud (Ohuchi, 1997a).
Vertebrate limb formation has been known to be initiated by a factor(s) secreted from the lateral plate mesoderm. A member of the fibroblast growth factor (FGF) family, FGF10, emanates from the prospective limb mesoderm to serve as an endogenous initiator for limb bud formation. Fgf10 expression in the prospective limb mesenchyme precedes Fgf8 expression in the nascent apical ectoderm. Ectopic application of FGF10 to the chick embryonic flank can induce Fgf8 expression in the adjacent ectoderm, resulting in the formation of an additional complete limb. Expression of Fgf10 persists in the mesenchyme of the established limb bud and appears to interact with Fgf8 in the apical ectoderm and Sonic hedgehog in the zone of polarizing activity. These results suggest that FGF10 is a key mesenchymal factor involved in the initial budding as well as the continuous outgrowth of vertebrate limbs (Ohuchi, 1997b).
FGFR2 is a membrane-spanning tyrosine kinase that serves as a high affinity receptor for several members of the fibroblast growth factor (FGF) family. To explore functions of FGF/FGFR2 signals in development, FGFR2 has been mutated by deleting the entire immunoglobin-like domain III of the receptor. Murine FGFR2 is essential for chorioallantoic fusion and placenta trophoblast cell proliferation. Fgfr2 mutant embryos display two distinct defects that result in failure to form a functional placenta. About one third of the mutants fail to form the chorioallantoic fusion junction and the remaining mutants do not have the labyrinthine portion of the placenta. Consequently, all mutants die at 10-11 days of gestation. Interestingly, mutant embryos do not form limb buds. Consistent with this defect, the expression of Fgf8, an apical ectodermal factor, is absent in the mutant presumptive limb ectoderm, and the expression of Fgf10, a mesenchymally expressed limb bud initiator, is down regulated in the underlying mesoderm. These findings provide direct genetic evidence that FGF/FGFR2 signals are absolutely required for vertebrate limb induction and that an FGFR2 signal is essential for the reciprocal regulation loop between FGF8 and FGF10 during limb induction (Xu, 1998).
During limb development, several signaling centers organize limb pattern. One of these, the apical ectodermal ridge (AER), is critical for proximodistal limb outgrowth mediated by FGFs. Signals from the underlying mesoderm, including WNTs and FGFs, regulate early steps of AER induction. Ectodermal factors, particularly En1, play a critical role in regulating morphogenesis of a mature, compact AER along the distal limb apex, from a broad ventral ectodermal precursor domain. Contribution of mesodermal factors to the morphogenesis of a mature AER is less clear. The chick T gene (Brachyury), the prototypical T-box transcription factor, is expressed in the limb bud as well as axial mesoderm and primitive streak. T is expressed in lateral plate mesoderm at the onset of limb bud formation and subsequently in the subridge mesoderm beneath the AER. Retroviral misexpression of T in chick results in anterior extension of the AER and subsequent limb phenotypes consistent with augmented AER extent and function. Analysis of markers for functional AER in mouse T-/- null mutant limb buds reveals disrupted AER morphogenesis. These data also suggest that FGF and WNT signals may operate both upstream and downstream of T. During limb induction, WNT signals maintain high Fgf10 expression in prospective limb and FGF10 activates ectodermal Wnt3a and Fgf8 expression, initiating AER formation. AER signals subsequently also maintain mesodermal Fgf10 expression. T transcripts are first clearly detected at stage 15, at the onset of Wnt3a and Fgf8 activation in the ectoderm. Both the ability of WNT3a and FGF8 to induce T expression, and the ability of T to increase subridge expression of Fgf10 early after misexpression suggest that T may be a component of the mesodermal response to developing AER signals that maintains high Fgf10 apically and thereby also maintains the forming AER, establishing a regulatory loop between ectoderm and mesoderm. Taken together, the results show that T plays a role in the regulation of AER formation, particularly maturation, and suggest that T may also be a component of the epithelial-mesenchymal regulatory loop involved in maintenance of a mature functioning AER (Y. Liu, 2002).
In vertebrate limbs that lack webbing, the embryonic interdigit region is removed by programmed cell death (PCD). Established models suggest that bone morphogenetic proteins (BMPs) directly trigger such PCD, although no direct genetic evidence exists for this. Alternatively, BMPs might indirectly affect PCD by regulating fibroblast growth factors (FGFs), which act as cell survival factors. The mouse BMP receptor gene Bmpr1a was inactivated specifically in the limb bud apical ectodermal ridge (AER), a source of FGF activity. Early inactivation completely prevents AER formation. However, inactivation after limb bud initiation causes an upregulation of two AER-FGFs, Fgf4 and Fgf8, and a loss of interdigital PCD leading to webbed limbs. To determine whether excess FGF signaling inhibits interdigit PCD in these Bmpr1a mutant limbs, double and triple AER-specific inactivations of Bmpr1a, Fgf4 and Fgf8 were performed. Webbing persists in AER-specific inactivations of Bmpr1a and Fgf8 owing to elevated Fgf4 expression. Inactivation of Bmpr1a, Fgf8 and one copy of Fgf4 eliminates webbing. It is concluded that during normal embryogenesis, BMP signaling to the AER indirectly regulates interdigit PCD by regulating AER-FGFs, which act as survival factors for the interdigit mesenchyme (Pajni-Underwood, 2007).
The complete cohort of molecules involved in interdigital cell death (ICD) and their interactions are yet to be defined. Bmp proteins, retinoic acid (RA) and Fgf8 have been previously identified as relevant factors in the control of ICD. This study determined that downregulation of Fgf8 expression in the ectoderm overlying the interdigital areas is the event that triggers ICD, whereas RA is the persistent cell death-inducing molecule that acts on the distal mesenchyme by a mechanism involving the induction of Bax expression. Inhibition of the mitogen-activated protein kinase (Mapk) pathway prevents the survival effect of Fgf8 on interdigital cells and the accompanying Erk1/2 phosphorylation and induction of Mkp3 expression. Fgf8 regulates the levels of RA by both decreasing the expression of Raldh2 and increasing the expression of Cyp26b1, whereas RA reduces Fgfr1 expression and Erk1/2 phosphorylation. In the mouse limb, inhibition of Bmp signaling in the mesenchyme does not affect ICD. However, noggin in the distal ectoderm induces Fgf8 expression and reduces interdigit regression. In the chick limb, exogenous noggin reduces ICD, but, when applied to the distal mesenchyme, this reduction is associated with an increase in Fgf8 expression. In agreement with the critical decline in Fgf8 expression for the activation of ICD, distal interdigital cells acquire a proximal position as interdigit regression occurs. Proliferating distal mesenchymal cells were identified as those that give rise to the interdigital cells fated to die. Thus, ICD is determined by the antagonistic regulation of cell death by Fgf8 and RA and occurs through a progressive, rather than massive, cell death mechanism (Hernandez-Martinez, 2009).
Members of the Pitx/RIEG family of homeodomain-containing transcription factors have been implicated in vertebrate organogenesis. In this study, the expression and regulation of Pitx1 and Pitx2 during mouse tooth development was examined. Pitx1 expression is detected in early development in a widespread pattern, in both epithelium and mesenchyme, covering the tooth-forming region in the mandible, and is then maintained in the dental epithelium from the bud stage to the late bell stage. Pitx2 expression, on the other hand, is restricted to the dental epithelium throughout odontogenesis. Interestingly, from E9.5 to E10.5, the expression domains of Pitx1 and Pitx2, in the developing mandible, overlap with that of Fgf8 but are exclusive to the zone of Bmp4 expression. Bead implantation experiments demonstrate that ectopic expression of Fgf8 can induce/maintain the expression of both Pitx1 and Pitx2 at E9.5. In contrast, Bmp4-expressing tissues and BMP4-soaked beads are able to repress Pitx1 expression in mandibular mesenchyme and Pitx2 expression in the presumptive dental epithelium, respectively. However, the effects of FGF8 and BMP4 are transient. It thus appears that the early expression patterns of Pitx1 and Pitx2 in the developing mandible are regulated by the antagonistic effects of FGF8 and BMP4 such that the Pitx1 and Pitx2 expression patterns are defined. These results indicate that the epithelial-derived signaling molecules are responsible not only for restricting specific gene expression in the dental mesenchyme, but also for defining gene expression in the dental epithelium (St.Amand, 2000).
In mammalian embryos, male and female external genitalia develop from the genital tubercle. Outgrowth of the genital tubercle is maintained by the urethral epithelium, and it has been reported that Fgf8 mediates this activity. To test directly whether Fgf8 is required for external genital development, Fgf8 was conditionally removed from the cloacal/urethral epithelium. Surprisingly, Fgf8 is not necessary for initiation, outgrowth or normal patterning of the external genitalia. In early genital tubercles, no redundant Fgf expression was found in the urethral epithelium, which contrasts with the situation in the apical ectodermal ridge (AER) of the limb. Analysis of Fgf8 pathway activity showed that four putative targets are either absent from early genital tubercles or are not regulated by Fgf8. Therefore the distribution of Fgf8 protein was examined; although it is present in the AER, Fgf8 is undetectable in the genital tubercle. Thus, Fgf8 is transcribed, but the signaling pathway is not activated during normal genital development. A phylogenetic survey of amniotes revealed Fgf8 expression in genital tubercles of eutherian and metatherian mammals, but not turtles or alligators, indicating that Fgf8 expression is neither a required nor a conserved feature of amniote external genital development. The results indicate that Fgf8 expression is an early readout of the genital initiation signal rather than the signal itself. It is proposed that induction of external genitalia involves an epithelial-epithelial interaction at the cloacal membrane, and it is suggested that the cloacal ectoderm may be the source of the genital initiation signal (Seifert, 2009).
The developing vertebrate mesencephalon shows a rostrocaudal gradient in the expression of a number of molecular markers and in the cytoarchitectonic differentiation of the tectum, where cells cease proliferating and differentiate in a rostral to caudal progression. Tissue grafting experiments have implicated cell signaling by the mesencephalic-metencephalic (mid-hindbrain) junction (or isthmus) in orchestrating these events. The role of Wnt-1 (Drosophila homolog: Wingless) and FGF8 signaling has been explored in the regulation of mesencephalic polarity. FGF8 is expressed in cardiac mesoderm underlying the presumptive mesencephalic/metencephalic region and may play a role in mesencephalic induction. Fgf8 is also expressed in the neural plate itself, in the most rostral metencephalon. Wnt-1 is expressed in the caudal mesencephalon. Wnt-1 regulates Fgf8 expression in the adjacent metencephalon, most likely via a secondary mesencephalic signal. Ectopic expression of Fgf8 in the mesencephalon is sufficient to activate expression of Engrailed-2 (Drosophila homolog: Engrailed) and ELF-1, two genes normally expressed in a decreasing caudal to rostral gradient in the posterior mesencephalon. ELF-1 is a ligand for a EPH-like receptor tyrosine kinase expressed in rostrocaudally increasing gradients across the caudal tectum and may function to inhibit temporal axon ingrowth and/or to attract nasal axons. Ectopic expression of Engrailed-1, a functionally equivalent homolog of En-2 is sufficient to activate ELF-1 expression by itself. These results indicate the existence of a molecular hierarchy in which FGF8 signaling establishes the graded expression of En-2 within the tectum. This in turn may act to specify other aspects of A-P polarity such as graded ELF-1 expression. FGF8 is a potent mitogen within the mesencephalon: when ectopically expressed, neural precursors continue to proliferate and neurogenesis is prevented. Taken together these results suggest that FGF8 signalling from the isthmus has a key role in coordinately regulating growth and polarity in the developing mesencephalon. It is unlikely that the normal sourse of FGF8 is the brain, as expression is not initiated in the mes/met region until 3-somites. This is after the regional activation of several genes in the presumptive mes/met region, including Pax-2, Wnt-1 and En-1. The issue of whether FGF8 plays a direct role in mesencephalon induction from cardiogenic mesodermal cells remains an open question. Engrailed itself may play an earlier role in mesencephalic specification. It is clear at least that FGF8 signaling plays an important role in the regulation of growth and polarity in the mesencephalon (Lee, 1997).
The patterns of the Gbx2, Pax2, Wnt1, and Fgf8 gene expression were analyzed in the chick with respect to the caudal limit of the Otx2 anterior domain, taken as a landmark of the midbrain/hindbrain (MH) boundary. The Gbx2 anterior boundary is always concomitant with the Otx2 posterior boundary. The ring of Wnt1 expression is included within the Otx2 domain and Fgf8 transcripts included within the Gbx2 neuroepithelium. Pax2 expression is centered on the MH boundary with a double decreasing gradient. A new nomenclature is proposed to differentiate the vesicles and constrictions observed in the avian MH domain at stage HH10 and HH20, based on the localization of the Gbx2/Otx2 common boundary (Hidalgo-Snachez, 1999).
The vertebrate central nervous system (CNS) contains a small group (~24,000 in human, ~3,200 in rodent, and ~7-10 in zebrafish) of evolutionary conserved noradrenergic (NA) neurons known as the locus coeruleus (LC). These neurons reside in the ventro-lateral region of the first hindbrain rhombomere and project to regions throughout the CNS. Their degeneration is associated with Parkinson's and Alzheimer's disease, whereas their abnormal function is thought to play a role in depression, sleep disorders, and schizophrenia. The zebrafish mutation soulless, in which the development of locus coeruleus noradrenergic neurons fails to occur, disrupts the homeodomain protein Phox2a. Phox2a is not only necessary but also sufficient to induce Phox2b+ dopamine-beta-hydroxylase+ and tyrosine hydroxylase+ NA neurons in ectopic locations. Phox2a is first detected in LC progenitors in the dorsal anterior hindbrain, and its expression there is dependent on FGF8 from the mid/hindbrain boundary and on optimal concentrations of BMP signal from the epidermal ectoderm/future dorsal neural plate junction. These findings suggest that Phox2a coordinates the specification of LC in part through the induction of Phox2b and in response to cooperating signals that operate along the mediolateral and anteroposterior axes of the neural plate (Guo, 1999).
The mid/hindbrain junction region, which expresses Fgf8, can act as an organizer to transform caudal forebrain or hindbrain tissue into midbrain or cerebellar structures, respectively. FGF8-soaked beads placed in the chick forebrain can similarly induce ectopic expression of mid/hindbrain genes and development of midbrain structures. In contrast, ectopic expression of Fgf8a in the mouse midbrain and caudal forebrain using a Wnt1 regulatory element produces no apparent patterning defects in the embryos examined. FGF8b-soaked beads can not only induce expression of the mid/hindbrain genes En1, En2 and Pax5 in mouse embryonic day 9.5 (E9.5) caudal forebrain explants, but also can induce the hindbrain gene Gbx2 and alter the expression of Wnt1 in both midbrain and caudal forebrain explants. FGF8b-soaked beads can repress Otx2 in midbrain explants. Furthermore, Wnt1-Fgf8b transgenic embryos in which the same Wnt1 regulatory element is used to express Fgf8b, have ectopic expression of En1, En2, Pax5 and Gbx2 in the dorsal hindbrain and spinal cord at E10.5, as well as exencephaly and abnormal spinal cord morphology. More strikingly, Fgf8b expression in more rostral brain regions appears to transform the midbrain and caudal forebrain into an anterior hindbrain fate through expansion of the Gbx2 domain and repression of Otx2 as early as the 7-somite stage. These findings suggest that normal Fgf8 expression in the anterior hindbrain not only functions to maintain development of the entire mid/hindbrain by regulating genes like En1, En2 and Pax5, but also might function to maintain a metencephalic identity by regulating Gbx2 and Otx2 expression (Liu, 1999).
It is interesting that the phenotype observed in early Wnt1-Fgf8b transgenics is similar to that seen in Otx1+/-Otx2+/- or Otx1-/-Otx2+/- double mutants; an early induction of Gbx2 and repression of Otx2 in the midbrain and caudal forebrain. In Otx1-/-;Otx2+/- embryos, an anterior expansion of Fgf8 expression precedes an anterior shift of Wnt1 and En1 expression and an anterior retraction of Otx2 expression. The Otx mutant studies suggest a certain level of Otx2 expression is necessary to repress expression of Fgf8 in the midbrain and forebrain, and these results suggest that, in addition, expanded Fgf8 expression could contribute to repression of Otx2 expression in the midbrain. A reciprocal negative regulation between Otx2 and Fgf8 might therefore normally contribute to maintaining the Otx2 caudal boundary and positioning the organizer (Liu, 1999 and references therein).
Beads containing recombinant FGF8 (FGF8-beads) were implanted in the prospective caudal diencephalon or midbrain of chick embryos at stages 9-12. This induces the neuroepithelium rostral and caudal to the FGF8-bead to form two ectopic, mirror-image midbrains. Furthermore, cells in direct contact with the bead form an outgrowth that protruded laterally from the neural tube. Tissue within such lateral outgrowths developed proximally into isthmic nuclei and distally into a cerebellum-like structure. These morphogenetic effects are apparently due to FGF8-mediated changes in gene expression in the vicinity of the bead, including a repressive effect on Otx2 and an inductive effect on En1, Fgf8 and Wnt1 expression. The ectopic Fgf8 and Wnt1 expression domains form nearly complete concentric rings around the FGF8-bead, with the Wnt1 ring outermost. These observations suggest that FGF8 induces the formation of a ring-like ectopic signaling center (organizer) in the lateral wall of the brain, similar to the one that normally encircles the neural tube at the isthmic constriction, which is located at the boundary between the prospective midbrain and hindbrain. This ectopic isthmic organizer apparently sends long-range patterning signals both rostrally and caudally, resulting in the development of the two ectopic midbrains. Interestingly, the data suggest that these inductive signals spread readily in a caudal direction, but are inhibited from spreading rostrally across diencephalic neuromere boundaries. These results provide insights into the mechanism by which FGF8 induces an ectopic organizer and suggest that a negative feedback loop between Fgf8 and Otx2 plays a key role in patterning the midbrain and anterior hindbrain (Martinez, 1999).
Specification and polarization of the midbrain and anterior hindbrain involves planar signals originating from the isthmus. Current evidence suggests that FGF8, expressed at the isthmus, provides this patterning influence. In this study, novel genes were sought that are involved in the process by which regional identity is imparted to midbrain and anterior hindbrain (rhombomere 1). An enhanced differential display reverse transcription method was used to clone cDNAs derived from transcripts expressed specifically in either rhombomere 1 or midbrain during the period of isthmic patterning activity. This gene expression screen has identified 28 differentially expressed cDNAs. A clone upregulated in cDNA derived from rhombomere 1 tissue shows a 91% identity at the nucleotide level to the putative human receptor tyrosine kinase antagonist: sprouty2. In situ hybridization on whole chick embryos shows chick sprouty2 to be expressed initially within the isthmus and rhombomere 1, spatially and temporally coincident with Fgf8 expression. However, at later stages this domain is more extensive than that of Fgf8. Introduction of ligand-coated beads into either midbrain or hindbrain region reveal that sprouty2 can be rapidly induced by FGF8. These data suggest that sprouty2 participates in a negative feedback regulatory loop to modulate the patterning activity of FGF8 at the isthmus (Chambers, 2000).
The most studied secondary neural organizer is the isthmic organizer, which is localized at the mid-hindbrain transition of the neural tube and controls the anterior hindbrain and midbrain regionalization. Otx2 and Gbx2 expressions are fundamental for positioning the organizer and the establishment of molecular interactions that induce Fgf8. Evidence in this study demonstrates that Otx2 and Gbx2 have an overlapping expression in the isthmic region. This area is the transversal domain where expression of Fgf8 is induced. The Fgf8 protein produced in the isthmus stabilizes and up-regulates Gbx2 expression, which, in turn, down-regulates Otx2 expression. The inductive effect of the Gbx2/Otx2 limit keeps Fgf8 expression stable and thus maintains its positive role in the expression of Pax2, En1,2 and Wnt1 (Garda, 2001).
Fibroblast growth factors (Fgfs) form a large family of secreted signalling proteins that have a wide variety of roles during embryonic development. Within the central nervous system (CNS) Fgf8 is implicated in patterning neural tissue adjacent to the midbrain-hindbrain boundary. However, the roles of Fgfs in CNS tissue rostral to the midbrain are less clear. The patterning of the forebrain was examined in zebrafish embryos that lack functional Fgf8/Ace. Ace is required for the development of midline structures in the forebrain. In the absence of Ace activity, midline cells fail to adopt their normal morphology and exhibit altered patterns of gene expression. This disruption to midline tissue leads to severe commissural axon pathway defects, including misprojections from the eye to ectopic ipsilateral and contralateral targets. Ace is also required for the differentiation of the basal telencephalon and several populations of putative telencephalic neurons but not for overall regional patterning of forebrain derivatives. ace expression co-localizes with anterior neural plate cells that have previously been shown to have forebrain patterning activity. Removal of these cells leads to a failure in induction of ace expression indicating that loss of Ace activity may contribute to the phenotypes observed when anterior neural plate cells are ablated. However, since ace mutant neural plate cells still retain at least some inductive activity, then other signals must also be produced by the anterior margin of the neural plate. In ace minus embryos, there are severe defects in the establishment of both the postoptic commissure and the anterior commissure such that both commissures are usually initially absent and fused at later stages. This indicates that Ace has a crucial role in the development of commissural neuroepithelium. In support of this, alterations in expression of midline genes encoding proteins that are likely to directly influence axon extension (netrin1, sema3D), and in genes more likely to indirectly influence guidance cues (no-isthmus embryos that carry mutations in the pax2.1 gene, six3 and Tiggywinkle hedgehog) (Shanmugalingam, 2000).
Fgf8, which is expressed at the embryonic mid/hindbrain junction, is required for and sufficient to induce the formation of midbrain and cerebellar structures. To address the genetic pathways through which FGF8 acts, the epistatic relationships of mid/hindbrain genes that respond to FGF8 were examined, using a novel mouse brain explant culture system. En2 and Gbx2 are the first genes to be induced by FGF8 in wild-type E9.5 diencephalic and midbrain explants treated with FGF8-soaked beads. By examining gene expression in En1/2 double mutant mouse embryos, it was found that Fgf8, Wnt1 and Pax5 do not require the En genes for initiation of expression, but do for their maintenance, and Pax6 expression is expanded caudally into the midbrain in the absence of EN function. Since E9.5 En1/2 double mutants lack the mid/hindbrain region, forebrain mutant explants were treated with FGF8 and, significantly, the EN transcription factors were found to be required for induction of Pax5. Thus, FGF8-regulated expression of Pax5 is dependent on EN proteins, and a factor other than FGF8 could be involved in initiating normal Pax5 expression in the mesencephalon/metencephalon. The En genes also play an important, but not absolute, role in repression of Pax6 in forebrain explants by FGF8. Gbx2 gain-of-function studies have shown that misexpression of Gbx2 in the midbrain can lead to repression of Otx2. However, in the absence of Gbx2, FGF8 can nevertheless repress Otx2 expression in midbrain explants. In contrast, Wnt1 is initially broadly induced in Gbx2 mutant explants, as in wild-type explants, but not subsequently repressed in cells near FGF8 that normally express Gbx2. Thus GBX2 acts upstream of, or parallel to, FGF8 in repressing Otx2, and acts downstream of FGF8 in repression of Wnt1. This is the first such epistatic study performed in mouse that combines gain-of-function and loss-of-function approaches to reveal aspects of mouse gene regulation in the mesencephalon/metencephalon that have been difficult to address using either approach alone (Liu, 2001).
The segmentation of the vertebrate hindbrain into rhombomeres is highly conserved, but how early hindbrain patterning is established is not well understood. Rhombomere 4 (r4) functions as an early-differentiating signaling center in the zebrafish hindbrain. Time-lapse analyses of zebrafish hindbrain development show that r4 forms first and hindbrain neuronal differentiation occurs first in r4. Two signaling molecules, FGF3 and FGF8, which are both expressed early in r4, are together required for the development of rhombomeres adjacent to r4, particularly r5 and r6. Transplantation of r4 cells can induce expression of r5/r6 markers, as can misexpression of either FGF3 or FGF8. Genetic mosaic analyses also support a role for FGF signaling acting from r4. Taken together, these findings demonstrate a crucial role for FGF-mediated inter-rhombomere signaling in promoting early hindbrain patterning and underscore the significance of organizing centers in patterning the vertebrate neural plate (Maves, 2002).
The roles were investigated of bare morphogenetic protein (BMP), sonic hedgehog (SHH) and fibroblast growth factor (FGF)-expressing signaling centers in regulating the patterned outgrowth of the telencephalic and optic vesicles. Implantation of BMP4 beads in the anterior neuropore of stage 10 chicken embryos repressed FGF8 and SHH expression. Similarly, loss of SHH expression in Shh mutant mice leads to increased BMP signaling and loss of Fgf8 expression in the prosencephalon. Increased BMP signaling and loss of FGF and SHH expression was correlated with decreased proliferation, increased cell death, and hypoplasia of the telencephalic and optic vesicles. However, decreased BMP signaling, through ectopic expression of Noggin, a BMP-binding protein, also caused decreased proliferation and hypoplasia of the telencephalic and optic vesicles, but with maintenance of Fgf8 and Shh expression, and no detectable increase in cell death. These results suggest that optimal growth requires a balance of BMP, FGF8 and SHH signaling. It is suggested that the juxtaposition of Fgf8, Bmp4 and Shh expression domains generate patterning centers that coordinate the growth of the telencephalic and optic vesicles, similar to how Fgf8, Bmp4 and Shh regulate growth of the limb bud. Furthermore, these patterning centers regulate regional specification within the forebrain and eye, as exemplified by the regulation of Emx2 expression by different levels of BMP signaling. In summary, evidence is presented that there is cross-regulation between BMP-, FGF- and SHH-expressing signaling centers in the prosencephalon which regulate morphogenesis of, and regional specification within, the telencephalic and optic vesicles (Ohkubo, 2002).
Hindbrain (brainstem) 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. A novel signaling center has been identified in the zebrafish embryo that arises prior to establishment of segmental patterning; it is located centrally within the hindbrain territory in a region that corresponds to the presumptive rhombomere 4. 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 (Walshe, 2002).
Previous work on signals that regulate establishment of hindbrain segmental identity has mostly focused on the role of retinoic acid (RA) released from paraxial mesoderm at a distance from the hindbrain primordium. While RA clearly directly regulates expression of Hox genes, another role may be to position the Fgf signaling center within the presumptive hindbrain. Ectopic RA application to zebrafish embryos results in respecification of r2 to an r4 phenotype, and Fgf3 expression is induced in r2. By contrast, inhibition of RA function results in posterior expansion of anterior hindbrain such that the Fgf3 domain lies at somitic levels (Walshe, 2002).
These studies identify the presumptive r4 territory as a source of planar signals that serve to pattern the neural plate. Other such signaling centers are the isthmus, where Fgf8 functions at later stages to pattern midbrain and r1, and the anterior neural ridge, which patterns the telencephalon, in part by Fgf3 and Fgf8 signaling. Thus, the common feature of all three is that Fgf provides a planar signal to pattern adjacent neural territories, indicating that Fgf signaling has been coopted to impart regional identity multiple times during evolution of the vertebrate brain (Walshe, 2002).
Complex spatiotemporal expression patterns of fgf3 and fgf8 within the developing zebrafish forebrain suggest their involvement in its regionalization and early development. These factors have unique and combinatorial roles during development of more posterior brain regions, and similar findings have been made for the developing forebrain. Fgf8 and Fgf3 regulate different aspects of telencephalic development, and 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 zona limitans intrathalamica development. Forebrain commissure formation is abnormal in the absence of either Fgf3 or Fgf8; however, most severe defects are observed in the absence of both. Defects are 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 (Walshe, 2003).
Numerous studies have demonstrated that the midbrain and cerebellum develop from a region of the early neural tube comprising two distinct territories known as the mesencephalon (mes) and rostral metencephalon (met; rhombomere1), respectively. Development of the mes and met is thought to be regulated by molecules produced by a signaling center, termed the isthmic organizer (IsO), which is localized at the boundary between them. FGF8 and WNT1 have been implicated as key components of IsO signaling activity, and previous studies have shown that in Wnt1-/- embryos, the mes/met is deleted by the 30 somite stage. The function of FGF8 in mouse mes/met development has been studied using a conditional gene inactivation approach. In mutant embryos, Fgf8 expression is transiently detected, but then is eliminated in the mes/met by the 10 somite stage. This results in a failure to maintain expression of Wnt1 as well as Fgf17, Fgf18, and Gbx2 in the mes/met at early somite stages, and in the absence of the midbrain and cerebellum at E17.5. A major cause of the deletion of these structures is ectopic cell death in the mes/met between the 7 and 30 somite stages. Interestingly, the prospective midbrain is deleted at an earlier stage than the prospective cerebellum. A remarkably similar pattern of cell death is observed in Wnt1 null homozygotes, and also ectopic mes/met cell death in En1 is detected in null homozygotes. These data show that Fgf8 is part of a complex gene regulatory network that is essential for cell survival in the mes/met (Chi, 2003).
The neocortex is divided into multiple areas with specific architecture, molecular identity and pattern of connectivity with the dorsal thalamus. Gradients of transcription factor expression in the cortical primordium regulate molecular regionalization and potentially the patterning of thalamic projections. Reduction of Fgf8 levels in hypomorphic mouse mutants shifts early gradients of gene expression rostrally, thereby modifying the molecular identity of rostral cortical progenitors. This shift correlates with a reduction in the size of a molecularly defined rostral neocortical domain and a corresponding rostral expansion of more caudal regions. Despite these molecular changes, the topography of projections between the dorsal thalamus and rostral neocortex in mutant neonates appears the same as the topography of wild-type littermates. Overall, this study demonstrates the role of endogenous Fgf8 in regulating early gradients of transcription factors in cortical progenitor cells and in molecular regionalization of the cortical plate (Garel, 2003).
Specification of the forebrain, midbrain and hindbrain primordia occurs during gastrulation in response to signals that pattern the gastrula embryo. Following establishment of the primordia, each brain part is thought to develop largely independently from the others under the influence of local organizing centers like the midbrain-hindbrain boundary (MHB, or isthmic) organizer. Mechanisms that maintain the integrity of brain subdivisions at later stages are not yet known. To examine such mechanisms in the anterior neural tube, the establishment and maintenance of the diencephalic-mesencephalic boundary (DMB) was studied. Maintenance of the DMB requires both the presence of a specified midbrain and a functional MHB organizer. Expression of pax6.1, a key regulator of forebrain development, is posteriorly suppressed by the Engrailed proteins, Eng2 and Eng3. Mis-expression of eng3 in the forebrain primordium causes downregulation of pax6.1, and forebrain cells correspondingly change their fate and acquire midbrain identity. Conversely, in embryos lacking both eng2 and eng3, the DMB shifts caudally into the midbrain territory. However, a patch of midbrain tissue remains between the forebrain and the hindbrain primordia in such embryos. This suggests that an additional factor maintains midbrain cell fate. Fgf8 is a candidate for this signal; it is both necessary and sufficient to repress pax6.1 and hence to shift the DMB anteriorly independently of the expression status of eng2/eng3. By examining small cell clones that are unable to receive an Fgf signal, cells in the presumptive midbrain neural plate were shown to require an Fgf signal to keep them from following a forebrain fate. Combined loss of both Eng2/Eng3 and Fgf8 leads to complete loss of midbrain identity, resulting in fusion of the forebrain and the hindbrain primordia. Thus, Eng2/Eng3 and Fgf8 are necessary to maintain midbrain identity in the neural plate and thereby position the DMB. This provides an example of a mechanism needed to maintain the subdivision of the anterior neural plate into forebrain and midbrain (Scholpp, 2003).
FGFR1 is an important signalling molecule during embryogenesis and in adulthood. FGFR1 mutations in human may lead to developmental defects and pathological conditions, including cancer and Alzheimer's disease. Cloning and expression analysis of the zebrafish fibroblast growth factor receptor 1 (fgfr1) is described. Initially, fgfr1 is expressed in the adaxial mesoderm with transcripts distinctly localised to the anterior portion of each half-somite. Hereupon, fgfr1 is also strongly expressed in the otic vesicles, branchial arches and the brain, especially at the midbrain-hindbrain boundary (MHB). The expression patterns of fgfr1 and fgf8 are strikingly similar and knock-down of fgfr1 phenocopies many aspects observed in the fgf8 mutant acerebellar, suggesting that Fgf8 exerts its function mainly by binding to FgfR1 (Scholpp, 2004).
The organisation of the telencephalon into its major structures depends on its early regionalisation along the dorsoventral axis. Previous studies have provided evidence that sonic hedgehog (SHH) is required for the generation of telencephalic cells of ventral character, and that sequential WNT and fibroblast growth factor (FGF) signalling specifies cells of dorsal telencephalic character. However, the signalling mechanisms that specify telencephalic cells of an intermediate character remain to be defined. Evidence is presented that retinoic acid has a crucial role in specifying telencephalic progenitor cells of intermediate character (Marklund, 2004).
These results provide insights into the sequential steps involved in assigning an initial DV regional identity to telencephalic neural progenitor cells. The following model emerges from these findings. At gastrula stages, most or all prospective telencephalic cells become specified as ventral (NKX2.1+) cells in response to node-derived SHH signals. At neural fold and early neural plate stages, cells in the prospective dorsal and intermediate regions of the telencephalon cells are exposed to WNT signals that induce PAX6+ cells. The head ectoderm adjacent to the telencephalon then starts to express retinaldehyde dehydrogenase 3, exposing telencephalic cells to RA signals that promote the generation of intermediate (MEIS2+) cells. From the neural plate stage, prospective ventral telencephalic cells are exposed to FGF8 derived from the anterior neural ridge, and FGF8 maintains ventral telencephalic character by opposing the influence of RA signals in ventral cells. At early neural tube stages, the domain of Fgf8 expression expands dorsally and FGF signals derived from the dorsal midline region induce definitive dorsal/precortical (EMX1+) cells, and cells that are exposed to RA and low levels of FGF8 acquire intermediate character (Marklund, 2004).
The mouse homeobox gene Gbx2 is first expressed throughout the posterior region of the embryo during gastrulation, and becomes restricted to rhombomeres 1-3 (r1-3) by embryonic day 8.5 (E8.5). Previous studies have shown that r1-3 do not develop in Gbx2 mutants and that there is an early caudal expansion of the midbrain gene Otx2 to the anterior border of r4. Furthermore, expression of Wnt1 and Fgf8, two crucial components of the isthmic organizer, is no longer segregated to adjacent domains in Gbx2 mutants. In this study, the phenotypic analysis of Gbx2 mutants has been extended by showing that Gbx2 is not only required for development of r1-3, but also for normal gene expression in r4-6. To determine whether Gbx2 can alter hindbrain development, Hoxb1-Gbx2 (HG) transgenic mice were generated in whichGbx2 is ectopically expressed in r4. Gbx2 was shown to be insufficient to induce r1-3 development in r4. To test whether an Otx2/Gbx2 interface can induce r1-3 development, the HG transgene was introduced onto a Gbx2-null mutant background and a new Otx2/Gbx2 border was recreated in the anterior hindbrain. Development of r3, but not r1 and r2, is rescued in Gbx2–/–; HG embryos. In addition, the normal spatial relationship of Wnt1 and Fgf8 is established at the new Otx2/Gbx2 border, demonstrating that an interaction between Otx2 and Gbx2 is sufficient to produce the normal pattern of Wnt1 and Fgf8 expression. However, the expression domains of Fgf8 and Spry1, a downstream target of Fgf8, are greatly reduced in mid/hindbrain junction area of Gbx2–/–; HG embryos and the posterior midbrain is truncated because of abnormal cell death. Interestingly, it was shown that increased cell death and a partial loss of the midbrain are associated with increased expression of Fgf8 and Spry1 in Gbx2 conditional mutants that lack Gbx2 in r1 after E9.0. These results together suggest that cell survival in the posterior midbrain is positively or negatively regulated by Fgf8, depending on Fgf8 expression level. These studies provide new insights into the regulatory interactions that maintain isthmic organizer gene expression and the consequences of altered levels of organizer gene expression on cell survival (Li, 2005).
The organizing center located at the midbrain-hindbrain boundary (MHB) patterns the midbrain and hindbrain primordia of the neural plate. Studies in several vertebrates have shown that the interface between cells expressing Otx and Gbx transcription factors marks the location in the neural plate where the organizer forms, but it is unclear how this location is set up. Using mutant analyses and shield ablation experiments in zebrafish, it has been found that axial mesendoderm, as a candidate tissue, has only a minor role in positioning the MHB. Instead, the blastoderm margin of the gastrula embryo acts as a source of signal(s) involved in this process. Positioning of the MHB organizer is tightly linked to overall neuroectodermal posteriorization, and specifically depends on Wnt8 signaling emanating from lateral mesendodermal precursors. Wnt8 is required for the initial subdivision of the neuroectoderm, including onset of posterior gbx1 expression and establishment of the posterior border of otx2 expression. Cell transplantation experiments further show that Wnt8 signaling acts directly and non-cell-autonomously. Consistent with these findings, a GFP-Wnt8 fusion protein travels from donor cells through early neural plate tissue. These findings argue that graded Wnt8 activity mediates overall neuroectodermal posteriorization and thus determines the location of the MHB organize (Rhinn, 2005).
How does Wnt8 participate in positioning of the MHB organizer? wnt8 is expressed in the marginal cells and hypoblast and two receptors, fz8c and fz9, are detected in both hypoblast and epiblast. Conceivably, Wnt8 is transmitted in a planar fashion through the neuroectoderm. This idea is supported by the clonal analysis of wnt8 overexpressing cells: gbx1 is activated in the host tissue one or two cells distant from the transplanted cells, and otx2 is repressed four or five cells distant from the transplanted cells. In unmanipulated neuroectoderm, the onset of gbx1 expression occurs close to the wnt8 domain with little or no overlap, and the otx2 expression domain is situated eight to ten cell diameters away from the wnt8 domain at 60% of epiboly. Thus, the wnt8 expression domain is appropriately located to generate a graded morphogenetic Wnt8 signal that regulates the expression of gbx1 and otx2 genes in vivo. This finding is more generally consistent with the ability of Wnt molecules to form gradients and to activate target genes in a concentration-dependent manner, as in the Drosophila wing imaginal disc, where expression of wingless target genes like neuralized, distalless and vestigial depends on the distance from wingless-expressing cells. Similarly, in the unmanipulated zebrafish neuroectoderm, the otx2 and the gbx1 domains are located at different distances from the Wnt8 source at the lateral blastoderm margin. Following global misexpression experiments, different Wnt8 doses can differentially regulate otx2 and gbx1 expression: wnt8 ectopic expression can induce gbx1 expression at low/intermediate doses, but represses at high doses. Conversely, otx2 is increasingly repressed with increasing wnt8 concentration. Similarly, around wnt8-expressing clones, gbx1 is induced at a distance of one or two cells around the clone, whereas otx2 is repressed at a distance of four or five cells. This suggests that a lower Wnt8 concentration is needed to repress otx2 than to induce gbx1. Altogether, these observations suggest that Wnt8 has properties of a morphogen whose activity is required to correctly position the otx2/gbx1 interface, and probably other target genes in the forming neural plate. The observation of secreted Wnt8-GFP protein emanating from clones of producing cells is generally consistent with this possibility. Distribution of another signaling molecule in the early neural plate, Fgf8, is carefully controlled by endocytosis. It will be interesting to determine if Wnt8 protein is indeed distributed in a graded fashion, and which mechanisms control this distribution. In mice, Wnt8 is expressed in the posterior epiblast of early primitive streak-stage embryos; although its function is unknown, Wnt8 may therefore serve a similar function as proposed in this study (Rhinn, 2005).
Cajal-Retzius (CR) cells play a key role in the formation of the cerebral cortex. These pioneer neurons are distributed throughout the cortical marginal zone in distinct graded distributions. Fate mapping and cell lineage tracing studies have recently shown that CR cells arise from restricted domains of the pallial ventricular zone, which are associated with signalling centres involved in the early regionalisation of the telencephalic vesicles. This study identified a subpopulation of CR cells in the rostral telencephalon that expresses Er81, a downstream target of Fgf8 signalling. The role of the rostral telencephalic patterning centre, which secretes FGF molecules, in the specification of these cells, was investigated. Using pharmacological inhibitors and genetic inactivation of Fgf8, it was shown that production of Fgf8 by the rostral telencephalic signalling centre is required for the specification of the Er81+ CR cell population. Moreover, the analysis of Fgf8 gain-of-function in cultivated mouse embryos and of Emx2 and Gli3 mutant embryos revealed that ectopic Fgf8 signalling promotes the generation of CR cells with a rostral phenotype from the dorsal pallium. These data showed that Fgf8 signalling is both required and sufficient to induce rostral CR cells. Together, these results shed light on the mechanisms specifying rostral CR cells and further emphasise the crucial role of telencephalic signalling centres in the generation of distinct CR cell populations (Zimmer, 2010).
The isthmic organizer and its key effector molecule, fibroblast growth factor 8 (Fgf8), have been cornerstones in studies of how organizing centers differentially pattern tissues. Studies have implicated different levels of Fgf8 signaling from the mid/hindbrain boundary (isthmus) as being responsible for induction of different structures within the tectal-isthmo-cerebellum region. However, the role of Fgf8 signaling for different durations in patterning tissues has not been studied. To address this, Fgf8 was conditionally ablated in the isthmus, and prolonged expression of Fgf8 was shown to be is required for the structures found progressively closer to the isthmus to form. Cell death cannot be the main factor accounting for the loss of brain structures near the isthmus, and instead it was demonstrated that tissue transformation underlies the observed phenotypes. It is suggested that the remaining Fgf8 and Fgf17 signaling in temporal Fgf8 conditional mutants is sufficient to ensure survival of most midbrain/hindbrain cells near the isthmus. One crucial role for sustained Fgf8 function is in repressing Otx2 in the hindbrain, thereby allowing the isthmus and cerebellum to form. A second requirement for sustained Fgf8 signaling is to induce formation of a posterior tectum. Finally, Fgf8 is also required to maintain the borders of expression of a number of key genes involved in tectal-isthmo-cerebellum development. Thus, the duration as well as the strength of Fgf8 signaling is key to patterning of the mid/hindbrain region. By extrapolation, the length of Fgf8 expression could be crucial to Fgf8 function in other embryonic organizers (Sato, 2009).
Recent findings implicate embryonic signaling centers in patterning the mammalian cerebral cortex. Mouse in utero electroporation and mutant analysis was used to test whether cortical signaling sources interact to regulate one another. Interactions were identified between the cortical hem (part of the dorsomedial edge of each cerebral cortical hemisphere), rich in Wingless-Int (WNT) proteins and bone morphogenetic proteins (BMPs), and an anterior telencephalic source of fibroblast growth factors (FGFs). Expanding the FGF8 domain suppressed Wnt2b, Wnt3a and Wnt5a expression in the hem. Next to the hem, the hippocampus was shrunken, consistent with its dependence for growth on a hem-derived WNT signal. Maintenance of hem WNT signaling and hippocampal development thus require a constraint on the FGF8 source, which is likely to be supplied by BMP activity. When endogenous BMP signaling is inhibited by noggin, robust Fgf8 expression appears ectopically in the cortical primordium. Abnormal signaling centers were further investigated in mice lacking the transcription factor EMX2, in which FGF8 activity is increased, WNT expression is reduced, and the hippocampus is defective. Suggesting that these defects are causally related, sequestering FGF8 in Emx2 homozygous mutants substantially recovered WNT expression in the hem and partially rescued hippocampal development. Because noggin can induce Fgf8 expression, noggin and BMP signaling were examined in the Emx2 mutant. As the telencephalic vesicle closed, Nog expression expanded and BMP activity reduced, potentially leading to FGF8 upregulation. These findings point to a cross-regulation of BMP, FGF, and WNT signaling in the early telencephalon, integrated by EMX2, and required for normal cortical development (Shimogori, 2004).
The Emx2 mutant mouse line provides an informative illustration of the consequences of signaling center defects. Homozygous mutants display an expanded FGF8 domain, and predictably, given the present findings, a partial loss of WNT gene expression in the hem. Evidence has been provided that shifts in region-specific gene expression in the Emx2 mutant neocortex are in part caused by excess FGF8. Findings from the present study indicate that the expanded FGF8 source in the mutant reduces WNT signaling from the cortical hem, which in turn could contribute to defective development of the hippocampus (Shimogori, 2004).
Emx2 is expressed broadly in the cortical primordium, but its loss does not lead to a broad expansion of Fgf8 expression. Instead, the normally medial and anterior FGF8 domain is enlarged laterally and posteriorly, but retains clear boundaries. Findings from the present study suggest a partial explanation. A likely cause of the expanded FGF8 domain in the Emx2 mutant is early overexpression of noggin at the telencephalic midline, decreasing local BMP activity. BMP inhibition of Fgf8 expression is thereby relieved close to the midline, but not at a distance. Remaining BMP activity may contain further lateral spread of Fgf8 expression (Shimogori, 2004).
It is suggested that cortically expressed EMX2 influences signaling centers by direct gene regulation in the cortical primordium. However, an indirect influence by EMX2 function outside the cortical primordium remains a formal possibility. Emx2 expression appears at E8-8.5 in rostral brain, and continues in both the cortical and subcortical forebrain, where EMX2 has diverse roles in development. These complexities challenge easy interpretation of specific defects in the Emx2 mutant. For example, a misrouting of thalamocortical axons, first ascribed to the absence of EMX2 in the neocortex, may be partially due to loss of gene function in the ventral telencephalon where the thalamocortical pathway begins (Shimogori, 2004).
Ultimately, the timing and sites of Emx2 expression that are crucial to particular aspects of development will be resolved by appropriate conditional deletions, or regional misexpression, of the gene. A recent advance has been the generation of a mouse that overexpresses Emx2 under the control of the nestin promotor. FGF8 levels appear unaffected, perhaps because Emx2 is overexpressed too late, yet area boundaries are shifted. These findings, together with the current ones, indicate a primary effect of EMX2 on cortical patterning, and a secondary effect via two signaling sources (Shimogori, 2004).
It is proposed that early in telencephalic development, EMX2 acts directly or indirectly on noggin to derepress BMP activity. BMP activity constrains expansion of the anterior FGF8 source, and keeps the cortical hem clear of FGF8, protecting local WNT gene expression. Meanwhile, normal levels of midline noggin allow the FGF8 source to be established and maintained. Effectively completing a negative feedback loop, FGF8 downregulates Emx2 expression. These interactions help to ensure FGF and WNT/BMP sources of appropriate size, position and duration to regulate cortical patterning and growth (Shimogori, 2004).
Effective induction of midbrain-specific dopamine (mDA) neurons from stem cells is fundamental for realizing their potential in biomedical applications relevant to Parkinson's disease. During early development, the Otx2-positive neural tissues are patterned anterior-posteriorly to form the forebrain and midbrain under the influence of extracellular signaling such as FGF and Wnt. In the mesencephalon, sonic hedgehog (Shh) specifies a ventral progenitor fate in the floor plate region that later gives rise to mDA neurons. This study systematically investigated the temporal actions of FGF signaling in mDA neuron fate specification of mouse and human pluripotent stem cells and mouse induced pluripotent stem cells. A brief blockade of FGF signaling on exit of the lineage-primed epiblast pluripotent state initiates an early induction of Lmx1a and Foxa2 in nascent neural progenitors. In addition to inducing ventral midbrain characteristics, the FGF signaling blockade during neural induction also directs a midbrain fate in the anterior-posterior axis by suppressing caudalization as well as forebrain induction, leading to the maintenance of midbrain Otx2. Following a period of endogenous FGF signaling, subsequent enhancement of FGF signaling by Fgf8, in combination with Shh, promotes mDA neurogenesis and restricts alternative fates. Thus, a stepwise control of FGF signaling during distinct stages of stem cell neural fate conversion is crucial for reliable and highly efficient production of functional, authentic midbrain-specific dopaminergic neurons. Importantly, evidence is provided that this novel, small-molecule-based strategy applies to both mouse and human pluripotent stem cells (Jaeger, 2011).
This study demonstrates a functional impact of the FGF/ERK signaling level on the course of mDA neuron differentiation of mouse and human pluripotent stem cells. Pharmacological inactivation of FGF/ERK activity upon exit of the lineage-primed epiblast pluripotent state initiates transcription activities that govern early mesencephalic patterning of both the anterior-posterior and dorsal-ventral axes, leading to the induction of mDA neural progenitor characteristics and maintenance of dopaminergic competence. The consolidation of these characteristics, however, requires a period of autocrine/paracrine FGF/ERK signaling immediately after neural induction. Either continued FGF/ERK blockade in newly derived neural progenitors, or enhancing FGF signaling activity by exogenous FGF8 in these cells, abolishes the effects of FGF receptor inhibitor PD173074. These findings demonstrate a previously unrecognized inhibitory role of FGF/ERK in the induction of ventral midbrain neural progenitors and offer a novel strategy for mDA neuron production from mouse and human pluripotent stem cells and iPSCs. Furthermore, the current method represents a simple, small-molecule-based paradigm for significantly improved efficiency and high reproducibility compared with previously reported transgene-free protocols. Importantly, this strategy directs a midbrain regional identity in the derived dopamine neurons, a property that is essential for functional integration of transplanted dopamine neurons in the Parkinsonian brain (Jaeger, 2011).
Stimulation of embryonic stem cell-derived neural progenitors with Shh and FGF8 is used by almost all dopamine differentiation protocols. However, unless combined with genetic manipulation of mDA transcription factors, such as Pitx3 or Lmx1a, the midbrain regional identity of the dopamine neurons generated has remained uncertain. Furthermore, the yield of Th+ neurons has often proved unreliable between experiments and even highly variable between different microscopic fields within a single culture. A major limiting factor is the temporal and spatial heterogeneity of embryonic stem cell-derived neural progenitors. The current findings demonstrate that the above issues can be addressed using epiblast stem cells (EpiSCs). in the absence of FGF/ERK signaling manipulation, nearly 40% of Th+ neurons generated by EpiSCs already co-expressed Pitx3. This represents a significant improvement over ESC-derived monolayer cultures, where Pitx3+ neurons are rarely observed. This improvement is likely to be due to the more synchronous conversion of EpiSCs to the neuroepithelial fate, which would allow for the effective capture of mDA-competent progenitors (Jaeger, 2011).
However, without additional FGF/ERK inhibitor treatment at the neural induction phase, the total numbers of Th+ Pitx3+ cells remained low due to the overall poor efficiency in producing Th+ cells. The early induction of both Lmx1a and Foxa2 by inhibiting FGF receptor or ERK is likely to be a key factor in the observed high efficiency in these experiments. This hypothesis is based on the following observations: (1) d5 PD-treated (EpiSC) MD cultures are highly enriched for Foxa2+ Lmx1a+ neural progenitors compared with untreated controls; (2) although Shh treatment in d5-9 MD results in comparable numbers of Foxa2+ Lmx1a+ cells in PD-primed and no-PD cultures, mDA neuron production was not enhanced in the manner observed with PD treatment; (3) replacing PD with Shh, which turned out to be a slower and less effective inducer of Lmx1a and Foxa2, also led to poor mDA production; and (4) previous reports have credited the dopaminergic-promoting activity of Lmx1a to its early transgene expression in ESC-derived neural progenitors and indicated that Lmx1a functions by cooperating with Foxa2 in specifying mDA fate during midbrain development (Jaeger, 2011).
The robust induction of Wnt1 and its targets in naïve neural progenitors is likely to be a key downstream mediator that confers the observed early induction of Lmx1a, in light of the recent finding that it can be directly regulated by Wnt1/β-catenin signaling. The same study also showed that, although Otx2 itself had no effect in promoting the expression of terminal mDA neuronal marker genes such as Th, Pitx3 and Nurr1, it significantly enhanced the regulatory effect of Lmx1a and Foxa2 on the expression of these genes. Thus, Otx2 plays a permissive role in Lmx1a/Foxa2-mediated mDA neuronal production. It is worth noting that a significant effect of FGF/ERK blockade is the maintenance of Otx2 in derived neural progenitors (Jaeger, 2011).
This study also shows that, in addition to inducing a regulatory cascade for ventralizing nascent neural progenitors, FGF/ERK inhibition suppresses forebrain specification while promoting anterior neural induction, as demonstrated by the strong and consistent repression of the forebrain regulator genes Six3 and Foxg1 and the hindbrain marker Gbx2. Thus, blocking FGF/ERK at the onset of neural induction leads to a direct and early induction of the midbrain fate at the expense of forebrain and caudal neural fates. This finding is consistent with the developmental role of FGF signaling in regionalization of the forebrain (Jaeger, 2011).
Furthermore, this study demonstrated the importance of precise temporal control of cell signaling and its cross-regulation with other signaling pathways in mDA neuronal fate specification. During development, Fgf8-mediated signaling can induce the patterned expression of many midbrain/rostral hindbrain genes and is required for normal development of the midbrain and cerebellum. Fgf8-induced Wnt1 and engrailed are key regulators of midbrain and cerebellum patterning, as well as of the differentiation and survival of dopamine neurons. In EpiSC-derived neural cultures, after an initial burst of upregulation induced by PD exposure, Wnt1 expression was subsequently reduced to a level below the no-PD control by unknown factors in the newly generated neural progenitors in d3-5 MD. This is the period when Shh, Lmx1a and Foxa2 expression levels continued to rise. Given that Shh and Wnt1 play opposing roles with regard to mDA neurogenesis, these findings suggest that the delay in FGF reactivation, which suppresses Wnt1 levels, might be crucial for achieving high numbers of Th+ neurons by consolidating Lmx1a and Foxa2 expression via Shh signaling (Jaeger, 2011).
From a technological standpoint, this study describes a novel method of mDA neuron differentiation that employs temporally controlled exposure of human and mouse pluripotent stem cells to an FGF/ERK-deficient environment. The highly reliable nature of this method was demonstrated using five independent mouse EpiSC lines, a mouse iPS cell line and two human ESC lines. This protocol offers several advantages over current methods of generating midbrain-specific DA neurons in that it is adherent culture-based and free from genetic manipulation and thus could be readily applied to other cell lines of interest. Furthermore, because it is fully chemically defined, this paradigm could be readily adapted for use in a clinical setting or scaled up for toxicity and drug screening relevant to developing new therapeutics for Parkinson's disease (Jaeger, 2011).
The midbrain-hindbrain (MHB) junction has the properties of an organizer that patterns the MHB region early in vertebrate development. Classical transplantation experiments demonstrate that MHB tissue can induce midbrain structures when transplanted into the diencephalon and cerebellar tissue when transplanted to the myelencephalon. Fgf8 is thought to mediate this organizer function. In addition to Fgf8, Fgf17 and Fgf18 are also expressed in the MHB junction. Fgf17 is expressed later and broader than either Fgf8 or Fgf18. Disrupting the Fgf17 gene in the mouse decreases precursor cell proliferation in the medial cerebellar (vermis) anlage after E11.5. Loss of an additional copy of Fgf8 enhances the phenotype and accelerates its onset, demonstrating that both molecules cooperate to regulate the size of the precursor pool of cells that develop into the cerebellar vermis. However, expression patterns of Wnt1, En2, Pax5 and Otx2 are not altered, suggesting that specification and patterning of MHB tissue is not perturbed and that these FGFs are not required to pattern the vermis at this stage of development. The consequence of this developmental defect is a progressive, dose-dependent loss of the most anterior lobe of the vermis in mice lacking Fgf17 and in mice lacking Fgf17 and one copy of Fgf8. Significantly, the differentiation of anterior vermis neuroepithelium is shifted rostrally and medially, demonstrating that FGF also regulates the polarized progression of differentiation in the vermis anlage. Finally, this developmental defect results in an ataxic gait in some mice (Xu, 2000).
Current evidence suggests that the anterior segment of the vertebrate hindbrain, rhombomere 1, gives rise to the entire cerebellum. It is situated where two distinct developmental patterning mechanisms converge: graded signaling from an organizing center (the isthmus) located where the midbrain/hindbrain boundary confronts segmentation of the hindbrain. The unique developmental fate of rhombomere 1 is reflected by its being the only hindbrain segment in which no Hox genes are expressed. Ectopic FGF8 protein, a candidate for the isthmic organizing activity, is able to induce and repress gene expression within the hindbrain in a manner appropriate to rhombomere 1. Using a heterotopic, heterospecific grafting strategy it has been demonstrated that rhombomere 1 is able to express Hox genes but that both isthmic tissue and FGF8 inhibit their expression. Inhibition of FGF8 function in vivo shows that it is responsible for defining the anterior limit of Hox gene expression within the developing brain and thereby specifies the extent of the r1 territory. A retinoid morphogen gradient determines the axial limit of expression of individual Hox genes within the hindbrain. A model is proposed wherein activation by retinoids is antagonized by FGF8, acting as an inhibitor in the anterior hindbrain, to set aside the territory from which the cerebellum will develop (Irving, 2000).
The cerebellum develops from the rhombic lip of the rostral hindbrain and is organized by fibroblast growth factor 8 (FGF8) expressed by the isthmus. Irx2, a member of the Iroquois (Iro) and Irx class of homeobox genes is expressed in the presumptive cerebellum. When Irx2 is misexpressed with Fgf8a in the chick midbrain, the midbrain develops into cerebellum in conjunction with repression of Otx2 and induction of Gbx2. During this event, signaling by the FGF8 and mitogen-activated protein (MAP) kinase cascade modulates the activity of Irx2 by phosphorylation. These data identify a link between the isthmic organizer and Irx2, thereby shedding light on the roles of Iro and Irx genes, which are conserved in both vertebrates and invertebrates (Matsumoto, 2004).
The mes/metencephalic boundary (isthmus) is an organizing center for the optic tectum and cerebellum. Fgf8 is accepted as a crucial organizing signal. Fgf8b can induce cerebellum in the mesencephalon, while Fgf8a transforms the presumptive diencephalon into mesencephalon. Since lower doses of Fgf8b exert similar effects to those of Fgf8a, the type difference can be attributed to the difference in the strength of the signal. It is of great interest to uncover mechanisms of signal transduction pathways downstream of the Fgf8 signal in tectal and cerebellar development, and this report concentrates on the Ras-ERK pathway. In normal embryos, extracellular-signal-regulated kinase (ERK) is activated at the site where Fgf8 mRNA is expressed. Fgf8b activates ERK while Fgf8a or a lower dose of Fgf8b does not activate ERK in the mes/metencephalon. Disruption of the Ras-ERK signaling pathway by a dominant negative form of Ras (RasS17N) changes the fate of the metencephalic alar plate from cerebellum to tectum. RasS17N cancels the effects of Fgf8b, while co-transfection of Fgf8a and RasS17N exerts additive effects. Disruption of Fgf8b, not Fgf8a, by siRNA results in posterior extension of the Otx2 expression domain. These results indicate that the presumptive metencephalon receives a strong Fgf8 signal that activates the Ras-ERK pathway and differentiates into the cerebellum (Satol, 2004).
Early patterning of the vertebrate midbrain and cerebellum is regulated by a mid/hindbrain organizer that produces three fibroblast growth factors (FGF8, FGF17 and FGF18). The mechanism by which each FGF contributes to patterning the midbrain, and induces a cerebellum in rhombomere 1 (r1) is not clear. FGF8b can transform the midbrain into a cerebellum fate, whereas FGF8a can promote midbrain development. A chick electroporation assay and in vitro mouse brain explant experiments have been used to compare the activity of FGF17b and FGF18 to FGF8a and FGF8b. (1) FGF8b is the only protein that can induce the r1 gene Gbx2 and strongly activate the pathway inhibitors Spry1/2, as well as repress the midbrain gene Otx2. Consistent with previous studies that indicated high level FGF signaling is required to induce these gene expression changes, electroporation of activated FGFRs produce similar gene expression changes to FGF8b. (2) FGF8b extends the organizer along the junction between the induced Gbx2 domain and the remaining Otx2 region in the midbrain, correlating with cerebellum development. By contrast, FGF17b and FGF18 mimic FGF8a by causing expansion of the midbrain and upregulating midbrain gene expression. This result is consistent with Fgf17 and Fgf18 being expressed in the midbrain and not just in r1 as is Fgf8. (3) Analysis of gene expression in mouse brain explants with beads soaked in FGF8b or FGF17b shows that the distinct activities of FGF17b and FGF8b are not due to differences in the amount of FGF17b protein produced in vivo. Finally, brain explants were used to define a positive feedback loop involving FGF8b mediated upregulation of Fgf18, and two negative feedback loops that include repression of Fgfr2/3 and direct induction of Spry1/2. Since Fgf17 and Fgf18 are co-expressed with Fgf8 in many tissues, these studies have broad implications for how these FGFs differentially control development (Liu, 2003).
The following steps in midbrain and cerebellum development in mouse are proposed. At the four-somite stage, Fgf8 is induced in the presumptive r1 territory by an unknown factor. Pax2 is required for this induction and OTX2 inhibits Fgf8 from being induced in the midbrain. FGF8b then induces Fgf18 in the surrounding cells, producing a larger domain and gradient of Fgf mRNA that extends into the midbrain. FGF8b also maintains two negative feedback loops by inducing Spry1 and Spry2 expression and inhibiting Fgfr2 and Fgfr3. Fgf17 is then induced by an unknown mechanism that is dependent on Fgf8 in a broader domain than Fgf18, further extending the gradient of Fgf mRNA expression. FGF17 and FGF18 protein, and possibly FGF8a and a low level of FGF8b, then regulate proliferation of the midbrain and cerebellum and En expression. The narrow domain where Fgf8 is expressed becomes the isthmus because of the activity of FGF8b, and the adjacent Otx2-negative r1 cells become the cerebellum. By the 15-somite stage Gbx2 is not required in r1 for cerebellum development, but is required earlier to specify r1. Thus, once Fgf8 expression in r1 is stabilized, perhaps by a secreted factor from the midbrain, a key function of high level signaling by FGF8b is to maintain a cascade of gene expression in the midbrain/r1 that maintains an Otx2-negative domain in r1 in which the cerebellum develops (Liu, 2003).
Members of the fibroblast growth factor (FGF) family of peptide ligands have been implicated in otic placode induction in several vertebrate species. Roles of fgf3 and fgf8 in zebrafish otic development have been functionally analyzed. The role of fgf8 was assessed by analyzing acerebellar (ace) mutants. fgf3 function was disrupted by injecting embryos with antisense morpholino oligomers (MO) specifically designed to block translation of fgf3 transcripts. Disruption of either fgf3 or fgf8 causes moderate reduction in the size of the otic vesicle. Injection of fgf3-MO into ace/ace mutants causes much more severe reduction or complete loss of otic tissue. Moreover, preplacode cells fail to express pax8 and pax2.1, indicating disruption of early stages of otic induction in fgf3-depleted ace/ace mutants. Both fgf3 and fgf8 are normally expressed in the germring by 50% epiboly and are induced in the primordium of rhombomere 4 by 80% epibloy. In addition, fgf3 is expressed during the latter half of gastrulation in the prechordal plate and paraxial cephalic mesendoderm, tissues that either pass beneath or persist near the prospective otic ectoderm. Conditions that alter the pattern of expression of fgf3 and/or fgf8 cause corresponding changes in otic induction. Loss of maternal and zygotic one-eyed pinhead (oep) does not alter expression of fgf3 or fgf8 in the hindbrain, but ablates mesendodermal sources of fgf signaling and delays otic induction by several hours. Conversely, treatment of wild-type embryos with retinoic acid greatly expands the periotic domains of expression of fgf3, fgf8, and pax8 and leads to formation of supernumerary and ectopic otic vesicles. These data support the hypothesis that fgf3 and fgf8 cooperate during the latter half of gastrulation to induce differentiation of otic placodes (Phillips, 2001).
Fgf3 has long been implicated in otic placode induction and early development of the otocyst; however, the results of experiments in mouse and chick embryos to determine its function have proved to be conflicting. Fgf3 expression was determined in relation to otic development in the zebrafish and antisense morpholino oligonucleotides were used to inhibit Fgf3 translation. Successful knockdown of Fgf3 protein was demonstrated and this resulted in a reduction of otocyst size together with reduction in expression of early markers of the otic placode. Fgf3 is co-expressed with Fgf8 in the hindbrain prior to otic induction and, strikingly, when Fgf3 morpholinos were co-injected together with Fgf8 morpholinos, a significant number of embryos failed to form otocysts. These effects were made manifest at early stages of otic development by an absence of early placode markers (pax2.1 and dlx3) but were not accompanied by effects on cell division or death. The temporal requirement for Fgf signalling was established as being between 60% epiboly and tailbud stages using the Fgf receptor inhibitor SU5402. However, the earliest molecular event in induction of the otic territory, pax8 expression, did not require Fgf signalling, indicating an inductive event upstream of signalling by Fgf3 and Fgf8. It is proposed that Fgf3 and Fgf8 are required together for formation of the otic placode and act during the earliest stages of its induction (Maroon, 2002).
In both chick and mouse, the otic placode, the rudiment of the inner ear, is induced by at least two signals, one from the cephalic paraxial mesoderm and the other from the neural ectoderm. In chick, the mesodermal signal, FGF19, induces neural ectoderm to express additional signals, including WNT8c and FGF3, resulting in induction of the otic placode. In mouse, mesodermal Fgf10 acting redundantly with neural Fgf3 is required for induction of the placode. To determine how the mesodermal inducers of the otic placode are localized, advantage was taken of the unique strengths of the two model organisms. Endoderm is shown to be necessary for otic induction in the chick and Fgf8, expressed in the chick endoderm subjacent to Fgf19, is both sufficient and necessary for the expression of Fgf19 in the mesoderm. In the mouse, Fgf8 is also expressed in endoderm as well as in other germ layers in the periotic placode region. Otic induction fails in embryos null for Fgf3 and hypomorphic for Fgf8 and expression of mesodermal Fgf10 is reduced. Thus, Fgf8 plays a critical upstream role in an FGF signaling cascade required for otic induction in chick and mouse (Ladher, 2005).
The auditory sensory epithelium (organ of Corti), where sound waves are converted to electrical signals, comprises a highly ordered array of sensory receptor (hair) cells and nonsensory supporting cells. Sprouty2, which encodes a negative regulator of signaling via receptor tyrosine kinases, is required for normal hearing in mice, and lack of SPRY2 results in dramatic perturbations in organ of Corti cytoarchitecture: instead of two pillar cells, supporting cells of the organ of Corti, there are three, resulting in the formation of an ectopic tunnel of Corti. These effects are due to a postnatal cell fate transformation of a Deiters’ cell into a pillar cell. Both this cell fate change and hearing loss can be partially rescued by reducing Fgf8 gene dosage in Spry2 null mutant mice. These results provide evidence that antagonism of FGF signaling by SPRY2 is essential for establishing the cytoarchitecture of the organ of Corti and for hearing (Shim, 2005).
Hair cells of the inner ear develop from an equivalence group marked by expression of the proneural gene Atoh1. In mouse, Atoh1 is necessary for hair cell differentiation, but its role in specifying the equivalence group (proneural function) has been questioned and little is known about its upstream activators. These issues have been addressed in zebrafish. Two zebrafish homologs, atoh1a and atoh1b, are together necessary for hair cell development. These genes crossregulate each other but are differentially required during distinct developmental periods, first in the preotic placode and later in the otic vesicle. Interactions with the Notch pathway confirm that atoh1 genes have early proneural function. Fgf3 and Fgf8 are upstream activators of atoh1 genes during both phases, and foxi1, pax8 and dlx genes regulate atoh1b in the preplacode. A model is presented in which zebrafish atoh1 genes operate in a complex network leading to hair cell development (Millimaki, 2007).
There have been differing opinions as to whether vertebrate Atoh1 genes act as classic proneural genes or only as terminal differentiation factors. Specific comparisons between zebrafish atoh1 genes and Drosophila ato reveal striking parallels. More generally, various authors have used four criteria to define proneural function that can be applied to zebrafish atoh1 genes. (1) Proneural genes are expressed before sensory fate specification. atoh1b is induced broadly in the preotic placode at 10.5 hpf, whereas specification of tether cells (stabilization of atoh1 expression) does not occur until 14 hpf. (2) Proneural genes are subject to lateral inhibition (and the related process of domain restriction) via Notch-mediated repression. Zebrafish atoh1 genes, once induced, are readily repressed by Notch activity. Moreover, both atoh1 genes facilitate their own repression by autonomously activating delta expression. (3) Proneural function is necessary for producing the equivalence group for the entire sensory structure. atoh1a;atoh1b morphants produce only a simple epithelium lacking hair cells; and while support cell markers are not known in zebrafish, it is important to note that the epithelium continues to express atoh1a. Since loss of atoh1 expression marks the first step in support cell specification, these cannot be support cells. (4) Proneural function is sufficient to induce ectopic sensory development. Misexpression of atoh1a induces ectopic hair cells, although only in limited regions near the otic vesicle or endogenous sensory epithelia, as has been shown for Atoh1 in mammals. Competence to respond appropriately to Atoh1 may require a unique combination of additional factors. The zone of competence could be influenced by pax2-5-8 genes, which are co-regulated with atoh1 genes by Fgf signaling. Other signaling pathways have also been implicated in this process. Misexpressing components of the Notch or Wnt pathways in chick can also induce ectopic sensory patches, but only in restricted regions near endogenous sensory patches. Combinatorial signaling and restricted zones of competence also influence the functions of proneural genes in Drosophila. Thus, while many additional details need to be resolved, zebrafish atoh1 genes meet all four criteria used to define proneural function (Millimaki, 2007).
In vertebrates, midline-derived sonic hedgehog and nodal are crucial for the initial proximal-distal patterning of the eye. The establishment of the distal optic stalk is in turn a prerequisite to initiate retinogenesis. However, the signal that activates this process is unknown. In both chick and fish, the initiation of retinal differentiation is triggered by a species-specific localized Fgf signaling center that acts as mediator of the midline signals. The concerted activity of Fgf8 and Fgf3 is both necessary and sufficient to coordinate retinal differentiation independent of the connecting optic stalk (Martinez-Morales, 2005).
The different positions of the organizing center found in different vertebrate species are likely due to species-specific adaptations. In zebrafish, similar to chick, an early Fgf8 expression domain in the central retina has been reported. This weak, early expression, detectable at 20 hpf, is transient and vanishes before the retinal progenitor cells are competent enough to respond at the onset of neurogenesis. In chick embryos, conversely, Fgf8 is initially expressed strongly in the optic stalk at stage 15 and is later downregulated distally, prior to the onset of neurogenesis. This specific downregulation of Fgf8 in both the central retina of zebrafish and the distal optic stalk of chick ensures the initiation of retinal differentiation from a single center. Although some subtle differences in the regulation of the promoter certainly exist, the Fgf8 expression profile is largely conserved among vertebrates. These minor differences explain how the central Fgf8 domain is active in chick embryos during neuronal differentiation (thus, coordinating differentiation from a central position), whereas it is repressed in the retina of zebrafish and vice versa (Martinez-Morales, 2005).
The cooperative activity of Fgf family members with overlapping expression domains seems to be a common theme for patterning in different regions of the nervous system. The sequential action of Fgf8 and Fgf17 during the development of the midbrain-hindbrain organizer illustrates this concept. At least three different Fgf family members, Fgf3, Fgf8, and Fgf17, have been identified during development in the optic stalk of several vertebrate models. A morpholino knockdown approach had indicated that Fgf8 and Fgf3 cooperate in the patterning and neurogenesis of the hindbrain, and forebrain. In line with these data, the results show that, though Fgf8 application is itself sufficient to initiate retinal differentiation, the simultaneous inactivation of Fgf8 and Fgf3 (in ace/lia double mutants) is required to block differentiation completely, thus yielding similar results to those obtained after the pharmacological inactivation of Fgf signaling with SU5402. The fact that in the double mutant neuronal differentiation is blocked, clearly argues against a redundant function of a so far not-identified paralog. Also, in the genomic sequence of zebrafish and other teleosts, no paralogs of Fgf3 or Fgf8 are detectable. In the chick embryo, similar to the situation in fish, Fgf3 and Fgf8 expression overlap temporally and spatially in the central retina and at least another Fgf8-related factor, Fgf18, is expressed there earlier during the initiation of neurogenesis, hinting at a cooperative activity also in this species (Martinez-Morales, 2005).
Axial eye patterning determines the positional code of retinal ganglion cells (RGCs), which is crucial for their topographic projection to the midbrain. Several asymmetrically expressed determinants of retinal patterning are known, but it is unclear how axial polarity is first established. This study finds that Fgf signals, including Fgf8, determine retinal patterning along the nasotemporal (NT) axis during early zebrafish embryogenesis: Fgf8 induces nasal and/or suppresses temporal retinal cell fates; and inhibition of all Fgf-receptor signaling leads to complete retinal temporalization and concomitant loss of all nasal fates. Misprojections of RGCs with Fgf-dependent alterations in retinal patterning to the midbrain demonstrate the importance of this early patterning process for late topographic map formation. The crucial period of Fgf-dependent patterning is at the onset of eye morphogenesis. Fgf8 expression, the restricted temporal requirement for Fgf-receptor signaling and target gene expression at this stage suggests that the telencephalic primordium is the source of Fgf8 and acts as novel signaling center for non-autonomous axial patterning of the prospective neural retina (Picker, 2005).
The cavefish morph of the Mexican tetra (Astyanax mexicanus) is blind at adult stage, although an eye that includes a retina and a lens develops during embryogenesis. There are, however, two major defects in cavefish eye development. One is lens apoptosis, a phenomenon that is indirectly linked to the expansion of ventral midline sonic hedgehog (Shh) expression during gastrulation and that induces eye degeneration. The other is the lack of the ventral quadrant of the retina. This study shows that such ventralisation is not extended to the entire forebrain because fibroblast growth factor 8 (Fgf8), which is expressed in the forebrain rostral signalling centre, is activated 2 hours earlier in cavefish embryos than in their surface fish counterparts, in response to stronger Shh signalling in cavefish. It was also shown that neural plate patterning and morphogenesis are modified in cavefish, as assessed by Lhx2 and Lhx9 expression. Inhibition of Fgf receptor signalling in cavefish with SU5402 during gastrulation/early neurulation mimics the typical surface fish phenotype for both Shh and Lhx2/9 gene expression. Fate-mapping experiments show that posterior medial cells of the anterior neural plate, which lack Lhx2 expression in cavefish, contribute to the ventral quadrant of the retina in surface fish, whereas they contribute to the hypothalamus in cavefish. Furthermore, when Lhx2 expression is rescued in cavefish after SU5402 treatment, the ventral quadrant of the retina is also rescued. It is proposed that increased Shh signalling in cavefish causes earlier Fgf8 expression, a crucial heterochrony that is responsible for Lhx2 expression and retina morphogenesis defect (Pottin, 2011).
Formation of the trochlear nerve within the anterior hindbrain provides a model system to study a simple axonal projection within the vertebrate central nervous system. Trochlear motor neurons are born within the isthmic organizer and also immediately posterior to it in anterior rhombomere 1. Axons of the most anterior cells follow a dorsal projection, which circumnavigates the isthmus, while those of more posterior trochlear neurons project anterodorsally to enter the isthmus. Once within the isthmus, axons form large fascicles that extend to a dorsal exit point. The possibility that the projection of trochlear axons towards the isthmus and their subsequent growth within that tissue might depend upon chemoattraction was investigate. Both isthmic tissue and Fgf8 protein are attractants for trochlear axons in vitro, while ectopic Fgf8 causes turning of these axons away from their normal routes in vivo. Both inhibition of FGF receptor activation and inhibition of Fgf8 function in vitro affect formation of the trochlear projection within explants in a manner consistent with a guidance function of Fgf8 during trochlear axon navigation (Irving, 2002).
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