Signaling by FGF4 and FGF8 is required for axial elongation of the mouse embryo

Fibroblast growth factor (FGF) signaling has been shown to play critical roles in vertebrate segmentation and elongation of the embryonic axis. Neither the exact roles of FGF signaling, nor the identity of the FGF ligands involved in these processes, has been conclusively determined. Fgf8 is required for cell migration away from the primitive streak when gastrulation initiates, but previous studies have shown that drastically reducing the level of FGF8 later in gastrulation has no apparent effect on somitogenesis or elongation of the embryo. In this study, we demonstrate that loss of both Fgf8 and Fgf4 expression during late gastrulation resulted in a dramatic skeletal phenotype. Thoracic vertebrae and ribs had abnormal morphology, lumbar and sacral vertebrae were malformed or completely absent, and no tail vertebrae were present. The expression of Wnt3a in the tail and the amount of nascent mesoderm expressing Brachyury were both severely reduced. Expression of genes in the NOTCH signaling pathway involved in segmentation was significantly affected, and somite formation ceased after the production of about 15-20 somites. Defects seen in the mutants appear to result from a failure to produce sufficient paraxial mesoderm, rather than a failure of mesoderm precursors to migrate away from the primitive streak. Although the epiblast prematurely decreases in size, we did not detect evidence of a change in the proliferation rate of cells in the tail region or excessive apoptosis of epiblast or mesoderm cells. We propose that FGF4 and FGF8 are required to maintain a population of progenitor cells in the epiblast that generates mesoderm and contributes to the stem cell population that is incorporated in the tailbud and required for axial elongation of the mouse embryo after gastrulation.     

Zebrafish Tbx16 regulates intermediate mesoderm cell fate by attenuating Fgf activity

Progenitors of the zebrafish pronephros, red blood and trunk endothelium all originate from the ventral mesoderm and often share lineage with one another, suggesting that their initial patterning is linked. Previous studies have shown that spadetail (spt) mutant embryos, defective in tbx16 gene function, fail to produce red blood cells, but retain the normal number of endothelial and pronephric cells. We report here that spt mutants are deficient in all the types of early blood, have fewer endothelial cells as well as far more pronephric cells compared to wildtype. In vivo cell tracing experiments reveal that blood and endothelium originate in spt mutants almost exclusive from the dorsal mesoderm whereas, pronephros and tail originate from both dorsal and ventral mesoderm. Together these findings suggest possible defects in posterior patterning. In accord with this, gene expression analysis shows that mesodermal derivatives within the trunk and tail of spt mutants have acquired more posterior identity. Secreted signaling molecules belonging to the Fgf, Wnt and Bmp families have been implicated as patterning factors of the posterior mesoderm. Further investigation demonstrates that Fgf and Wnt signaling are elevated throughout the nonaxial region of the spt gastrula. By manipulating Fgf signaling we show that Fgfs both promote pronephric fate and repress blood and endothelial fate. We conclude that Tbx16 plays an important role in regulating the balance of intermediate mesoderm fates by attenuating Fgf activity.     

Fgf8a induces neural crest indirectly through the activation of Wnt8 in the paraxial mesoderm

Two independent signals are necessary for neural crest (NC) induction in Xenopus: a Bmp signal, which must be partially attenuated by Bmp antagonists, and a separate signal mediated by either a canonical Wnt or an Fgf. The mesoderm underlying the NC-forming region has been proposed as a source of this second signal. Wnt8 and Fgf8a are expressed in this tissue around the time of NC induction and are therefore good candidate NC inducers. Loss-of-function studies indicate that both of these ligands are necessary to specify the NC; however, it is unclear whether these signaling molecules are operating in the same or in parallel pathways to generate the NC. Here, we describe experiments addressing this outstanding question. We show that although Wnt8 expression can restore NC progenitors in Fgf8a-deficient embryos, Fgf8a is unable to rescue NC formation in Wnt8-depleted embryos. Moreover, the NC-inducing activity of Fgf8a in neuralized explants is strongly repressed by co-injection of a Wnt8 or a beta-catenin morpholino, suggesting that the activity of these two signaling molecules is linked. Consistent with these observations, Fgf8a is a potent inducer of Wnt8 in both whole embryos and animal explants, and Fgf8a knockdown results in a dramatic loss of Wnt8 expression in the mesoderm. We propose that Fgf8a induces NC indirectly through the activation of Wnt8 in the paraxial mesoderm, which in turn promotes NC formation in the overlying ectoderm primed by Bmp antagonists.     

Ectodermal Wnt/β-catenin signaling shapes the mouse face

The canonical Wnt/β-catenin pathway is an essential component of multiple developmental processes. To investigate the role of this pathway in the ectoderm during facial morphogenesis, we generated conditional β-catenin mouse mutants using a novel ectoderm-specific Cre recombinase transgenic line. Our results demonstrate that ablating or stabilizing β-catenin in the embryonic ectoderm causes dramatic changes in facial morphology. There are accompanying alterations in the expression of Fgf8 and Shh, key molecules that establish a signaling center critical for facial patterning, the frontonasal ectodermal zone (FEZ). These data indicate that Wnt/β-catenin signaling within the ectoderm is critical for facial development and further suggest that this pathway is an important mechanism for generating the diverse facial shapes of vertebrates during evolution.     

FGF8 dose-dependent regulation of embryonic submandibular salivary gland morphogenesis

FGF8 has been shown to play important morphoregulatory roles during embryonic development. The observation that craniofacial, cardiovascular, pharyngeal, and neural phenotypes vary with Fgf8 gene dosage suggests that FGF8 signaling induces differences in downstream responses in a dose-dependent manner. In this study, we investigated if FGF8 plays a dose-dependent regulatory role during embryonic submandibular salivary gland (SMG) morphogenesis. We evaluated SMG phenotypes of Fgf8 hypomorphic mice, which have decreased Fgf8 gene function throughout embryogenesis. We also evaluated SMG phenotypes of Fgf8 conditional mutants in which Fgf8 function has been completely ablated in its expression domain in the first pharyngeal arch ectoderm from the time of arch formation. Fgf8 hypomorphs have hypoplastic SMGs, whereas conditional mutant SMGs exhibit ontogenic arrest followed by involution and are absent by E18.5. SMG aplasia in Fgf8 ectoderm conditional mutants indicates that FGF8 signaling is essential for the morphogenesis and survival of Pseudoglandular Stage and older SMGs. Equally important, the presence of an initial SMG bud in Fgf8 conditional mutants indicates that initial bud formation is FGF8 independent. Mice heterozygous for either the Fgf8 null allele (Fgf8(+/N)) or the hypomorphic allele (Fgf8(+/H)) have SMGs that are indistinguishable from wild-type (Fgf8(+/+)) mice which suggest that there is not only an FGF8 dose-dependent phenotypic response, but a nonlinear, threshold-like, epistatic response as well. We also found that enhanced FGF8 signaling induced, and abrogated FGF8 signaling decreased, SMG branching morphogenesis in vitro. Furthermore, since FGF10 and Shh expression is modulated by Fgf8 levels, we postulated that exogenous FGF10, Shh, or FGF10 + Shh peptide supplementation in vitro would largely "rescue" the abnormal SMG phenotype associated with decreased FGF8 signaling. This is as expected, though there is no synergistic effect with FGF10 + Shh peptide supplementation. These in vitro experiments model the principle that mutations have different effects in the context of different epigenotypes.     

Early thyroid development requires a Tbx1-Fgf8 pathway

The thyroid develops within the pharyngeal apparatus from endodermally-derived cells. The many derivatives of the pharyngeal apparatus develop at similar times and sometimes from common cell types, explaining why many syndromic disorders express multiple birth defects affecting different structures that share a common pharyngeal origin. Thus, different derivatives may share common genetic networks during their development. Tbx1, the major gene associated with DiGeorge syndrome, is a key player in the global development of the pharyngeal apparatus, being required for virtually all its derivatives, including the thyroid. Here we show that Tbx1 regulates the size of the early thyroid primordium through its expression in the adjacent mesoderm. Because Tbx1 regulates the expression of Fgf8 in the mesoderm, we postulated that Fgf8 mediates critical Tbx1-dependent interactions between mesodermal cells and endodermal thyrocyte progenitors. Indeed, conditional ablation of Fgf8 in Tbx1-expressing cells caused an early thyroid phenotype similar to that of Tbx1 mutant mice. In addition, expression of an Fgf8 cDNA in the Tbx1 domain rescued the early size defect of the thyroid primordium in Tbx1 mutants. Thus, we have established that a Tbx1->Fgf8 pathway in the pharyngeal mesoderm is a key size regulator of mammalian thyroid.     

Shh and Fgf8 act synergistically to drive cartilage outgrowth during cranial development

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. Here, we directly examine the ability of FGF8 and SHH signaling, singly and in combination, to regulate cranial skeletogenesis, both in vitro and in vivo. We find that SHH and FGF8 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.     

An FGF3-BMP Signaling Axis Regulates Caudal Neural Tube Closure,Neural Crest Specification and Anterior-Posterior Axis Extension

During vertebrate axis extension, adjacent tissue layers undergo profound morphological changes: within the neuroepithelium, neural tube closure and neural crest formation are occurring, while within the paraxial mesoderm somites are segmenting from the presomitic mesoderm (PSM). Little is known about the signals between these tissues that regulate their coordinated morphogenesis. Here, we analyze the posterior axis truncation of mouse Fgf3 null homozygotes and demonstrate that the earliest role of PSM-derived FGF3 is to regulate BMP signals in the adjacent neuroepithelium. FGF3 loss causes elevated BMP signals leading to increased neuroepithelium proliferation, delay in neural tube closure and premature neural crest specification. We demonstrate that elevated BMP4 depletes PSM progenitors in vitro, phenocopying the Fgf3 mutant, suggesting that excessive BMP signals cause the Fgf3 axis defect. To test this in vivo we increased BMP signaling in Fgf3 mutants by removing one copy of Noggin, which encodes a BMP antagonist. In such mutants, all parameters of the Fgf3 phenotype were exacerbated: neural tube closure delay, premature neural crest specification, and premature axis termination. Conversely, genetically decreasing BMP signaling in Fgf3 mutants, via loss of BMP receptor activity, alleviates morphological defects. Aberrant apoptosis is observed in the Fgf3 mutant tailbud. However, we demonstrate that cell death does not cause the Fgf3 phenotype: blocking apoptosis via deletion of pro-apoptotic genes surprisingly increases all Fgf3 defects including causing spina bifida. We demonstrate that this counterintuitive consequence of blocking apoptosis is caused by the increased survival of BMP-producing cells in the neuroepithelium. Thus, we show that FGF3 in the caudal vertebrate embryo regulates BMP signaling in the neuroepithelium, which in turn regulates neural tube closure, neural crest specification and axis termination. Uncovering this FGF3-BMP signaling axis is a major advance toward understanding how these tissue layers interact during axis extension with important implications in human disease.     

Retinoic Acid Activity in Undifferentiated Neural Progenitors Is Sufficient to Fulfill Its Role in Restricting Fgf8 Expression for Somitogenesis

Bipotent axial stem cells residing in the caudal epiblast during late gastrulation generate neuroectodermal and presomitic mesodermal progeny that coordinate somitogenesis with neural tube formation, but the mechanism that controls these two fates is not fully understood. Retinoic acid (RA) restricts the anterior extent of caudal fibroblast growth factor 8 (Fgf8) expression in both mesoderm and neural plate to control somitogenesis and neurogenesis, however it remains unclear where RA acts to control the spatial expression of caudal Fgf8. Here, we found that mouse Raldh2-/- embryos, lacking RA synthesis and displaying a consistent small somite defect, exhibited abnormal expression of key markers of axial stem cell progeny, with decreased Sox2+ and Sox1+ neuroectodermal progeny and increased Tbx6+ presomitic mesodermal progeny. The Raldh2-/- small somite defect was rescued by treatment with an FGF receptor antagonist. Rdh10 mutants, with a less severe RA synthesis defect, were found to exhibit a small somite defect and anterior expansion of caudal Fgf8 expression only for somites 1–6, with normal somite size and Fgf8 expression thereafter. Rdh10 mutants were found to lack RA activity during the early phase when somites are small, but at the 6-somite stage RA activity was detected in neural plate although not in presomitic mesoderm. Expression of a dominant-negative RA receptor in mesoderm eliminated RA activity in presomitic mesoderm but did not affect somitogenesis. Thus, RA activity in the neural plate is sufficient to prevent anterior expansion of caudal Fgf8 expression associated with a small somite defect. Our studies provide evidence that RA restriction of Fgf8 expression in undifferentiated neural progenitors stimulates neurogenesis while also restricting the anterior extent of the mesodermal Fgf8 mRNA gradient that controls somite size, providing new insight into the mechanism that coordinates somitogenesis with neurogenesis.     

Directed Bmp4 expression in neural crest cells generates a genetic model for the rare human bony syngnathia birth defect

Congenital bony syngnathia, a rare but severe human birth defect, is characterized by bony fusion of the mandible to the maxilla. However, the genetic mechanisms underlying this birth defect are poorly understood, largely due to limitation of available animal models. Here we present evidence that transgenic expression of Bmp4 in neural crest cells causes a series of craniofacial malformations in mice, including a bony fusion between the maxilla and hypoplastic mandible, resembling the bony syngnathia syndrome in humans. In addition, the anterior portion of the palatal shelves emerged from the mandibular arch instead of the maxilla in the mutants. Gene expression assays showed an altered expression of several facial patterning genes, including Hand2, Dlx2, Msx1, Barx1, Foxc2 and Fgf8, in the maxillary and mandibular processes of the mutants, indicating mis-patterned cranial neural crest (CNC) derived cells in the facial region. However, despite of formation of cleft palate and ectopic cartilage, forced expression of a constitutively active form of BMP receptor-Ia (caBmprIa) in CNC lineage did not produce the syngnathia phenotype, suggesting a non-cell autonomous effect of the augmented BMP4 signaling. Our studies demonstrate that aberrant BMP4-mediated signaling in CNC cells leads to mis-patterned facial skeleton and congenital bony syngnathia, and suggest an implication of mutations in BMP signaling pathway in human bony syngnathia.     

Hindbrain-derived Wnt and Fgf signals cooperate to specify the otic placode in Xenopus

Induction of the otic placode, the rudiment of the inner ear, is believed to depend on signals derived from surrounding tissues, the head mesoderm and the prospective hindbrain. Here we report the first attempt to define the specific contribution of the neuroectoderm to this inductive process in Xenopus. To this end we tested the ability of segments of the neural plate (NP), isolated from different axial levels, to induce the otic marker Pax8 when recombined with blastula stage animal caps. We found that one single domain of the NP, corresponding to the prospective anterior hindbrain, had Pax8-inducing activity in this assay. Surprisingly, more than half of these recombinants formed otic vesicle-like structures. Lineage tracing experiments indicate that these vesicle-like structures are entirely derived from the animal cap and express several pan-otic markers. Pax8 activation in these recombinants requires active Fgf and canonical Wnt signaling, as interference with either pathway blocks Pax8 induction. Furthermore, we demonstrate that Fgf and canonical Wnt signaling cooperate to activate Pax8 expression in isolated animal caps. We propose that in the absence of mesoderm cues the combined activity of hindbrain-derived Wnt and Fgf signals specifies the otic placode in Xenopus, and promotes its morphogenesis into an otocyst.     

Level-specific role of paraxial mesoderm in regulation of Tbx5/Tbx4 expression and limb initiation

Tetrapod limbs, forelimbs and hindlimbs, emerge as limb buds during development from appropriate positions along the rostro-caudal axis of the main body. In this study, tissue interactions by which rostro-caudal level-specific limb initiation is established were analyzed. The limb bud originates from the lateral plate located laterally to the paraxial mesoderm, and we obtained evidence that level-specific tissue interactions between the paraxial mesoderm and the lateral plate mesoderm are important for the determination of the limb-type-specific gene expression and limb outgrowth. When the wing-level paraxial mesoderm was transplanted into the presumptive leg region, the wing-level paraxial mesoderm upregulated the expression of Tbx5, a wing marker gene, and down regulated the expression of Tbx4 and Pitx1, leg marker genes, in the leg-level lateral plate. The wing-level paraxial mesoderm relocated into the leg level also inhibited outgrowth of the hindlimb bud and down regulated Fgf10 and Fgf8 expression, demonstrating that the wing-level paraxial mesoderm cannot substitute for the function of the leg-level paraxial mesoderm in initiation and outgrowth of the hindlimb. The paraxial mesoderm taken from the neck- and flank-level regions also had effects on Tbx5/Tbx4 expression with different efficiencies. These findings suggest that the paraxial mesoderm has level-specific abilities along the rostro-caudal axis in the limb-type-specific mechanism for limb initiation.