Coordinated directional outgrowth and pattern formation by integration of Wnt5a and Fgf signaling in planar cell polarity.

Embryonic morphogenesis of a complex organism requires proper regulation of patterning and directional growth. Planar cell polarity (PCP) signaling is emerging as a crucial evolutionarily conserved mechanism whereby directional information is conveyed. PCP is thought to be established by global cues, and recent studies have revealed an instructive role of a Wnt signaling gradient in epithelial tissues of both invertebrates and vertebrates. However, it remains unclear whether Wnt/PCP signaling is regulated in a coordinated manner with embryonic patterning during morphogenesis. Here, in mouse developing limbs, we find that apical ectoderm ridge-derived Fgfs required for limb patterning regulate PCP along the proximal-distal axis in a Wnt5a-dependent manner. We demonstrate with genetic evidence that the Wnt5a gradient acts as a global cue that is instructive in establishing PCP in the limb mesenchyme, and that Wnt5a also plays a permissive role to allow Fgf signaling to orient PCP. Our results indicate that limb morphogenesis is regulated by coordination of directional growth and patterning through integration of Wnt5a and Fgf signaling.

Fibroblast growth factor deficiencies impact anxiety-like behavior and the serotonergic system.

Serotonergic neurons in the dorsal raphe nucleus (DR) are organized in anatomically distinct subregions that form connections with specific brain structures to modulate diverse behaviors, including anxiety-like behavior. It is unclear if the functional heterogeneity of these neurons is coupled to their developmental heterogeneity, and if abnormal development of specific DR serotonergic subregions can permanently impact anxiety circuits and behavior. The goal of this study was to examine if deficiencies in different components of fibroblast growth factor (Fgf) signaling could preferentially impact the development of specific populations of DR serotonergic neurons to alter anxiety-like behavior in adulthood. Wild-type and heterozygous male mice globally hypomorphic for Fgf8, Fgfr1, or both (Fgfr1/Fgf8) were tested in an anxiety-related behavioral battery. Both Fgf8- and Fgfr1/Fgf8-deficient mice display increased anxiety-like behavior as measured in the elevated plus-maze and the open-field tests. Immunohistochemical staining of a serotonergic marker, tryptophan hydroxylase (Tph), revealed reductions in specific populations of serotonergic neurons in the ventral, interfascicular, and ventrolateral/ventrolateral periaqueductal gray subregions of the DR in all Fgf-deficient mice, suggesting a neuroanatomical basis for increased anxiety-like behavior. Overall, this study suggests Fgf signaling selectively modulates the development of different serotonergic neuron subpopulations. Further, it suggests anxiety-like behavior may stem from developmental disruption of these neurons, and individuals with inactivating mutations in Fgf signaling genes may be predisposed to anxiety disorders.

A computational model of the effect of gene misexpression on the development of cortical areas.

Brain function depends on the specialisation of brain areas. In the murine cerebral cortex, the development of these areas depends on the coordinated expression of several genes in precise spatial patterns in the telencephalon during embryogenesis. Manipulating the expression of these genes during development alters the positions and sizes of cortical areas in the adult. Qualitative data also show that these genes regulate each other's expression during development so that they form a regulatory network with many feedback loops. However, it is currently unknown which regulatory interactions are critical to generating the correct expression patterns to lead to normal cortical development. Here, we formalise the relationships inferred from genetic manipulations into computational models. We simulate many different networks potentially consistent with the experimental data and show that a surprising diversity of networks produce similar results. This demonstrates that existing data cannot uniquely specify the network. We conclude by suggesting experiments necessary to constrain the model and help identify and understand the true structure of this regulatory network.

Distinct populations within Isl1 lineages contribute to appendicular and facial skeletogenesis through the ß-catenin pathway.

Isl1 expression marks progenitor populations in developing embryos. In this study, we investigated the contribution of Isl1-expressing cells that utilize the ß-catenin pathway to skeletal development. Inactivation of ß-catenin in Isl1-expressing cells caused agenesis of the hindlimb skeleton and absence of the lower jaw (agnathia). In the hindlimb, Isl1-lineages broadly contributed to the mesenchyme; however, deletion of ß-catenin in the Isl1-lineage caused cell death only in a discrete posterior domain of nascent hindlimb bud mesenchyme. We found that the loss of posterior mesenchyme, which gives rise to Shh-expressing posterior organizer tissue, caused loss of posterior gene expression and failure to expand chondrogenic precursor cells, leading to severe truncation of the hindlimb. In facial tissues, Isl1-expressing cells broadly contributed to facial epithelium. We found reduced nuclear ß-catenin accumulation and loss of Fgf8 expression in mandibular epithelium of Isl1(-/-) embryos. Inactivating ß-catenin in Isl1-expressing epithelium caused both loss of epithelial Fgf8 expression and death of mesenchymal cells in the mandibular arch without affecting epithelial proliferation and survival. These results suggest a Isl1→ß-catenin→Fgf8 pathway that regulates mesenchymal survival and development of the lower jaw in the mandibular epithelium. By contrast, activating ß-catenin signaling in Isl1-lineages caused activation of Fgf8 broadly in facial epithelium. Our results provide evidence that, despite its broad contribution to hindlimb mesenchyme and facial epithelium, the Isl1-ß-catenin pathway regulates skeletal development of the hindlimb and lower jaw through discrete populations of cells that give rise to Shh-expressing posterior hindlimb mesenchyme and Fgf8-expressing mandibular epithelium.

Deregulated FGF and homeotic gene expression underlies cerebellar vermis hypoplasia in CHARGE syndrome.

Mutations in CHD7 are the major cause of CHARGE syndrome, an autosomal dominant disorder with an estimated prevalence of 1/15,000. We have little understanding of the disruptions in the developmental programme that underpin brain defects associated with this syndrome. Using mouse models, we show that Chd7 haploinsufficiency results in reduced Fgf8 expression in the isthmus organiser (IsO), an embryonic signalling centre that directs early cerebellar development. Consistent with this observation, Chd7 and Fgf8 loss-of-function alleles interact during cerebellar development. CHD7 associates with Otx2 and Gbx2 regulatory elements and altered expression of these homeobox genes implicates CHD7 in the maintenance of cerebellar identity during embryogenesis. Finally, we report cerebellar vermis hypoplasia in 35% of CHARGE syndrome patients with a proven CHD7 mutation. These observations provide key insights into the molecular aetiology of cerebellar defects in CHARGE syndrome and link reduced FGF signalling to cerebellar vermis hypoplasia in a human syndrome. DOI: http://dx.doi.org/10.7554/eLife.01305.001.

A Tbx1-Six1/Eya1-Fgf8 genetic pathway controls mammalian cardiovascular and craniofacial morphogenesis.

Shared molecular programs govern the formation of heart and head during mammalian embryogenesis. Development of both structures is disrupted in human chromosomal microdeletion of 22q11.2 (del22q11), which causes DiGeorge syndrome (DGS) and velo-cardio-facial syndrome (VCFS). Here, we have identified a genetic pathway involving the Six1/Eya1 transcription complex that regulates cardiovascular and craniofacial development. We demonstrate that murine mutation of both Six1 and Eya1 recapitulated most features of human del22q11 syndromes, including craniofacial, cardiac outflow tract, and aortic arch malformations. The mutant phenotypes were attributable in part to a reduction of fibroblast growth factor 8 (Fgf8), which was shown to be a direct downstream effector of Six1 and Eya1. Furthermore, we showed that Six1 and Eya1 genetically interacted with Fgf8 and the critical del22q11 gene T-box transcription factor 1 (Tbx1) in mice. Together, these findings reveal a Tbx1-Six1/Eya1-Fgf8 genetic pathway that is crucial for mammalian cardiocraniofacial morphogenesis and provide insights into the pathogenesis of human del22q11 syndromes.

Fetal and postnatal lung defects reveal a novel and required role for Fgf8 in lung development.

The fibroblast growth factor, FGF8, has been shown to be essential for vertebrate cardiovascular, craniofacial, brain and limb development. Here we report that Fgf8 function is required for normal progression through the late fetal stages of lung development that culminate in alveolar formation. Budding, lobation and branching morphogenesis are unaffected in early stage Fgf8 hypomorphic and conditional mutant lungs. Excess proliferation during fetal development disrupts distal airspace formation, mesenchymal and vascular remodeling, and Type I epithelial cell differentiation resulting in postnatal respiratory failure and death. Our findings reveal a previously unknown, critical role for Fgf8 function in fetal lung development and suggest that this factor may also contribute to postnatal alveologenesis. Given the high number of premature infants with alveolar dysgenesis and lung dysplasia, and the accumulating evidence that short-term benefits of available therapies may be outweighed by long-term detrimental effects on postnatal alveologenesis, the therapeutic implications of identifying a factor or pathway that can be targeted to stimulate normal alveolar development are profound.

Role of mesodermal FGF8 and FGF10 overlaps in the development of the arterial pole of the heart and pharyngeal arch arteries.

RATIONALE: The genes encoding fibroblast growth factor (FGF) 8 and 10 are expressed in the anterior part of the second heart field that constitutes a population of cardiac progenitor cells contributing to the arterial pole of the heart. Previous studies of hypomorphic and conditional Fgf8 mutants show disrupted outflow tract (OFT) and right ventricle (RV) development, whereas Fgf10 mutants do not have detectable OFT defects. OBJECTIVES: Our aim was to investigate functional overlap between Fgf8 and Fgf10 during formation of the arterial pole. METHODS AND RESULTS: We generated mesodermal Fgf8; Fgf10 compound mutants with MesP1Cre. The OFT/RV morphology in these mutants was affected with variable penetrance; however, the incidence of embryos with severely affected OFT/RV morphology was significantly increased in response to decreasing Fgf8 and Fgf10 gene dosage. Fgf8 expression in the pharyngeal arch ectoderm is important for development of the pharyngeal arch arteries and their derivatives. We now show that Fgf8 deletion in the mesoderm alone leads to pharyngeal arch artery phenotypes and that these vascular phenotypes are exacerbated by loss of Fgf10 function in the mesodermal core of the arches. CONCLUSIONS: These results show functional overlap of FGF8 and FGF10 signaling from second heart field mesoderm during development of the OFT/RV, and from pharyngeal arch mesoderm during pharyngeal arch artery formation, highlighting the sensitivity of these key aspects of cardiovascular development to FGF dosage.

Genetic evidence that FGFs have an instructive role in limb proximal-distal patterning.

Half a century ago, the apical ectodermal ridge (AER) at the distal tip of the tetrapod limb bud was shown to produce signals necessary for development along the proximal-distal (P-D) axis, but how these signals influence limb patterning is still much debated. Fibroblast growth factor (FGF) gene family members are key AER-derived signals, with Fgf4, Fgf8, Fgf9 and Fgf17 expressed specifically in the mouse AER. Here we demonstrate that mouse limbs lacking Fgf4, Fgf9 and Fgf17 have normal skeletal pattern, indicating that Fgf8 is sufficient among AER-FGFs to sustain normal limb formation. Inactivation of Fgf8 alone causes a mild skeletal phenotype; however, when we also removed different combinations of the other AER-FGF genes, we obtained unexpected skeletal phenotypes of increasing severity, reflecting the contribution that each FGF can make to the total AER-FGF signal. Analysis of the compound mutant limb buds revealed that, in addition to sustaining cell survival, AER-FGFs regulate P-D-patterning gene expression during early limb bud development, providing genetic evidence that AER-FGFs function to specify a distal domain and challenging the long-standing hypothesis that AER-FGF signalling is permissive rather than instructive for limb patterning. We discuss how a two-signal model for P-D patterning can be integrated with the concept of early specification to explain the genetic data presented here.

Frontal cortex subdivision patterning is coordinately regulated by Fgf8, Fgf17, and Emx2.

The frontal cortex (FC) plays a major role in cognition, movement and behavior. However, little is known about the genetic mechanisms that govern its development. We recently described a panel of gene expression markers that delineate neonatal FC subdivisions and identified FC regionalization defects in Fgf17-/- mutant mice (Cholfin and Rubenstein [2007] Proc. Natl. Acad. Sci. U. S. A. [in press]). In the present study, we applied this FC gene expression panel to examine regionalization phenotypes in Fgf8(neo/neo), Emx2-/-, and Emx2-/-;Fgf17-/- newborn mice. We report that Fgf8, Fgf17 and Emx2 play distinct roles in the molecular regionalization of FC subdivisions. The changes in regionalization are presaged by differential effects of rostral patterning center Fgf8 and Fgf17 signaling on the rostral cortical neuroepithelium, revealed by altered expression of Spry1, Spry2, and "rostral" transcription factors Er81, Erm, Pea3, and Sp8. We used Emx2-/-;Fgf17-/- double mutants to provide direct evidence that Emx2 and Fgf17 antagonistically regulate the expression of Erm, Pea3, and Er81 in the rostral cortical neuroepithelium and FC regionalization. We have integrated our results to propose a model for how fibroblast growth factors regulate FC patterning through regulation of regional transcription factor expression within the FC anlage.

Differential requirements for FGF3, FGF8 and FGF10 during inner ear development.

FGF signaling is required during multiple stages of inner ear development in many different vertebrates, where it is involved in induction of the otic placode, in formation and morphogenesis of the otic vesicle as well as for cellular differentiation within the sensory epithelia. In this study we have looked to define the redundant and conserved roles of FGF3, FGF8 and FGF10 during the development of the murine and avian inner ear. In the mouse, hindbrain-derived FGF10 ectopically induces FGF8 and rescues otic vesicle formation in Fgf3 and Fgf10 homozygous double mutants. Conditional inactivation of Fgf8 after induction of the placode does not interfere with otic vesicle formation and morphogenesis but affects cellular differentiation in the inner ear. In contrast, inactivation of Fgf8 during induction of the placode in a homozygous Fgf3 null background leads to a reduced size otic vesicle or the complete absence of otic tissue. This latter phenotype is more severe than the one observed in mutants carrying null mutations for both Fgf3 and Fgf10 that develop microvesicles. However, FGF3 and FGF10 are redundantly required for morphogenesis of the otic vesicle and the formation of semicircular ducts. In the chicken embryo, misexpression of Fgf3 in the hindbrain induces ectopic otic vesicles in vivo. On the other hand, Fgf3 expression in the hindbrain or pharyngeal endoderm is required for formation of the otic vesicle from the otic placode. Together these results provide important insights into how the spatial and temporal expression of various FGFs controls different steps of inner ear formation during vertebrate development.