Requirement For FGF Signaling Research Articles

Activation of germline-specific genes is required for limb regeneration in the Mexican axolotl

The capacity for tissue and organ regeneration in humans is dwarfed by comparison to that of salamanders. Emerging evidence suggests that mechanisms learned from the early phase of salamander limb regeneration—wound healing, cellular dedifferentiation and blastemal formation–will reveal therapeutic approaches for tissue regeneration in humans. Here we describe a unique transcriptional fingerprint of regenerating limb tissue in the Mexican axolotl (Ambystoma mexicanum) that is indicative of cellular reprogramming of differentiated cells to a germline-like state. Two genes that are required for self-renewal of germ cells in mice and flies, Piwi-like 1 (PL1) and Piwi-like 2 (PL2), are expressed in limb blastemal cells, the basal layer keratinocytes and the thickened apical epithelial cap in the wound epidermis in the regenerating limb. Depletion of PL1 and PL2 by morpholino oligonucleotides decreased cell proliferation and increased cell death in the blastema leading to a significant retardation of regeneration. Examination of key molecules that are known to be required for limb development or regeneration further revealed that FGF8 is transcriptionally downregulated in the presence of the morpholino oligos, indicating PL1 and PL2 might participate in FGF signaling during limb regeneration. Given the requirement for FGF signaling in limb development and regeneration, the results suggest that PL1 and PL2 function to establish a unique germline-like state that is associated with successful regeneration.

Amphioxus FGF signaling predicts the acquisition of vertebrate morphological traits

FGF signaling is one of the few cell-cell signaling pathways conserved among all metazoans. The diversity of FGF gene content among different phyla suggests that evolution of FGF signaling may have participated in generating the current variety of animal forms. Vertebrates possess the greatest number of FGF genes, the functional evolution of which may have been implicated in the acquisition of vertebrate-specific morphological traits. In this study, we have investigated the roles of the FGF signal during embryogenesis of the cephalochordate amphioxus, the best proxy for the chordate ancestor. We first isolate the full FGF gene complement and determine the evolutionary relationships between amphioxus and vertebrate FGFs via phylogenetic and synteny conservation analysis. Using pharmacological treatments, we inhibit the FGF signaling pathway in amphioxus embryos in different time windows. Our results show that the requirement for FGF signaling during gastrulation is a conserved character among chordates, whereas this signal is not necessary for neural induction in amphioxus, in contrast to what is known in vertebrates. We also show that FGF signal, acting through the MAPK pathway, is necessary for the formation of the most anterior somites in amphioxus, whereas more posterior somite formation is not FGF-dependent. This result leads us to propose that modification of the FGF signal function in the anterior paraxial mesoderm in an amphioxus-like vertebrate ancestor might have contributed to the loss of segmentation in the preotic paraxial mesoderm of the vertebrate head.

Specification of Region-Specific Neurons Including Forebrain Glutamatergic Neurons from Human Induced Pluripotent Stem Cells

BackgroundDirected differentiation of human induced pluripotent stem cells (hiPSC) into functional, region-specific neural cells is a key step to realizing their therapeutic promise to treat various neural disorders, which awaits detailed elucidation.Methodology/Principal FindingsWe analyzed neural differentiation from various hiPSC lines generated by others and ourselves. Although heterogeneity in efficiency of neuroepithelial (NE) cell differentiation was observed among different hiPSC lines, the NE differentiation process resembles that from human embryonic stem cells (hESC) in morphology, timing, transcriptional profile, and requirement for FGF signaling. NE cells differentiated from hiPSC, like those from hESC, can also form rostral phenotypes by default, and form the midbrain or spinal progenitors upon caudalization by morphogens. The rostrocaudal neural progenitors can further mature to develop forebrain glutamatergic projection neurons, midbrain dopaminergic neurons, and spinal motor neurons, respectively. Typical ion channels and action potentials were recorded in the hiPSC-derived neurons.Conclusions/SignificanceOur results demonstrate that hiPSC, regardless of how they were derived, can differentiate into a spectrum of rostrocaudal neurons with functionality, which supports the considerable value of hiPSC for study and treatment of patient-specific neural disorders.

Heterogeneity and Fgf dependence of adult neural progenitors in the zebrafish telencephalon

Adult telencephalic neurogenesis is a conserved trait of all vertebrates studied. It has been investigated in detail in rodents, but very little is known about the composition of neurogenic niches and the cellular nature of progenitors in nonmammalian vertebrates. To understand the components of the progenitor zones in the adult zebrafish telencephalon and the link between glial characteristics and progenitor state, we examined whether canonical glial markers are colocalized with proliferation markers. In the adult zebrafish telencephalon, we identify heterogeneous progenitors that reside in two distinct glial domains. We find that the glial composition of the progenitor zone is linked to its proliferative behavior. Analyzing both fast-cycling proliferating cells as well as slowly cycling progenitors, we find four distinct progenitor types characterized by differential expression of glial markers. Importantly, a significant proportion of progenitors do not display typical radial glia characteristics. By blocking or activating Fgf signaling by misexpression of a dominant negative Fgf-receptor 1 or Fgf8a, respectively, we find that ventral and dorsal progenitors in the telencephalon also differ in their requirement for Fgf signaling. Together with data on the expression of Fgf signaling components in the ventricular zone of the telencephalon, this suggests that Fgf signaling directly regulates proliferation of specific subsets of adult telencephalic progenitors in vivo. Taken together our results show that adult neural progenitor cells are heterogeneous with their respect to distribution into two distinct glial domains and their dependence upon Fgf signaling as a proliferative cue in the zebrafish telencephalon.

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

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

Development of midline cell types and commissural axon tracts requires Fgfr1 in the cerebrum

The adult cerebral hemispheres are connected to each other by specialized midline cell types and by three axonal tracts: the corpus callosum, the hippocampal commissure, and the anterior commissure. Many steps are required for these tracts to form, including early patterning and later axon pathfinding steps. Here, the requirement for FGF signaling in forming midline cell types and commissural axon tracts of the cerebral hemispheres is examined. Fgfr1, but not Fgfr3, is found to be essential for establishing all three commissural tracts. In an Fgfr1 mutant, commissural neurons are present and initially project their axons, but these fail to cross the midline that separates the hemispheres. Moreover, midline patterning defects are observed in the mutant. These defects include the loss of the septum and three specialized glial cell types, the indusium griseum glia, midline zipper glia, and glial wedge. Our findings demonstrate that FGF signaling is required for generating telencephalic midline structures, in particular septal and glial cell types and all three cerebral commissures. In addition, analysis of the Fgfr1 heterozygous mutant, in which midline patterning is normal but commissural defects still occur, suggests that at least two distinct FGF-dependent mechanisms underlie the formation of the cerebral commissures.

Formation of sensory pigment cells requires fibroblast growth factor signaling during ascidian embryonic development

The tadpole larva of the ascidian Halocynthia roretzi has two sensory pigment cells in its brain vesicle. To elucidate the temporal requirement for FGF signaling in formation of the pigment cells, embryos were treated with an FGF receptor 1 inhibitor, SU5402, or an MEK inhibitor, U0126 during various embryonic stages. In the present study, it is shown that the embryos treated with SU5402 from the 16‐cell stage to the early gastrula stage do not form pigment cells, whereas those treated after the early gastrula stage form pigment cells. In pigment cell formation, embryos suddenly exhibited the sensitivity to SU5402 only for 1 h at the neural plate stage (∼4 h after the beginning of gastrulation). When U0126 treatment was carried out at various stages between the 8‐cell and late neurula stages, the embryos scarcely formed pigment cells. Pigment cell formation occurred when the embryos were placed in U0126 at early tailbud stage. These results indicate that FGF signaling is involved in pigment cell formation...

FGF Signaling Restricts the Primary Blood Islands to Ventral Mesoderm

According to the three-signal model of mesoderm patterning in Xenopus, all mesoderm, with the exception of the Spemann organizer, is originally specified as ventral type, such as lateral plate and primary blood islands. It is proposed that the blood islands become restricted to the ventralmost mesoderm because they are not exposed to the BMP-inhibiting activity of the Spemann organizer. We present evidence here that, contrary to predictions of this model, the blood islands remain ventrally restricted even in the absence of Spemann organizer signaling. We further observed that inhibition of FGF signaling with a dominant negative receptor resulted in the expansion of the blood island-forming territory with a concomitant loss of somite. The requirement for FGF signaling in specifying somite versus blood island territories was observed as early as midgastrulation. The nonoverlapping expression domains of Xnr-2 and Xbra in the gastrula marginal zone appear to mark presumptive blood island and somite, respectively. Inhibition of FGF signaling with dominant negative receptor leads to an expansion of Xnr-2 expression and to a corresponding reduction in Xbra expression. On the other hand, we found no evidence that manipulation of BMP signaling, either positively or negatively, altered the expression domains of Xnr-2 and Xbra. These results suggest that FGF signaling, rather than BMP-inhibiting activity, is essential for restriction of the ventral blood islands to ventral mesoderm.

Fibroblast growth factor signaling is required for the proliferation and patterning of progenitor cells in the developing anterior pituitary

The proliferation and patterning of progenitor cells in the anterior pituitary require signals derived from the neuroepithelium of the juxtaposed infundibulum. The infundibulum expresses Fibroblast growth factor ( Fgf) 8 and Fgf 18, and FGFs can mimic some of the activities of the infundibulum. The requirement for FGF signaling during growth and patterning of the anterior pituitary has not, however, been established. By blocking FGF receptor signaling in explants of the anterior pituitary cultured in vitro we provide evidence that FGF signaling derived from the infundibulum is required for the proliferation and patterning of progenitor cells in the anterior pituitary.

The roles of FGFs in the early development of vertebrate limbs.

‘‘Fibroblast growth factor’’ (FGF) was first identified 25 years ago as a mitogenic activity in pituitary extracts (Armelin 1973; Gospodarowicz 1974). This modest observation subsequently led to the identification of a large family of proteins that affect cell proliferation, differentiation, survival, and motility (for review, see Basilico and Moscatelli 1992; Baird 1994). Recently, evidence has been accumulating that specific members of the FGF family function as key intercellular signaling molecules in embryogenesis (for review, see Goldfarb 1996). Indeed, it may be no exaggeration to say that, in conjunction with the members of a small number of other signaling molecule families [including WNT (Parr and McMahon 1994), Hedgehog (HH) (Hammerschmidt et al. 1997), and bone morphogenetic protein (BMP) (Hogan 1996)], FGFs are responsible for inducing and/or regulating the subsequent development of most organs in the vertebrate body. The purpose of this review is to discuss the functions performed by members of the FGF family in one of the best-studied vertebrate developmental systems—limb formation. It focuses initially on what is known about FGF function in the established limb bud and then discusses the mechanisms by which the signaling centers that control outgrowth and patterning of the established limb bud are formed, emphasizing possible roles played by FGFs in these processes. Finally, it discusses the potential role of FGFs in the induction of limb development. The insights into the requirement for FGF signaling in bone development that have been gained from analyses of mutations in human and mouse FGF receptor (FGFR) genes will not be discussed, as those studies have been the subject of several recent reviews (Wilkie et al. 1995; Yamaguchi and Rossant 1995; De Moerlooze and Dickson 1997; Webster and Donoghue 1997).