Fibroblast Growth Factor Receptor-induced Phosphorylation of EphrinB1 Modulates Its Interaction with Dishevelled

The Eph family of receptor tyrosine kinases and their membrane-bound ligands, the ephrins, have been implicated in regulating cell adhesion and migration during development by mediating cell-to-cell signaling events. The transmembrane ephrinB1 protein is a bidirectional signaling molecule that signals through its cytoplasmic domain to promote cellular movements into the eye field, whereas activation of the fibroblast growth factor receptor (FGFR) represses these movements and retinal fate. In Xenopus embryos, ephrinB1 plays a role in retinal progenitor cell movement into the eye field through an interaction with the scaffold protein Dishevelled (Dsh). However, the mechanism by which the FGFR may regulate this cell movement is unknown. Here, we present evidence that FGFR-induced repression of retinal fate is dependent upon phosphorylation within the intracellular domain of ephrinB1. We demonstrate that phosphorylation of tyrosines 324 and 325 disrupts the ephrinB1/Dsh interaction, thus modulating retinal progenitor movement that is dependent on the planar cell polarity pathway. These results provide mechanistic insight into how fibroblast growth factor signaling modulates ephrinB1 control of retinal progenitor movement within the eye field.     

Early stages of zebrafish eye formation require the coordinated activity of Wnt11, Fz5, and the Wnt/beta-catenin pathway

During regional patterning of the anterior neural plate, a medially positioned domain of cells is specified to adopt retinal identity. These eye field cells remain coherent as they undergo morphogenetic events distinct from other prospective forebrain domains. We show that two branches of the Wnt signaling pathway coordinate cell fate determination with cell behavior during eye field formation. Wnt/beta-catenin signaling antagonizes eye specification through the activity of Wnt8b and Fz8a. In contrast, Wnt11 and Fz5 promote eye field development, at least in part, through local antagonism of Wnt/beta-catenin signaling. Additionally, Wnt11 regulates the behavior of eye field cells, promoting their cohesion. Together, these results allow us to postulate a model in which Wnt11 and Fz5 signaling promotes early eye development through the coordinated antagonism of signals that suppress retinal identity and promotion of coherence of eye field cells.     

Roles of cell-extrinsic growth factors in vertebrate eye pattern formation and retinogenesis

Formation of the vertebrate visual system involves complex interplays of cell-extrinsic cues and cell-intrinsic determinants. Studies in several vertebrate species demonstrate that multiple classes of signaling molecules participate in pattern formation of the eye and neurogenesis of the retina. Certain signals, such as hedgehog, BMP, and FGF molecules, are repeatedly deployed at varying concentration thresholds and in different cellular contexts. Accumulating evidence reveals a striking conservation of molecular mechanisms regulating the neurogenic process between Drosophila and vertebrate retinas. The remaining challenge is to understand how these well-characterized signaling pathways are activated and integrated to impact eye morphogenesis and retinal progenitor cell fate determination.     

Back and forth between cell fate specification and movement during vertebrate gastrulation

Animal body plan arises during gastrulation and organogenesis by the coordination of inductive events and cell movements. Several signaling pathways, such as BMP, FGF, Hedgehog, Nodal, and Wnt have well-recognized instructive roles in cell fate specification during vertebrate embryogenesis. Growing evidence indicates that BMP, Nodal, and FGF signaling also regulate cell movements, and that they do so through mechanisms distinct from those that specify cell fates. Moreover, pathways controlling cell movements can also indirectly influence cell fate specification by regulating dimensions and relative positions of interacting tissues. The current challenge is to delineate the molecular mechanisms via which the major signaling pathways regulate cell fate specification and movements, and how these two processes are coordinated to ensure normal development.     

rugose (rg), a Drosophila A kinase anchor protein,is required for retinal pattern formation and interacts genetically with multiple signaling pathways

In the developing Drosophila eye, cell fate determination and pattern formation are directed by cell-cell interactions mediated by signal transduction cascades. Mutations at the rugose locus (rg) result in a rough eye phenotype due to a disorganized retina and aberrant cone cell differentiation, which leads to reduction or complete loss of cone cells. The cone cell phenotype is sensitive to the level of rugose gene function. Molecular analyses show that rugose encodes a Drosophila A kinase anchor protein (DAKAP 550). Genetic interaction studies show that rugose interacts with the components of the EGFR- and Notch-mediated signaling pathways. Our results suggest that rg is required for correct retinal pattern formation and may function in cell fate determination through its interactions with the EGFR and Notch signaling pathways.     

Animal-vegetal asymmetries influence the earliest steps in retina fate commitment in Xenopus.

An individual retina descends from a restricted and invariant group of nine animal blastomeres at the 32-cell stage. We tested which molecular signaling pathways are responsible for the competence of animal blastomeres to contribute to the retina. Inactivation of activin/Vg1 or fibroblast growth factor (FGF) signaling by expression of dominant-negative receptors does not prevent an animal blastomere from contributing to the retina. However, increasing bone morphogenetic protein (BMP) signaling in the retina-producing blastomeres significantly reduces their contribution. Conversely, reducing BMP signaling by expression of a dominant-negative BMP receptor or Noggin allows other animal blastomeres to contribute to the retina. Thus, the initial step in the retinal lineage is regulated by position within the BMP/Noggin field of epidermal versus neural induction. Vegetal tier blastomeres, in contrast, cannot contribute to the retina even when given access to the appropriate position and signaling fields by transplantation to the dorsal animal pole. We tested whether expression of molecules within the mesoderm inducing (activin, FGF), mesoderm-modifying (Wnt), or neural-inducing (BMP, Noggin) pathways impart a retinal fate on vegetal cell descendants. None of these, several of which induce secondary head structures, caused vegetal cells to contribute to retina. This was true even if the injected blastomeres were transplanted to the dorsal animal pole. Two pathways that specifically induce head tissues also were investigated. The simultaneous blockade of Wnt and BMP signaling, which results in the formation of a complete secondary axis with head and eyes, did not cause the vegetal clone to give rise to retina. However, Cerberus, a secreted protein that also induces an ectopic head with eyes, redirected vegetal progeny into the retina. These experiments indicate that vegetal blastomere incompetence to express a retinal fate is not due to a lack of components of known signaling pathways, but relies on a specific pathway of head induction.     

Targeted expression of the dominant-negative FGFR4a in the eye using Xrx1A regulatory sequences interferes with normal retinal development

Molecular analysis of vertebrate eye development has been hampered by the availability of sequences that can selectively direct gene expression in the developing eye. We report the characterization of the regulatory sequences of the Xenopus laevis Rx1A gene that can direct gene expression in the retinal progenitor cells. We have used these sequences to investigate the role of Fibroblast Growth Factor (FGF) signaling in the development of retinal cell types. FGFs are signaling molecules that are crucial for correct patterning of the embryo and that play important roles in the development of several embryonic tissues. FGFs and their receptors are expressed in the developing retina, and FGF receptor-mediated signaling has been implicated to have a role in the specification and survival of retinal cell types. We investigated the role of FGF signaling mediated by FGF receptor 4a in the development of retinal cell types in Xenopus laevis. For this purpose, we have made transgenic Xenopus tadpoles in which the dominant-negative FGFR4a (Delta FGFR4a) coding region was linked to the newly characterized regulatory sequences of the Xrx1A gene. We found that the expression of Delta FGFR4a in retinal progenitor cells results in abnormal retinal development. The retinas of transgenic animals expressing Delta FGFR4a show disorganized cell layering and specifically lack photoreceptor cells. These experiments show that FGFR4a-mediated FGF signaling is necessary for the correct specification of retinal cell types. Furthermore, they demonstrate that constructs using Xrx1A regulatory sequences are excellent tools with which to study the developmental processes involved in retinal formation.     

A complex choreography of cell movements shapes the vertebrate eye

Optic cup morphogenesis (OCM) generates the basic structure of the vertebrate eye. Although it is commonly depicted as a series of epithelial sheet folding events, this does not represent an empirically supported model. Here, we combine four-dimensional imaging with custom cell tracking software and photoactivatable fluorophore labeling to determine the cellular dynamics underlying OCM in zebrafish. Although cell division contributes to growth, we find it dispensable for eye formation. OCM depends instead on a complex set of cell movements coordinated between the prospective neural retina, retinal pigmented epithelium (RPE) and lens. Optic vesicle evagination persists for longer than expected; cells move in a pinwheel pattern during optic vesicle elongation and retinal precursors involute around the rim of the invaginating optic cup. We identify unanticipated movements, particularly of central and peripheral retina, RPE and lens. From cell tracking data, we generate retina, RPE and lens subdomain fate maps, which reveal novel adjacencies that might determine corresponding developmental signaling events. Finally, we find that similar movements also occur during chick eye morphogenesis, suggesting that the underlying choreography is conserved among vertebrates.     

Membrane‐mediated regulation of vascular identity

Vascular diseases span diverse pathology, but frequently arise from aberrant signaling attributed to specific membrane‐associated molecules, particularly the Eph‐ephrin family. Originally recognized as markers of embryonic vessel identity, Eph receptors and their membrane‐associated ligands, ephrins, are now known to have a range of vital functions in vascular physiology. Interactions of Ephs with ephrins at cell‐to‐cell interfaces promote a variety of cellular responses such as repulsion, adhesion, attraction, and migration, and frequently occur during organ development, including vessel formation. Elaborate coordination of Eph‐ and ephrin‐related signaling among different cell populations is required for proper formation of the embryonic vessel network. There is growing evidence supporting the idea that Eph and ephrin proteins also have postnatal interactions with a number of other membrane‐associated signal transduction pathways, coordinating translation of environmental signals into cells. This article provides an overview of membrane‐bound signaling mechanisms that define vascular identity in both the embryo and the adult, focusing on Eph‐ and ephrin‐related signaling. We also discuss the role and clinical significance of this signaling system in normal organ development, neoplasms, and vascular pathologies. Birth Defects Research (Part C) 108:65–84, 2016. © 2016 Wiley Periodicals, Inc.     

Fibroblast Growth Factor Signaling Is Essential for Self-renewal of Dental Epithelial Stem Cells

A constant supply of epithelial cells from dental epithelial stem cell (DESC) niches in the cervical loop (CL) enables mouse incisors to grow continuously throughout life. Elucidation of the cellular and molecular mechanisms underlying this unlimited growth potential is of broad interest for tooth regenerative therapies. Fibroblast growth factor (FGF) signaling is essential for the development of mouse incisors and for maintenance of the CL during prenatal development. However, how FGF signaling in DESCs controls the self-renewal and differentiation of the cells is not well understood. Herein, we report that FGF signaling is essential for self-renewal and the prevention of cell differentiation of DESCs in the CL as well as in DESC spheres. Inhibiting the FGF signaling pathway decreased proliferation and increased apoptosis of the cells in DESC spheres. Suppressing FGFR or its downstream signal transduction pathways diminished Lgr5-expressing cells in the CL and promoted cell differentiation both in DESC spheres and the CL. Furthermore, disruption of the FGF pathway abrogated Wnt signaling to promote Lgr5 expression in DESCs both in vitro and in vivo. This study sheds new light on understanding the mechanism by which the homeostasis, expansion, and differentiation of DESCs are regulated.     

FGF信号通路在内耳发育调控和毛细胞再生中的作用

内耳是感受听觉和平衡觉的复杂器官。在内耳发育过程中,成纤维生长因子(fibroblast growth factor, FGF)信号通路参与了听基板的诱导、螺旋神经节(statoacoustic ganglion, SAG)的发育以及Corti器感觉上皮的分化。FGF信号开启了内耳早期发育的基因调控网络,诱导前基板区域以及听基板的形成。正常表达的FGF信号分子可促进听囊腹侧成神经细胞的特化,但成熟SAG神经元释放的过量FGF5可抑制此过程,形成负反馈环路使SAG在稳定状态下发育。FGF20在Notch信号通路的调控下参与了前感觉上皮区域向毛细胞和支持细胞的分化过程,而内毛细胞分泌的FGF8可调控局部支持细胞分化为柱细胞。人类FGF信号通路异常可导致多种耳聋相关遗传病。此外,FGF信号通路在低等脊椎动物毛细胞自发再生以及干细胞向内耳毛细胞诱导过程中都起到了关键作用。本文综述了FGF信号通路在内耳发育调控以及毛细胞再生中的作用及其相关研究进展,以期为毛细胞再生中FGF信号通路调控机制的阐明奠定理论基础。     

Ephrins in reverse,park and drive

Eph receptors and their membrane-anchored ephrin ligands are thought to orchestrate cell movements by transducing bidirectional tyrosine-kinase-mediated signals into both cells expressing the receptors and cells expressing the ligands. Whether the resulting event is repulsion of an axonal growth cone, directing the orderly segmentation of hindbrain rhombomere cells or controlling angiogenic remodelling, such elaborate and diverse cell movements require intricate changes in the actin cytoskeleton, as well as precise regulation of cellular adhesion. Recent work by several groups has begun to link ephrin reverse signals to intracellular pathways that regulate actin dynamics and might help to explain how these ligands function as receptors to direct cell movement, adhesion and de-adhesion events.     

Retinal fate and ganglion cell differentiation are potentiated by acidic FGF in an in vitro assay of early retinal development.

One of the earliest events in vertebrate eye development is the establishment of the pigmented epithelium and neural retina. These fundamentally different tissues derive from the invaginated optic vesicle, or optic cup. Even after achieving a fairly advanced state of differentiation, the pigmented epithelium exhibits the same potential as the optic cup in that it can "transdifferentiate" into neural retina. C. M. Park and M. J. Hollenberg (Dev. Biol. 134, 201-205, 1989) discovered that administration of basic fibroblast growth factor, coupled with retinal removal, could trigger this transformation in vivo. We have developed a quantitative in vitro assay to study the role(s) of the fibroblast growth factor (FGF) family in this phenomenon and more generally in early retinal development. We found that several aspects of the process, including inhibition of pigmented epithelium differentiation, proliferation, and conversion to a retinal fate, were not strictly correlated. Both acidic and basic FGFs were found to potentiate all aspects of the process, with acidic FGF being 4 to 20 times more potent than basic FGF for inhibition of pigmentation and induction of retinal antigens. Depending upon its concentration, acidic FGF induced from 40% to 80% of the cells in the explants to produce antigens normally expressed by retinal ganglion cells, the first cell type to be generated in retinal development. Expression of such a ganglion cell marker could be directly stimulated in non-dividing cells as well as in dividing cells, indicating that conversion from the pigmented epithelial to retinal fate did not require cell division. These data suggest that acidic FGF, or a related molecule, may function in establishment of retinal fate from the optic cup. This effect may be directly or indirectly mediated by induction of retinal ganglion cell fate among multipotent progenitor cells.