Epiboly generates the epidermal basal monolayer and spreads the nascent mammalian skin to enclose the embryonic body.

Epiboly is a morphogenetic process that is employed in the surface ectoderm of anamniotes during gastrulation to cover the entire embryo. We propose here that mammals also utilise this process to expand the epidermis and enclose the body cavity and spinal cord with a protective surface covering. Our data supports a model whereby epidermal spreading is driven by the primary establishment of the epidermal basal progenitor monolayer through radial cell intercalation of a multi-layered epithelium towards the basal lamina. By using a suspension organotypic culture strategy, we find that this process is fibronectin-dependent and autonomous to the skin. The radial cell rearrangements that drive epidermal spreading also require ROCK activity but are driven by cell protrusions and not myosin II contractility. Epidermal progenitor monolayer formation and epidermal spreading are delayed in Crash mice, which possess a dominant mutation in Celsr1, an orthologue of the core planar cell polarity (PCP) Drosophila protein Flamingo (also known as Stan). We observe a failure of ventral enclosure in Crash mutants suggesting that defective epidermal spreading might underlie some ventral wall birth defects.

Basal enrichment within neuroepithelia suggests novel function(s) for Celsr1 protein.

A characteristic of the 7TM-cadherins, Flamingo and Celsr1, is their asymmetric protein distribution and polarized activity at neighboring epithelial cell interfaces along defined axes of planar cell polarity. Here, we describe a novel distribution of Celsr1 protein to the basal surface of neuroepithelial cells within both the early neural tube and a less well-defined group of ventricular zone cells at the midline of the developing spinal cord. Importantly, this basal enrichment is lost in embryos homozygous for a mutant Celsr1 allele. We also demonstrate an intimate association between basal enrichment of Celsr1 protein and dorsal sensory tract morphogenesis, an intriguing spatio-temporal organization of Celsr1 protein along the apico-basal neuroepithelial axis suggestive of multiple Celsr1 protein isoforms and the existence of distinct cell surface Celsr1 protein species with direct signaling potential. Together, these data raise compelling new questions concerning the role of Celsr1 during neural development.

Critical role of FLRT1 phosphorylation in the interdependent regulation of FLRT1 function and FGF receptor signalling.

BACKGROUND: Fibronectin leucine rich transmembrane (FLRT) proteins have dual properties as regulators of cell adhesion and potentiators of fibroblast growth factor (FGF) mediated signalling. The mechanism by which the latter is achieved is still unknown and is the subject of this investigation. PRINCIPAL FINDINGS: Here we show that FLRT1 is a target for tyrosine phosphorylation mediated by FGFR1 and implicate a non-receptor Src family kinase (SFK). We identify the target tyrosine residues in the cytoplasmic domain of FLRT1 and show that these are not direct substrates for Src kinase suggesting that the SFK may exert effects via potentiation of FGFR1 kinase activity. We show that whilst FLRT1 expression results in a ligand-dependent elevation of MAP kinase activity, a mutant version of FLRT1, defective as an FGFR1 kinase substrate (Y3F-FLRT1), has the property of eliciting ligand-independent chronic activation of the MAP kinase pathway which is suppressed by pharmacological inhibition of either FGFR1 or Src kinase. Functional investigation of FGFR1 and FLRT1 signalling in SH-SY5Y neuroblastoma cells reveals that FLRT1 alone acts to induce a multi-polar phenotype whereas the combination of FLRT1 and FGFR activation, or expression of Y3F-FLRT1, acts to induce neurite outgrowth via MAPK activation. Similar results were obtained in a dendrite outgrowth assay in primary hippocampal neurons. We also show that FGFR1, FLRT1 and activated Src are co-localized and this complex is trafficked toward the soma of the cell. The presence of Y3F-FLRT1 rather than FLRT1 resulted in prolonged localization of this complex within the neuritic arbour. CONCLUSIONS: This study shows that the phosphorylation state of FLRT1, which is itself FGFR1 dependent, may play a critical role in the potentiation of FGFR1 signalling and may also depend on a SFK-dependent phosphorylation mechanism acting via the FGFR. This is consistent with an 'in vivo' role for FLRT1 regulation of FGF signalling via SFKs. Furthermore, the phosphorylation-dependent futile cycle mechanism controlling FGFR1 signalling is concurrently crucial for regulation of FLRT1-mediated neurite outgrowth.

Complex and dynamic patterns of Wnt pathway gene expression in the developing chick forebrain.

BACKGROUND: Wnt signalling regulates multiple aspects of brain development in vertebrate embryos. A large number of Wnts are expressed in the embryonic forebrain; however, it is poorly understood which specific Wnt performs which function and how they interact. Wnts are able to activate different intracellular pathways, but which of these pathways become activated in different brain subdivisions also remains enigmatic. RESULTS: We have compiled the first comprehensive spatiotemporal atlas of Wnt pathway gene expression at critical stages of forebrain regionalisation in the chick embryo and found that most of these genes are expressed in strikingly dynamic and complex patterns. Several expression domains do not respect proposed compartment boundaries in the developing forebrain, suggesting that areal identities are more dynamic than previously thought. Using an in ovo electroporation approach, we show that Wnt4 expression in the thalamus is negatively regulated by Sonic hedgehog (Shh) signalling from the zona limitans intrathalamica (ZLI), a known organising centre of forebrain development. CONCLUSION: The forebrain is exposed to a multitude of Wnts and Wnt inhibitors that are expressed in a highly dynamic and complex fashion, precluding simple correlative conclusions about their respective functions or signalling mechanisms. In various biological systems, Wnts are antagonised by Shh signalling. By demonstrating that Wnt4 expression in the thalamus is repressed by Shh from the ZLI we reveal an additional level of interaction between these two pathways and provide an example for the cross-regulation between patterning centres during forebrain regionalisation.

Fibroblast growth factor (FGF) gene expression in the developing cerebellum suggests multiple roles for FGF signaling during cerebellar morphogenesis and development.

The cerebellum is derived from the anterior-most segment of the embryonic hindbrain, rhombomere 1 (r1). Previous studies have shown that the early development and patterning of r1 requires fibroblast growth factor (FGF) signaling. However, many of the developmental processes that shape cerebellar morphogenesis take place later in embryonic development and during the first 2 weeks of postnatal life in the mouse. Here, we present a more comprehensive analysis of the expression patterns of genes encoding FGF receptors and secreted FGF ligands during these later stages of cerebellar development. We show that these genes are expressed in multiple cell types in the developing cerebellum, in an astonishing array of distinct patterns. These data suggest that FGF signaling functions throughout cerebellar development to regulate many processes that shape the formation of a functional cerebellum.

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.

The emergence of ectomesenchyme.

In the head, neural crest cells generate ectomesenchymal derivatives: cartilage, bone, and connective tissue. Indeed, these cells generate much of the cranial skeleton. There have, however, been few studies of how this lineage is established. Here, we show that neural crest cells stop expressing early neural crest markers upon entering the pharyngeal arches and switch to become ectomesenchymal. By contrast, those neural crest cells that do not enter the arches persist in their expression of early neural crest markers. We further show that fibroblast growth factor (FGF) signaling is involved in directing neural crest cells to become ectomesenchymal. If neural crest cells are rendered insensitive to FGFs, they persist in their expression of early neural crest markers, even after entering the pharyngeal arches. However, our results further suggest that, although FGF signaling is required for the realization of the ectomesenchymal lineages, other cues from the pharyngeal epithelia are also likely to be involved.

Expression of Shisa2, a modulator of both Wnt and Fgf signaling, in the chick embryo.

Shisa proteins are a recently-identified family of modulators of both FGF and Wnt signaling that block both maturation and transport to the cell surface of their respective receptors. The latter are retained within the endoplasmic reticulum, thereby inhibiting or reducing cellular responses to the ligands. We describe expression of a Shisa2 orthologue in an amniote: the chick embryo. We show that Shisa2 transcripts are expressed in a dynamic manner along the anteroposterior axis, in a manner consistent with a role in head development as demonstrated for Xenopus Shisa, being ubiquitously expressed in anterior tissues. However, expression is progressively restricted anteriorly within the developing neural tube and adjacent mesenchyme and ectoderm, eventually becoming restricted to the telencephalic lobes. Similarly, from being ubiquitous within the optic cups, transcripts become restricted to the prospective ciliary margin. A similar process is evident in the somites, where expression is initially ubiquitous but remains at high levels first in dermamyotome and subsequently is only detected in myotome. During the initial stages of organogenesis, Shisa2 transcripts are detected in cardiac and lung bud mesenchyme and in nephric ducts and tubules. Within the pharyngeal region, expression is observed in pharyngeal pouches from their first appearance and later in mesenchyme of all pharyngeal arches, as well as in cranial ganglia. Transcripts are also detected in the dorsal mesenchyme of the limb bud.

Initiation to end point: the multiple roles of fibroblast growth factors in neural development.

From a wealth of experimental findings, derived from both in vitro and in vivo experiments, it is becoming clear that fibroblast growth factors regulate processes that are central to all aspects of nervous system development. Some of these functions are well known, whereas others, such as the roles of these proteins in axon guidance and synaptogenesis, have been established only recently. The emergent picture is one of remarkable economy, in which this family of ligands is deployed and redeployed at successive developmental stages to sculpt the nervous system.

Sustained interactive Wnt and FGF signaling is required to maintain isthmic identity.

Fibroblast growth factor 8 (FGF8) is expressed at the mid-hindbrain boundary and is an important signal emanating from the isthmic organizer. Wnt1 is expressed in the caudal midbrain juxtaposed to Fgf8 expression and has been implicated in its regulation. In this study, we examine the requirement for continuous Wnt signaling in the maintenance of Fgf8 expression at the isthmus. We demonstrate that prior to HH10, ongoing Wnt signaling is required to maintain the normal pattern of isthmic Fgf8 expression in ovo. Similarly, in explant assays, sustained Wnt signaling is essential to maintain Fgf8 expression in rhombomere 1. The mechanism by which Wnt signaling regulates isthmic Fgf8 expression is likely to be a maintenance response rather than an inductive effect. Finally, we show that Wnt maintenance of Fgf8 expression is dependent upon positive feedback by FGF signaling itself, and that rhombomere 1 does not receive instructive cues from the posterior hindbrain. In summary, these findings establish that a sustained reciprocal interaction between Wnt and FGF signaling is essential to maintain isthmic identity.

Branching morphogenesis of the ureteric epithelium during kidney development is coordinated by the opposing functions of GDNF and Sprouty1.

Branching of ureteric bud-derived epithelial tubes is a key morphogenetic process that shapes development of the kidney. Glial cell line-derived neurotrophic factor (GDNF) initiates ureteric bud formation and promotes subsequent branching morphogenesis. Exactly how GDNF coordinates branching morphogenesis is unclear. Here we show that the absence of the receptor tyrosine kinase antagonist Sprouty1 (Spry1) results in irregular branching morphogenesis characterized by both increased number and size of ureteric bud tips. Deletion of Spry1 specifically in the epithelium is associated with increased epithelial Wnt11 expression as well as increased mesenchymal Gdnf expression. We propose that Spry1 regulates a Gdnf/Ret/Wnt11-positive feedback loop that coordinates mesenchymal-epithelial dialogue during branching morphogenesis. Genetic experiments indicate that the positive (GDNF) and inhibitory (Sprouty1) signals have to be finely balanced throughout renal development to prevent hypoplasia or cystic hyperplasia. Epithelial cysts develop in Spry1-deficient kidneys that share several molecular characteristics with those observed in human disease, suggesting that Spry1 null mice may be useful animal models for cystic hyperplasia.

A high throughput messenger RNA differential display screen identifies discrete domains of gene expression and novel patterning processes along the developing neural tube.

BACKGROUND: During early development the vertebrate neural tube is broadly organized into the forebrain, midbrain, hindbrain and spinal cord regions. Each of these embryonic zones is patterned by a combination of genetic pathways and the influences of local signaling centres. However, it is clear that much remains to be learned about the complete set of molecular cues that are employed to establish the identity and intrinsic neuronal diversity of these territories. In order to address this, we performed a high-resolution messenger RNA differential display screen to identify molecules whose expression is regionally restricted along the anteroposterior (AP) neuraxis during early chick development, with particular focus on the midbrain and hindbrain vesicles. RESULTS: This approach identified 44 different genes, with both known and unknown functions, whose transcription is differentially regulated along the AP axis. The identity and ontological classification of these genes is presented. The wide variety of functional classes of transcripts isolated in this screen reflects the diverse spectrum of known influences operating across these embryonic regions. Of these 44 genes, several have been selected for detailed in situ hybridization analysis to validate the screen and accurately define the expression domains. Many of the identified cDNAs showed no identity to the current databases of known or predicted genes or ESTs. Others represent genes whose embryonic expression has not been previously reported. Expression studies confirmed the predictions of the primary differential display data. Moreover, the nature of identified genes, not previously associated with regionalisation of the brain, identifies novel potential mechanisms in that process. CONCLUSION: This study provides an insight into some of the varied and novel molecular networks that operate during the regionalization of embryonic neural tissue and expands our knowledge of molecular repertoire used during development.

Fgf8 expression defines a morphogenetic center required for olfactory neurogenesis and nasal cavity development in the mouse.

In vertebrate olfactory epithelium (OE), neurogenesis proceeds continuously, suggesting that endogenous signals support survival and proliferation of stem and progenitor cells. We used a genetic approach 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.

Combinatorial activity of Flamingo proteins directs convergence and extension within the early zebrafish embryo via the planar cell polarity pathway.

The seven-transmembrane protocadherin, Flamingo, functions in a number of processes during Drosophila development, including planar cell polarity (PCP). To assess the role(s) of Flamingo1/Celsr1 (Fmi1) during vertebrate embryogenesis we have exploited the zebrafish system, identifying two Fmi1 orthologues (zFmi1a and zFmi1b) and employing morpholinos to induce mis-splicing of zebrafish fmi1 mRNAs, to both imitate mutations identified in Drosophila flamingo and generate novel aberrant Flamingo proteins. We demonstrate that in the zebrafish gastrula, Fmi1 proteins function in concert with each other and with the vertebrate PCP proteins, Wnt11 and Strabismus, to mediate convergence and extension during gastrulation, without altering early dorso-ventral patterning. We show that zebrafish Fmi1a promotes extension of the entire antero-posterior axis of the zebrafish gastrula including prechordal plate and ventral diencephalic precursors. However, while we show that control over axial extension is autonomous, we find that Fmi1a is not required within lateral cells undergoing dorsal convergence.

Planar polarity of hair cells in the chick inner ear is correlated with polarized distribution of c-flamingo-1 protein.

Hair cells of the vertebrate inner ear are directional mechanosensors: they have a polarity, defined by a vector in the plane of the sensory epithelium. It has been suggested that this polarity might be controlled by genes homologous to those that control planar cell polarity (PCP) in Drosophila, and vertebrate homologues of the Drosophila PCP genes Van Gogh/strabismus and flamingo/starry night are indeed essential for normal hair cell PCP. The underlying molecular mechanism is unclear, however. Although the PCP protein Flamingo shows a polarized intracellular distribution in the fly, it is unknown whether this is necessary for its function. Here, we describe the expression pattern of a flamingo homologue, c-flamingo-1 (c-fmi-1), in the developing chick ear and show that its protein product, like that of flamingo in the fly, has a polarized distribution in each hair cell, defining an axis that corresponds to the structural PCP axis. This conservation between fly and vertebrate suggests that the polarized protein localization is functionally important. In the basilar papilla, the same localization is seen in supporting cells also, suggesting that supporting cells are cryptically polarized, despite having no overt structural polarity; they may thus participate in PCP signal transmission across the sensory patch.