Biomedical subjects

Kate G Storey

Publications and source records attributed to Kate G Storey.

8 recordsLinked to original sources

PubMed2006

A spatial and temporal map of FGF/Erk1/2 activity and response repertoires in the early chick embryo.

During early vertebrate development Fibroblast Growth Factor (FGF) signalling is required for multiple activities including specification of mesodermal, neural and heart tissue, as well as gastrulation movements and regulation of differentiation and pattern onset in the extending body axis. A current challenge is to understand how FGF signalling generates such diverse outcomes. A key FGF downstream pathway is the Ras-MAPK/Erk1/2 cascade, which culminates in the phosphorylation of target proteins, such as the Ets family of transcription factors. To begin to assess specificity downstream of FGF in the chick embryo we have characterised the patterns of Fgfr1-4 expression and Erk1/2 activation, as well as expression of the Erk1/2 specific phosphatase, Mkp3 and of three Ets factor genes (Erm, Pea3 and Er81) from early blastula to the 10 somite stage. We identify new sites of Fgfr expression and show that nearly all regions of Erk1/2 activity are within Fgfr expression domains and require FGF signalling. Differences in intensity, duration, distribution and sub-cellular localisation of activated Erk1/2 are observed in distinct cell populations within the embryo and during wound healing. With few exceptions, a tight correspondence between Erk1/2 activation and Mkp3 expression is found, while specific combinations of Ets factors are associated with distinct regions of Erk1/2 activation. These findings provide a comprehensive spatial and temporal map of FGF/Erk1/2 activity during early chick development and identify region and tissue specific differences in expression of Fgfrs as well as Erk1/2 phosphorylation and transcriptional targets which help to define response specificity.

Animals↗

PubMed2006

Negative feedback predominates over cross-regulation to control ERK MAPK activity in response to FGF signalling in embryos.

Expression of the gene encoding the MKP-3/Pyst1 protein phosphatase, which inactivates ERK MAPK, is induced by FGF. However, which intracellular signalling pathway mediates this expression is unclear, with essential roles proposed for both ERK and PI(3)K in chick embryonic limb. Here, we report that MKP-3/Pyst1 expression is sensitive to inhibition of ERK or MAPKK, that endogenous MKP-3/Pyst1 co-localizes with activated ERK, and expression of MKP-3/Pyst1 in mice lacking PDK1, an essential mediator of PI(3)K signalling. We conclude that MKP-3/Pyst1 expression is mediated by ERK activation and that negative feedback control predominates in limiting the extent of FGF-induced ERK activity.

Animals↗

PubMed2006

A robust system for RNA interference in the chicken using a modified microRNA operon.

RNA interference (RNAi) provides an effective method to silence gene expression and investigate gene function. However, RNAi tools for the chicken embryo have largely been adapted from vectors designed for mammalian cells. Here we present plasmid and retroviral RNAi vectors specifically designed for optimal gene silencing in chicken cells. The vectors use a chicken U6 promoter to express RNAs modelled on microRNA30, which are embedded within chicken microRNA operon sequences to ensure optimal Drosha and Dicer processing of transcripts. The chicken U6 promoter works significantly better than promoters of mammalian origin and in combination with a microRNA operon expression cassette (MOEC), achieves up to 90% silencing of target genes. By using a MOEC, we show that it is also possible to simultaneously silence two genes with a single vector. The vectors express either RFP or GFP markers, allowing simple in vivo tracking of vector delivery. Using these plasmids, we demonstrate effective silencing of Pax3, Pax6, Nkx2.1, Nkx2.2, Notch1 and Shh in discrete regions of the chicken embryonic nervous system. The efficiency and ease of use of this RNAi system paves the way for large-scale genetic screens in the chicken embryo.

Animals↗

PubMed2005

FGF-dependent Notch signaling maintains the spinal cord stem zone.

Generation of the spinal cord relies on proliferation of undifferentiated cells located in a caudal stem zone. Although fibroblast growth factor (FGF) signaling is required to maintain this cell group, we do not know how it controls cell behavior in this context. Here we characterize an overlooked expression domain of the Notch ligand, Delta1, in the stem zone and demonstrate that this constitutes a proliferative cell group in which Notch signaling is active. We show that FGF signaling is required for expression of the proneural gene cash4 in the stem zone, which in turn induces Delta1. We further demonstrate that Notch signaling is required for cell proliferation within the stem zone; however, it does not regulate cell movement out of this region, nor is loss of Notch signaling sufficient to drive neuronal differentiation within this tissue. These data identify a novel role for the Notch pathway during vertebrate neurogenesis in which signaling between high Delta1-expressing cells maintains the neural precursor pool that generates the spinal cord. Our findings also suggest a mechanism for the establishment of the cell selection process, lateral inhibition: Mutual inhibition between Delta/Notch-expressing stem zone cells switches to single Delta1-presenting neurons as FGF activity declines in the newly formed neuroepithelium.

Animals↗

PubMed2005

In vivo role of the phosphate groove of PDK1 defined by knockin mutation.

AGC kinases are mediators of signalling responses stimulated by agonists and are activated following phosphorylation at their T-loop residue by the 3-phosphoinositide-dependent protein kinase-1 (PDK1). Agonists stimulate the activation of the AGC kinases p70 ribosomal S6 kinase (S6K), p90 ribosomal S6 kinase (RSK) and serum and glucocorticoid-induced protein kinase (SGK), by inducing the phosphorylation of these enzymes at a non-catalytic regulatory site termed the hydrophobic motif. This creates a high-affinity docking site enabling PDK1 to bind and phosphorylate the T-loop of these enzymes. The site that interacts with these substrates is located on the small lobe of the catalytic domain of PDK1 and is composed of a hydrophobic groove next to a basic phosphate groove. The disruption of the hydrophobic groove ablates activation of S6K, RSK and SGK, but the role of the phosphate groove in regulating the function of PDK1 has not been explored in vivo. We generated knockin ES cells, in which both copies of the gene encoding PDK1 were altered to express a form of PDK1 that retains catalytic activity and integrity of the hydrophobic groove, but in which the phosphate groove was disrupted. The knockin ES cells were viable, mutant PDK1 was expressed at normal levels and IGF1 induced activation of protein kinase B (PKB/Akt), which is a PDK1 substrate that does not require hydrophobic motif phosphorylation to be activated. In the phosphate-groove-knockin ES cells, the activation of S6K, RSK and SGK by agonists, although markedly impaired, was not abolished. PDK1 also phosphorylates the T-loop of protein kinase C (PKC) isoforms, which stabilizes these enzymes. However, in contrast to S6K, RSK and SGK, hydrophobic motif phosphorylation of these enzymes is not thought to control their activation by PDK1. Consistent with this notion, we employed appropriate PDK1-knockin ES cells to demonstrate that the hydrophobic groove of PDK1, but not the phosphate groove, is required for the stabilization of PKC isoforms. These findings provide genetic evidence that the phosphate groove of PDK1 is required for maximal activation of isoforms of S6K, SGK and RSK, but not PKC. We also found that no live births of homozygous phosphate-groove-knockin mice are observed, indicating a key role for this regulatory motif in normal development. The knockin embryos develop to a greater extent than PDK1-knockout and hydrophobic-groove-knockin embryos, which died between E9.5-E11.5. The knockin embryos are observed until E19.5 and displayed general growth retardation and craniofacial developmental defects.

3-Phosphoinositide-Dependent Protein Kinases↗

PubMed2005

Specification and maintenance of the spinal cord stem zone.

Epiblast cells adjacent to the regressing primitive streak behave as a stem zone that progressively generates the entire spinal cord and also contributes to paraxial mesoderm. Despite this fundamental task, this cell population is poorly characterised, and the tissue interactions and signalling pathways that specify this unique region are unknown. Fibroblast growth factor (FGF) is implicated but it is unclear whether it is sufficient and/or directly required for stem zone specification. It is also not understood how establishment of the stem zone relates to the acquisition of spinal cord identity as indicated by expression of caudal Hox genes. Here, we show that many cells in the chick stem zone express both early neural and mesodermal genes; however, stem zone-specific gene expression can be induced by signals from underlying paraxial mesoderm without concomitant induction of an ambivalent neural/mesodermal cell state. The stem zone is a site of FGF/MAPK signalling and we show that although FGF alone does not mimic paraxial mesoderm signals, it is directly required in epiblast cells for stem zone specification and maintenance. We further demonstrate that caudal Hox gene expression in the stem zone also depends on FGF and that neither stem zone specification nor caudal Hox gene onset requires retinoid signalling. These findings thus support a two step model for spinal cord generation - FGF-dependent establishment of the stem zone in which progressively more caudal Hox genes are expressed, followed by the retinoid-dependent assignment of spinal cord identity.

Animals↗

PubMed2004

Opposing FGF and retinoid pathways: a signalling switch that controls differentiation and patterning onset in the extending vertebrate body axis.

Construction of the trunk/caudal region of the vertebrate embryo involves a set of distinct molecules and processes whose relationships are just coming into focus. In addition to the subdivision of the embryo into head and trunk domains, this "caudalisation" process requires the establishment and maintenance of a stem zone. This sequentially generates caudal tissues over a long period which then undergo differentiation and patterning in the extending body axis. Here we review recent studies that show that changes in the signalling properties of the paraxial mesoderm act as a switch that controls onset of differentiation and pattern in the spinal cord. These findings identify distinct roles for different caudalising factors; in particular, Fibroblast Growth Factor (FGF) inhibits differentiation in the caudal stem zone, while Retinoic acid (RA) provided rostrally by somitic mesoderm is required for neuronal differentiation and establishment of ventral neural pattern. Furthermore, the mutual opposition of FGF and RA pathways controls not only neural differentiation but also mesoderm segmentation and might also underlie the progressive assignment of rostrocaudal identity by regulating Hox gene availability and activation.

Animals↗

PubMed2002

Onset of neuronal differentiation is regulated by paraxial mesoderm and requires attenuation of FGF signalling.

While many neuronal differentiation genes have been identified, we know little about what determines when and where neurons will form and how this process is coordinated with the differentiation of neighbouring tissues. In most vertebrates the onset of neuronal differentiation takes place in the spinal cord in a head to tail sequence. Here we demonstrate that the changing signalling properties of the adjacent paraxial mesoderm control the progression of neurogenesis in the chick spinal cord. We find an inverse relationship between the expression of caudal neural genes in the prospective spinal cord, which is maintained by underlying presomitic mesoderm and FGF signalling, and neuronal differentiation, which is repressed by such signals and accelerated by somitic mesoderm. We show that key to this interaction is the ability of somitic mesoderm to repress Fgf8 transcription in the prospective spinal cord. Our findings further indicate that attenuation of FGF signalling in the prospective spinal cord is a prerequisite for the onset of neuronal differentiation and may also help to resolve mesodermal and neural cell fates. However, inhibition of FGF signalling alone does not promote the formation of neurons, which requires still further somite signalling. We propose a model in which signalling from somitic tissue promotes the differentiation of the spinal cord and serves to co-ordinate neural and mesodermal development.

Animals↗