Grant Wheeler | University of East Anglia (original) (raw)

Papers by Grant Wheeler

Cold Spring Harbor Protocols

Biology

Homologous long non-coding RNAs (lncRNAs) are elusive to identify by sequence similarity due to t... more Homologous long non-coding RNAs (lncRNAs) are elusive to identify by sequence similarity due to their fast-evolutionary rate. Here we develop LincOFinder, a pipeline that finds conserved intergenic lncRNAs (lincRNAs) between distant related species by means of microsynteny analyses. Using this tool, we have identified 16 bona fide homologous lincRNAs between the amphioxus and human genomes. We characterized and compared in amphioxus and Xenopus the expression domain of one of them, Hotairm1, located in the anterior part of the Hox cluster. In addition, we analyzed the function of this lincRNA in Xenopus, showing that its disruption produces a severe headless phenotype, most probably by interfering with the regulation of the Hox cluster. Our results strongly suggest that this lincRNA has probably been regulating the Hox cluster since the early origin of chordates. Our work pioneers the use of syntenic searches to identify non-coding genes over long evolutionary distances and helps to...

European Journal of Pharmaceutics and Biopharmaceutics

Novel alternatives to antibiotics are urgently needed for the successful treatment of antimicrobi... more Novel alternatives to antibiotics are urgently needed for the successful treatment of antimicrobial resistant (AMR) infections. Experimental antibacterial oligonucleotide therapeutics, such as transcription factor decoys (TFD), are a promising approach to circumvent AMR. However, the therapeutic potential of TFD is contingent upon the development of carriers that afford efficient DNA protection against nucleases and delivery of DNA to the target infection site. As a carrier for TFD, here we present three prototypes of anionic solid lipid nanoparticles that were coated with either the cationic bolaamphiphile 12-bistetrahydroacridinium or with protamine. Both compounds switched particles zeta potential to positive values, showing efficient complexation with TFD and demonstrable protection from deoxyribonuclease. The effective delivery of TFD into bacteria was confirmed by confocal microscopy while SLN-bacteria interactions were studied by flow cytometry. Antibacterial efficacy was confirmed using a model TFD

Wnt/FZD signalling activity is required for spinal cord development, including the dorsal-ventral... more Wnt/FZD signalling activity is required for spinal cord development, including the dorsal-ventral patterning of the neural tube, where it affects proliferation and specification of neurons. Wnt ligands initiate canonical, β-catenin-dependent, signaling by binding to Frizzled receptors. However, in many developmental contexts the cognate FZD receptor for a particular Wnt ligand remains to be identified. Here, we characterized FZD10 expression in the dorsal neural tube where it overlaps with both Wnt1 and Wnt3a, as well as markers of dorsal progenitors and interneurons. We show FZD10 expression is sensitive to Wnt1, but not Wnt3a expression, and FZD10 plays a role in neural tube patterning. Knockdown approaches show that Wnt1 induced ventral expansion of dorsal neural markes, Pax6 and Pax7, requires FZD10. In contrast, Wnt3a induced dorsalization of the neural tube is not affected by FZD10 knockdown. Gain of function experiments show that FZD10 is not sufficient on its own to mediate ...

Developmental Biology, 2015

Journal of Cell Biology, 1999

␣ 6 ␤ 4 and the bullous pemphigoid antigen 180 (BP180) 1 , are transmembrane proteins that are in... more ␣ 6 ␤ 4 and the bullous pemphigoid antigen 180 (BP180) 1 , are transmembrane proteins that are involved in anchoring the basal epithelial cells to the underlying basement membrane. The hemidesmosomal plaque proteins, BP230 and plectin, are localized in the cytoplasm, and play a major role in the binding of intermediate filaments to

Biochemical Society Transactions, 1989

Genome biology, 2015

The ADAMTS (A Disintegrin and Metalloproteinase with Thrombospondin motifs) enzymes are secreted,... more The ADAMTS (A Disintegrin and Metalloproteinase with Thrombospondin motifs) enzymes are secreted, multi-domain matrix-associated zinc metalloendopeptidases that have diverse roles in tissue morphogenesis and patho-physiological remodeling, in inflammation and in vascular biology. The human family includes 19 members that can be sub-grouped on the basis of their known substrates, namely the aggrecanases or proteoglycanases (ADAMTS1, 4, 5, 8, 9, 15 and 20), the procollagen N-propeptidases (ADAMTS2, 3 and 14), the cartilage oligomeric matrix protein-cleaving enzymes (ADAMTS7 and 12), the von-Willebrand Factor proteinase (ADAMTS13) and a group of orphan enzymes (ADAMTS6, 10, 16, 17, 18 and 19). Control of the structure and function of the extracellular matrix (ECM) is a central theme of the biology of the ADAMTS, as exemplified by the actions of the procollagen-N-propeptidases in collagen fibril assembly and of the aggrecanases in the cleavage or modification of ECM proteoglycans. Defec...

Biochemical Society transactions, 1991

... Grant N. Wheeler, Roger S. Buxton, Andrew E. Parker, Joachim Arnemann, David A. Rees, Ian A. ... more ... Grant N. Wheeler, Roger S. Buxton, Andrew E. Parker, Joachim Arnemann, David A. Rees, Ian A. King and Anthony I. Magee*. National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK and Clinical Research Centre, Harrow, Middx., HA1 3UJ, UK. ...

Proceedings of the National Academy of Sciences, 1991

Among the variety of specialized intercellular junctions, those of the adherens type have the mos... more Among the variety of specialized intercellular junctions, those of the adherens type have the most obvious association with cytoskeletal elements. This may be with the actin microfilanent system as in the zonula adherens or with intermediate ifiaments as in the macula adherens, or desmosome. In the former case, it is clear that transmembrane glycoproteins of the cadherin family are important adhesive components of the molecular assembly. We now show for desmosomes that a major glycoprotein component (desmosomal glycoprotein DGI) has extensive homology with the cadherins, defining an extended fmily, but also has unique features in its cytoplasmic domain that are likely to be relevant to the association with intermediate rather than actin filaments. A novel 282-residue extension contains repeats of --29 amino acid residues predicted to have an antiparallel P-sheet structure, followed by a glycine-rich sequence. As in the cadherins, the extracellular domain contains possible Ca2+-binding sequences and a potential protease processing site. The cell adhesion recognition region (His-Ala-Val) of the cadherins is modified to Arg-Ala-Leu.

Genomics, 1991

We have established PCR assays for the genes coding for the major proteins of the desmosome type ... more We have established PCR assays for the genes coding for the major proteins of the desmosome type of cell junction, the desmosomal cadherins DGI (desmoglein) and DGII/III (desmocollins), and the plaque proteins DPI/II (desmoplakin) and DPIII (plakoglobin) and used them to test human-mouse and human-rat somatic cell hybrids with different contents of human chromosomes. From these data we were able to assign DGI to chromosome 18 (DSG), DGII/ III to chromosome 9p (DSC), DPI/II to chromosome 6p21ter(DSP), and DPIII to chromosome 7 (JUP).

Nanomedicine, 2016

With the rise in production of nanoparticles (NPs) for an ever-increasing number of applications,... more With the rise in production of nanoparticles (NPs) for an ever-increasing number of applications, there is an urgent need to efficiently assess their potential toxicity. We propose a NP hazard assessment protocol that combines mammalian cytotoxicity data with embryonic vertebrate abnormality scoring to determine an overall toxicity index. We observed that, after exposure to a range of NPs, Xenopus phenotypic scoring showed a strong correlation with cell based in vitro assays. Magnetite-cored NPs, negative for toxicity in vitro and Xenopus, were further confirmed as nontoxic in mice. The results highlight the potential of Xenopus embryo analysis as a fast screening approach for toxicity assessment of NPs, which could be introduced for the routine testing of nanomaterials.

PloS one, 2015

MicroRNAs (miRNAs) are short, non-coding RNAs around 22 nucleotides long. They inhibit gene expre... more MicroRNAs (miRNAs) are short, non-coding RNAs around 22 nucleotides long. They inhibit gene expression either by translational repression or by causing the degradation of the mRNAs they bind to. Many are highly conserved amongst diverse organisms and have restricted spatio-temporal expression patterns during embryonic development where they are thought to be involved in generating accuracy of developmental timing and in supporting cell fate decisions and tissue identity. We determined the expression patterns of 180 miRNAs in Xenopus laevis embryos using LNA oligonucleotides. In addition we carried out small RNA-seq on different stages of early Xenopus development, identified 44 miRNAs belonging to 29 new families and characterized the expression of 5 of these. Our analyses identified miRNA expression in many organs of the developing embryo. In particular a large number were expressed in neural tissue and in the somites. Surprisingly none of the miRNAs we have looked at show expressi...

BMC medical genomics, 2015

Negative regulators of signal transduction cascades play critical roles in controlling different ... more Negative regulators of signal transduction cascades play critical roles in controlling different aspects of normal embryonic development. Sprouty2 (Spry2) negatively regulates receptor tyrosine kinases (RTK) and FGF signalling and is important in differentiation, cell migration and proliferation. In vertebrate embryos, Spry2 is expressed in paraxial mesoderm and in forming somites. Expression is maintained in the myotome until late stages of somite differentiation. However, its role and mode of action during somite myogenesis is still unclear. Here, we analysed chick Spry2 expression and showed that it overlaps with that of myogenic regulatory factors MyoD and Mgn. Targeted mis-expression of Spry2 led to inhibition of myogenesis, whilst its C-terminal domain led to an increased number of myogenic cells by stimulating cell proliferation. Spry2 is expressed in somite myotomes and its expression overlaps with myogenic regulatory factors. Overexpression and dominant-negative interferenc...

Development (Cambridge, England), 2014

Myogenesis involves the stable commitment of progenitor cells followed by the execution of myogen... more Myogenesis involves the stable commitment of progenitor cells followed by the execution of myogenic differentiation, processes that are coordinated by myogenic regulatory factors, microRNAs and BAF chromatin remodeling complexes. BAF60a, BAF60b and BAF60c are structural subunits of the BAF complex that bind to the core ATPase Brg1 to provide functional specificity. BAF60c is essential for myogenesis; however, the mechanisms regulating the subunit composition of BAF/Brg1 complexes, in particular the incorporation of different BAF60 variants, are not understood. Here we reveal their dynamic expression during embryo myogenesis and uncover the concerted negative regulation of BAF60a and BAF60b by the muscle-specific microRNAs (myomiRs) miR-133 and miR-1/206 during somite differentiation. MicroRNA inhibition in chick embryos leads to increased BAF60a or BAF60b levels, a concomitant switch in BAF/Brg1 subunit composition and delayed myogenesis. The phenotypes are mimicked by sustained BAF...

Developmental Biology, 2015

Methods in Molecular Biology, 2008

The amphibian Xenopus laevis has been successfully used for many years as a model system for stud... more The amphibian Xenopus laevis has been successfully used for many years as a model system for studying vertebrate development. Because of technical limitations, however, molecular investigations have mainly concentrated on early stages. We have developed a straightforward method for stage-specific induction of gene expression in transgenic Xenopus embryos [1,2]. This method is based on the Xenopus heat shock protein 70 (Xhsp70 [3]) promoter driving the expression of desired gene products. We found that ubiquitous expression of the transgene is induced upon relatively mild heat treatment. Green fluorescent protein (GFP) was used as a marker to monitor successful induction of gene expression in transgenic embryos. We used this method to study the stage specificity of Wnt signalling function. Transient ectopic Wnt-8 expression during early neurulation was sufficient to repress anterior head development and this capacity was restricted to early stages of neurulation. By transient over-expression at different stages of development, we show that frizzled-7 disrupted morphogenesis sequentially from anterior to posterior along the dorsal axis as development proceeds. These results demonstrate that this method for inducible gene expression in transgenic Xenopus embryos will be a very powerful tool for temporal analysis of gene function and for studying molecular mechanisms of vertebrate organogenesis.

Cold Spring Harbor Protocols

Biology

Homologous long non-coding RNAs (lncRNAs) are elusive to identify by sequence similarity due to t... more Homologous long non-coding RNAs (lncRNAs) are elusive to identify by sequence similarity due to their fast-evolutionary rate. Here we develop LincOFinder, a pipeline that finds conserved intergenic lncRNAs (lincRNAs) between distant related species by means of microsynteny analyses. Using this tool, we have identified 16 bona fide homologous lincRNAs between the amphioxus and human genomes. We characterized and compared in amphioxus and Xenopus the expression domain of one of them, Hotairm1, located in the anterior part of the Hox cluster. In addition, we analyzed the function of this lincRNA in Xenopus, showing that its disruption produces a severe headless phenotype, most probably by interfering with the regulation of the Hox cluster. Our results strongly suggest that this lincRNA has probably been regulating the Hox cluster since the early origin of chordates. Our work pioneers the use of syntenic searches to identify non-coding genes over long evolutionary distances and helps to...

European Journal of Pharmaceutics and Biopharmaceutics

Novel alternatives to antibiotics are urgently needed for the successful treatment of antimicrobi... more Novel alternatives to antibiotics are urgently needed for the successful treatment of antimicrobial resistant (AMR) infections. Experimental antibacterial oligonucleotide therapeutics, such as transcription factor decoys (TFD), are a promising approach to circumvent AMR. However, the therapeutic potential of TFD is contingent upon the development of carriers that afford efficient DNA protection against nucleases and delivery of DNA to the target infection site. As a carrier for TFD, here we present three prototypes of anionic solid lipid nanoparticles that were coated with either the cationic bolaamphiphile 12-bistetrahydroacridinium or with protamine. Both compounds switched particles zeta potential to positive values, showing efficient complexation with TFD and demonstrable protection from deoxyribonuclease. The effective delivery of TFD into bacteria was confirmed by confocal microscopy while SLN-bacteria interactions were studied by flow cytometry. Antibacterial efficacy was confirmed using a model TFD

Wnt/FZD signalling activity is required for spinal cord development, including the dorsal-ventral... more Wnt/FZD signalling activity is required for spinal cord development, including the dorsal-ventral patterning of the neural tube, where it affects proliferation and specification of neurons. Wnt ligands initiate canonical, β-catenin-dependent, signaling by binding to Frizzled receptors. However, in many developmental contexts the cognate FZD receptor for a particular Wnt ligand remains to be identified. Here, we characterized FZD10 expression in the dorsal neural tube where it overlaps with both Wnt1 and Wnt3a, as well as markers of dorsal progenitors and interneurons. We show FZD10 expression is sensitive to Wnt1, but not Wnt3a expression, and FZD10 plays a role in neural tube patterning. Knockdown approaches show that Wnt1 induced ventral expansion of dorsal neural markes, Pax6 and Pax7, requires FZD10. In contrast, Wnt3a induced dorsalization of the neural tube is not affected by FZD10 knockdown. Gain of function experiments show that FZD10 is not sufficient on its own to mediate ...

Developmental Biology, 2015

Journal of Cell Biology, 1999

␣ 6 ␤ 4 and the bullous pemphigoid antigen 180 (BP180) 1 , are transmembrane proteins that are in... more ␣ 6 ␤ 4 and the bullous pemphigoid antigen 180 (BP180) 1 , are transmembrane proteins that are involved in anchoring the basal epithelial cells to the underlying basement membrane. The hemidesmosomal plaque proteins, BP230 and plectin, are localized in the cytoplasm, and play a major role in the binding of intermediate filaments to

Biochemical Society Transactions, 1989

Genome biology, 2015

The ADAMTS (A Disintegrin and Metalloproteinase with Thrombospondin motifs) enzymes are secreted,... more The ADAMTS (A Disintegrin and Metalloproteinase with Thrombospondin motifs) enzymes are secreted, multi-domain matrix-associated zinc metalloendopeptidases that have diverse roles in tissue morphogenesis and patho-physiological remodeling, in inflammation and in vascular biology. The human family includes 19 members that can be sub-grouped on the basis of their known substrates, namely the aggrecanases or proteoglycanases (ADAMTS1, 4, 5, 8, 9, 15 and 20), the procollagen N-propeptidases (ADAMTS2, 3 and 14), the cartilage oligomeric matrix protein-cleaving enzymes (ADAMTS7 and 12), the von-Willebrand Factor proteinase (ADAMTS13) and a group of orphan enzymes (ADAMTS6, 10, 16, 17, 18 and 19). Control of the structure and function of the extracellular matrix (ECM) is a central theme of the biology of the ADAMTS, as exemplified by the actions of the procollagen-N-propeptidases in collagen fibril assembly and of the aggrecanases in the cleavage or modification of ECM proteoglycans. Defec...

Biochemical Society transactions, 1991

... Grant N. Wheeler, Roger S. Buxton, Andrew E. Parker, Joachim Arnemann, David A. Rees, Ian A. ... more ... Grant N. Wheeler, Roger S. Buxton, Andrew E. Parker, Joachim Arnemann, David A. Rees, Ian A. King and Anthony I. Magee*. National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK and Clinical Research Centre, Harrow, Middx., HA1 3UJ, UK. ...

Proceedings of the National Academy of Sciences, 1991

Among the variety of specialized intercellular junctions, those of the adherens type have the mos... more Among the variety of specialized intercellular junctions, those of the adherens type have the most obvious association with cytoskeletal elements. This may be with the actin microfilanent system as in the zonula adherens or with intermediate ifiaments as in the macula adherens, or desmosome. In the former case, it is clear that transmembrane glycoproteins of the cadherin family are important adhesive components of the molecular assembly. We now show for desmosomes that a major glycoprotein component (desmosomal glycoprotein DGI) has extensive homology with the cadherins, defining an extended fmily, but also has unique features in its cytoplasmic domain that are likely to be relevant to the association with intermediate rather than actin filaments. A novel 282-residue extension contains repeats of --29 amino acid residues predicted to have an antiparallel P-sheet structure, followed by a glycine-rich sequence. As in the cadherins, the extracellular domain contains possible Ca2+-binding sequences and a potential protease processing site. The cell adhesion recognition region (His-Ala-Val) of the cadherins is modified to Arg-Ala-Leu.

Genomics, 1991

We have established PCR assays for the genes coding for the major proteins of the desmosome type ... more We have established PCR assays for the genes coding for the major proteins of the desmosome type of cell junction, the desmosomal cadherins DGI (desmoglein) and DGII/III (desmocollins), and the plaque proteins DPI/II (desmoplakin) and DPIII (plakoglobin) and used them to test human-mouse and human-rat somatic cell hybrids with different contents of human chromosomes. From these data we were able to assign DGI to chromosome 18 (DSG), DGII/ III to chromosome 9p (DSC), DPI/II to chromosome 6p21ter(DSP), and DPIII to chromosome 7 (JUP).

Nanomedicine, 2016

With the rise in production of nanoparticles (NPs) for an ever-increasing number of applications,... more With the rise in production of nanoparticles (NPs) for an ever-increasing number of applications, there is an urgent need to efficiently assess their potential toxicity. We propose a NP hazard assessment protocol that combines mammalian cytotoxicity data with embryonic vertebrate abnormality scoring to determine an overall toxicity index. We observed that, after exposure to a range of NPs, Xenopus phenotypic scoring showed a strong correlation with cell based in vitro assays. Magnetite-cored NPs, negative for toxicity in vitro and Xenopus, were further confirmed as nontoxic in mice. The results highlight the potential of Xenopus embryo analysis as a fast screening approach for toxicity assessment of NPs, which could be introduced for the routine testing of nanomaterials.

PloS one, 2015

MicroRNAs (miRNAs) are short, non-coding RNAs around 22 nucleotides long. They inhibit gene expre... more MicroRNAs (miRNAs) are short, non-coding RNAs around 22 nucleotides long. They inhibit gene expression either by translational repression or by causing the degradation of the mRNAs they bind to. Many are highly conserved amongst diverse organisms and have restricted spatio-temporal expression patterns during embryonic development where they are thought to be involved in generating accuracy of developmental timing and in supporting cell fate decisions and tissue identity. We determined the expression patterns of 180 miRNAs in Xenopus laevis embryos using LNA oligonucleotides. In addition we carried out small RNA-seq on different stages of early Xenopus development, identified 44 miRNAs belonging to 29 new families and characterized the expression of 5 of these. Our analyses identified miRNA expression in many organs of the developing embryo. In particular a large number were expressed in neural tissue and in the somites. Surprisingly none of the miRNAs we have looked at show expressi...

BMC medical genomics, 2015

Negative regulators of signal transduction cascades play critical roles in controlling different ... more Negative regulators of signal transduction cascades play critical roles in controlling different aspects of normal embryonic development. Sprouty2 (Spry2) negatively regulates receptor tyrosine kinases (RTK) and FGF signalling and is important in differentiation, cell migration and proliferation. In vertebrate embryos, Spry2 is expressed in paraxial mesoderm and in forming somites. Expression is maintained in the myotome until late stages of somite differentiation. However, its role and mode of action during somite myogenesis is still unclear. Here, we analysed chick Spry2 expression and showed that it overlaps with that of myogenic regulatory factors MyoD and Mgn. Targeted mis-expression of Spry2 led to inhibition of myogenesis, whilst its C-terminal domain led to an increased number of myogenic cells by stimulating cell proliferation. Spry2 is expressed in somite myotomes and its expression overlaps with myogenic regulatory factors. Overexpression and dominant-negative interferenc...

Development (Cambridge, England), 2014

Myogenesis involves the stable commitment of progenitor cells followed by the execution of myogen... more Myogenesis involves the stable commitment of progenitor cells followed by the execution of myogenic differentiation, processes that are coordinated by myogenic regulatory factors, microRNAs and BAF chromatin remodeling complexes. BAF60a, BAF60b and BAF60c are structural subunits of the BAF complex that bind to the core ATPase Brg1 to provide functional specificity. BAF60c is essential for myogenesis; however, the mechanisms regulating the subunit composition of BAF/Brg1 complexes, in particular the incorporation of different BAF60 variants, are not understood. Here we reveal their dynamic expression during embryo myogenesis and uncover the concerted negative regulation of BAF60a and BAF60b by the muscle-specific microRNAs (myomiRs) miR-133 and miR-1/206 during somite differentiation. MicroRNA inhibition in chick embryos leads to increased BAF60a or BAF60b levels, a concomitant switch in BAF/Brg1 subunit composition and delayed myogenesis. The phenotypes are mimicked by sustained BAF...

Developmental Biology, 2015

Methods in Molecular Biology, 2008

The amphibian Xenopus laevis has been successfully used for many years as a model system for stud... more The amphibian Xenopus laevis has been successfully used for many years as a model system for studying vertebrate development. Because of technical limitations, however, molecular investigations have mainly concentrated on early stages. We have developed a straightforward method for stage-specific induction of gene expression in transgenic Xenopus embryos [1,2]. This method is based on the Xenopus heat shock protein 70 (Xhsp70 [3]) promoter driving the expression of desired gene products. We found that ubiquitous expression of the transgene is induced upon relatively mild heat treatment. Green fluorescent protein (GFP) was used as a marker to monitor successful induction of gene expression in transgenic embryos. We used this method to study the stage specificity of Wnt signalling function. Transient ectopic Wnt-8 expression during early neurulation was sufficient to repress anterior head development and this capacity was restricted to early stages of neurulation. By transient over-expression at different stages of development, we show that frizzled-7 disrupted morphogenesis sequentially from anterior to posterior along the dorsal axis as development proceeds. These results demonstrate that this method for inducible gene expression in transgenic Xenopus embryos will be a very powerful tool for temporal analysis of gene function and for studying molecular mechanisms of vertebrate organogenesis.