The construction of a small library of mouse repetitive DNA has been previously reported (Pietras... more The construction of a small library of mouse repetitive DNA has been previously reported (Pietras et al., Nucleic Acids Res. 11:6965-6983, 1983). Here we report that the 35 plasmids in this library corresponding to highly repeated (greater than 30,000 copies per genome) dispersed DNA sequences can be grouped into no more than 5 distinct families. These families together comprise 8 to 10% of the mouse genome. They include the previously described small elements B1, B2, and R and the large MIF-1 element. Twelve of the 35 clones contain evolutionarily conserved (EC) sequences. One EC clone in our library mostly consists of alternating dCdT residues; another consists of tandem repeats of the sequence CCTCT. The majority of B1s and B2s in the genome appear to be homogeneous, whereas R sequences, ECs, and MIF-1s are heterogeneous. Two earlier reports showed highly repeated mammalian DNA sequences in the herpesvirus genome (Peden et al., Cell 31:71-80, 1982; Puga et al., Cell 31:81-87, 198...
Confocal microscopy is a powerful tool for the study of cellular receptor trafficking and endocyt... more Confocal microscopy is a powerful tool for the study of cellular receptor trafficking and endocytosis. Unbiased and robust image analysis workflows are required for the identification, and study, of aberrant trafficking. After a brief review of related strategies, identifying both good and bad practice, custom workflows for the analysis of live cell 3D time-lapse data are presented. Strategies for data pre-processing, including denoising and background subtraction are considered. We use a 3D level set protocol to accurately segment cells using only the signal from fluorescently labelled receptor. A protocol for the quantification of changes to subcellular receptor distribution over time is then presented. As an example, ligand stimulated trafficking of epidermal growth factor receptor (EGFR) is shown to be significantly reduced in both AG1478 and Dynasore treated cells. Protocols for the quantitative analysis of colocalization between receptor and endosomes are also introduced, incl...
The insulin-receptor binding activity and insulin-stimulated growth response of PC13 clone 5 cell... more The insulin-receptor binding activity and insulin-stimulated growth response of PC13 clone 5 cells were investigated for both the embryo carcinoma (EC) and retinoic acid-induced differentiated derivatives of this cell line. Whereas the EC cell was found to have very few, if any, receptors and showed no demonstrable dependence on insulin for growth, the differentiated derivative cell expressed a large number of insulin receptors and, when challenged with the hormone, showed stimulation of both DNA synthesis and cell division. The same data were obtained for five independent PC13 clones. These results, coupled with previous observations, lend weight to the suggestion that the appearance of specific receptors for growth regulatory substances may be a manifestation of a general change in growth-regulatory mechanisms accompanying EC cell differentiation and loss of malignancy.
Uteri ofLifnull mice do not support embryo implantation. Since deletion of some genes often preve... more Uteri ofLifnull mice do not support embryo implantation. Since deletion of some genes often prevents the survival of null mice to adulthood, we have used a proven inhibitor of leukaemia inhibitory factor (LIF) signalling to identify the precise window of time during which LIF is requiredin vivo, and assessed the cellular expression of several LIF-associated targets. On day 4 of pregnancy, mice were injected with hLIF-05 (inhibitor) into the uterine lumen, with corresponding volumes of PBS (vehicle) injected into the contralateral horn. On days 5 and 6, the number of implantation sites was recorded and the uteri processed for immunohistochemistry. Blockade of LIF on day 4 reduced embryo implantation by 50% (P≤0.0001) and was effective maximally between 0930 and 1230 h. Antagonism of LIF signalling was evidenced by a lack of phosphorylated STAT3 in the luminal epithelium (LE). Amphiregulin was absent from the LE on day 4 evening and H-type-1 antigen expression was retained in the LE o...
The apical ectodermal ridge (AER), a thickening of the limb epithelium at its distal tip, is pivo... more The apical ectodermal ridge (AER), a thickening of the limb epithelium at its distal tip, is pivotal for maintaining vertebrate limb outgrowth. In a recent study, Flrt3 has been involved in the activation of ERK by FGFs. Moreover, Flrt3 has been described as a transmembrane protein able to interact with FGF receptors modulating, this way, FGFR signaling, either directly or recruiting cytoplasmic cofactors that enhance the activation of this pathway. Preliminary results show that Flrt3 expression is restricted to the AER, colocalizing its expression with fgf8 and pERK activity. Moreover, our data indicate that Flrt3 expression is not regulated by FGF activity, although ectopic Wnt3a is able to induce Flrt3 expression. Loss-of-function studies have shown that the silencing of Flrt3 affects the integrity of AER and, subsequently, its proper activity during limb bud outgrowth. We are performing studies in order to uncover the downstream targets o the Flrt3/Erk activity.
Activation of signalling by fibroblast growth factor receptor leads to phosphorylation of the sig... more Activation of signalling by fibroblast growth factor receptor leads to phosphorylation of the signalling attenuator human Sprouty 2 (hSpry2) on residue Y55. This event requires the presence of the signalling adaptor fibroblast growth factor receptor substrate 2 (FRS2). The phosphorylation of hSpry2 is therefore mediated by an intermediate kinase. Using a SRC family kinase-specific inhibitor and mutant cells, we show that hSpry2 is a direct substrate for SRC family kinases, including SRC itself. Activation of SRC via fibroblast growth factor signalling is dependent upon FRS2 and fibroblast growth factor receptor kinase activity. SRC forms a complex with hSpry2 and this interaction is enhanced by hSpry2 phosphorylation. Phosphorylation of hSpry2 is required for hSpry2 to inhibit activation of the extracellular signal-regulated kinase pathway. These results show that recruitment of SRC to FRS2 leads to activation of signal attenuation pathways.
A major challenge in computational systems biology is the articulation of a biological process in... more A major challenge in computational systems biology is the articulation of a biological process in a form which can be understood by the biologist yet is amenable to computational execution. Process calculi have proved to especially powerful computational tools for modelling and reasoning about biological processes and we have previously described, and implemented, a Narrative approach to describing biological models
Summary The analysis of factors which regulate cell proliferation and differentiation in early ma... more Summary The analysis of factors which regulate cell proliferation and differentiation in early mammalian development has been facilitated by the existence of cell lines derived from pluripotential stem cells of the early embryo; embryonal carcinoma (EC) cells and embryonic stem (ES) cells. EC cells have proved to be a useful source of embryonic growth factors. A potent mitogen, ECDGF has been isolated from EC cell conditioned medium. ECDGF appears to be a novel member of the heparin binding growth factor family. A remarkable feature of heparin binding growth factors is their ability to induce mesodermal differentiation in Xenopus laevis animal pole explants. ES cell differentiation in vitro is controlled by exogenous factors, in particular a novel differentiation inhibitory factor DIA. Controlled bipotential differentiation of ES cells can be achieved by exposing ES cells to different combinations of regulatory signals.
Summary The mammalian embryo develops as a quasi-stem cell system whose differentiation and pluri... more Summary The mammalian embryo develops as a quasi-stem cell system whose differentiation and pluripotentiality in vitro is controlled by a single regulatory factor, Differentiation Inhibiting Activity/Leukemia Inhibitory Factor (DIA/LIF). DIA/LIF is expressed in two distinct functional forms, derived from the use of alternate transcriptional start sites, one of which is freely diffusible and the other tightly associated with the extracellular matrix. The dissemination of the DIA/LIF signal is therefore under specific molecular control. The expression of DIA/LIF in vitro is both developmentally programmed and controlled by the action of other growth factors, the most notable of which are members of the fibroblast growth factor family expressed by the stem cells themselves. This indicates that differentiation and proliferation in early development of the mouse are controlled, at least in part, by an interactive network of specific growth and differentiation regulatory factors.
The construction of a small library of mouse repetitive DNA has been previously reported (Pietras... more The construction of a small library of mouse repetitive DNA has been previously reported (Pietras et al., Nucleic Acids Res. 11:6965-6983, 1983). Here we report that the 35 plasmids in this library corresponding to highly repeated (greater than 30,000 copies per genome) dispersed DNA sequences can be grouped into no more than 5 distinct families. These families together comprise 8 to 10% of the mouse genome. They include the previously described small elements B1, B2, and R and the large MIF-1 element. Twelve of the 35 clones contain evolutionarily conserved (EC) sequences. One EC clone in our library mostly consists of alternating dCdT residues; another consists of tandem repeats of the sequence CCTCT. The majority of B1s and B2s in the genome appear to be homogeneous, whereas R sequences, ECs, and MIF-1s are heterogeneous. Two earlier reports showed highly repeated mammalian DNA sequences in the herpesvirus genome (Peden et al., Cell 31:71-80, 1982; Puga et al., Cell 31:81-87, 198...
Confocal microscopy is a powerful tool for the study of cellular receptor trafficking and endocyt... more Confocal microscopy is a powerful tool for the study of cellular receptor trafficking and endocytosis. Unbiased and robust image analysis workflows are required for the identification, and study, of aberrant trafficking. After a brief review of related strategies, identifying both good and bad practice, custom workflows for the analysis of live cell 3D time-lapse data are presented. Strategies for data pre-processing, including denoising and background subtraction are considered. We use a 3D level set protocol to accurately segment cells using only the signal from fluorescently labelled receptor. A protocol for the quantification of changes to subcellular receptor distribution over time is then presented. As an example, ligand stimulated trafficking of epidermal growth factor receptor (EGFR) is shown to be significantly reduced in both AG1478 and Dynasore treated cells. Protocols for the quantitative analysis of colocalization between receptor and endosomes are also introduced, incl...
The insulin-receptor binding activity and insulin-stimulated growth response of PC13 clone 5 cell... more The insulin-receptor binding activity and insulin-stimulated growth response of PC13 clone 5 cells were investigated for both the embryo carcinoma (EC) and retinoic acid-induced differentiated derivatives of this cell line. Whereas the EC cell was found to have very few, if any, receptors and showed no demonstrable dependence on insulin for growth, the differentiated derivative cell expressed a large number of insulin receptors and, when challenged with the hormone, showed stimulation of both DNA synthesis and cell division. The same data were obtained for five independent PC13 clones. These results, coupled with previous observations, lend weight to the suggestion that the appearance of specific receptors for growth regulatory substances may be a manifestation of a general change in growth-regulatory mechanisms accompanying EC cell differentiation and loss of malignancy.
Uteri ofLifnull mice do not support embryo implantation. Since deletion of some genes often preve... more Uteri ofLifnull mice do not support embryo implantation. Since deletion of some genes often prevents the survival of null mice to adulthood, we have used a proven inhibitor of leukaemia inhibitory factor (LIF) signalling to identify the precise window of time during which LIF is requiredin vivo, and assessed the cellular expression of several LIF-associated targets. On day 4 of pregnancy, mice were injected with hLIF-05 (inhibitor) into the uterine lumen, with corresponding volumes of PBS (vehicle) injected into the contralateral horn. On days 5 and 6, the number of implantation sites was recorded and the uteri processed for immunohistochemistry. Blockade of LIF on day 4 reduced embryo implantation by 50% (P≤0.0001) and was effective maximally between 0930 and 1230 h. Antagonism of LIF signalling was evidenced by a lack of phosphorylated STAT3 in the luminal epithelium (LE). Amphiregulin was absent from the LE on day 4 evening and H-type-1 antigen expression was retained in the LE o...
The apical ectodermal ridge (AER), a thickening of the limb epithelium at its distal tip, is pivo... more The apical ectodermal ridge (AER), a thickening of the limb epithelium at its distal tip, is pivotal for maintaining vertebrate limb outgrowth. In a recent study, Flrt3 has been involved in the activation of ERK by FGFs. Moreover, Flrt3 has been described as a transmembrane protein able to interact with FGF receptors modulating, this way, FGFR signaling, either directly or recruiting cytoplasmic cofactors that enhance the activation of this pathway. Preliminary results show that Flrt3 expression is restricted to the AER, colocalizing its expression with fgf8 and pERK activity. Moreover, our data indicate that Flrt3 expression is not regulated by FGF activity, although ectopic Wnt3a is able to induce Flrt3 expression. Loss-of-function studies have shown that the silencing of Flrt3 affects the integrity of AER and, subsequently, its proper activity during limb bud outgrowth. We are performing studies in order to uncover the downstream targets o the Flrt3/Erk activity.
Activation of signalling by fibroblast growth factor receptor leads to phosphorylation of the sig... more Activation of signalling by fibroblast growth factor receptor leads to phosphorylation of the signalling attenuator human Sprouty 2 (hSpry2) on residue Y55. This event requires the presence of the signalling adaptor fibroblast growth factor receptor substrate 2 (FRS2). The phosphorylation of hSpry2 is therefore mediated by an intermediate kinase. Using a SRC family kinase-specific inhibitor and mutant cells, we show that hSpry2 is a direct substrate for SRC family kinases, including SRC itself. Activation of SRC via fibroblast growth factor signalling is dependent upon FRS2 and fibroblast growth factor receptor kinase activity. SRC forms a complex with hSpry2 and this interaction is enhanced by hSpry2 phosphorylation. Phosphorylation of hSpry2 is required for hSpry2 to inhibit activation of the extracellular signal-regulated kinase pathway. These results show that recruitment of SRC to FRS2 leads to activation of signal attenuation pathways.
A major challenge in computational systems biology is the articulation of a biological process in... more A major challenge in computational systems biology is the articulation of a biological process in a form which can be understood by the biologist yet is amenable to computational execution. Process calculi have proved to especially powerful computational tools for modelling and reasoning about biological processes and we have previously described, and implemented, a Narrative approach to describing biological models
Summary The analysis of factors which regulate cell proliferation and differentiation in early ma... more Summary The analysis of factors which regulate cell proliferation and differentiation in early mammalian development has been facilitated by the existence of cell lines derived from pluripotential stem cells of the early embryo; embryonal carcinoma (EC) cells and embryonic stem (ES) cells. EC cells have proved to be a useful source of embryonic growth factors. A potent mitogen, ECDGF has been isolated from EC cell conditioned medium. ECDGF appears to be a novel member of the heparin binding growth factor family. A remarkable feature of heparin binding growth factors is their ability to induce mesodermal differentiation in Xenopus laevis animal pole explants. ES cell differentiation in vitro is controlled by exogenous factors, in particular a novel differentiation inhibitory factor DIA. Controlled bipotential differentiation of ES cells can be achieved by exposing ES cells to different combinations of regulatory signals.
Summary The mammalian embryo develops as a quasi-stem cell system whose differentiation and pluri... more Summary The mammalian embryo develops as a quasi-stem cell system whose differentiation and pluripotentiality in vitro is controlled by a single regulatory factor, Differentiation Inhibiting Activity/Leukemia Inhibitory Factor (DIA/LIF). DIA/LIF is expressed in two distinct functional forms, derived from the use of alternate transcriptional start sites, one of which is freely diffusible and the other tightly associated with the extracellular matrix. The dissemination of the DIA/LIF signal is therefore under specific molecular control. The expression of DIA/LIF in vitro is both developmentally programmed and controlled by the action of other growth factors, the most notable of which are members of the fibroblast growth factor family expressed by the stem cells themselves. This indicates that differentiation and proliferation in early development of the mouse are controlled, at least in part, by an interactive network of specific growth and differentiation regulatory factors.
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