title: The Effect of Pax3 Over-expression on Myoblast Function

authors: Brijs, Erik

subjects: Pax3 over-expression

abstract: The paired-box transcription factor 3 (Pax3), is a powerful myogenic regulatory factor during embryo-myogenesis. The expression of Pax3 is involved in determining somitic cell fate, resulting in the formation of the first differentiated muscle, the myotome. In addition, Pax3 expression in embryonic hypaxial precursor cells prevents premature differentiation, whilst promoting cellular migration and thus ensuring the proper development of limb muscles. Subsequent muscle growth in the developing embryo is provided by myogenic precursor cells marked by the expression of Pax3 and its paralogue, Pax7. Following foetal development, these precursor cells establish the distinct lineage of myogenic stem cells referred to as satellite cells, responsible for post-natal development and regeneration. However, satellite cells are not the only population of cells that generate myogenic precursor cells in adult muscle for regeneration and growth. Recently, it has been shown that in adult muscle, Pax3 is expressed in a population of novel, interstitial cells, and is up-regulated upon muscle injury.
To determine the role played by Pax3 in post-natal muscle development and regeneration, three Pax3 over-expressing myoblast (C2C12) cell lines were generated and experimentally compared with control cells. Four facets of myoblast function were assessed: proliferation, differentiation, survival and migration. 
The proliferation assay showed that Pax3 over-expression significantly (Plt0.001) slowed C2C12 myoblast proliferation, Pax3 over-expressing clones only reached 63% of the proliferative rate of control C2C12 cells. Furthermore, this study clearly showed that over-expression of Pax3 significantly (Plt0.01) delayed the differentiation of C2C12 myoblasts. Analysis showed that following 72 h of low serum induced differentiation 52% of the control C2C12 cell population had fused to form myofibres, in comparison only 16-32% of the myoblast population that over-expressed Pax3 had fused to from myofibres. Taken together, these data suggest that following satellite cell activation, Pax3 expression could be maintained in precursor cells to expand the myogenic population, as their myogenic commitment is delayed by Pax3 expression and this would facilitate increased cell cycling, albeit at a reduced rate. Furthermore, a low percentage of fusion strongly indicates a higher proportion of cells that are renewing the quiescent satellite cell population.
Furthermore, over-expression of Pax3 also conferred a dose-dependent anti-apoptotic function to C2C12 myoblasts. Myoblasts that over-expressed a low level of Pax3 were more resistant to apoptosis in comparison to control C2C12 cells and to myoblasts that over-expressed Pax3 at higher level. The anti-apoptotic function conferred by Pax3 expression would come into effect within the Pax3+ self-renewing satellite cell lineage. 
Finally, Pax3 over-expression was shown to increase the migratory ability of C2C12 myoblasts. In a chemotaxis assay, there was a strong indication that an intermediate over-expression of Pax3 increased myoblast sensitivity to chemotactic signals, priming cells for migration.  The delay in differentiation also exhibited by Pax3 over-expression may function to allow satellite cell-derived progenitors to migrate to sites of injury, therefore enhancing skeletal muscle regenerative capacity.

text: The Effect of Pax3 Over-expression on Myoblast Function The paired-box transcription factor 3 (Pax3), is a powerful myogenic regulatory factor during embryo-myogenesis. The expression of Pax3 is involved in determining somitic cell fate, resulting in the formation of the first differentiated muscle, the myotome. In addition, Pax3 expression in embryonic hypaxial precursor cells prevents premature differentiation, whilst promoting cellular migration and thus ensuring the proper development of limb muscles. Subsequent muscle growth in the developing embryo is provided by myogenic precursor cells marked by the expression of Pax3 and its paralogue, Pax7. Following foetal development, these precursor cells establish the distinct lineage of myogenic stem cells referred to as satellite cells, responsible for post-natal development and regeneration. However, satellite cells are not the only population of cells that generate myogenic precursor cells in adult muscle for regeneration and growth. Recently, it has been shown that in adult muscle, Pax3 is expressed in a population of novel, interstitial cells, and is up-regulated upon muscle injury.
To determine the role played by Pax3 in post-natal muscle development and regeneration, three Pax3 over-expressing myoblast (C2C12) cell lines were generated and experimentally compared with control cells. Four facets of myoblast function were assessed: proliferation, differentiation, survival and migration. 
The proliferation assay showed that Pax3 over-expression significantly (Plt0.001) slowed C2C12 myoblast proliferation, Pax3 over-expressing clones only reached 63% of the proliferative rate of control C2C12 cells. Furthermore, this study clearly showed that over-expression of Pax3 significantly (Plt0.01) delayed the differentiation of C2C12 myoblasts. Analysis showed that following 72 h of low serum induced differentiation 52% of the control C2C12 cell population had fused to form myofibres, in comparison only 16-32% of the myoblast population that over-expressed Pax3 had fused to from myofibres. Taken together, these data suggest that following satellite cell activation, Pax3 expression could be maintained in precursor cells to expand the myogenic population, as their myogenic commitment is delayed by Pax3 expression and this would facilitate increased cell cycling, albeit at a reduced rate. Furthermore, a low percentage of fusion strongly indicates a higher proportion of cells that are renewing the quiescent satellite cell population.
Furthermore, over-expression of Pax3 also conferred a dose-dependent anti-apoptotic function to C2C12 myoblasts. Myoblasts that over-expressed a low level of Pax3 were more resistant to apoptosis in comparison to control C2C12 cells and to myoblasts that over-expressed Pax3 at higher level. The anti-apoptotic function conferred by Pax3 expression would come into effect within the Pax3+ self-renewing satellite cell lineage. 
Finally, Pax3 over-expression was shown to increase the migratory ability of C2C12 myoblasts. In a chemotaxis assay, there was a strong indication that an intermediate over-expression of Pax3 increased myoblast sensitivity to chemotactic signals, priming cells for migration.  The delay in differentiation also exhibited by Pax3 over-expression may function to allow satellite cell-derived progenitors to migrate to sites of injury, therefore enhancing skeletal muscle regenerative capacity.

year: 2009

title: The synthesis of triangulo-trimetal complexes containing both iron(II) and vanadium(II)

authors: Evans, David J.

subjects: Crystal structures

abstract: Reactions between a variety of vanadium(II) and iron(II) starting materials in tetrahydrofuran (thf) solution afforded di-, tri-, and tetra-nuclear complexes containing thf or N,N,N′,N′-tetramethylethylenediamine (tmen) as ligands. Most products were ionic and contained the two metals in different structural units, cation or anion. Trinuclear clusters formulated as [V3−xFexCl₅(tmen)₃][BPh₄](x=0–3) were obtained and characterised by C, H, N and metal analyses, ¹H NMR, Mössbauer, and positive ion FAB and electrospray mass spectroscopies, and magnetic moment measurements in solution. We characterised fully by low-temperature single-crystal X-ray diffraction analysis the mixed-metal triangulo-complex salt [V₂Fe(μ-Cl)₃(μ₃-Cl)₂(tmen)₃][BPh₄].

text: The synthesis of triangulo-trimetal complexes containing both iron(II) and vanadium(II) Reactions between a variety of vanadium(II) and iron(II) starting materials in tetrahydrofuran (thf) solution afforded di-, tri-, and tetra-nuclear complexes containing thf or N,N,N′,N′-tetramethylethylenediamine (tmen) as ligands. Most products were ionic and contained the two metals in different structural units, cation or anion. Trinuclear clusters formulated as [V3−xFexCl₅(tmen)₃][BPh₄](x=0–3) were obtained and characterised by C, H, N and metal analyses, ¹H NMR, Mössbauer, and positive ion FAB and electrospray mass spectroscopies, and magnetic moment measurements in solution. We characterised fully by low-temperature single-crystal X-ray diffraction analysis the mixed-metal triangulo-complex salt [V₂Fe(μ-Cl)₃(μ₃-Cl)₂(tmen)₃][BPh₄].

year: 2001-07-01

title: Using the curriculum guidelines for the teaching and learning of te reo Māori in Years 1 - 13 of New Zealand schools to create lessons for young learners

authors: Diane, Johnson

subjects: New Zealand

abstract: In 2009, The New Zealand Ministry of Education published curriculum guidelines for te reo Māori in Years 1 - 13 of English-medium New Zealand schools, that is, from age 5 upwards. These guidelines recommend a communicative approach to the teaching and learning of te reo Māori. In this article, we discuss communicative approaches and provide examples of ways in which teachers can design communicative language lessons that are appropriate for very young learners, lessons that are not only fun but also effective in developing genuine communicative competence in te reo Māori.

text: Using the curriculum guidelines for the teaching and learning of te reo Māori in Years 1 - 13 of New Zealand schools to create lessons for young learners In 2009, The New Zealand Ministry of Education published curriculum guidelines for te reo Māori in Years 1 - 13 of English-medium New Zealand schools, that is, from age 5 upwards. These guidelines recommend a communicative approach to the teaching and learning of te reo Māori. In this article, we discuss communicative approaches and provide examples of ways in which teachers can design communicative language lessons that are appropriate for very young learners, lessons that are not only fun but also effective in developing genuine communicative competence in te reo Māori.

year: 2009-09