I: Controle foetal des metabolites du cholecalciferol chez le rat : incidence du diabete maternel. II: Regulation de la calcemie chez le rat

SIGLECNRS T 62327 / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Effect of 25-hydroxyvitamin D3 and 1,25-dihydroxyvitamin D3 maternal loads on maternal and fetal vitamin D metabolite levels in the rat

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Six homeoproteins directly activate Myod expression in the gene regulatory networks that control early myogenesis.

International audienceIn mammals, several genetic pathways have been characterized that govern engagement of multipotent embryonic progenitors into the myogenic program through the control of the key myogenic regulatory gene Myod. Here we demonstrate the involvement of Six homeoproteins. We first targeted into a Pax3 allele a sequence encoding a negative form of Six4 that binds DNA but cannot interact with essential Eya co-factors. The resulting embryos present hypoplasic skeletal muscles and impaired Myod activation in the trunk in the absence of Myf5/Mrf4. At the axial level, we further show that Myod is still expressed in compound Six1/Six4:Pax3 but not in Six1/Six4:Myf5 triple mutant embryos, demonstrating that Six1/4 participates in the Pax3-Myod genetic pathway. Myod expression and head myogenesis is preserved in Six1/Six4:Myf5 triple mutant embryos, illustrating that upstream regulators of Myod in different embryonic territories are distinct. We show that Myod regulatory regions are directly controlled by Six proteins and that, in the absence of Six1 and Six4, Six2 can compensate

Six1 and Six4 gene expression is necessary to activate the fast-type muscle gene program in the mouse primary myotome

International audienceWhile the signaling pathways and transcription factors active in adult slow- and fast-type muscles begin to be characterized, genesis of muscle fiber-type diversity during mammalian development remains unexplained. We provide evidence showing that Six homeoproteins are required to activate the fast-type muscle program in the mouse primary myotome. Affymetrix transcriptomal analysis of Six1(-/-)Six4(-/-) E10.5 somites revealed the specific down-regulation of many genes of the fast-type muscle program. This data was confirmed by in situ hybridization performed on Six1(-/-)Six4(-/-) embryos. The first mouse myocytes express both fast-type and slow-type muscle genes. In these fibers, Six1 and Six4 expression is required to specifically activate fast-type muscle genes. Chromatin immunoprecipitation experiments confirm the binding of Six1 and Six4 on the regulatory regions of these muscle genes, and transfection experiments show the ability of these homeoproteins to activate specifically identified fast-type muscle genes. This in vivo wide transcriptomal analysis of the function of the master myogenic determinants, Six, identifies them as novel markers for the differential activation of a specific muscle program during mammalian somitic myogenesis

Six1 and Eya1 Expression Can Reprogram Adult Muscle from the Slow-Twitch Phenotype into the Fast-Twitch Phenotype

Muscle fibers show great differences in their contractile and metabolic properties. This diversity enables skeletal muscles to fulfill and adapt to different tasks. In this report, we show that the Six/Eya pathway is implicated in the establishment and maintenance of the fast-twitch skeletal muscle phenotype. We demonstrate that the MEF3/Six DNA binding element present in the aldolase A pM promoter mediates the high level of activation of this promoter in fast-twitch glycolytic (but not in slow-twitch) muscle fibers. We also show that among the Six and Eya gene products expressed in mouse skeletal muscle, Six1 and Eya1 proteins accumulate preferentially in the nuclei of fast-twitch muscles. The forced expression of Six1 and Eya1 together in the slow-twitch soleus muscle induced a fiber-type transition characterized by the replacement of myosin heavy chain I and IIA isoforms by the faster IIB and/or IIX isoforms, the activation of fast-twitch fiber-specific genes, and a switch toward glycolytic metabolism. Collectively, these data identify Six1 and Eya1 as the first transcriptional complex that is able to reprogram adult slow-twitch oxidative fibers toward a fast-twitch glycolytic phenotype

Six proteins are required for <i>Myod</i> expression in the mouse embryo.

<p>A- Chromatin Immunoprecipitation (ChIP) experiments performed with Eya antibodies or control IgG, on chromatin prepared from Pax3-GFP cells separated by flow cytometry from the trunk region of <i>Pax3^GFP/+</i> embryos <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003425#pgen.1003425-Relaix1" target="_blank">[32]</a> at E11.5. ChIP experiments reveal association of Eya proteins with the core enhancer (CE) and distal regulatory region (DRR) 5′ of the <i>Myod</i> gene. B- Sequence of mouse <i>Myod</i> core enhancer (CE) and DRR. MEF3 sites are in red, E boxes in blue, Pitx sites in purple and Pax3 site in green. Underlined sequences correspond to the LS4 and LS15 linker-scanner mutagenesis performed on the human core enhancer <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003425#pgen.1003425-Kucharczuk1" target="_blank">[37]</a>. C- Electromobility shift assays showing the interaction of Six1 and Six4 proteins with three distinct MEF3 DNA elements present in the regulatory regions of <i>Myod</i>. Radioactively labelled oligonucleotides with the <i>Myogenin</i> MEF3 site (Myog) were incubated with in vitro translated Six1 and Six4 proteins as a control (1). A 60 or 300 fold excess of unlabelled oligonucleotides containing the MEF3 <i>Myogenin</i> site (2,3), the MEF3 DRR site (4,5), the MEF3 CE1 site (6,7), the MEF3 CE2 site (8,9) or a 300 fold excess of unrelated <i>Myogenin</i> NFI oligonucleotides (10) were added in the reaction mix. D- Wild type and MEF3 mutant <i>Myod</i> transgenes used in the study, (not to scale). PRR, proximal regulatory region corresponding to the <i>Myod</i> promoter. E-Transient transgenic embryos with wild type or mutant <i>Myod</i> sequences at E12-E12.5. X-Gal staining of transgenic embryos with wt <i>CE-MD6.0-nLacZ</i> (a–c) or <i>mut3MEF3-CE-MD6.0-nLacZ</i> (d–f) transgenes. Six out of ten wild type transgenes expressed the <i>LacZ</i> reporter with the same expression pattern, three of them are shown. The number of transgenes inserted varied between 3 and 34 for X-Gal-positive (X-Gal+) embryos, and from 1 to 14 for X-Gal-negative (X-Gal−) embryos. Three out of eight mutant transgenic embryos expressed the <i>LacZ</i> reporter, all three are shown. The number of transgenes inserted was 23 (f), 39 (d) and 40 (e) for X-Gal+ embryos, and from 1 to 51 for X-Gal− embryos. F- Sections for one wild type (c) and for the three mutant transgenic embryos expressing the <i>LacZ</i> transgene were analysed for Myod protein by immunohistochemistry at the thoracic (Th) (c–f), and eye (c″–f″) levels to detect myogenic cells, thus revealing the % of transgene expression (X-Gal+ cells, c′–f′ and c′″–f′″) in the myogenic cell population (Myod-positive cells). While most Myod+ cells express the wt <i>Myod</i> transgene (c′, c′″), very few are marked by expression of the mutant <i>Myod</i> transgene (d′–f′, d′″–f′″).</p

Six4Δ affects <i>Myod</i> expression and myogenesis in the absence of Myf5.

<p>A–D′, Whole mount <i>in situ</i> hybridization experiments using a <i>Myod</i> probe on <i>Myf5^+/−</i> (A, A′), <i>Myf5^+/−</i> : <i>Pax3^Six4Δ/+</i> (B, B′), <i>Myf5^−/−</i> (C, C′) and <i>Myf5^−/−</i> : <i>Pax3^Six4Δ/+</i> (D, D′) embryos at E11.5. At this stage, in <i>Myf5^−/−</i> embryos (C, C′), <i>Myod</i> is activated and begins to rescue the formation of the myotome (arrowheads in C′). However, in <i>Myf5</i> deficient embryos which express <i>Six4Δ</i> under the control of <i>Pax3</i> regulatory elements, <i>Myf5^−/−</i> : <i>Pax3^Six4Δ/+</i> (D, D′), <i>Myod</i> expression is reduced, affecting the rescue of myotome formation (D′, arrowheads). In contrast, in thoracic somites of <i>Myf5^+/−</i> : <i>Pax3^Six4Δ/+</i> (B, B′) <i>Myod</i> expression is not altered compared to <i>Myf5^+/−</i> embryos (A,A′). A′–D′, show enlargements in the interlimb region of A–D. E–H′, co-immunohistochemistry on transverse sections of hypaxial somites from <i>Myf5^+/−</i> (E, E′), <i>Myf5^+/−</i> : <i>Pax3^Six4Δ/+</i> (F, F′), <i>Myf5^−/−</i> (G, G′) and <i>Myf5^−/−</i> : <i>Pax3^Six4Δ/+</i> (H, H′) embryos at E11.5 using anti-β-Galactosidase (β-Gal) (green, E–H) and anti-Myod (red, E′–H′) antibodies confirms the severe reduction of Myod expression in <i>Myf5^−/−</i> : <i>Pax3^Six4Δ/+</i> (H, H′) embryos. Arrowheads indicate examples of cells in which the β-Gal reporter from the <i>Myf5^nLacZ</i> allele is expressed and which co-express Myod.</p

Targeting of a sequence encoding dominant negative Six4 into the <i>Pax3</i> locus.

<p>A, Alignment of Six protein sequences shows conservation of the N-terminal-most regions of the Six-domain. This region is absent in the Six4Δ mRNA splicing variant. B, Bandshift assays show that Six4 and Six4Δ bind the MEF3 site, but that only Six4 can interact with Eya2 protein to form a larger complex. C, Transfection experiments performed in primary cultures of chick myoblasts show that Six4 and Eya2 synergistically activate transcription of a luciferase reporter driven by the multimerized MEF3 sequence. In contrast, Six4Δ and Eya2 display no functional synergy, and increasing amounts of Six4Δ compete for Six4-Eya2 transcriptional activation. Y axis, ratio between Luciferase and Renilla activities in arbitrary units. D-G, Strategy for targeting the Six4Δ coding sequence into an allele of <i>Pax3</i>. The probes and restriction enzymes (EcoRV: RV) are indicated, with the size of the resulting wild-type and recombined restriction fragments. The targeting construct (E) contains 2.4 kb and 4 kb of 5′ and 3′ genomic flanking sequences of the mouse <i>Pax3</i> gene. A floxed <i>puromycin-pA</i> selection marker (Puro), replaces the coding sequence in exon 1 of <i>Pax3</i> (D), followed by a di-cistronic cassette containing the murine <i>Six4Δ</i> cDNA comprising the whole coding region, followed by an <i>IRESnLacZ</i> cassette and by a final <i>pA</i> signal. The <i>IRESnLacZ</i> allows easy detection of <i>Six4Δ</i> expression <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003425#pgen.1003425-Relaix1" target="_blank">[32]</a>. A counter-selection cassette encoding the A subunit of Diptheria Toxin (DTA) was inserted at the 5′end of the vector. After homologous recombination in embryonic stem (ES) cells, <i>Six4Δ-IRESnLacZ</i> expression from the <i>Pax3^(Six4</i>^{Δ<i>-IRESnLacZ)</i>} allele is blocked by the floxed <i>puromycin-pA</i> cassette (F) and is therefore conditional to removal by crossing with a <i>PGK</i>-<i>Cre</i> mouse <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003425#pgen.1003425-Lallemand1" target="_blank">[52]</a>. This generates the <i>Pax3^{Six4Δ-IRESnLacZ}</i> allele (abbreviated <i>Pax3^Six4Δ)</i> (G).</p