Riassunto analitico
Facioscapulohumeral muscular dystrophy (FSHD) is the third most common hereditary muscle disorder. A reduced number of tandemly arrayed repetitive DNA elements, named D4Z4, located at the chromosome 4q35 region, have been found in the majority of FSHD patients, possibly interfering with the expression of nearby genes. Consistently we proved that the transcriptional activation of FSHD region gene 1 (FRG1), a gene located upstream of the D4Z4 array at 4q35, causes muscular dystrophy in mice However, the correlation between FRG1 misregulation and FSHD is controversial and its precise function is still unknown. Thus, for the first time in mammals, we studied the endogenous spatio-temporal expression of Frg1 in wild-type mice as well the effects of its ablation during embryonic development using a conditional Cre/loxP system. Our study revealed that Frg1 is expressed throughout the entire embryogenesis at different levels with a peak at 12.5 d.p.c. The immunohistological analysis of Frg1 expression at 12.5 d.p.c showed that Frg1 is highly expressed in early precursors of neural, muscle and cartilage cells and in the developing liver. Thus Frg1 expression is not restricted to muscle cells precursors, but is present in subset of cells originating from the three embryonic layers. Thus Frg1 might exert its function in very early stages of development prior to gastrulation and the reorganization of single-layered blastula into a trilaminar ("three-layered") structure. To further investigate the role of Frg1 during embryogenesis, we induced Frg1 ablation in Frg1 cond/cond mice at different time points. Interestingly, lack of Frg1 at E7.5 and E8.5 leads to smaller embryos in comparison with wild-type, whereas at later stages no phenotypic differences were evident between Frg1-/- and wild-type embryos, suggesting that Frg1 function is critical in early embryonic stages. Interestingly, we determined that the reduced size of Frg1-/- embryos is not due to a developmental delay, because both wild-type and knock-out littermates present the same staging criteria. Moreover, the detailed analysis of Frg1-/- embryos at E11.5, after Frg1 ablation at E7.5, failed to reveal morphological abnormalities of specific organs. Instead we detected that the number of cells was reduced in almost all tissues analyzed. Notably, this reduction was statistically significant only in the limb buds, structures of early limb development. Consequently we investigated whether Frg1 might be involved in limb morphogenesis and/or muscular differentiation. To this aim we analyzed the expression of myogenic factors throughout development. Firstly, we found that in early stages, up to E12.5, there is a pronounced downregulation of Pax3, known as upstream regulator of the primary myogenesis, and other myogenic regulatory factors (MyoD and myogenin), necessary for muscular differentiation progress, indicating that Frg1 act on the Myf5-independent lineage as an upstream regulator of Pax3 which in turn affects MyoD and myogenin. Analysis of limb muscle precursor cells at E11.5, a day after their entry into the limb bud, showed Pax3 positive cells both in the ventral and dorsal muscle masses. This observation excludes a defect in migration into the limb and suggests a failure in proliferation. In support of this hypothesis we found upregulation of Meox2 which influences G1/S check-point determining a cell cycle arrest. Secondly, starting from E11.5 we found a remarkable up regulation of Pax7, a marker of satellite cells, responsible for secondary myogenesis. This result together with the reduction of Pax3, MyoD and myogenin expression suggest that Frg1 absence results in the inhibition of differentiation and maintenance of a quiescent/self renewing phenotype. Collectively our studies demonstrate that Frg1 plays a key role in muscle development and differentiation throughout embryogenesis.
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