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Facioscapulohumeral muscular dystrophy (FSHD) region gene 1 (FRG1) is a candidate gene for mediating FSHD pathophysiology, however, very little is known about the endogenous FRG1 protein. This study uses immunocytochemistry (ICC) and histology to provide insight into FRG1's role in vertebrate muscle development and address its potential involvement in FSHD pathophysiology. In cell culture, primary myoblast/myotube cultures, and mouse and human muscle sections, FRG1 showed distinct nuclear and cytoplasmic localizations and nuclear shuttling assays indicated the subcellular pools of FRG1 are linked. During myoblast differentiation, FRG1's subcellular distribution changed dramatically with FRG1 eventually associating with the matured Z-discs. This Z-disc localization was confirmed using isolated mouse myofibers and found to be maintained in adult human skeletal muscle biopsies. Thus, FRG1 is not likely involved in the initial assembly and alignment of the Z-disc but may be involved in sarcomere maintenance or signaling. Further analysis of human tissue showed FRG1 is strongly expressed in arteries, veins, and capillaries, the other prominently affected tissue in FSHD. Overall, we show that in mammalian cells, FRG1 is a dynamic nuclear and cytoplasmic protein, however in muscle, FRG1 is also a developmentally regulated sarcomeric protein suggesting FRG1 may perform a muscle-specific function. Thus, FRG1 is the only FSHD candidate protein linked to the muscle contractile machinery and may address why the musculature and vasculature are specifically susceptible in FSHD., (Copyright © 2010 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.)

The genetic lesion leading to facioscapulohumeral muscular dystrophy (FSHD) is a dominant deletion at the 4q35 locus. The generally accepted disease model involves an epigenetic dysregulation in the region resulting in the upregulation of one or more proximal genes whose overexpression specifically affects skeletal muscle. However, multiple FSHD candidate genes have been proposed without clear consensus. Using Xenopus laevis as a model for vertebrate development our lab has studied the effects of overexpression of the FSHD candidate gene ortholog, frg1 (FSHD region gene 1), showing that increased levels of frg1 systemically led specifically to an abnormal musculature and increased angiogenesis, the two most prominent clinical features of FSHD. Here we studied the overexpression effects of three other promising FSHD candidate genes, DUX4, DUX4c, and PITX1 using the same model system and methods for direct comparison. Expression of even very low levels of either DUX4 or pitx1 early in development led to massive cellular loss and severely abnormal development. These abnormalities were not muscle specific. In contrast, elevated levels of DUX4c resulted in no detectable adverse affects on muscle and DUX4c levels did not alter the expression of myogenic regulators. This data supports a model for DUX4 and PITX1 in FSHD only as pro-apoptotic factors if their expression in FSHD is confined to cells within the myogenic pathway; neither could account for the vascular pathology prevalent in FSHD. Taken together, increased frg1 expression alone leads to a phenotype that most closely resembles the pathophysiology observed in FSHD patients.

The leading candidate gene responsible for facioscapulohumeral muscular dystrophy (FSHD) is FRG1 (FSHD region gene 1). However, the correlation of altered FRG1 expression levels with disease pathology has remained controversial and the precise function of FRG1 is unknown. Here, we carried out a detailed analysis of the normal expression patterns and effects of FRG1 misexpression during vertebrate embryonic development using Xenopus laevis. We show that frg1 is expressed in and essential for the development of the tadpole musculature. FRG1 morpholino injection disrupted myotome organization and led to inhibited myotome growth, while elevated FRG1 led to abnormal epaxial and hypaxial muscle formation. Thus, maintenance of normal FRG1 levels is critical for proper muscle development, supportive of FSHD disease models whereby misregulation of FRG1 plays a causal role underlying the pathology exhibited in FSHD patients. Developmental Dynamics 238:1502-1512, 2009. (c) 2008 Wiley-Liss, Inc.

The genetic lesion that is diagnostic for facioscapulohumeral muscular dystrophy (FSHD) results in an epigenetic misregulation of gene expression, which ultimately leads to the disease pathology. FRG1 (FSHD region gene 1) is a leading candidate for a gene whose misexpression might lead to FSHD. Because FSHD pathology is most prominent in the musculature, most research and therapy efforts focus on muscle cells. Previously, using Xenopus development as a model, we showed that altering frg1 expression levels systemically leads to aberrant muscle development, illustrating the potential for aberrant FRG1 levels to disrupt the musculature. However, 50-75% of FSHD patients also exhibit retinal vasculopathy and FSHD muscles have increased levels of vascular- and endothelial-related FRG1 transcripts, illustrating an underlying vascular component to the disease. To date, no FSHD candidate gene has been proposed to affect the vasculature. Here, we focus on a role for FRG1 expression in the vasculature. We found that endogenous frg1 is expressed in both the developing and adult vasculature in Xenopus. Furthermore, expression of FRG1 was found to be essential for the development of the vasculature, as a knockdown of FRG1 resulted in decreased angiogenesis and reduced expression of the angiogenic regulator DAB2. Conversely, tadpoles subjected to frg1 overexpression displayed the pro-angiogenic phenotypes of increased blood vessel branching and dilation of blood vessels, and developed edemas, suggesting that their circulation was disrupted. Thus, the systemic upregulation of the FRG1 protein shows the potential for acquiring a disrupted vascular phenotype, providing the first link between a FSHD candidate gene and the vascular component of FSHD pathology. Overall, in conjunction with our previous analysis, we show that FRG1 overexpression is capable of disrupting both the musculature and vasculature, recapitulating the two most prominent features of FSHD.

Background: In Xenopus the bone morphogenetic protein growth and differentiation factor 6 (GDF6) is expressed at the edge of the neural plate, and within the anterior neural plate including the eye fields. Here we address the role of GDF6 in neural and eye development by morpholino knockdown experiments., Results: We show that depletion of GDF6 (BMP13) resulted in a reduction in eye size, loss of laminar structure and a reduction in differentiated neural cell types within the retina. This correlated with a reduction in staining for Smad1/5/8 phosphorylation indicating a decrease in GDF6 signalling through loss of phosphorylation of these intracellular mediators of bone morphogenetic protein (BMP) signalling. In addition, the Pax6 expression domain is reduced in size at early optic vesicle stages. Neural cell adhesion molecule (NCAM) is generally reduced in intensity along the neural tube, while in the retina and brain discreet patches of NCAM expression are also lost. GDF6 knock down resulted in an increase in cell death along the neural tube and within the retina as determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining., Conclusion: Our data demonstrate that GDF6 has an important role in neural differentiation in the eye as well as within the central nervous system, and that GDF6 may act in some way to maintain cell survival within the ectoderm, during the normal waves of programmed cell death.

Allele-specific transcription is a characteristic feature of imprinted genes. Many imprinted genes are also transcribed in a tissue- or cell type-specific manner. Overlapping epigenetic signals must, therefore, modulate allele-specific and tissue-specific expression at imprinted loci. In addition, long-range interactions with an Imprinting Center (IC) may influence transcription, in an allele-specific or cell-type specific manner. The IC on human chromosome 15q11 controls parent-of-origin specific allelic identity of a set of genes located in cis configuration within 2 Mb. We have now examined the chromatin accessibility of the promoter region of one of the Imprinting Centre-controlled genes, NDN encoding necdin, using in vivo DNA footprinting to identify sites of DNA-protein interaction and altered chromatin configuration. We identified sites of modified chromatin that mark the parental alleles in NDN-expressing cells, and in cells in which NDN is not expressed. Our results suggest that long-lasting allele-specific marks and more labile tissue-specific marks layer epigenetic information that can be discriminated using DNA footprinting methodologies. Sites of modified chromatin mark the parental alleles in NDN-expressing cells, and in cells in which NDN is not expressed. Our results suggest that a layering of epigenetic information controls allele- and tissue-specific gene expression of this imprinted gene., ((c) 2005 Wiley-Liss, Inc.)

Allele-specific DNA methylation, histone acetylation and histone methylation are recognized as epigenetic characteristics of imprinted genes and imprinting centers (ICs). These epigenetic modifications are also used to regulate tissue-specific gene expression. Epigenetic differences between alleles can be significant either in the function of the IC or in the cis-acting effect of the IC on 'target' genes responding to it. We have now examined the epigenetic characteristics of NDN, a target gene of the chromosome 15q11-q13 Prader-Willi Syndrome IC, using sodium bisulfite sequencing to analyze DNA methylation and chromatin immunoprecipitation to analyze histone modifications. We observed a bias towards maternal allele-specific DNA hypermethylation of the promoter CpG island of NDN, independent of tissue-specific transcriptional activity. We also found that NDN lies in a domain of paternal allele-specific histone hyperacetylation that correlates with transcriptional state, and a domain of differential histone H3 lysine 4 di- and tri-methylation that persists independent of transcription. These results suggest that DNA methylation and histone H3 lysine 4 methylation are persistent markers of imprinted gene regulation while histone acetylation participates in tissue-specific activity and silencing in somatic cells.

Ndn is located on chromosome 7C, an imprinted region of the mouse genome. Imprinting of Ndn and adjacent paternally expressed genes is regulated by a regional imprinting control element known as the imprinting center (IC). An IC also controls imprint resetting of target genes in the region of conserved synteny on human chromosome 15q11-q13, which is deleted or rearranged in the neurodevelopmental disorder Prader-Willi syndrome. Epigenetic modifications such as DNA methylation, which occur in gametes and can be stably propagated, are presumed to establish and maintain the imprint in target genes of the IC. While most DNA becomes substantially demethylated by the blastocyst stage, some imprinted genes have regions that escape global demethylation and may maintain the imprint. We have now analyzed the methylation of 39 CpG dinucleotide sequences in the 5' end of Ndn by sodium bisulfite sequencing in gametes and in preimplantation and adult tissues. While sperm DNA is completely unmethylated across this region, oocyte DNA is partially methylated. A distinctive but unstable maternal methylation pattern persists until the morula stage and is lost in the blastocyst stage, where low levels of methylation are present on most DNA strands of either parental origin. The methylation pattern is then substantially remodeled, and fewer than half of maternally derived DNA strands in adult brain resemble the oocyte pattern. We postulate that for Ndn, DNA methylation may initially preserve a gametic imprint during preimplantation development, but other epigenetic events may maintain the imprint later in embryonic development.