Biomedical subjects

M J McGrew

Publications and source records attributed to M J McGrew.

10 recordsLinked to original sources

PubMed2001

FGF signaling controls somite boundary position and regulates segmentation clock control of spatiotemporal Hox gene activation.

Vertebrate segmentation requires a molecular oscillator, the segmentation clock, acting in presomitic mesoderm (PSM) cells to set the pace at which segmental boundaries are laid down. However, the signals that position each boundary remain unclear. Here, we report that FGF8 which is expressed in the posterior PSM, generates a moving wavefront at which level both segment boundary position and axial identity become determined. Furthermore, by manipulating boundary position in the chick embryo, we show that Hox gene expression is maintained in the appropriately numbered somite rather than at an absolute axial position. These results implicate FGF8 in ensuring tight coordination of the segmentation process and spatiotemporal Hox gene activation.

Animals↗

PubMed2000

Oscillating expression of c-Hey2 in the presomitic mesoderm suggests that the segmentation clock may use combinatorial signaling through multiple interacting bHLH factors.

Vertebrate somitogenesis comprises the generation of a temporal periodicity, the establishment of anteroposterior compartment identity, and the translation of the temporal periodicity into the metameric pattern of somites. Molecular players at each of these steps are beginning to be identified. Especially, members of the Notch signaling cascade appear to be involved in setting up the somitogenesis clock and subsequent events. We had previously demonstrated specific expression of the mHey1 and mHey2 basic helix-loop-helix (bHLH) factors during somitogenesis. Here we show that perturbed Notch signaling in Dll1 and Notch1 knockout mutants affects this expression in the presomitic mesoderm (PSM) and the somites. In the caudal PSM, however, mHey2 expression is maintained and thus is likely to be independent of Notch signaling. Furthermore, we analysed the dynamic expression of the respective chicken c-Hey1 and c-Hey2 genes during somitogenesis. Not only is c-Hey2 rhythmically expressed across the chicken presomitic mesoderm like c-hairy1, but its transcription is similarly independent of de novo protein synthesis. In contrast, the dynamic expression of c-Hey1 is restricted to the anterior segmental plate. Both c-Hey genes are coexpressed with c-hairy1 in the posterior somite half. Further in vitro and in vivo interaction assays demonstrated direct homo- and heterodimerisation between these hairy-related bHLH proteins, suggesting a combinatorial action in both the generation of a temporal periodicity and the anterior-posterior somite compartmentalisation.

Amino Acid Sequence↗

PubMed1999

An E box comprises a positional sensor for regional differences in skeletal muscle gene expression and methylation.

To dissect the molecular mechanisms conferring positional information in skeletal muscles, we characterized the control elements responsible for the positionally restricted expression patterns of a muscle-specific transgene reporter, driven by regulatory sequences from the MLC1/3 locus. These sequences have previously been shown to generate graded transgene expression in the segmented axial muscles and their myotomal precursors, fortuitously marking their positional address. An evolutionarily conserved E box in the MLC enhancer core, not recognized by MyoD, is a target for a nuclear protein complex, present in a variety of tissues, which includes Hox proteins and Zbu1, a DNA-binding member of the SW12/SNF2 gene family. Mutation of this E box in the MLC enhancer has only a modest positive effect on linked CAT gene expression in transfected muscle cells, but when introduced into transgenic mice the same mutation elevates CAT transgene expression in skeletal muscles, specifically releasing the rostral restriction on MLC-CAT transgene expression in the segmented axial musculature. Increased transgene activity resulting from the E box mutation in the MLC enhancer correlates with reduced DNA methylation of the distal transgenic MLC1 promoter as well as in the enhancer itself. These results identify an E box and the proteins that bind to it as a positional sensor responsible for regional differences in axial skeletal muscle gene expression and accessibility.

Animals↗

PubMed1998

The lunatic fringe gene is a target of the molecular clock linked to somite segmentation in avian embryos.

The most obvious segments of the vertebrate embryo are the trunk mesodermal somites which give rise to the segmented vertebral column and the skeletal muscles of the body. Mechanistic insights into vertebrate somitogenesis have recently been gained from observations of rhythmic expression of the avian hairy-related gene (c-hairy1) in chick presomitic mesoderm (PSM), suggesting the existence of a molecular clock linked to somite segmentation ([1]; reviewed in [2]). Here, we show that lunatic Fringe (IFng), a vertebrate homolog of the Drosophila Fringe gene, is also expressed rhythmically in PSM. The PSM expression of IFng was observed as coordinated pulses of mRNA resembling the expression of c-hairy1. We show that c-hairy1 and IFng expression in the PSM are coincident, indicating that both genes are responding to the same segmentation clock. The genes were found to differ in their regulation, however; in contrast to c-hairy1, IFng mRNA oscillations required continued protein synthesis, suggesting that IFng could be acting downstream of c-hairy1 in the clock mechanism. In Drosophila, Fringe has been shown to play a role in modulating Notch-Delta signalling [3,4], a pathway which in vertebrates has been implicated in defining somite boundaries [5-9]. These observations place the segmentation clock upstream of the Notch-Delta pathway during vertebrate somitogenesis.

Animals↗

PubMed1998

Somitogenesis: segmenting a vertebrate.

The partitioning of the vertebrate body into a repetitive series of segments, or somites, requires the spatially and temporally co-ordinated behaviour of mesodermal cells. To date, it remains unknown how applicable our knowledge of the genetic mechanisms governing Drosophila segmentation will be to that of vertebrates, though recent results indicate some degree of conservation. Genetic studies in the mouse point to a major role for the Notch-Delta signalling pathway in somite formation. Furthermore, a molecular clock may be 'ticking' in the presomitic mesoderm.

Animals↗

PubMed1996

Modular elements of the MLC 1f/3f locus confer fiber-specific transcription regulation in transgenic mice.

The two proteins encoded by the fast alkali myosin light chain (MLC) 1f/3f locus are developmentally regulated, muscle specific, and expressed exclusively in fast-twitch fibers. Their expression is independently regulated by two separate promoters and a downstream enhancer. Previous studies showed a reporter gene directed by the rat MLC If promoter and MLC enhancer to exhibit correct skeletal muscle-specific expression in transgenic mice during development and to be preferentially expressed in fast-twitch Type IIB fibers [Donoghue et al., (1991) J. Cell B.ol. 115:423-434]. The MLC 3f promoter also directed muscle-specific expression of a CAT reporter gene in adult transgenic mice and showed little dependence upon the enhancer. Here, we show that the MLC 3f promoter also directs transgene expression in the fast-twitch fibers of adult skeletal muscle, but almost exclusively to fiber Types IIA and IIX. MLC 3f transgene expression occurs in only a subset of the fiber types that express the endogenous locus, indicating modular elements included in the transgene confer fiber-specific transcription regulation. MyoD protein was also found to be restricted to fiber Types IIA and IIX, providing evidence for its possible role in mediating fiber-specific gene expression.

Animals↗

PubMed1996

Distinct gene expression patterns in skeletal and cardiac muscle are dependent on common regulatory sequences in the MLC1/3 locus.

The myosin light-chain 1/3 locus (MLC1/3) is regulated by two promoters and a downstream enhancer element which produce two protein isoforms in fast skeletal muscle at distinct stages of mouse embryogenesis. We have analyzed the expression of transcripts from the internal MLC3 promoter and determined that it is also expressed in the atria of the heart. Expression from the MLC3 promoter in these striated muscle lineages is differentially regulated during development. In transgenic mice, the MLC3 promoter is responsible for cardiac-specific reporter gene expression while the downstream enhancer augments expression in skeletal muscle. Examination of the methylation status of endogenous and transgenic promoter and enhancer elements indicates that the internal promoter is not regulated in a manner similar to that of the MLC1 promoter or the downstream enhancer. A GATA protein consensus sequence in the proximal MLC3 promoter but not the MLC1 promoter binds with high affinity to GATA-4, a cardiac muscle- and gut-specific transcription factor. Mutation of either the MEF2 or GATA motifs in the MLC3 promoter attenuates its activity in both heart and skeletal muscles, demonstrating that MLC3 expression in these two diverse muscle types is dependent on common regulatory elements.

Animals↗

PubMed1995

Role of methylation in maintenance of positionally restricted transgene expression in developing muscle.

In transgenic mouse embryos, expression of a muscle-specific reporter, consisting of a chloramphenicol acetyltransferase gene linked to regulatory sequences from the rat myosin light chain 1/3 locus (MLC-CAT), is graded in developing axial muscles along the rostrocaudal axis and in cell cultures derived from these muscles. Here we demonstrate that maintenance of positional differences in MLC-CAT transgene expression cannot be attributed to differences in the transcriptional competence of corresponding muscles. Rather, patterns of transgene expression are reflected in the extent of CpG demethylation of both MLC1 promoter and MLC enhancer sequences. Variations in reporter gene expression can be reconstituted by in vitro methylation of specific CpGs in transfected MLC-CAT DNA. As the MLC-CAT transgene is activated during embryogenesis, demethylation of the MLC1 promoter lags behind that of the downstream MLC enhancer, which appears to be the initial target for epigenetic modification. In developing somites, demethylation of the transgenic MLC enhancer is not graded and therefore does not reflect early regional differences in MLC-CAT transgene expression patterns. These studies implicate selective methylation in the maintenance rather than in the establishment of transcriptional differences in developing muscles.

Animals↗

PubMed1993

Quantitation of genomic methylation using ligation-mediated PCR.

We have developed a new technique for the quantitation of CpG methylation of genomic DNA. This method measures the conversion of a larger amplified DNA fragment to a shorter DNA product correlating with demethylation. The procedure uses pairs of non-isoschizomeric enzymes, one of which is methylation-sensitive, to cleave genomic DNA at closely spaced sites. The extent of cleavage by the methylation-sensitive restriction enzyme is quantitated by amplification of these digestion products with ligation-mediated PCR and radioactive labeling of the product. The ratio of the two amplified fragments correlates with the degree of methylation at the restriction site. The analysis is rapid, quantitative, internally controlled and requires small quantities of genomic DNA.

Animals↗

PubMed1987

A profile of male and female applicants for a special college program for learning-disabled students.

Intellectual, achievement, and personality profiles were developed for a sample of 245 applicants to a special college program for learning-disabled students. Only 15% of the applicants had WAIS IQs above 115; the most depressed subtests were Arithmetic, Digit Span, Information, Vocabulary, and Digit Symbol. For both sexes, mean WRAT reading levels were higher than spelling and arithmetic (roughly seventh- vs. sixth-grade competence). Although the male applicants (N = 174) had significantly higher WAIS Verbal IQs than the female applicants (N = 71), their WRAT spelling achievement levels were significantly lower. A higher percentage of the females than males exhibited specific arithmetic disability. For both sexes, but more robustly for females, specific arithmetic disability was associated with more elevated MMPI profiles. Dyslexic students, by contrast, admitted to fewer problem areas on the MMPI. Relatively few applicants had MMPI scaled scores greater than or equal to 70.

Achievement↗