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M Buckingham

Publications and source records attributed to M Buckingham.

At least 19 recordsLinked to original sources
PubMed2001

MLC3F transgene expression in iv mutant mice reveals the importance of left-right signalling pathways for the acquisition of left and right atrial but not ventricular compartment identity.

Abstract Transcriptional differences between left and right cardiac chambers are revealed by an nlacZ reporter transgene controlled by regulatory sequences of the MLC3F gene, which is expressed in the left ventricle (LV), atrioventricular canal (AVC), and right atrium (RA). To examine the role of left-right signalling in the acquisition of left and right chamber identity, we have investigated MLC3F transgene expression in iv mutant mice. iv/iv mice exhibit randomised direction of heart looping and an elevated frequency of associated laterality defects, including atrial isomerism. At fetal stages, 3F-nlacZ-2E transgene expression remains confined to the morphological LV, AVC, and RA in L-loop hearts, although these appear on the opposite side of the body. In cases of morphologically distinguishable right atrial appendage isomerism, both atrial appendages show strong transgene expression. Conversely, specimens with morphological left atrial appendage isomerism show only weak expression in both atrial appendages. The earliest left-right atrial differences in the expression of the 3F-nlacZ-2E transgene are observed at E8.5. DiI labelling experiments confirmed that transcriptional regionalisation of the 3F-nlacZ-2E transgene at this stage reflects future atrial chamber identity. In some iv/iv embryos at E8.5, the asymmetry of 3F-nlacZ-2E expression was lost, suggesting atrial isomerism at the transcriptional level prior to chamber formation. These data suggest that molecular specification of left and right atrial but not ventricular chambers is dependent on left-right axial cues.

Animals↗
PubMed2001

Skeletal muscle formation in vertebrates.

Research in the past year has added to our understanding of the signalling systems that specify myogenic identity in the embryo and of the regulation and roles of MyoD family members. New insights into the movement of muscle precursor cells include the demonstration that Lbx1 is essential for their migration from the somite to some but not all sites of muscle formation elsewhere. Later in development, ras as well as calcineurin signalling is now implicated in the definition of slow versus fast fibre types. The myogenic identity of precursor cells in the adult depends on Pax7, the orthologue of Pax3 which is required for early myogenesis; this finding is of major importance for muscle regeneration and the active field of stem cell research.

Animals↗
PubMed2000

Multiple transcriptional domains, with distinct left and right components, in the atrial chambers of the developing heart.

During heart development, 2 fast-conducting regions of working myocardium balloon out from the slow-conducting primary myocardium of the tubular heart. Three regions of primary myocardium persist: the outflow tract, atrioventricular canal, and inflow tract, which are contiguous throughout the inner curvature of the heart. The contribution of the inflow tract to the definitive atrial chambers has remained enigmatic largely because of the lack of molecular markers that permit unambiguous identification of this myocardial domain. We now report that the genes encoding atrial natriuretic factor, myosin light chain (MLC) 3F, MLC2V, and Pitx-2, and transgenic mouse lines expressing nlacZ under the control of regulatory sequences of the mouse MLC1F/3F gene, display regionalized patterns of expression in the atrial component of the developing mouse heart. These data distinguish 4 broad transcriptional domains in the atrial myocardium: (1) the atrioventricular canal that will form the smooth-walled lower atrial rim proximal to the ventricles; (2) the atrial appendages; (3) the caval vein myocardium (systemic inlet); and (4) the mediastinal myocardium (pulmonary inlet), including the atrial septa. The pattern of expression of Pitx-2 reveals that each of these transcriptional domains has a distinct left and right component. This study reveals for the first time differential gene expression in the systemic and pulmonary inlets, which is not shared by the contiguous atrial appendages and provides evidence for multiple molecular compartments within the atrial chambers. Furthermore, this work will allow the contribution of each of these myocardial components to be studied in congenitally malformed hearts, such as those with abnormal venous return.

Aging↗
PubMed2000

Myf5 is a novel early axonal marker in the mouse brain and is subjected to post-transcriptional regulation in neurons.

Myf5 is a key basic Helix-Loop-Helix transcription factor capable of converting many non-muscle cells into muscle. Together with MyoD it is essential for initiating the skeletal muscle programme in the embryo. We previously identified unexpected restricted domains of Myf5 transcription in the embryonic mouse brain, first revealed by Myf5-nlacZ(+/)(-) embryos (Tajbakhsh, S. and Buckingham, M. (1995) Development 121, 4077-4083). We have now further characterized these Myf5 expressing neurons. Retrograde labeling with diI, and the use of a transgenic mouse line expressing lacZ under the control of Myf5 regulatory sequences, show that Myf5 transcription provides a novel axonal marker of the medial longitudinal fasciculus (mlf) and the mammillotegmental tract (mtt), the earliest longitudinal tracts to be established in the embryonic mouse brain. Tracts projecting caudally from the developing olfactory system are also labelled. nlacZ and lacZ expression persist in the adult brain, in a few ventral domains such as the mammillary bodies of the hypothalamus and the interpeduncular nucleus, potentially derived from the embryonic structures where the Myf5 gene is transcribed. To investigate the role of Myf5 in the brain, we monitored Myf5 protein accumulation by immunofluorescence and immunoblotting in neurons transcribing the gene. Although Myf5 was detected in muscle myotomal cells, it was absent in neurons. This would account for the lack of myogenic conversion in brain structures and the absence of a neural phenotype in homozygous null mutants. RT-PCR experiments show that the splicing of Myf5 primary transcripts occurs correctly in neurons, suggesting that the lack of Myf5 protein accumulation is due to regulation at the level of mRNA translation or protein stability. In the embryonic neuroepithelium, Myf5 is transcribed in differentiated neurons after the expression of neural basic Helix-Loop-Helix transcription factors. The signalling molecules Wnt1 and Sonic hedgehog, implicated in the activation of Myf5 in myogenic progenitor cells in the somite, are also produced in the viscinity of the Myf5 expression domain in the mesencephalon. We show that cells expressing Wnt1 can activate neuronal Myf5-nlacZ gene expression in dissected head explants isolated from E9.5 embryos. Furthermore, the gene encoding the basic Helix-Loop-Helix transcription factor mSim1 is expressed in adjacent cells in both the somite and the brain, suggesting that signalling molecules necessary for the activation of mSim1 as well as Myf5 are present at these different sites in the embryo. This phenomenon may be widespread and it remains to be seen how many other potentially potent regulatory genes, in addition to Myf5, when activated do not accumulate protein at inappropriate sites in the embryo.

Animals↗
PubMed2000

Modular long-range regulation of Myf5 reveals unexpected heterogeneity between skeletal muscles in the mouse embryo.

The myogenic factor Myf5 plays a key role in muscle cell determination, in response to signalling cascades that lead to the specification of muscle progenitor cells. We have adopted a YAC transgenic approach to identify regulatory sequences that direct the complex spatiotemporal expression of this gene during myogenesis in the mouse embryo. Important regulatory regions with distinct properties are distributed over 96 kb upstream of the Myf5 gene. The proximal 23 kb region directs early expression in the branchial arches, epaxial dermomyotome and in a central part of the myotome, the epaxial intercalated domain. Robust expression at most sites in the embryo where skeletal muscle forms depends on an enhancer-like sequence located between -58 and -48 kb from the Myf5 gene. This element is active in the epaxial and hypaxial myotome, in limb muscles, in the hypoglossal chord and also at the sites of Myf5 transcription in prosomeres p1 and p4 of the brain. However later expression of Myf5 depends on a more distal region between -96 and -63 kb, which does not behave as an enhancer. This element is necessary for expression in head muscles but strikingly only plays a role in a subset of trunk muscles, notably the hypaxially derived ventral body muscles and also those of the diaphragm and tongue. Transgene expression in limb muscle masses is not affected by removal of the -96/-63 region. Epaxially derived muscles and some hypaxial muscles, such as the intercostals and those of the limb girdles, are also unaffected. This region therefore reveals unexpected heterogeneity between muscle masses, which may be related to different facets of myogenesis at these sites. Such regulatory heterogeneity may underlie the observed restriction of myopathies to particular muscle subgroups.

Animals↗
PubMed1999

Sonic hedgehog controls epaxial muscle determination through Myf5 activation.

Sonic hedgehog (Shh), produced by the notochord and floor plate, is proposed to function as an inductive and trophic signal that controls somite and neural tube patterning and differentiation. To investigate Shh functions during somite myogenesis in the mouse embryo, we have analyzed the expression of the myogenic determination genes, Myf5 and MyoD, and other regulatory genes in somites of Shh null embryos and in explants of presomitic mesoderm from wild-type and Myf5 null embryos. Our findings establish that Shh has an essential inductive function in the early activation of the myogenic determination genes, Myf5 and MyoD, in the epaxial somite cells that give rise to the progenitors of the deep back muscles. Shh is not required for the activation of Myf5 and MyoD at any of the other sites of myogenesis in the mouse embryo, including the hypaxial dermomyotomal cells that give rise to the abdominal and body wall muscles, or the myogenic progenitor cells that form the limb and head muscles. Shh also functions in somites to establish and maintain the medio-lateral boundaries of epaxial and hypaxial gene expression. Myf5, and not MyoD, is the target of Shh signaling in the epaxial dermomyotome, as MyoD activation by recombinant Shh protein in presomitic mesoderm explants is defective in Myf5 null embryos. In further support of the inductive function of Shh in epaxial myogenesis, we show that Shh is not essential for the survival or the proliferation of epaxial myogenic progenitors. However, Shh is required specifically for the survival of sclerotomal cells in the ventral somite as well as for the survival of ventral and dorsal neural tube cells. We conclude, therefore, that Shh has multiple functions in the somite, including inductive functions in the activation of Myf5, leading to the determination of epaxial dermomyotomal cells to myogenesis, as well as trophic functions in the maintenance of cell survival in the sclerotome and adjacent neural tube.

Animals↗
PubMed1999

Transplacental delivery of the Wnt antagonist Frzb1 inhibits development of caudal paraxial mesoderm and skeletal myogenesis in mouse embryos.

Axial structures (neural tube/notochord) and surface ectoderm activate myogenesis in the mouse embryo; their action can be reproduced, at least in part, by several molecules such as Sonic hedgehog and Wnts. Recently, soluble Wnt antagonists have been identified. Among those examined only Frzb1 was found to be expressed in the presomitic mesoderm and newly formed somites and thus its possible role in regulating myogenesis was investigated in detail. When presomitic mesoderm or newly formed somites were cultured with axial structures and surface ectoderm on a feeder layer of C3H10T1/2 cells expressing Frzb1, myogenesis was abolished or severely reduced in presomitic mesoderm and the three most recently formed somites. In contrast, no effect was observed on more mature somites. Inhibition of myogenesis did not appear to be associated with increased cell death since the final number of cells in the explants grown in the presence of Frzb1 was only slightly reduced in comparison with controls. In order to examine the possible function of Frzb1 in vivo, we developed a method based on the overexpression of the soluble antagonist by transient transfection of WOP cells with a Frzb1 expression vector and injection of transfected cells into the placenta of pregnant females before the onset of maternofoetal circulation. Frzb1, secreted by WOP cells, accumulated in the embryo and caused a marked reduction in size of caudal structures. Myogenesis was strongly reduced and, in the most severe cases, abolished. This was not due to a generalized toxic effect since only several genes downstream of the Wnt signaling pathway such as En1, Noggin and Myf5 were downregulated; in contrast, Pax3 and Mox1 expression levels were not affected even in embryos exhibiting the most severe phenotypes. Taken together, these results suggest that Wnt signals may act by regulating both myogenic commitment and expansion of committed cells in the mouse mesoderm.

Animals↗
PubMed1999

In vivo satellite cell activation via Myf5 and MyoD in regenerating mouse skeletal muscle.

Regeneration of adult skeletal muscle is an asynchronous process requiring the activation, proliferation and fusion of satellite cells, to form new muscle fibres. This study was designed to determine the pattern of expression in vivo of the two myogenic regulatory factors, Myf5 and MyoD during this process. Cardiotoxin was used to induce regeneration in the gastrocnemius and soleus muscles of heterozygous Myf5-nlacZ mice, and the muscles were assayed for the presence of (beta)-galactosidase (Myf5) and MyoD. Adult satellite cells identified by M-cadherin labelling, when activated, initially express either MyoD or Myf5 or both myogenic factors. Subsequently all proliferating myoblasts express MyoD and part of the population is (beta)-galactosidase (Myf5) positive. Furthermore, we demonstrate that activated satellite cells, which express either Myf5 or MyoD, do not accumulate selectively on fast or slow muscle fibres.

Animals↗
PubMed1998

A novel GFPneo vector designed for the isolation and analysis of enhancer elements in transfected mammalian cells.

We have designed a new approach to the direct cloning and rapid analysis of mammalian enhancer elements by fusing green fluorescent protein and neomycinphosphotransferase under the control of a thymidine kinase minimal promoter. DNA fragments containing known or potential enhancer elements can be inserted into a polylinker upstream of GFPneo and re-isolated from stably transfected cell lines by a direct transgene-specific polymerase chain reaction (PCR), for further analysis. C2C12 muscle cells were transfected with four vectors containing the GFPneo fusion gene regulated by the cytomegalovirus promoter, the myoD distal core enhancer and myoblast- and myotube-specific enhancers from the desmin gene. GFPneo shows robust epifluorescence by microscopy and flow cytometry and retains sufficient neo activity to permit selection of G418-resistant clones. The fluorescence signal pattern of GFPneo expressed under the control of the desmin enhancers mirrors their transcriptional profile during myogenic differentiation. This finding demonstrates the value of GFPneo as a tool to analyse differentiation stage-specific regulatory DNA elements in stably transfected mammalian cell lines. We were able to re-isolate the myoD enhancer mediating GFPneo expression from a stably transfected C2C12 clone by a transgene-specific PCR reaction, demonstrating the feasibility of using this new vector system for the isolation of regulatory sequences.

Animals↗
PubMed1998

DAN is a secreted glycoprotein related to Xenopus cerberus.

We report that DAN, a potential cell cycle regulator and tumour suppressor, is a secreted glycoprotein related to Xenopus cerberus. DAN, cerberus, its mouse relative Cer-1/cer-l/Cerberus-like/Cerr1, and the recently described factor DRM/Gremlin, appear to be members of the cystine knot superfamily, which includes TGFbetas and BMPs. Like cerberus and mCer-1, DAN-induced cement glands as well as markers of anterior neural tissue and endoderm in Xenopus animal cap assays, features of BMP signalling blockade. During mouse embryogenesis, Dan was expressed from E8.5 in cranial mesenchyme and somites, then later in limb and facial mesenchyme. The pattern in somites was highly dynamic, with transcripts initially localized to the caudal half of the nascent epithelial somite, then, after maturation, to sclerotomal cells adjacent to the neural tube. Dan was also expressed in the developing myotome. The expression domains include sites in which BMP inhibition is known to be important for development. Thus, DAN appears to be a secreted factor belonging to the cystine knot superfamily, and one of a growing number of antagonists acting to modulate BMP signalling during development.

Amino Acid Sequence↗
PubMed1998

Differential activation of Myf5 and MyoD by different Wnts in explants of mouse paraxial mesoderm and the later activation of myogenesis in the absence of Myf5.

Activation of myogenesis in newly formed somites is dependent upon signals derived from neighboring tissues, namely axial structures (neural tube and notochord) and dorsal ectoderm. In explants of paraxial mesoderm from mouse embryos, axial structures preferentially activate myogenesis through a Myf5-dependent pathway and dorsal ectoderm preferentially through a MyoD-dependent pathway. Here we report that cells expressing Wnt1 will preferentially activate Myf5 while cells expressing Wnt7a will preferentially activate MyoD. Wnt1 is expressed in the dorsal neural tube and Wnt7a in dorsal ectoderm in the early embryo, therefore both can potentially act in vivo to activate Myf5 and MyoD, respectively. Wnt4, Wnt5a and Wnt6 exert an intermediate effect activating both Myf5 and MyoD equivalently in paraxial mesoderm. Sonic Hedgehog synergises with both Wnt1 and Wnt7a in explants from E8.5 paraxial mesoderm but not in explants from E9.5 embryos. Signaling through different myogenic pathways may explain the rescue of muscle formation in Myf5 null embryos, which do not form an early myotome but later develop both epaxial and hypaxial musculature. Explants of unsegmented paraxial mesoderm contain myogenic precursors capable of expressing MyoD in response to signaling from a neural tube isolated from E10.5 embryos, the developmental stage when MyoD is present throughout the embryo. Myogenic cells cannot activate MyoD in response to signaling from a less mature neural tube. Together these data suggest that different Wnt molecules can activate myogenesis through different pathways such that commitment of myogenic precursors is precisely regulated in space and time to achieve the correct pattern of skeletal muscle development.

Animals↗
PubMed1997

Regionalized transcriptional domains of myosin light chain 3f transgenes in the embryonic mouse heart: morphogenetic implications.

Within the embryonic heart, five segments can be distinguished: two fast-conducting atrial and ventricular compartments flanked by slow-conducting segments, the inflow tract, the atrioventricular canal, and the outflow tract. These compartments assume morphological identity as a result of looping of the linear heart tube. Subsequently, the formation of interatrial, interventricular, and outflow tract septa generates a four-chambered heart. The lack of markers that distinguish right and left compartments within the heart has prevented a precise understanding of these processes. Transgenic mice carrying an nlacZ reporter gene under transcriptional control of regulatory sequences from the MLC1F/3F gene provide specific markers to investigate such regionalization. Our results show that transgene expression is restricted to distinct regions of the myocardium: beta-galactosidase activity in 3F-nlacZ-2E mice is confined predominantly to the embryonic right atrium, atrioventricular canal, and left ventricle, whereas, in 3F-nlacZ-9 mice, the transgene is expressed in both atrial and ventricular segments (right/left) and in the atrioventricular canal, but not in the inflow and outflow tracts. These lines of mice illustrate that distinct embryonic cardiac regions have different transcriptional specificities and provide early markers of myocardial subdivisions. Regional differences in transgene expression are not detected in the linear heart tube but become apparent as the heart begins to loop. Subsequent regionalization of transgene expression provides new insights into later morphogenetic events, including the development of the atrioventricular canal and the fate of the outflow tract.

Animals↗
PubMed1997

Redefining the genetic hierarchies controlling skeletal myogenesis: Pax-3 and Myf-5 act upstream of MyoD.

We analyzed Pax-3 (splotch), Myf-5 (targeted with nlacZ), and splotch/Myf-5 homozygous mutant mice to investigate the roles that these genes play in programming skeletal myogenesis. In splotch and Myf-5 homozygous embryos, myogenic progenitor cell perturbations and early muscle defects are distinct. Remarkably, splotch/Myf-5 double homozygotes have a dramatic phenotype not seen in the individual mutants: body muscles are absent. MyoD does not rescue this double mutant phenotype since activation of this gene proves to be dependent on either Pax-3 or Myf-5. Therefore, Pax-3 and Myf-5 define two distinct myogenic pathways, and MyoD acts genetically downstream of these genes for myogenesis in the body. This genetic hierarchy does not appear to operate for head muscle formation.

Animals↗
PubMed1997

The role of PCR in the diagnosis and management of CMV in solid organ recipients: what is the predictive value for the development of disease and should PCR be used to guide antiviral therapy?

Cytomegalovirus remains a significant source of morbidity and mortality in immunocompromised hosts. The increased sensitivity of molecular diagnostic techniques (PCR, antigenemia) has resulted in our ability to detect viral replication earlier in the posttransplant period, before the onset of symptoms. With the advent of effective antiviral therapy, "preemptive therapy," guided by sensitive, early and specific predictors of CMV disease, has become a realistic objective. Although multiple studies have analyzed the sensitivity and specificity of these tests, their predictive value for the development of disease has not been defined. The purpose of this study was to evaluate the predictive value of a positive CMV PCR in the setting of solid abdominal organ transplantation. A total of 476 PCR assays were performed on 134 transplant recipients (102 kidney, 19 kidney/pancreas, 11 liver, 2 other) either as protocol serial samples or as dictated by clinical events. All samples were concomitantly analyzed using standard virological assays for CMV including culture, shell vial, and serology. Patients with any CMV seropositive donor/recipient (D/R) combination received ganciclovir prophylaxis in conjunction with antilymphocyte induction for 14 days. No subsequent CMV prophylaxis was used. The positive predictive value was 55% in all seropositive donor/recipient combinations. The highest risk group (seronegative recipient of seropositive donor) showed the highest positive predictive value, whereas seropositive recipients of either seropositive or seronegative donors showed positive predictive values of 45% and 25%, respectively. Negative predictive value was 100% for all groups. Early detection of CMV infection has important implications for patient management, including preemptive therapy, which can be guided by PCR, especially in high risk (D+/R-) patients.

Acyclovir↗