The arterial pole of the mouse heart forms from Fgf10-expressing cells in pharyngeal mesoderm.

Development of the arterial pole of the heart is a critical step in cardiogenesis, yet its embryological origin remains obscure. We have analyzed a transgenic mouse line in which beta-galactosidase activity is observed in the embryonic right ventricle and outflow tract of the heart and in contiguous splanchnic and pharyngeal mesoderm. The nlacZ transgene has integrated upstream of the fibroblast growth factor 10 (Fgf10) gene and comparison with the expression pattern of Fgf10 in pharyngeal mesoderm indicates transgene control by Fgf10 regulatory sequences. Dil labeling shows a progressive movement of cells from the pharyngeal arch region into the growing heart tube between embryonic days 8.25 and 10.5. These data suggest that arterial pole myocardium originates outside the classical heart field.

Localization of dystrophin gene transcripts during mouse embryogenesis.

The spatial and temporal expression of the dystrophin gene has been examined during mouse embryogenesis, using in situ hybridization on tissue sections with a probe from the 5' end of the dystrophin coding sequence. In striated muscle, dystrophin transcripts are detectable from about 9 d in the heart and slightly later in skeletal muscle. However, there is an important difference between the two types of muscle: the heart is already functional as a contractile organ before the appearance of dystrophin transcripts, whereas this is not the case in skeletal muscle, where dystrophin and myosin heavy chain transcripts are first detectable at the same time. In the heart, dystrophin transcripts accumulate initially in the outflow tract and, at later stages, in both the atria and ventricles. In skeletal muscle, the gene is expressed in all myocytes irrespective of fiber type. In smooth muscle dystrophin transcripts are first detectable from 11 d post coitum in blood vessels, and subsequently in lung bronchi and in the digestive tract. The other major tissue where the dystrophin gene is expressed is the brain, where transcripts are clearly detectable in the cerebellum from 13 d. High-level expression of the gene is also seen in particular regions of the forebrain involved in the regulation of circadian rhythms, the endocrine system, and olfactory function, not previously identified in this context. The findings are discussed in the context of the pathology of Duchenne muscular dystrophy.

Expression of CD34 and Myf5 defines the majority of quiescent adult skeletal muscle satellite cells.

Skeletal muscle is one of a several adult post-mitotic tissues that retain the capacity to regenerate. This relies on a population of quiescent precursors, termed satellite cells. Here we describe two novel markers of quiescent satellite cells: CD34, an established marker of hematopoietic stem cells, and Myf5, the earliest marker of myogenic commitment. CD34(+ve) myoblasts can be detected in proliferating C2C12 cultures. In differentiating cultures, CD34(+ve) cells do not fuse into myotubes, nor express MyoD. Using isolated myofibers as a model of synchronous precursor cell activation, we show that quiescent satellite cells express CD34. An early feature of their activation is alternate splicing followed by complete transcriptional shutdown of CD34. This data implicates CD34 in the maintenance of satellite cell quiescence. In heterozygous Myf5(nlacZ/+) mice, all CD34(+ve) satellite cells also express beta-galactosidase, a marker of activation of Myf5, showing that quiescent satellite cells are committed to myogenesis. All such cells are positive for the accepted satellite cell marker, M-cadherin. We also show that satellite cells can be identified on isolated myofibers of the myosin light chain 3F-nlacZ-2E mouse as those that do not express the transgene. The numbers of satellite cells detected in this way are significantly greater than those identified by the other three markers. We conclude that the expression of CD34, Myf5, and M-cadherin defines quiescent, committed precursors and speculate that the CD34(-ve), Myf5(-ve) minority may be involved in maintaining the lineage-committed majority.

Muscle: the regulation of myogenesis.

The study of myogenesis in the embryo is a rapidly expanding field. In this context, the consequences of mutating different members of the MyoD family, together with an increasing number of observations that point to the importance of the MEF2 or RSRF family as myogenic regulators, and the identification of Pax-3 as a marker of early myogenic cells, have advanced our understanding of the molecular embryology of skeletal muscle. Novel cardiac regulatory factors such as Nkx-2.5 and GATA-4, in addition to MEF2 isoforms, are also beginning to be identified. At the molecular level, crystallographic studies have led to a structural model of the actinomyosin complex and also to information about how MyoD contacts DNA.

Number and organization of actin-related sequences in the mouse genome.

Recombinant plasmids containing cDNA sequences complementary to the two mouse striated-muscle actin messenger RNAs (pAF81, pAM91) and to a non-muscle actin mRNA (pAL41) have been used to examine the number and organization of actin-related sequences in the mouse genome. A large number (greater than 20) of actin-related sequences are detected on Southern blots of restricted mouse DNA, the majority of which hybridize to both the 5' and 3' ends of the actin-coding sequence, even under conditions revealing only sequences greater than 80% homologous to the actin cDNA probes. More stringent washing of these blots indicates that the two striated muscle actins are each encoded by single genes, and that a non-muscle (beta or gamma) actin cDNA detects one homologous and two closely related sequences in mouse DNA. The segregation of the two striated-muscle actin genes in recombinant inbred mouse strains shows that these genes are not closely linked (greater than 1 centimorgan), and that the skeletal muscle actin gene is not linked to a non-muscle actin gene. Screening a bank of mouse genomic DNA, cloned in Charon 4A, indicates that the number of actin-related sequences in the mouse genome is much higher than 20. In particular, five phages have been isolated representing part of a sub-family of 20 to 50 similar but non-identical sequences, only weakly homologous to actin cDNA probes (probably a family of actin pseudogenes), which are the result of a recent amplification of a greater than 17 X 10(3) base region of mouse DNA.

Insertional mutation of the mouse Msx1 homeobox gene by an nlacZ reporter gene.

We have generated a null allele of the mouse Msx1 homeobox gene by insertion of an nlacZ reporter gene into its homeobox. The sensitivity of beta-galactosidase detection permitted us to reveal novel aspects of Msx1 gene expression in heterozygous embryos, in particular in ectoderm and mesoderm during gastrulation, and in migrating neural crest cells. Homozygous mutant mice die at birth with facial defects (see Satokata, I. and Maas, R. (1994) Msx1 deficient mice exhibit cleft palate and abnormalities of craniofacial and tooth development. Nat. Genet. 6, 348-356). To investigate the reason for this limited phenotype, we compared the pattern of Msx1 expression with that of the closely related Msx2 gene in wild type embryos and in Msx1-/- mutants. Notably, whereas the expression of Msx1 and Msx2 overlap in the developing limb, this is not the case in the facial regions most affected in the mutant.

Cardiosensor mice and transcriptional subdomains of the vertebrate heart.

Differential regulation of cardiac gene expression in vertebrates has been extensively documented in the context of atrial and ventricular morphogenesis. Recent data, largely from the analysis of transgene and endogenous gene expression patterns, have revealed transcriptional differences between left and right cardiac chambers which suggest that the heart is composed of a series of distinct transcriptional domains. Such phenomena provide regional markers (cardiosensors) for the fine analysis of normal and abnormal heart development. Regional subdivisions and transcriptional heterogeneity within the myocardium emerge in response to patterning of the precardiac mesoderm and early heart tube on the anterior-posterior axis, and to embryonic left-right signals which dictate the direction of cardiac looping. Several families of transcription factors have been characterized which may be implicated in the regionalization of myocardial gene expression.

Modular regulation of the MLC1F/3F gene and striated muscle diversity.

Isoform diversity in striated muscle is largely controlled at the level of transcription. In this review we will concentrate on studies concerning transcriptional regulation of the alkali myosin light chain 1F/3F gene. Uncoupled activity of the MLC1F and 3F promoters, together with complex patterns of transcription in developing skeletal and cardiac muscle, combine to make analysis of this gene particularly intriguing. In vitro and transgenic studies of MLC1F/3F regulatory elements have revealed an array of cis-acting modules that each drive a subset of the expression pattern of the two promoters. These cis-acting regulatory modules, including the MLC1F and 3F promoter regions and two skeletal muscle enhancers, control tissue-specificity, cell or fibre-type specificity, and the spatiotemporal regulation of gene expression, including positional information. How each of these regulatory modules acts and how their individual activites are integrated to coordinate transcription at this locus are discussed.

The control of muscle gene expression: a review of molecular studies on the production and processing of primary transcripts.

Many muscle proteins exist as multiple isoforms. This diversity is generated by the presence of multigene families and by alternative splicing of individual genes. Examples are given of different modes of alternative splicing undergone by the primary transcripts of muscle genes, and preliminary studies on the mechanism are mentioned. The chromosomal organization of muscle genes is discussed briefly. Studies on their transcriptional regulation are reviewed first in terms of cis-acting sequences in the proximal promoter region and elsewhere in the vicinity of the gene, which are necessary for its expression, and, secondly, in terms of trans-acting transcriptional factors which interact with such sequences. Molecular regulation of splicing and of transcription is discussed mainly with reference to the muscle genes of mammals.

Actin and myosin genes are transcriptionally regulated during mouse skeletal muscle development.

During primary and secondary myotube formation in utero and subsequent maturation of muscle fibers after birth there are complex changes in the pattern of contractile protein gene expression at the RNA and protein levels. In order to determine the degree of transcriptional regulation of actin and myosin genes we have carried out "nuclear run-on" experiments using nuclei prepared from the limb muscle of mice at 14.5, 15.5, 17.5, and 18.5 days in utero and at 10-12 and 12.5 days after birth. We show that transitions in the expression of these genes in vivo are regulated transcriptionally. Transcription of the sarcomeric alpha-actins changes from cardiac to predominantly skeletal actin over this time period; transcription of the beta-actin gene is repressed. The myosin heavy chain and myosin light chain genes also undergo transcriptional transitions during muscle development. Notably, transcription from the MLC3F promoter is activated after that of the MLC1F promoter, which is part of the same gene. These results are discussed in the context of published RNA data.

Mouse limb muscle is determined in the absence of the earliest myogenic factor myf-5.

myf-5 is the only member of the MyoD family of myogenic regulatory genes to be expressed in the mouse embryo prior to muscle cell differentiation. We have used the developing limb as a model in which to follow the formation of peripheral muscle, to address the question of whether myogenic precursor cells are already present in the limb bud before expression of myf-5. The lacZ reporter gene has been introduced into the myf-5 gene by homologous recombination so that its expression is under the control of the endogenous myf-5 locus. beta-Galactosidase (beta-gal) coloration provides a sensitive assay for myf-5+ cells. Embryos were generated from embryonic stem cells carrying this mutation, and the appearance of beta-gal+ (myf-5+) cells was followed during limb development in vivo. Limb buds, at a stage when they are beta-gal-, were cultured in vitro. After several days, beta-gal+ cells accumulated in the premuscle mass. We conclude that determined muscle precursor cells in the limb bud do not initially express any member of the MyoD family. Furthermore, myogenic precursor cells in the somite, which, according to the avian model, migrate from the ventral/lateral edge of the dermomyotome to form peripheral muscle masses, are also negative for these factors.

Lineage restriction of the myogenic conversion factor myf-5 in the brain.

myf-5 is one of four transcription factors belonging to the MyoD family that play key roles in skeletal muscle determination and differentiation. We have shown earlier by gene targeting nlacZ into the murine myf-5 locus that myf-5 expression in the developing mouse embryo is closely associated with the restriction of precursor muscle cells to the myogenic lineage. We now identify unexpected expression of this myogenic factor in subdomains of the brain. myf-5 expression begins to be detected at embryonic day 8 (E8) in the mesencephalon and coincides with the appearance of the first differentiated neurons; expression in the secondary prosencephalon initiates at E10 and is confined to the ventral domain of prosomere p4, later becoming restricted to the posterior hypothalamus. This expression is observed throughout embryogenesis. No other member of the MyoD family is detected in these regions, consistent with the lack of myogenic conversion. Furthermore, embryonic stem cells expressing the myf-5/nlacZ allele yield both skeletal muscle and neuronal cells when differentiated in vitro. These observations raise questions about the role of myf-5 in neurogenesis as well as myogenesis, and introduce a new lineage marker for the developing brain.