Arlette Cohen

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.

Cytoplasmic distribution of pulse-labelled poly(A)-containing RNA, particularly 26 S RNA, during myoblast growth and differentiation☆

Abstract Total poly(A)-containing RNA in different polysomal and supernatant cytoplasmic fractions was analysed after pulse-labelling in dividing myoblasts and fused myotubes. In particular, the peak of 26 S RNA (putative messenger for the large subunit of myosin) is located in a light region of the gradient coinciding with the monosome-trisome fractions prior to fusion, and is found in the heavy polysomes only after fusion. These heavy polysomes are free (i.e. not membrane bound). Treatment of the light part of the polysome gradient with EDTA shows that the 26 S RNA found here does not exist as part of a polysomal complex, but is present as a ribonucleoprotein particle cosedimenting in this region. Previous experiments had indicated that in actively dividing myoblasts 26 S RNA has a relatively short half-life but that it becomes “stable” after the cessation of mitosis just prior to fusion. RNA chase experiments performed in the present study show that the “short-lived” 26 S RNA from dividing myoblasts, which is present as a ribonucleoprotein particle, does not enter the heavy polysomes. In contrast, the more stable 26 S RNA also initially present as a ribonucleoprotein, just prior to and in the early stages of fusion, can be shown by chase experiments to enter the heavy polysomes later in fusion. Hence accumulation of 26 S RNA seems to precede its activation as a messenger.

A skeletal muscle cell line isolated from a mouse teratocarcinoma undergoes apparently normal terminal differentiation in vitro

Abstract A muscle cell line was obtained as a clonal isolate from a contractile zone of cultured cells derived from a mouse teratocarcinoma. The cell line (C17-S1-D-T984) differentiates in vitro when the cells become confluent, with the formation of multinucleated myotubes. The muscle fibres contract spontaneously and electron microscopy demonstrates the presence of organized sarcomeric structures. In addition to the presence of the contractile proteins, there is also an accumulation of acetylcholine receptors and the appearance of the M form of creatine phosphokinase, all characteristic markers of differential skeletal muscle.

A modified assay procedure for revealing the M form of creatine kinase in cultured muscle cells

Abstract A convenient standard procedure for identifying the different isozymes of creatine kinase is that of electrophoresis on cellulose acetate strips with subsequent colorimetric revelation of the enzyme by a series of reactions coupled to the initial production of ATP. Inclusion of the myokinase inhibitor diadenosine pentaphosphate in the reaction mixture eliminates the confusion in interpretation which may arise due to the similar migration properties of the M form of creatine kinase and of myokinase which is also revealed by the procedure. Use of the inhibitor permits the clear demonstration of the muscle form of the enzyme in differentiated muscle cell cultures and particularly in rat myoblast cell lines where relatively high levels of myokinase complicated previous estimations.

Chromosomal assignment of two myosin alkali light-chain genes encoding the ventricular/slow skeletal muscle isoform and the atrial/fetal muscle isoform (MYL3, MYL4)

SummaryIn all eukaryotes, myosin plays a major role in the maintenance of cell shape and in cellular movement; in association with actin and other contractile proteins it is also a major structural component of the muscle sarcomere. Several isoforms of myosin alkali light chain have been identified, associated with different muscle types. We have recently localized the gene encoding the fast skeletal muscle alkali light-chain isoforms MLC1F and MLC3F (HGM symbol, MYL1) to human chromosome 2q32.1-qter (Cohen-Haguenauer 1988). We present here the chromosomal assignment of two loci encoding the ventricular muscle isoform MLC1V (equivalent to the slow skeletal muscle isoform MLC1Sb) and the atrial muscle isoform MLC1A (equivalent to the fetal isoform MLC1emb) using a panel of 25 independent man-rodent somatic cell hybrids. The MLC1V gene (HGM symbol, MYL3) was mapped to human chromosome 3 using a human full-length cDNA probe that hybridizes to a single major human TaqI 2.8-kb fragment. The MLC1A probe (HGM symbol, MYL4) was a 360-bp mouse cDNA fragment that gave a distinct signal with human DNA using low stringency conditions of hybridization and washings and after presaturation of the Southern blots with rodent DNA. A single PstI 7.8-kb fragment gives an intense signal, and its presence correlates with the presence of chromosome 17 among the hybrids. These data are in keeping with the localizations of the MLC1V gene to mouse chromosome 9, and of the MLC1A gene to mouse chromosome 11, which share some markers in common with human chromosomes 3 and 17 respectively.

The Actin and Myosin Multigene Families

The Actin and Myosin Multigene Families: a) a study of the accumulation of their RNA transcripts demonstrates different developmental strategies during skeletal muscle formation, b) a genetic analysis of their chromosomal organization indicates gene dispersion and permits some precise localizations on the genetic map of the mouse.

Msx1 is close but not allelic to either Hm or Hx on mouse chromosome 5.

The Msx1 homeobox locus has been mapped in relation to the mutations hammer-toe (Hm) and hemimelic extra toes (Hx). Msx1 is expressed in the developing limb, while limb development is affected by the Hm and Hx mutations. Hm and Hx are very tightly linked loci. In interspecific crosses, the segregation of either mutation was followed in relation to polymorphic alleles of Msx1, Il6, and En2, to give a fine map around the mutant loci. Our results show that Msx1 is not allelic to either of the mutations, but is located about 3 cM from them. Il6 did not recombine with either Hm or Hx and, therefore, provides a point of access for the analysis of these mutations at the molecular level.This locus was originally named Hox-7 and has been renamed by the Mouse Nomenclature Committee in accordance with the proposals set out by Scott (1992) for vertebrate homobox gene nomenclature.

Actin and Myosin Genes and Their Expression During Skeletal Muscle Myogenesis

The differentiation of skeletal muscle cells is characterized morphologically by the fusion of myoblasts to form multinucleated muscle fibres. This process takes place gradually during skeletal muscle development in vivo. It can also be followed in tissue culture. Mammalian myoblasts will grow in monolayers, either in primary culture or as established cell lines, and will fuse spontaneously when the culture becomes confluent (for review see Yaffe 1968, Buckingham 1977). The formation of muscle fibres is characterized biochemically by the increased synthesis of contractile proteins (e.g. Devlin and Emerson 1978, Garreis 1979) and their organization into sarcomeric structures (Fischman 1970), by the accumulation of enzymes important in muscle metabolism (e.g. Caravatti et al. 1979), and by the appearance of membrane components such as the acetylcholine receptor (e.g. Merlie et al. 1975), essential for nerve-muscle interaction.