GATA-2 is a maternal transcription factor present in Xenopus oocytes as a nuclear complex which is maintained throughout early development.

We show that Xenopus oocytes and embryos contain GATA-2, stored in nuclei as a non-chromatin-bound complex. Its binding site specificity is different from that of GATA-1, having a much higher affinity for the motif with a core GATC sequence. This binding site preference was markedly reduced by either release of the factor with deoxycholate or purification on a DNA affinity column, suggesting a role for a cofactor(s). The identity of the maternal GATA factor was established as GATA-2 in two ways: (1) binding to an oligonucleotide probe was abolished by inclusion of either of two GATA-2 monoclonal antibodies, and (2) a protein of correct molecular weight for GATA-2 was detected by immunoblotting with a polyclonal antibody raised against a Xenopus GATA-2-specific peptide. Although predominantly complexed, some of the oocyte GATA-2 is functional as a transcription factor because the transcriptional activity of the chicken betaH-globin promoter injected into oocytes was reduced by mutation of either of two GATA binding sites. This effect was more pronounced when the stronger of the two sites was mutated. Butyrate treatment of oocytes stimulated cap-site initiation by up to 17-fold with both normal promoter and GATA site mutant constructs, showing that the mechanism of butyrate stimulation is not via GATA-2. The possible significance of regulating the availability of maternal GATA-2 during early development is discussed.

GATA factors and the origins of adult and embryonic blood in Xenopus: responses to retinoic acid.

The transcription factors, GATA-1, -2 and -3 play essential roles in the differentiation of haematopoietic cells. To study the process of blood formation during vertebrate development we have used the expression of these GATA factors to locate haematopoietic cells in Xenopus embryos and to act as sensors for the effects of all-trans retinoic acid (RA), a signalling molecule which influences both anteroposterior patterning and haematopoietic differentiation. GATA factor expression was detected in the leading edge of the gastrulating mesoderm, in the ventral blood island (VBI) and dorsolateral plate (DLP) mesoderms and in a population of cells between the VBI and DLP. The VBI contributes to both embryonic and adult blood, whereas the DLP contains precursors of adult blood only, which have not been identified previously with molecular markers. The possibility that the GATA-2-expressing cells between the VBI and DLP were haematopoietic progenitors migrating from the VBI to the DLP was ruled out by transplantation analysis. Differential effects of RA on the expression of GATA-1 and GATA-2 suggest that RA has a direct action on haematopoietic differentiation, rather than on the formation of haematopoietic mesoderm.

Anteriorization of CRABP-I expression by retinoic acid in the developing mouse central nervous system and its relationship to teratogenesis.

We have investigated the role that cellular retinoic acid binding protein I (CRABP-I) may play in the development of the murine hindbrain. Since the central nervous system (CNS) represents a major site of the teratogenic action of retinoic acid (RA), we have also determined the effects of exposure of high levels of RA on CRABP-I expression within the CNS. Expression of CRABP-I can first be detected within the presumptive hindbrain of presomitic mouse embryos and later also appears in neural crest cells and neural crest derivatives; it is thus tissue specific at these early stages. Exposure of 7.75-day mouse embryos to RA induces two phenotypes: one is externally normal and the other is exencephalic. In the exencephalic embryos we show that there is abnormal crest migration, a fusion of the trigeminal and facial-acoustic ganglia, a rostral and lateral shift of the otic vesicle, and a loss of hindbrain rhombomeres. Furthermore, and in contrast to in vitro studies, we demonstrate that CRABP-I appears to be up-regulated in both phenotypes of mouse embryos treated with RA and that this up-regulation is accompanied by an anteriorization of its expression within the nervous system. This new CRABP-I expression domain thus retains its tissue specificity. The role that CRABP-I may play in normal development of the hindbrain and in teratogenesis and the similarity of these results to those obtained with various Hox genes are discussed.

Negative control of Xenopus GATA-2 by activin and noggin with eventual expression in precursors of the ventral blood islands.

To increase our understanding of haematopoiesis during early vertebrate development, we have studied the expression pattern of the transcription factor GATA-2 in Xenopus embryos, and asked how this is regulated. We show that the blood island precursors of the ventral mesoderm express GATA-2 RNA at neural tube stages, some 5 hours before globin RNA is detected in their derivatives. Prior to this however, GATA-2 is expressed much more widely within the embryo. Maternal transcripts are uniformly distributed, and zygotic transcription is activated during gastrulation throughout ventral and lateral regions of the embryo, with expression highest in the sensorial ectoderm and only weak in the ventral mesoderm. The domain of GATA-2 expression in neurulae outlines the region of the neural plate and suggests a possible wider role in dorsoventral patterning. To identify the signals involved in regulating this pattern of expression, we performed experiments with embryo explants. GATA-2 is activated autonomously in isolated animal caps and this activation is suppressed by the mesoderm-inducing factor activin, but not by FGF. Thus, the down-regulation of GATA-2 observed in the region of the Spemann organiser may be a response to an activin-like signal emanating from the dorsal-vegetal region or Nieuwkoop centre. GATA-2 activation in animal caps and ventral marginal zones was suppressed by co-culturing with dorsal marginal zones, suggesting that a signal from the Spemann organiser is involved in suppression of GATA-2 in the dorsal region of the embryo. Expression of a candidate for this signal, noggin, had the same effect. Taken together, the observations presented here suggest that GATA-2 activation occurs by default in the absence of signals, that the restriction of its expression within the early embryo is controlled by negative signals emanating from the Nieuwkoop centre and the organiser, and that noggin and activin-like molecules play a role in these signalling pathways.

Role of myelin P0 protein as a homophilic adhesion molecule.

Peripheral nervous system myelin is an extension of the Schwann cell's plasma membrane that tightly enwraps axons in many layers and permits nerve impulses to be rapidly conducted. It is not known how these multiple membrane layers are held together in this compact form. Here we present evidence supporting the hypothesis that the extracellular leaflets of myelin are held together by the most abundant protein of myelin of the peripheral nervous system, P0, by homophilic interaction of its extracellular domains. Transfected Chinese hamster ovary cells expressing P0 protein adhere to each other in suspension, to form large aggregates, whereas cells that are identical but which do not express P0 do not. We also show that this aggregation is mediated by homophilic binding between P0-expressing cells and that the apposing plasma membranes of these cells specifically form desmosomes, whereas control transfected cells do not. As the only difference between the two cell populations is the expression of P0, this protein is apparently responsible for the changes in morphology and adhesion in the cells that express it. The idea that P0 is a homophilic adhesion molecule is supported by its inclusion in the immunoglobulin supergene family, all members of which are involved in recognition and/or adhesion.

Intercellular adhesion mediated by human muscle neural cell adhesion molecule: effects of alternative exon use.

Mouse 3T3 fibroblasts were permanently transfected with cDNAs encoding isoforms of the neural cell adhesion molecule (N-CAM) present in human skeletal muscle and brain. Parental and transfected cells were then used in a range of adhesion assays. In the absence of external shear forces, transfection with cDNAs encoding either transmembrane or glycosylphosphatidylinositol (GPI)-linked N-CAM species significantly increased the intercellular adhesiveness of 3T3 cells in suspension. Transfection of a cDNA encoding a secreted N-CAM isoform was without effect on adhesion. Cells transfected with cDNAs containing or lacking the muscle-specific domain 1 sequence, a four-exon group spliced into the muscle but not the brain GPI-linked N-CAM species, were equally adhesive in the assays used. We also demonstrate that N-CAM-mediated intercellular adhesiveness is inhibited by 0.2 mg/ml heparin; but, at higher concentrations, reduced adhesion of parental cells was also seen. Coaggregation of fluorescently labeled and unlabeled cell populations was performed and measured by comparing their distribution within aggregates with distributions that assume nonspecific (random) aggregation. These studies demonstrate that random aggregation occurs between transfected cells expressing the transmembrane and GPI-linked N-CAM species and between parental cells and those expressing the secreted N-CAM isoform. Other combinations of these populations tested exhibited partial adhesive specificity, indicating homophilic binding between surface-bound N-CAM. Thus, the approach exploited here allows for a full analysis of the requirements, characteristics, and specificities of the adhesive behavior of individual N-CAM isoforms.

The kinetics of cell-substratum detachment mediated by trypsin: a comparison of normal and Duchenne fibroblasts.

In previous studies of cell-cell and cell-substratum adhesion, we have identified differences in the behaviour between human skin fibroblasts cultured from normal individuals and patients with Duchenne muscular dystrophy (DMD). In these studies, monolayer cultures were dissociated by trypsinization and no detectable difference was noted in the efficiency of cell dissociation between normal and DMD fibroblast cultures. However, a detailed study by Kent has suggested that Duchenne fibroblasts exhibit increased sensitivity to trypsin. We have re-investigated this finding using an assay that directly measures the number of cells remaining attached to a substratum following trypsinization. In a series of experiments using cultures derived from five normal and five DMD individuals, we can detect no significant difference in the trypsin-induced detachment rates between normal and DMD skin fibroblasts. This observation applies to both growth-phase and stationary-phase cell cultures. This inconsistency with previously reported data on the trypsin-sensitivity of DMD cells is considered in terms of the different assays used and the effects of trypsin on cell-cell and cell-substratum adhesion. The relationship between abnormalities in the behaviour of DMD cells and the localization and primary structure of the DMD gene product are also discussed.

Distinct dystrophin mRNA species are expressed in embryonic and adult mouse skeletal muscle.

We have examined dystrophin mRNA in embryonic, newborn and adult mouse skeletal muscle. A discrete nerve-independent increase in mRNA size was observed between embryonic and adult stages, indicating that a developmentally regulated mRNA isoform switch occurs in the expression of the Duchenne muscular dystrophy (DMD) gene in skeletal muscle. These distinct mRNAs are most likely generated via selection of alternative transcriptional start sites or RNA processing pathways. In addition, denervation of adult muscle was without effect on the expression pattern.

Requirements for the Ca2+-independent component in the initial intercellular adhesion of C2 myoblasts.

Using a sensitive and quantitative adhesion assay, we have studied the initial stages of the intercellular adhesion of the C2 mouse myoblast line. After dissociation in low levels of trypsin in EDTA, C2 cells can rapidly reaggregate by Ca2+-independent mechanisms to form large multicellular aggregates. If cells are allowed to recover from dissociation by incubation in defined media, this adhesive system is augmented by a Ca2+-dependent mechanism with maximum recovery seen after 4 h incubation. The Ca2+-independent adhesion system is inhibited by preincubation of cell monolayers with cycloheximide before dissociation. Aggregation is also reduced after exposure to monensin, implicating a role for surface-translocated glycoproteins in this mechanism of adhesion. In coaggregation experiments using C2 myoblasts and 3T3 fibroblasts in which the Ca2+-dependent adhesion system was inactivated, no adhesive specificity between the two cell types was seen. Although synthetic peptides containing the RGD sequence are known to inhibit cell-substratum adhesion in various cell types, incubation of C2 myoblasts with the integrin-binding tetrapeptide, RGDS, greatly stimulated the Ca2+-independent aggregation of these cells while control analogs had no effect. These results show that a Ca2+-independent mechanism alone is sufficient to allow for the rapid formation of multicellular aggregates in a mouse myoblast line, and that many of the requirements and perturbants of the Ca2+-independent system of intercellular myoblast adhesion are similar to those of the Ca2+-dependent adhesion mechanisms.

Spreading behaviour of cultured fibroblasts from carriers of Duchenne muscular dystrophy.

Cultured skin fibroblasts from patients with Duchenne muscular dystrophy (DMD) are more sensitive than normal cells to prolonged exposure to the ionophore monensin. In a cell spreading assay in which cells were preincubated with monensin and subsequently allowed to adhere to and spread on a glass substratum in serum-free medium for 100 min, the mean transformed cell area of normal and DMD cells was 5.97 +/- 0.11 and 5.29 +/- 0.03, respectively. Cultured fibroblasts from carriers of DMD yielded a value of 5.59 +/- 0.03, which is intermediate between, and significantly different from, the values for both normal and DMD cultures. This result would be predicted on the basis of random X-chromosome inactivation in female carriers of this disorder. However, comparison of DMD carrier cell spreading data with data obtained from pooled and summated measurements taken from separate experiments using either normal or DMD fibroblasts suggest a more complex situation. Examination of the variance of the means of cell area for the true carrier population and the summated normal and DMD population provides evidence suggesting that some form of cellular interaction may occur between the two cell genotypes in culture.

Neural control of the forms of acetylcholinesterase in slow mammalian muscles.

The 'heavy', collagen-tailed form of acetylcholinesterase (AChE), having a s(0)20,w of 16S in mammals, occurs at vertebrate muscle endplates and has been widely regarded as a marker of neuronal influence on muscle in vivo. However, an interesting exception has been described by Bacou et al., in a previous report in Nature. They found, in a slow-twitch muscle of the rabbit, that after denervation the 16S form of AChE increases markedly, rather than disappearing. Such a phenomenon would modify current concepts of neuromuscular regulation. We report here, however, that this exception is apparent rather than real in terms of endplate AChE regulation.

The effect of monensin on cell aggregation of normal and dystrophic human skin fibroblasts.

Measurements of aggregation kinetics using couette viscometry show that freshly trypsinized skin fibroblasts from patients with Duchenne muscular dystrophy have values of intercellular adhesiveness approx. 40% those of normal cells. If cells are allowed to recover from the effects of trypsinization (by incubation for 2 h at 37 degrees C in serum-containing medium) the intercellular adhesiveness of both cell types increases, and normal and Duchenne cells aggregate to the same extent. Exposure to the ionophore monensin during the recovery phase leads to suppression of recovery in both cell types, and this effect of the drug is greater in Duchenne fibroblasts. These results are discussed in relation to other data on the reported differential effects of trypsin and monensin on normal and Duchenne fibroblasts.

Adhesive interactions between normal and dystrophic human skin fibroblasts.

The adhesive properties of skin fibroblasts from patients with Duchenne muscular dystrophy (DMD) were studied by analysing cell aggregate formation in suspensions consisting of normal and DMD fibroblasts. By the use of aggregation kinetics and fluorescent labelling, the genotypic composition of aggregates in mixed-cell suspensions could be visualised. The distribution of normal and DMD cells within these aggregates could then be compared to theoretical binomial distributions which assume no difference in the specific adhesiveness between the two genotypes. Analysis of the 3- and 4-cell aggregates which were produced by co-aggregating normal and DMD cells demonstrate that there is no qualitative (specific) difference in the adhesiveness between normal and DMD fibroblasts. However, quantitative changes in the cell-cell adhesion of DMD fibroblasts may be present, and this is supported by the relatively small proportion of intermediate size heterotypic aggregates which were formed in mixed-genotype cell suspensions. In such mixtures, fewer aggregates consisting of 5 or more cells were formed compared to fibroblast suspensions derived from pairs of normal individuals. Furthermore, cell suspensions from pairs of DMD patients produced even less greater than or equal to 5-cell aggregates than were found in the mixed-genotype experiments. These findings are considered in relation to previous reports of abnormal cell adhesiveness and other adhesion-related mechanisms in DMD cells.

Involvement of fast and slow twitch muscle fibres in avian muscular dystrophy.

The extent of differential fibre type involvement in chicken muscular dystrophy can be assessed quantitatively by the statistical parameters of fibre area, nuclei content and nuclei distribution in the individual fibre types. Two muscles, the posterior latissimus dorsi (PLD) and the serratus metapatagialis (SMP), were found to have similar overall fibre type composition, although the latter contains two subtypes of type I fibres, one of which has not previously been recognised in avian muscle. In both muscles, type IIB fibres are most affected by the progressive pathology. Nuclear proliferation is one of the histopathological features which can be measured, and in the PLD, the mean number of total nuclei in type IIB fibre cross-sections (Nt) is increased from 2.23 in normal chickens to 3.70 in dystrophic chickens, by 60 days. The corresponding values for Nt in type IIB muscle fibres of the SMP at 50 days are 1.74 and 5.10. Likewise, statistical analyses of the distribution of the fibre areas and their variability demonstrate that the incidence of abnormality in chicken dystrophy is greatest in type IIB fibres in both these muscles. Although type I fibres in the PLD are resistant to dystrophic change, it is noteworthy that in the SMP the type I fibres, also, are severely affected from an early stage, by these quantitative criteria. On the other hand, all fibres in a tonic muscle, the metapatagialis latissimus dorsi, are unaffected, as is true of all other tonic muscles previously studied. It is concluded that any twitch fibre type can, in principle, be affected by the actions of the gene concerned, and that this expression can be greatly modified in individual muscles by various physiological features, for example their natural pattern of use or relative disuse.

Monensin inhibits initial spreading of cultured human fibroblasts.

The monovalent ionophore, monensin, inhibits the secretion of both pro-collagen and fibronectin in cultured human fibroblasts and other cell types. The block to secretion is due to the ability of monensin to suppress the export of these secretory proteins from the Golgi apparatus. As such proteins are known to be implicated in the adhesion, spreading and movement of cultured fibroblasts, it might be expected that monensin treatment would interfere with these processes. However, it has recently been reported that monensin-treated human embryonal fibroblasts attached and spread onto glass substrata to the same extent as untreated cells, although at later stages they fail to develop focal adhesion sites. However, these experiments were performed using medium supplemented with fetal calf serum (FCS). We now demonstrate that in the absence of FCS, while monensin has little or no effect on the initial adhesion of fibroblasts to the substratum, subsequent spreading is much reduced. The inhibition of spreading is noticeable within 30 min of plating and is maintained for at least 100 min in monensin-free medium following prolonged pre-incubation of the cells with monensin.

Structural change in muscles of the dystrophic chicken. II. Progression of the histopathology in the pectoralis muscle.

Dystrophic (D) and normal (N) chicken pectoral muscle was analysed for histopathological differences from the embryo (day 20) through to the mature adult stage. A variety of indices of structural changes were used, to express the progression quantitatively. At the embryonic stage, fibroblast numbers (but not satellite cell numbers) are increased in D. D shows fibre hypertrophy discernible from about 4 days after hatching. Other abnormalities of fibre size and shape appear in D from about 2 weeks onwards. Nuclei numbers and location change characteristically in D, compared to N, from about 4 days, and these differences subsequently become very prominent. Fibre splitting and phagocytosis become significant in D from about 2 weeks onwards and increase noticeably thereafter. The later stages in D, with gross loss of muscle fibres, are also described, in the pectoral and posterior latissimus dorsi muscles. The analyses show that some of the structural changes in D precede the discernible difference in fibre type composition which develops between D and N. The changes in the muscle used provide a basis for assessing the effects of drug treatments at various stages, including the early post-hatch period.

Structural change in muscles of the dystrophic chicken. I. Quantitative indices.

The pectoral and posterior latissimus dorsi (PLD) muscles in the dystrophic chicken show many pathological changes. At 7 weeks of age, in a strain of these birds bred for early onset of this inherited disorder, physical weakness is established and these changes can be clearly assessed. Quantitative indices of these progressive changes were obtained by light and electron microscopic analyses; these indices cover fibre hypertrophy, fibre area variation, nuclei distribution, phagocytosis and satellite cell and fibroblast populations. The dystrophic pectoral muscle at 7 weeks is more severely affected than the PLD. In the former, there is an increase of peripheral myonuclei per fibre cross-section of over 200%, a hypertrophy factor nearly 200-fold greater than normal and longitudinal fibre splitting in 11% of the fibres. Many foci of degeneration are also present at this early stage. Although regenerating fibres are uncommon, an increased satellite cell population is observed. These indices provide a basis for assessing the effects of in vivo drug treatments, at the level of muscle structure.