The specification of heart mesoderm occurs during gastrulation in Xenopus laevis

The establishment of heart mesoderm during Xenopus development has been examined using an assay for heart differentiation in explants and explant combinations in culture. Previous studies using urodele embryos have shown that the heart mesoderm is induced by the prospective pharyngeal endoderm during neurula and postneurula stages. In this study, we find that the specification of heart mesoderm must begin well before the end of gastrulation in Xenopus embryos. Explants of prospective heart mesoderm isolated from mid- or late neurula stages were capable of heart formation in nearly 100% of cases, indicating that the specification of heart mesoderm is complete by midneurula stages. Moreover, inclusion of pharyngeal endoderm had no statistically significant effect upon either the frequency of heart formation or the timing of the initiation of heartbeat in explants of prospective heart mesoderm isolated after the end of gastrulation. When the superficial pharyngeal endoderm was removed at the beginning of gastrulation, experimental embryos formed hearts, as did explants of prospective heart mesoderm from such embryos. These results indicate that the inductive interactions responsible for the establishment of heart mesoderm occur prior to the end of gastrulation and do not require the participation of the superficial pharyngeal endoderm.     

Regulation and function of tinman during dorsal mesoderm induction and heart specification in Drosophila

The homeobox gene tinman plays a key role in the specification of Drosophila heart progenitors and the visceral mesoderm of the midgut, both of which arise at defined positions within dorsal areas of the mesoderm. Here, we show that in addition to the heart and midgut visceral mesoderm, tinman is also required for the specification of all dorsal body wall muscles. Thus it appears that the precursors of the heart, visceral musculature, and dorsal somatic muscles are all specified within the same broad domain of dorsal mesodermal tinman expression. Locally restricted activities of tinman are also observed during its early, general mesodermal expression, where tinman is required for the activation of the homeobox gene buttonless in precursors of the “dorsal median” (DM) glial cells along the ventral midline. These observations, together with others showing only mild effects of ectopic tinman expression on heart development, indicate that tinman function is obligatory, but not sufficient to determine individual tissues within the mesoderm. Therefore, we propose that tinman has a role in integrating positional information that is provided by intersecting domains of additional regulators and signals, which may include Wingless, Sloppy Paired, and Hedgehog in the dorsal mesoderm and EGF-signaling at the ventral midline. Previous studies have shown that Dpp acts as an inductive signal from dorsal ectodermal cells to induce tinman expression in the dorsal mesoderm, which, in turn, is needed for heart and visceral mesoderm formation. In the present report, we show that Thickveins, a type I receptor of Dpp, is essential for the transmission of Dpp signals into the mesoderm. Constitutive activity of Tkv in the entire mesoderm induces ectopic tinman expression in the ventral mesoderm, and this results in the ectopic formation of heart precursors in a defined area of the ventrolateral mesoderm. We further show that Screw, a second BMP2/4-related gene product, Tolloid, a BMP1-related protein, and the zinc finger-containing protein Schnurri, are required to allow full levels of tinman induction during this process. It is likely that some of these functional and regulatory properties of tinman are shared by tinman-related genes from vertebrates that have similarly important roles in embryonic heart development. Dev. Genet. 22:187–200, 1998. © 1998 Wiley-Liss, Inc.     

The cesium-induced delay in myoblast membrane fusion is accompanied by changes in isolated membrane lipids

We have recently demonstrated that cesium ions delay the sharp decrease in both membrane conductivity and membrane permittivity of chick embryo myoblasts seen at fusion (Santini, M.T., Bonincontro, A., Cametti, C. and Indovina, P.L. (1988) Biochim. Biophys. Acta 945, 56-64). Analysis of the conductivity dispersion data (obtained in the radiowave frequency range) indicated that cesium delays fusion by about 30 h. We suggested that cesium is affecting both active ionic transport by blocking potassium channels as well as interfering with membrane lipid and/or protein charges. In the present study, we have investigated both the possible role of membrane lipids in myoblast fusion and the possible effects of cesium on these lipids. Our data indicate that lipid changes do occur in the isolated myoblast plasma membrane of controls during myogenic differentiation especially prior to fusion and that in cesium cultures these variations do not occur. These variations are in accordance with current membrane fusion theory. Specifically, there is a decrease in bilayer-stabilizing lipids (phosphatidylcholine) and an increase in bilayer-destabilizing ones (phosphatidylethanolamine and phosphatidic acid) and cholesterol during the fusion process. In addition, although slight, during fusion there appears to be a decrease in phosphatidylinositol which is believed to be involved in the inositol phosphate second messenger system. In cesium cultures, in which fusion is greatly delayed, the same lipid changes do not take place and those that are observed seem to reflect the fusion delay.     

The cesium-induced delay in myoblast membrane fusion is accompanied by changes in cellular subfraction lipid composition.

We have recently demonstrated that the delay in myoblast membrane fusion induced by cesium is accompanied by changes in isolated membrane lipids (Santini, M.T., Indovina, P.L., Cantafora, A. and Blotta, I. (1990) Biochim. Biophys. Acta 1023, 298-304). In the present study, we have investigated changes in the lipid profile of total cell homogenates and microsomal membrane fractions during myoblast membrane fusion as well as the effects that addition of cesium may have on these lipid variations in order to try to understand the production and translocation of lipids during this myogenic process. The data presented here indicate that the lipid composition of cell homogenates and microsomes varies in a different manner from isolated plasma membranes during myogenic fusion. In addition, cesium affects, in a different manner, the normally-occurring lipid production and distribution which takes place in each subcellular fraction.     

The role of micromere signaling in Notch activation and mesoderm specification during sea urchin embryogenesis.

In the sea urchin embryo, the micromeres act as a vegetal signaling center. These cells have been shown to induce endoderm; however, their role in mesoderm development has been less clear. We demonstrate that the micromeres play an important role in the induction of secondary mesenchyme cells (SMCs), possibly by activating the Notch signaling pathway. After removing the micromeres, we observed a significant delay in the formation of all mesodermal cell types examined. In addition, there was a marked reduction in the numbers of pigment cells, blastocoelar cells and cells expressing the SMC1 antigen, a marker for prospective SMCs. The development of skeletogenic cells and muscle cells, however, was not severely affected. Transplantation of micromeres to animal cells resulted in the induction of SMC1-positive cells, pigment cells, blastocoelar cells and muscle cells. The numbers of these cell types were less than those found in sham transplantation control embryos, suggesting that animal cells are less responsive to the micromere-derived signal than vegetal cells. Previous studies have demonstrated a role for Notch signaling in the development of SMCs. We show that the micromere-derived signal is necessary for the downregulation of the Notch protein, which is correlated with its activation, in prospective SMCs. We propose that the micromeres induce adjacent cells to form SMCs, possibly by presenting a ligand for the Notch receptor.     

Differentiation of the body wall musculature in Macrostomum and Hoploplana (Turbellaria,Platyhelminthes)

Using the Phalloidin-Rhodamine flourescence-labelling technique for F-actin, we have studied the development of the body wall musculature in Macrostomum hystricinum marinum and in thepolyclad Hoploplana inquilina. The structure of the muscle grid in the freshly hatched Macrostomum (see also Rieger & Salvenmoser, 1991) and the young larva of Holplana served as reference systems for the embryonic development of the body wall musculature. In Macrostomum muscle fiber differentiation starts around 60% of developmental time between egg-laying and hatching, and in Hoploplana around 80% of embryonic development.In Macrostomum, early stages show TV-antenna-like arrangements of one longitudinal and several circular fibers. In Hoploplana our preliminary results show a particularly large, longitudinal fiber on either side of the body. These primary longitudinal fibers may serve as a founder cell for other longitudinal fibers and as spatial guides for the circular muscles. Similar founder cells have been reported during early muscle differentiation in leeches (Jellies & Kristan, 1988; Jellies, 1990). In Hoploplana, a special muscle system is present at the outset under the apical organ. It consists of what seems to be a spirally toranged fiber — when seen in head-on view — and of two additional fibers crossing this spiral, from the later developing posterior to the anterior lobe.TEM-studies of embryos of Macrostomum suggest that the longitudinal nerve cords represent an important guide during early differentiation of the pattern within the body wall musculature. Young stages of myoblasts can be identified along the main lateral nerve cord. Commonly, the myoblasts are seen to alternate with young neurons in their position along the nerve cord. Embryonic stages of Macrostomum hystricinum marinum were obtained from our cultures (Rieger et al., 1988). Immediately prior to fixation (Paraformaldehyde, Stephanini's fixative) the eggshells were punctured with tungsten needles. We noted some variability of developmental time for certain embryonic stages, which we cannot explain. Developmental stages of Hoploplana inquilina were collected at the Marine Biological Laboratory, Woods Hole, MA, USA according to the procedure outlined in Boyer (1987) and Boyer (1989). They have been timed in relation to normal developmental time to an early Müller's larva at about 100 hours.     

Differential expression of an endogenous mannose-binding protein R1 during muscle development and regeneration delineating its role in myoblast fusion

The role of the endogenous brain carbohydrate-binding proteinR1 in muscle cell development and regeneration was analysedboth in vivo and in vitro. In vivo, R1 was developmentally regulated,with an embryonic 65 000 subunit and a neonatal 67 000 subunit,being replaced progressively by a 135 000 adult form. LectinR1 was intracellularly localized at birth and in the prenatalperiod. During development and at the time of myoblast fusion,the antigen was progressively found at the surface, where itremained at low levels in the adult. In vitro, in pure myoblastcultures, only the embryonic form was present. The ultrastructuralstudies indicated that the lectin could participate in the membranefusion process during myoblast fusion. The specific role inmyoblast fusion, derived from the ultrastructural localizationof R1, was evidenced by a strong inhibitory effect of anti-R1 Fab fragments (10–100 µg/ml), relative to controlFab fragments. In vivo, the embryonic subunit pattern and subcellulardistribution of R1 reappeared in muscle cells after lesion ofthe adult muscle. This suggested that, as observed in vitro,R1 participated in vivo in the phenomenon of myoblast fusion.Similar modifications in subunit expression were observed inmuscles after denemation (the embryonic form of lectin R1 reappearingafter lesion), suggesting that R1 could be involved in the processof neuromuscular junction formation. Thus, it is proposed thatthe carbohydrate-binding protein R1 is an important recognitionmolecule for the formation of myotubes. Its potential involvementin a recognition process between axons and muscle cells duringneuromuscular junction formation is discussed. culture development fusion N-glycan glycoprotein lectin mannose myoblast     

Features of glycogen phosphorylase from the body wall musculature of the lugworm Arenicola marina and the mode of activation during anoxia

The activities of glycogen phosphorylases a and b from the body wall musculature of the marine worm Arenicola marina (Annelida, Polychaeta) were determined after various periods of anoxia. Already under normoxic conditions one third of the total activity was produced from the a form. During anoxia the ratio of both forms as well as the total activity did not change. The activity of soluble phosphorylase kinase was comparatively low in this tissue 4.3 +/- 1.2 nmol . min-1 . (g wet wt.)-1; the fast twitching tail muscle of shrimps, e.g., had a 10-fold higher phosphorylase kinase activity, whereas phosphorylase activities in both tissues were about the same 2.3 +/- 0.5 mumol . min-1 . (g wet wt.)-1. Glycogen phosphorylase b was purified from the body wall tissue of the marine worm in one step by 5'-AMP-Sepharose resulting in a single protein band in SDS-PAGE. This preparation was accepted as substrate by the phosphorylase kinase from rabbit muscle but a complete phosphorylation could not be achieved. The molecular mass of native phosphorylase was approximately 216 kDa, that of subunits 95 kDa indicating that the enzyme exists as a dimer. There were no isozymes in this preparation, the RF-value (0.17) of the single band in PAGE ranged between those of the isozymes from mice hearts. The activities of phosphorylases b and a were similarly dependent on pH and temperature but differed drastically in the affinities to phosphate and AMP. In presence of 1 mM AMP the app. Km of phosphorylase a for phosphate was 16 mM, that of phosphorylase b above 100 mM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Anaerobic reduction of fumarate in the body wall musculature ofArenicola marina (Polychaeta)

Summary The reduction of fumarate, which is a characteristic feature of anoxic catabolism of some invertebrates, was investigated in mitochondria or mitochondrial fragments prepared from the body wall musculature of the lug-wormArenicola marina (Annelida, Polychaeta).A coupling of the reduction of fumarate to succinate to the oxidation of NADH was demonstrated.The pathway of hydrogen transfer from NADH to fumarate was studied by using specific inhibitors of the respiratory chain. From the results it is concluded that parts of the respiratory chain are involved.During anaerobiosis mitochondria formed succinate at a high rate from malate which had been added as substrate. The formation of succinate is coupled to oxidative phosphorylation. The ratio ATP-production/formation of succinate was found to be 0.6 to 0.8.Succinate formation from malate is inhibited by arsenite and monofluoroacetate.TheK _m for fumarate of the fumarate reductase inArenicola body wall musculature is 2.5×10^–5 M.Abbreviations Ap₅A P¹,P⁵-di(adenosine-5-)pentaphosphate - APAD acetylpyridine adenine dinucleotide - DNP 2,4-dinitrophenol - fw fresh weight (of body wall musculature) - NaFAc sodiummonofluoroacetic acid - PCA perchloric acid - PEP phosphoenolpyruvate Supported by Deutsche Forschungsgemeinschaft (Ze 40/13, Ze 40/14 and Gr 456/5)     

Variation in fructooligosaccharide contents during plant development and in different cultivars of durum wheat

Abstract

Fructooligosaccharides (FOS) have received a great deal of attention for their properties as prebiotic components. Several plants store FOS in their tissues as carbohydrate source or as osmoregulators. In comparison with the other natural FOS sources, cereals, in particular durum wheat, store a significant amount of these oligosaccharides during their growth. To gain a deeper insight on the relationships between FOS and plant/kernel development, FOS levels were analysed in the grains and stems of a widely cultivated durum wheat cultivar (Simeto), during the whole period of grain filling. In addition, a broad analysis of FOS contents was carried out by comparing their levels in the stems and grains of 45 cultivars of durum wheat harvested at two development stages. Results show a large variation among cultivars in their capability to metabolise FOS.     

Differentiation of the body wall musculature in Macrostomum hystricinum marinum and Hoploplana inquilina (Plathelminthes), as models for muscle development in lower Spiralia

Recent studies on the differentiation of the body wall musculature in a medicinal leech and in the free-living plathelminth Macrostomum hystricinum marinum, Beklemischev 1950 provide the first evidence of a complex developmental signalling pattern, possibly involving stem cells and the nervous system, in the organization of the muscle grid formed by developing myocytes. To enhance further our understanding of the ontogenetic and phylogenetic origin of such muscle grids, which consist of circular, longitudinal and diagonal muscle fibres, we have undertaken a study of muscle development in the polyclad flatworm Hoploplana inquilina Wheeler 1894 in collaboration with the Marine Biological Laboratory, Woods Hole. We have also continued our examination of the development of the body wall musculature in M. hystricinum. Both species were studied using rhodamine-phalloidin staining and transmission electron microscopy. Additional visualization of the fluorescent whole mount preparations was performed with confocal laser microscopy and digital image processing. The results of our investigation suggest that: (1) the mechanism of muscle development in H. inquilina supports the deeply rooted concept of bilateral symmetry (right and left longitudinal founder muscle), and (2) a first circular muscle in this species develops on the border between an anterior body unit and the main body; a caudalmost region is less obvious. The presence of a spiral muscle functioning as a circular muscle system of the head region points to a separate developmental mechanism for this region and the trunk. In contrast to H. inquilina, where the larval stage forces an intermediate restructuring of the musculature of the body wall before the adult body shape is finally developed, the formation of the body wall musculature of M. hystricinum already seems constrained by the adult body shape.     

Selective effects of actinomycin D on myosin biosynthesis and myoblast fusion during myogenesis in culture

The regulation of myosin biosynthesis in primary culture from embryonic chick muscle has been studied by measuring the residual myosin synthesis and the rate of fusion after actinomycin treatment. It is shown that while the rate of fusion is completely unaffected by an 8-h treatment with the inhibitor, throughout culture, the myosin biosynthesis shows different levels of inhibition during culture. In fact at onset of fusion (53 h) myosin synthesis is strongly inhibited, while at a later period following fusion (100 h) it is scarcely affected by the treatment.     

Functional dicer is necessary for appropriate specification of radial glia during early development of mouse telencephalon

Early telencephalic development involves transformation of neuroepithelial stem cells into radial glia, which are themselves neuronal progenitors, around the time when the tissue begins to generate postmitotic neurons. To achieve this transformation, radial precursors express a specific combination of proteins. We investigate the hypothesis that micro RNAs regulate the ability of the early telencephalic progenitors to establish radial glia. We ablate functional Dicer, which is required for the generation of mature micro RNAs, by conditionally mutating the Dicer1 gene in the early embryonic telencephalon and analyse the molecular specification of radial glia as well as their progeny, namely postmitotic neurons and basal progenitors. Conditional mutation of Dicer1 from the telencephalon at around embryonic day 8 does not prevent morphological development of radial glia, but their expression of Nestin, Sox9, and ErbB2 is abnormally low. The population of basal progenitors, which are generated by the radial glia, is disorganised and expanded in Dicer1^-/- dorsal telencephalon. While the proportion of cells expressing markers of postmitotic neurons is unchanged, their laminar organisation in the telencephalic wall is disrupted suggesting a defect in radial glial guided migration. We found that the laminar disruption could not be accounted for by a reduction of the population of Cajal Retzius neurons. Together, our data suggest novel roles for micro RNAs during early development of progenitor cells in the embryonic telencephalon.     

Group I PAKs function downstream of Rac to promote podosome invasion during myoblast fusion in vivo

The p21-activated kinases (PAKs) play essential roles in diverse cellular processes and are required for cell proliferation, apoptosis, polarity establishment, migration, and cell shape changes. Here, we have identified a novel function for the group I PAKs in cell–cell fusion. We show that the two Drosophila group I PAKs, DPak3 and DPak1, have partially redundant functions in myoblast fusion in vivo, with DPak3 playing a major role. DPak3 is enriched at the site of fusion colocalizing with the F-actin focus within a podosome-like structure (PLS), and promotes actin filament assembly during PLS invasion. Although the small GTPase Rac is involved in DPak3 activation and recruitment to the PLS, the kinase activity of DPak3 is required for effective PLS invasion. We propose a model whereby group I PAKs act downstream of Rac to organize the actin filaments within the PLS into a dense focus, which in turn promotes PLS invasion and fusion pore initiation during myoblast fusion.     

A critical function for the actin cytoskeleton in targeted exocytosis of prefusion vesicles during myoblast fusion

Myoblast fusion is an essential step during muscle differentiation. Previous studies in Drosophila have revealed a signaling pathway that relays the fusion signal from the plasma membrane to the actin cytoskeleton. However, the function for the actin cytoskeleton in myoblast fusion remains unclear. Here we describe the characterization of solitary (sltr), a component of the myoblast fusion signaling cascade. sltr encodes the Drosophila ortholog of the mammalian WASP-interacting protein. Sltr is recruited to sites of fusion by the fusion-competent cell-specific receptor Sns and acts as a positive regulator for actin polymerization at these sites. Electron microscopy analysis suggests that formation of F-actin-enriched foci at sites of fusion is involved in the proper targeting and coating of prefusion vesicles. These studies reveal a surprising cell-type specificity of Sltr-mediated actin polymerization in myoblast fusion, and demonstrate that targeted exocytosis of prefusion vesicles is a critical step prior to plasma membrane fusion.