The origin of skeletal muscle stem cells in the embryo and the adult.

Skeletal muscle progenitors are specified during embryogenesis and in addition have recently been found to be generated from either mesenchymal or neural stem cells in the adult. We review recent progress in identifying the signals and transcription factors that control skeletal muscle formation during embryogenesis and in the adult.

FGF is required for posterior neural patterning but not for neural induction.

Fibroblast growth factor (FGF) has been implicated in a variety of developmental processes including posterior mesoderm and neural patterning. Previous work has led to contradictory roles for FGF in neural induction and anteroposterior neural patterning. Launay et al. (Development 122, 869-880, 1996) suggested a requirement for FGF in anterior neural induction. In contrast, Kroll and Amaya (Development 122, 3173-3183, 1996) and Bang et al. (Development 124, 2075-2085, 1997) proposed that FGF is not required for early neural patterning. Here we use a loss-of-function assay to examine whether FGF is required for neural patterning in three experimental situations: (i) in Xenopus early embryos, (ii) in embryonic explants consisting of presumptive dorsal mesoderm and neurectoderm (Keller explants), and (iii) in explants of dorsal ectoderm and posterior mesoderm in which FGF signaling is specifically blocked in the ectoderm. When cultured until tailbud stages, Keller explants develop neural tissue with normal anteroposterior pattern. Overexpression of the dominant-negative FGF receptor (XFD) in Keller explants inhibited the posterior neural markers En-2, Krox-20, and HoxB9, but not the panneural marker nrp-1 and the anterior neurectodermal markers XAG-1 and Xotx-2. Similar results were seen in whole embryos, but only when XFD RNA was targeted to both the dorsal and lateral regions. In contrast, addition of FGF to Keller explants resulted in a shift of the midbrain-hindbrain boundary marker En-2 to a more anterior position normally fated to become cement gland. We also determined whether FGF is required specifically by the neurectoderm for anteroposterior neural patterning. Recombinants of dorsal ectoderm and posterior mesoderm were made in which FGF was specifically blocked in the ectoderm. Spinal cord and hindbrain markers were inhibited in these recombinants, whereas anterior markers and cement gland development were enhanced. Our results demonstrate that FGF is important for posterior development in both mesoderm and neurectoderm and that neural induction and posteriorization represent separable developmental events.

Properties of the dorsal activity found in the vegetal cortical cytoplasm of Xenopus eggs.

The Xenopus egg contains a maternal dorsal determinant that is specifically localized to the vegetal cortex. We have previously shown that vegetal cortical cytoplasm can generate a full dorsal axis when it is injected into ventral vegetal blastomeres of a cleavage-stage embryo. In this study, we have defined further the properties of the dorsal activity. The cortical dorsal activity arises during oocyte maturation after germinal vesicle breakdown. When injected into the four extreme animal pole blastomeres of ultraviolet-ventralized 32-cell embryos, vegetal cortical cytoplasm partially rescued dorsal axial structures. As revealed by lineage tracing, these axial structures formed ectopically from the progeny of the cells that were injected. Injection of animal cortical cytoplasm had no effect. When mid-blastula (stage 8) animal caps from these injected embryos were isolated and cultured, both vegetal cortex-enriched and animal cortex-enriched animal caps produced only epidermis. Therefore vegetal cortex, on its own, is not a mesoderm inducer. Between stage 8 (blastula) and stage 10 (gastrula), a ventral mesoderm-inducing signal spreads from vegetal cells towards the animal pole. We tested whether this natural mesoderm-inducing factor interacts with the activity found in the vegetal cortex. Injection of vegetal cortex enhanced the formation of neural tissue and cement gland when animal caps were isolated at stage 10. When cultured from stage 8 in the presence of the ventral mesoderm-inducing fibroblast growth factor, animal caps enriched in vegetal cortex developed significantly more neural tissue and cement gland than ones enriched in animal cortex. These results indicate that the dorsal activity localized to the egg vegetal cortex alters the response of cells to mesoderm inducers.

Cortical cytoplasm, which induces dorsal axis formation in Xenopus, is inactivated by UV irradiation of the oocyte.

Localized maternal determinants control the formation of dorsal axial structures in Xenopus embryos. To examine the spatial distribution of dorsal determinants, we injected cytoplasm from various regions of the egg and 16-cell embryo into the ventral vegetal cells of a 16-cell recipient embryo. Cortical cytoplasm from the egg vegetal surface induced the formation of a secondary dorsal axis in 53% of recipients. In contrast, animal cortical, equatorial cortical and vegetal deep cytoplasm never induced secondary axis formation. We also compared the axis-inducing ability of animal versus vegetal dorsal cortical cytoplasm from 16-cell embryos. Significantly more dorsalizing activity was found in vegetal dorsal cytoplasm compared to animal dorsal cytoplasm at this stage. Previous work has shown that UV irradiation of the vegetal surface of either prophase I oocytes, or fertilized eggs, leads to the development of embryos that lack dorsal structures. Egg vegetal cortical cytoplasm was capable of restoring the dorsal axis of 16-cell recipient embryos derived from UV-irradiated oocytes or fertilized eggs. We also tested the axis inducing ability of cytoplasm obtained when UV-irradiated oocytes and eggs were treated as donors of cytoplasm. While vegetal cortical cytoplasm from UV-irradiated fertilized eggs retains its dorsalizing activity, cytoplasm obtained from eggs, UV irradiated as oocytes, does not. The egg vegetal cortex provides a suitable source for the isolation of maternal dorsal determinants. In addition, since UV irradiation of the oocyte vegetal surface destroys the dorsalizing activity of transferred cytoplasm, UV can be used to further restrict possible candidates for such determinants.

Arrangement of kinetochore proteins and satellite DNA in neuronal interphase nuclei: changes induced by gamma-aminobutyric acid (GABA).

Discrete chromatin domains occupy specific nuclear compartments in neuronal interphase nuclei. Nuclear rotation, defined as the motion of interphase chromatin domains, has been proposed to function in the placement of specific chromatin domains to nuclear compartments which are appropriate to the physiological state or the state of differentiation of the cell. Rates of this chromatin motion may be increased by agents, including gamma-amino butyric acid (GABA), which may cause changes in gene expression. To test whether GABA also causes a spatial rearrangement of specific chromatin domains, the three-dimensional distribution of kinetochores in nuclei of mouse dorsal root ganglion neurons was determined by immunofluorescence. In addition, centromeric satellite DNA sequences were localized by in situ hybridization using a biotinylated mouse satellite DNA probe followed by immunofluorescence. Kinetochores occurred in clusters, associated with nucleoli or in intermediate nucleoplasmic regions, between the nucleolus and the nuclear membrane. Clusters of satellite DNA sequences were found either associated with nucleoli or throughout the nucleoplasm. Strikingly, nucleoplasmic kinetochores consistently occupied a 5-microns distance from the nuclear center, representing 70% of the spherical nuclear radius. Exposure of neurons to GABA induced a significant reorganization of kinetochores which may represent movement of chromosomes to alternate nuclear compartments to accommodate a new transcriptional state.