Applications of confocal microscopy to study the roles of the cytoskeleton during early embryogenesis in amphibians
No abstract available
NASA Discipline Developmental Biology↗
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Reduced receptor aggregation and altered cytoskeleton in cultured myocytes after space-flight
We carried out parallel experiments first on the slow clinostat and then in space-flight to examine the effects of altered gravity on the aggregation of the nicotinic acetylcholine receptors and the structure of the cytoskeleton in cultured Xenopus embryonic muscle cells. By examining the concordance between results from space flight and the clinostat, we tested whether the slow clinostat is a relevant simulation paradigm. Space-flown cells showed marked changes in the distribution and organization of actin filaments and had a reduced incidence of acetylcholine receptor aggregates at the site of contact with polystyrene beads. Similar effects were found after clinostat rotation. The sensitivity of synaptic receptor aggregation and cytoskeletal morphology suggests that in the microgravity of space cell behavior may be importantly altered.
STS-56 Shuttle Project↗
Vector-averaged gravity does not alter acetylcholine receptor single channel properties
To examine the physiological sensitivity of membrane receptors to altered gravity, we examined the single channel properties of the acetylcholine receptor (AChR), in co-cultures of Xenopus myocytes and neurons, to vector-averaged gravity in the clinostat. This experimental paradigm produces an environment in which, from the cell's perspective, the gravitational vector is "nulled" by continuous averaging. In that respect, the clinostat simulates one aspect of space microgravity where the gravity force is greatly reduced. After clinorotation, the AChR channel mean open-time and conductance were statistically not different from control values but showed a rotation-dependent trend that suggests a process of cellular adaptation to clinorotation. These findings therefore suggest that the ACHR channel function may not be affected in the microgravity of space despite changes in the receptor's cellular organization.
NASA Discipline Cell Biology↗
Blastomeres show differential fate changes in 8-cell Xenopus laevis embryos that are rotated 90 degrees before first cleavage
To study the mechanisms of dorsal axis specification, the alteration in dorsal cell fate of cleavage stage blastomeres in axis-respecified Xenopus laevis embryos was investigated. Fertilized eggs were rotated 90 degrees with the sperm entry point up or down with respect to the gravitational field. At the 8-cell stage, blastomeres were injected with the lineage tracers, Texas Red- or FITC-Dextran Amines. The distribution of the labeled progeny was mapped at the tail-bud stages (stages 35-38) and compared with the fate map of an 8-cell embryo raised in a normal orientation. As in the normal embryos, each blastomere in the rotated embryos has a characteristic and predictable cell fate. After 90 degrees rotation the blastomeres in the 8-cell stage embryo roughly switched their position by 90 degrees, but the fate of the blastomeres did not simply show a 90 degrees switch appropriate for their new location. Four types of fate change were observed: (i) the normal fate of the blastomere is conserved with little change; (ii) the normal fate is completely changed and a new fate is adopted according to the blastomere's new position: (iii) the normal fate is completely changed, but the new fate is not appropriate for its new position; and (4) the blastomere partially changed its fate and the new fate is a combination of its original fate and a fate appropriate to its new location. According to the changed fates, the blastomeres that adopt dorsal fates were identified in rotated embryos. This identification of dorsal blastomeres provides basic important information for further study of dorsal signaling in Xenopus embryos.
NASA Discipline Developmental Biology↗
Xwnt-8 and lithium can act upon either dorsal mesodermal or neurectodermal cells to cause a loss of forebrain in Xenopus embryos
When Xenopus gastrulae are made to misexpress Xwnt-8, or are exposed to lithium ions, they develop with a loss of anterior structures. In the current study, we have characterized the neural defects produced by either Xwnt-8 or lithium and have examined potential cellular mechanisms underlying this anterior truncation. We find that the primary defect in embryos exposed to lithium at successively earlier stages during gastrulation is a progressive rostral to caudal deletion of the forebrain, while hindbrain and spinal regions of the CNS remain intact. Misexpression of Xwnt-8 during gastrulation produces an identical loss of forebrain. Our results demonstrate that lithium and Wnts can act upon either prospective neural ectodermal cells, or upon dorsal mesodermal cells, to cause a loss of anterior pattern. Specifically, ectodermal cells isolated from lithium- or Wnt-exposed embryos are unable to form anterior neural tissue in response to inductive signals from normal dorsal mesoderm. In addition, although dorsal mesodermal cells from lithium- or Wnt-exposed embryos are specified properly, and produce normal levels of the anterior neural inducing molecules noggin and chordin, they show a greatly reduced capacity to induce anterior neural tissue in conjugated ectoderm. Taken together, our results are consistent with a model in which Wnt- or lithium-mediated signals can induce either mesodermal or ectodermal cells to produce a dominant posteriorizing morphogen which respecifies anterior neural tissue as posterior.
Non-NASA Center↗
Relocation of mitochondria to the prospective dorsal marginal zone during Xenopus embryogenesis
Dorsal-ventral axis formation in Xenopus laevis begins with a cytoplasmic rotation during the first cell cycle and culminates in a series of cell interactions and movements during gastrulation and neurulation that lead to the formation of dorsal-anterior structures. Evidence reported here indicates that mitochondria are differentially redistributed along the prospective dorsal-ventral axis as a consequence of the cortical-cytoplasmic rotation during the first cell cycle. This finding reinvigorates a possibility that has been considered for many years: asymmetries in cytoplasmic components and metabolic activities contribute to the development of morphological asymmetries.
Non-NASA Center↗
Deep cytoplasmic rearrangements in axis-respecified Xenopus embryos
In fertilized eggs of the frog Xenopus, the vegetal yolk mass rotates away from the future dorsal side (J. P. Vincent and J. Gerhart, 1987, Dev. Biol. 123, 526-539), and a major rearrangement of the deep animal hemisphere cytoplasm produces a characteristic swirl in the prospective dorsal side (M. V. Danilchik and J. M. Denegre, 1991, Development 111, 845-856). The relationship between this swirl and determination of the dorsal-ventral axis was further investigated by attempting to experimentally separate the positions of the swirl and the dorsal-ventral axis. Eggs were obliquely oriented in the gravity field to respecify the direction of yolk mass rotation and the position of the dorsal-ventral axis. When yolk mass rotation occurred in the absence of a sperm, as in activated eggs, a swirl pattern formed on the side away from which the yolk mass had rotated. In fertilized eggs tipped with the sperm entry point (SEP) down or to the side, swirl patterns were always found to form on the side away from which the yolk mass was displaced. However, in eggs tipped SEP up, in which the yolk mass was forced to rotate away from the SEP, more complicated rearrangements were observed in addition to the rotation-oriented swirl. Because the direction of yolk mass rotation was found to be influenced by both gravity and the actual position of the SEP in obliquely oriented eggs (SEP to the side), such complicated rearrangement patterns may result from opposing forces generated by both yolk mass rotation and the expanding sperm aster. Thus, except in cases in which the influences of SEP position and unit gravity opposed each other, it was not possible to experimentally separate the position of the deep cytoplasmic swirl from the direction of yolk mass rotation, and therefore the position of the prospective dorsal side.
Non-NASA Center↗
Planar induction of anteroposterior pattern in the developing central nervous system of Xenopus laevis
It has long been thought that anteroposterior (A-P) pattern in the vertebrate central nervous system is induced in the embryo's dorsal ectoderm exclusively by signals passing vertically from underlying, patterned dorsal mesoderm. Explants from early gastrulae of the frog Xenopus laevis were prepared in which vertical contact between dorsal ectoderm and mesoderm was prevented but planar contact was maintained. In these, four position-specific neural markers (engrailed-2, Krox-20, XlHbox 1, and XlHbox 6) were expressed in the ectoderm in the same A-P order as in the embryo. Thus, planar signals alone, following a path available in the normal embryo, can induce A-P neural pattern.
NASA Discipline Developmental Biology↗