Richard P. Elinson

The entire mesodermal mantle behaves as Spemann's organizer in dorsoanterior enhanced Xenopus laevis embryos

The body plan of Xenopus laevis can be respecified by briefly exposing early cleavage stage embryos to lithium. Such embryos develop exaggerated dorsoanterior structures such as a radial eye and cement gland (K.R. Kao, Y. Masui, and R.P. Elinson, 1986, Nature (London) 322, 371-373). In this paper, we demonstrate that the enhanced dorsoanterior phenotype results from an overcommitment of mesoderm to dorsoanterior mesoderm. Histological and immunohistochemical observations reveal that the embryos have a greatly enlarged notochord with very little muscle tissue. In addition, they develop a radial, beating heart, suggesting that lithium also specifies anterior mesoderm and pharyngeal endoderm. Randomly oriented diametrically opposed marginal zone grafts from lithium-treated embryos, when transplanted into ultraviolet (uv)-irradiated axis-deficient hosts, rescue dorsal axial structures. These transplantation experiments demonstrate that the entire marginal zone of the early gastrula consists of presumptive dorsal mesoderm. Vital dye marking experiments also indicate that the entire marginal zone maps to the prominent proboscis that is composed of chordamesoderm and represents the long axis of the embryo. These results suggest that lithium respecifies the mesoderm of Xenopus laevis embryos so that it differentiates into the Spemann organizer. We suggest that the origin of the dorsoanterior enhanced phenotypes generated by lithium and the dorsoanterior deficient phenotypes generated by uv irradiation are due to relative quantities of organizer. Our evidence demonstrates the existence of a continuum of body plan phenotypes based on this premise.

A transient array of parallel microtubules in frog eggs: potential tracks for a cytoplasmic rotation that specifies the dorso-ventral axis.

The dorsoventral axis of the frog embryo is specified by a rotation of the egg cytoplasm relative to the cortex. When eggs undergoing the cortical/cytoplasmic rotation were examined by immunocytochemistry and electron microscopy, an extensive array of parallel microtubules was found covering the vegetal hemisphere of the egg. The microtubules were 1-3 microns deep from the plasma membrane and were aligned parallel to the direction of rotation. They formed at the start of rotation and disappeared at its completion. Colchicine and uv irradiation, inhibitors of the rotation, prevented the formation of the parallel microtubules. Based on these properties, we suggest that the parallel microtubules serve as tracks for the cortical/cytoplasmic rotation which specifies the dorsoventral axis of the embryo.

A fast block to polyspermy in frogs mediated by changes in the membrane potential

Abstract The role of the egg membrane potential in the prevention of polyspermy in Rana pipiens was studied with intracellular microelectrodes and ion-substituted media. At fertilization, the egg membrane potential shifts from a resting value of −28 to +8 mV in a single step of less than 1 sec. A second, slower shift reaches a maximum amplitude of +17 mV; the membrane potential is positive for a total of 21 min. When the membrane potential of unfertilized eggs exposed to sperm was held at +1 to +22 mV for 30 min by injecting current through a second intracellular electrode, the initiation of the first cleavage furrow was delayed about 20 min, suggesting that the eggs were not fertilized while the membrane potential was positive. Injection of a similar amount of current after fertilization did not delay cleavage. Furthermore, fertilization in ion-substituted media suggests a correlation between the maximum amplitude of the positive-going shift and the incidence of polyspermy. Up to 25% of eggs were polyspermic when inseminated in the presence of NaI, and the maximum amplitude was reduced to −20 mV when eggs were fertilized in 40 mM NaI. In contrast, fertilization in 40 mM NaCl reduced the maximum amplitude only to +6 mV, and produced no polyspermy. In solutions of NaBr, intermediate effects on the membrane potential and polyspermy were seen. Comparable results were obtained with the toad, Bufo americanus. We conclude that the membrane potential shift prevents polyspermy.

β-TrCP is a negative regulator of the Wnt/β-catenin signaling pathway and dorsal axis formation in Xenopus embryos

The Wnt/beta-catenin signaling pathway is responsible for the establishment of dorsoventral axis of Xenopus embryos. The recent finding of the F-box/WD40-repeat protein slimb in Drosophila, whose loss-of-function mutation causes ectopic activation of wingless signaling (Jiang, J., Struhl, G., 1998. Nature 391, 493-496), led us to examine the role of its vertebrate homolog betaTrCp in the Wnt/beta-catenin signaling and dorsal axis formation in Xenopus embryos. Co-injection of betaTrCp mRNA diminished Xwnt8 mRNA-induced axis formation and expression of Siamois and Xnr3, suggesting that betaTrCP is a negative regulator of the Wnt/beta-catenin signaling pathway. An mRNA for a betaTrCp mutant construct (DeltaF), which lacked the F-box domain, induced an ectopic axis and expression of Siamois and Xnr3. Because this activity of DeltaF was suppressed by co-injection of DeltaF TrCP mRNA, DeltaF likely acts in a dominant negative fashion. The activity of DeltaF was diminished by C-cadherin, glycogen synthase kinase 3 and Axin, but not by a dominant negative dishevelled. These results suggest that betaTrCp can act as a negative regulator of dorsal axis formation in Xenopus embryos.

Antero-posterior tissue polarity links mesoderm convergent extension to axial patterning

Remodelling its shape, or morphogenesis, is a fundamental property of living tissue. It underlies much of embryonic development and numerous pathologies. Convergent extension (CE) of the axial mesoderm of vertebrates is an intensively studied model for morphogenetic processes that rely on cell rearrangement. It involves the intercalation of polarized cells perpendicular to the antero-posterior (AP) axis, which narrows and lengthens the tissue. Several genes have been identified that regulate cell behaviour underlying CE in zebrafish and Xenopus. Many of these are homologues of genes that control epithelial planar cell polarity in Drosophila. However, elongation of axial mesoderm must be also coordinated with the pattern of AP tissue specification to generate a normal larval morphology. At present, the long-range control that orients CE with respect to embryonic axes is not understood. Here we show that the chordamesoderm of Xenopus possesses an intrinsic AP polarity that is necessary for CE, functions in parallel to Wnt/planar cell polarity signalling, and determines the direction of tissue elongation. The mechanism that establishes AP polarity involves graded activin-like signalling and directly links mesoderm AP patterning to CE.

Site of sperm entry and a cortical contraction associated with egg activation in the frog Rana pipens

Abstract The region of the frog egg that is receptive to fertilization was determined. As an approximation to the site of sperm entry, the start of the male pronuclear penetration path within the egg was made visible externally by bleaching fixed eggs. A bleached egg had a pigment accumulation on its surface corresponding to the start of the penetration path. The accumulation characteristically changed shape with cortical movements prior to first cleavage, and most accumulations (path starts) were within 60° of the animal pole. Localized inseminations and an analysis of the distribution of failures of fertilization at the egg plasma membrane demonstrated that few if any sperm entered the vegetal region of the egg. Localized inseminations, however, demonstrated that sperm entered between 60° from the animal pole and the animal-vegetal margin. Although sperm entry occurred throughout the animal region, most penetration paths started within 60° of the animal pole. To account for this, the sperm nucleus must move towards the animal pole prior to starting the penetration path. This movement appeared to be due to a contraction of the cortex towards the animal pole that occurred 3–4 min after activation of the egg.

Dorsalization of mesoderm induction by lithium

Lithium dorsalizes the body plan of Xenopus embryos when administered at the 32-cell stage (K.R. Kao and R.P. Elinson, 1988, Dev. Biol. 127, 64-77). In this paper, we have attempted to determine the effects of lithium on mesoderm induction, in order to localize the target of action of lithium. In the 32-cell embryo, the vegetal-most tier 4 cells are able to induce dorsal development in the overlying, equatorial tier 3 cells (R.L. Gimlich and J.C. Gerhart, 1984, Dev. Biol. 104, 117-130). Our experiments show that microinjection of lithium into either tier 3 or tier 4 cells of ultraviolet-irradiated, dorsoanterior-deficient embryos rescues normal development. Lineage tracer studies show that only tier 3-injected cells contribute progeny to dorsal axial structures while tier 4-injected cells contribute progeny to endoderm. Sandwich explants between animal caps and ventral vegetal cells cause induction of large amounts of muscle in the explants if either caps or vegetal cells are pretreated with lithium. Similarly, fibroblast growth factor-mediated mesoderm induction is also modified by lithium so that muscle is induced instead of ventral mesoderm. We conclude that lithium dorsalizes the response of animal cells to mesoderm induction signals, while not acting directly as a mesoderm inducer itself. The target of action of lithium is likely the third tier of cells of the 32-cell embryo.

Frogs without polliwogs: Evolution of anuran direct development

Direct development is the assumption of the adult morphology without progression through an intervening, morphologically distinct, free‐living larval phase. We discuss the ecological factors contributing to the evolution of this derived life‐history strategy in frogs, and the developmental modifications that facilitate such an unusual mode of embryogenesis. Studies on the Puerto Rican tree frog, Eleutherodactylus coqui, have identified several such modifications, including developmental adaptations for dealing with increased egg size, and loss of tadpole structures. Surprisingly, this direct developer still undergoes a thyroid hormone‐dependent metamorphosis, which occurs before hatching. We suggest how the ancestral biphasic developmental pattern may have been rearranged during the evolution of direct development. BioEssays 23:233–241, 2001.

Fertilization in Amphibians: The Ancestry of the Block to Polyspermy

Publisher Summary Amphibians exhibit both styles of fertilization: monospermy and polyspermy. Monospermy is confined to tailless frogs and toads that comprise the order Anura, while physiological polyspermy is found in the tailed newts and salamanders of the order Urodela. This chapter reviews the process of fertilization in amphibians, particularly as it relates to polyspermy. The chapter discusses ways to explore the nature of the cytological ancestry. Amphibians exhibit a great diversity of reproductive patterns. This diversity makes the amphibians ideal animals for examining the cellular evolution of developmental processes. Fertilization in anurans and urodeles is so different that it represents an important character distinguishing the orders. Comparison between fertilization in anurans and urodeles shows a mirror image dichotomy with respect to polyspermy. The anuran egg has a fast and slow block to polyspermy, and it has no protection when more than one sperm enters. On the other hand, the urodele egg lacks both external blocks to polyspermy but can deal with accessory nuclei within the egg. The arrangement of components in the membranes and in the cortical granules is considered important for the polyspermy-blocking reactions.

Cytoplasmic phases in the first cell cycle of the activated frog egg

The first cell cycle of the activated frog egg is longer than subsequent cycles and several developmentally important events such as the determination of bilateral symmetry occur at this time. When eggs of Rana pipiens or Xenopus laevis are dissected at times after activation, differences in the consistency of the animal half cytoplasm can be detected visually, and the first cell cycle has been divided into four cytoplasmic phases on this basis. Phase 1 includes the events of activation and lasts about one-third of the first cycle. In phase 2, the cytoplasm becomes fluid except for the rigid, growing sperm aster, and most of the migration of the pronuclei occurs in phase 2. In phase 3, the cytoplasm becomes firm whether or not a sperm aster had been present, and the grey crescent forms, indicating the plane of bilateral symmetry. The firmness of the cytoplasm is colchicine sensitive but cytochalasin B insensitive as is grey crescent formation. In phase 4, the cortex detaches from the firm cytoplasm, and the firmness is now cytochalasin B sensitive and colchicine insensitive. The changes in cytoplasmic consistency during the first cell cycle probably reflect changes in the cytoskeleton, and the cytoplasmic consistency is functionally correlated with developmental events in the first cell cycle.