Modification of Dorsal-Ventral Polarity in Xenopus laevis Embryos Following Withdrawal of Egg Contents before First Cleavage

When fertilized Xenopus laevis eggs were pricked just beneath the marginal zone with a thick glass needle prior to the first cleavage, a small amount of cytoplasm escaped into the exudate. Those eggs were placed in a poly L-lysine-coated plastic dish filled with 10% Ficoll solution. The location of the sperm entrance site (SES) of each egg was marked by scratching the surface of the plastic dish. The pricked embryos were anchored to the dish through poly L-lysine, and developed, therefore, without changing their original position. Consequently, development of the dorsalventral polarity was conveniently monitored with respect to the location of the SES. Embryos which developed from eggs pricked on the side opposite the SES showed modification of the dorsal-ventral polarity: Semi-quantitative studies showed that an exudation approximately 1.5–12.5% of the whole egg contents from the presumptive dorsal side caused a reversal of the dorsal-ventral polarity. That is, the dorsal lip of the blastopore formed on the same side of the SES, whereas the dorsal lip formed on the side opposite the SES in the normal control and sham-operated embryos. Half of the embryos which had larger cytoplasmic exudates more than 12.5% of the whole egg contents failed to form the dorsal lip by the time all controls and the embryos with smaller exudates showed normal dorsal lip formation. When eggs were pricked on the SES side, the normal topographic relationship between the SES and future dorsal lip side was reinforced.     

Multiple extracellular activities in Drosophila egg perivitelline fluid are required for establishment of embryonic dorsal-ventral polarity.

Twelve maternal effect genes (the dorsal group and cactus) are required for the establishment of the embryonic dorsal-ventral axis in the Drosophila embryo. Embryonic dorsal-ventral polarity is defined within the perivitelline compartment surrounding the embryo by the ventral formation of a ligand for the Toll receptor. Here, by transplantation of perivitelline fluid we demonstrate the presence of three separate activities present in the perivitelline fluid that can restore dorsal-ventral polarity to mutant easter, snake, and sp?tzle embryos, respectively. These activities are not capable of defining the polarity of the dorsal-ventral axis; instead they restore structures according to the intrinsic dorsal-ventral polarity of the mutant embryos. They appear to be involved in the ventral formation of a ligand for the Toll protein. This process requires serine proteolytic activity; the injection of serine protease inhibitors into the perivitelline space of wild-type embryos results in the formation of dorsalized embryos.     

The neurogenic locus brainiac cooperates with the Drosophila EGF receptor to establish the ovarian follicle and to determine its dorsal-ventral polarity.

We have characterized the function of a new neurogenic locus, brainiac (brn), during oogenesis. Homozygous brn females lay eggs with fused dorsal appendages, a phenotype associated with torpedo (top) alleles of the Drosophila EGF receptor (DER) locus. By constructing double mutant females for both brn and top, we have found that brn is required for determining the dorsal-ventral polarity of the ovarian follicle. However, embryos from mature brn eggs develop a neurogenic phenotype which can be zygotically rescued if a wild-type sperm fertilizes the egg. This is the first instance of a Drosophila gene required for determination of dorsal-ventral follicle cell fates that is not required for determination of embryonic dorsal-ventral cell fates. The temperature-sensitive period for brn dorsal-ventral patterning begins at the inception of vitellogenesis. The interaction between brn and DER is also required for at least two earlier follicle cell activities which are necessary to establish the ovarian follicle. Prefollicular cells fail to migrate between each oocyte/nurse cell complex, resulting in follicles with multiple sets of oocytes and nurse cells. brn and DER function is also required for establishing and/or maintaining a continuous follicular epithelium around each oocyte/nurse cell complex. These brn functions as well as the brn requirement for determination of dorsal-ventral polarity appear to be genetically separable functions of the brn locus. Genetic mosaic experiments show that brn is required in the germline during these processes whereas the DER is required in the follicle cells. We propose that brn may be part of a germline signaling pathway differentially regulating successive DER-dependent follicle cell activities of migration, division and/or adhesion and determination during oogenesis. These experiments indicate that brn is required in both tyrosine kinase and neurogenic intercellular signaling pathways. Moreover, the functions of brn in oogenesis are distinct from those of Notch and Delta, two other neurogenic loci that are known to be required for follicular development.     

Relocalization of the dorsal protein from the cytoplasm to the nucleus correlates with its function

dorsal is one of the maternally active dorsal-ventral polarity genes of Drosophila and is homologous to the vertebrate proto-oncogene c-rel. In wild-type embryos, the dorsal protein is found in the cytoplasm during cleavage. After the nuclei migrate to the periphery of the embryo, a ventral-to-dorsal gradient of nuclear dorsal protein is established. The formation of the nuclear gradient is disrupted in mutant embryos from other maternally active dorsal-ventral polarity genes: in dorsalized embryos only cytoplasmic protein is observed, while in ventralized embryos the nuclear gradient is shifted dorsally. My findings suggest that nuclear localization is critical for dorsal to function as a morphogen and that the distribution of the dorsal protein determines cell fate along the dorsal-ventral axis.     

Restricting oxygen supply to the prospective dorsal side does not reverse axis polarity in embryos of Xenopus laevis

During the first cell cycle, the prospective dorsal side of the embryo of Xenopus laevis becomes enriched in mitochondria relative to the ventral side. This differential distribution of mitochondria persists throughout early development, but it is not known if it is of functional significance, since there do not appear to be dorsal-ventral differences in metabolic rate. However, the unilateral anaerobiosis experiments of Landström and Løvtrup do suggest a role for energy metabolism in determining axis polarity. These experiments apparently show that restricting oxygen supply to the prospective dorsal side causes a reversal of dorsal-ventral axis polarity. We have reinvestigated this point using cell-marking techniques. We find that although gastrulation is initiated at the open end of the tube, the polarity of neural plate development is unaffected. Thus, definitive dorsal-ventral polarity is not affected by the experimental treatment, and it is unlikely that gradients of energy metabolism have a role in specifying axis polarity in X. laevis.     

Establishment of dorsal-ventral polarity in the Drosophila embryo: the induction of polarity by the Toll gene product

Drosophila females that lack Toll gene activity produce dorsalized embryos, in which all embryonic cells behave like the dorsal cells of the wild-type embryo. Injection of wild-type cytoplasm into young Toll- embryos restores their ability to produce a normal dorsal-ventral pattern in a position-dependent manner. No matter where the cytoplasm is injected relative to the dorsal-ventral axis of the egg shell, the position of the injected cytoplasm defines the ventralmost part of the rescued pattern. Although injection of wild-type cytoplasm into mutants at six other dorsal-group loci also restores the ability to produce lateral and ventral structures, only Toll- embryos lack any residual dorsal-ventral polarity. Experiments suggest that the activity of the Toll product is normally regulated by other dorsal-group genes and that the function of the Toll product is to provide the source for a morphogen gradient in the dorsal-ventral axis of the wild-type embryo.     

The dorsal protein is distributed in a gradient in early Drosophila embryos

dorsal is one of the maternally active dorsal-ventral polarity genes of Drosophila and is closely related to the vertebrate proto-oncogene c-rel. Genetic experiments suggest that dorsal represents one of the last (if not the last) steps in the maternal pathway involved in establishing dorsal-ventral polarity in the early embryo. Even though the dorsal RNA is uniformly distributed in the embryo, we have found that the dorsal protein is specifically localized in peripheral nuclei of syncytial and cellular blastoderm stage embryos, and it is distributed in a ventral-to-dorsal gradient. These findings suggest possible mechanisms for how the dorsal protein may communicate maternal positional information to the zygotic genome.     

Left-right asymmetry in the alimentary canal of the Drosophila embryo

In many animal groups, left-right (LR) asymmetry within the body is observed. The left and right sides of the body are generally defined with reference to the anterior-posterior (AP) and dorsal-ventral (DV) axes. In this study, we investigated whether LR asymmetry is solely dependent on the AP and DV polarities in Drosophila embryos. We focused on the proventriculus, a posterior part of the foregut, and the hindgut because LR asymmetry in these body parts is highly stable in normal embryos. In embryos with a fully reversed AP polarity, LR asymmetry in both the proventriculus and the hindgut was re-oriented in relation to the reversed AP polarity. This demonstrates that inversion of AP polarity does not affect LR asymmetry of these tissues, and implies that LR asymmetry is specified in relation to the AP and DV polarities. Our findings were not consistent with the alternative hypothesis that LR asymmetry is predetermined by maternal signals that localize asymmetrically along the LR axis in the oocyte and/or early embryo.     

Examining pattern formation in mouse, chicken and frog embryos with an En-specific antiserum.

We have raised an antiserum, designated alpha Enhb-1, to a portion of the mouse En-2 protein containing the homeodomain. The antiserum detects both the En-1 and En-2 proteins in mouse, chick and Xenopus embryos by Western blot analysis. Using whole-mount immunohistochemistry, combined in some cases with scanning electron microscopy, we have examined the distribution of the proteins in the early embryos of these species. The major features of expression were similar. The initial production of En protein occurred, just before or during the formation of the first somites, in a band of the anterior neural plate in the prospective mid/hindbrain region. Later in development En-1 protein accumulated in the ventral ectoderm of the developing mouse and chick limb buds, indicating that a dorsal-ventral polarity is present as soon as any limb bud swelling is apparent and that, at least in the mouse, this polarity is established independently of the apical ectodermal ridge. In all three species, alpha Enhb-1 bound to a subset of ventro-lateral differentiating neurons in the spinal cord and hindbrain and their pattern of birth in the mouse reflected the division of the hindbrain into rhombomeres. En-1 protein also accumulated in a lateral stripe of dermatome in the mouse and chick, indicating a dorsal-ventral subdivision of this tissue. The results show that En expression is a good marker for pattern formation in a variety of tissues and will be useful in experimental studies designed to characterize further these processes.     

Characterization of intracellular ice formation in Drosophila melanogaster embryos

Cryomicroscopy and differential scanning calorimetry (DSC) were used to characterize the incidence of intracellular ice formation (IIF) in 12- to 13-hr-old embryos of Drosophila melanogaster (Oregon-R strain P2) as influenced by the state of the eggcase (untreated, dechorionated, or permeabilized), the composition of the suspending medium (with and without cryoprotectants), and the cooling rate. Untreated eggs underwent IIF over a very narrow temperature range when cooled at 4 or 16 degrees C/min with a median temperature of intracellular ice formation (TIIF50) of -28 degrees C. The freezable water volume of untreated eggs was approximately 5.4 nl as determined by DSC. IIF in dechorionated eggs occurred over a much broader temperature range (-13 to -31 degrees C), but the incidence of IIF increased sharply below -24 degrees C, and the cumulative incidence of IIF at -24 degrees C decreased with cooling rate. In permeabilized eggs without cryoprotectants (CPAs), IIF occurred at much warmer temperatures and over a much wider temperature range than in untreated eggs, and the TIIF50 was cooling rate dependent. At low cooling rates (1 to 2 degrees C/min), TIIF50 increased with cooling rate; at intermediate cooling rates (2 to 16 degrees C/min), TIIF50 decreased with cooling rate. The total incidence of IIF in permeabilized eggs was 54% at 1 degree C/min, and volumetric contraction almost always occurred during cooling. Decreasing the cooling rate to 0.5 degree C/min reduced the incidence of IIF to 43%. At a cooling rate of 4 degrees C/min, ethylene glycol reduced the TIIF50 by about 12 degrees C for each unit increase in molarity of CPA (up to 2.0 M) in the suspending medium. The TIIF50 was cooling rate dependent when embryos were preequilibrated with 1.0 M propylene glycol or ethylene glycol, but was not so in 1.0 M DMSO. For embryos equilibrated in 1.5 M ethylene glycol and then held at -5 degrees C for 1 min before further cooling at 1 degree C/min, the incidence of IIF was decreased to 31%. Increasing the duration of the isothermal hold to 10 min reduced the incidence of IIF to 22% and reduced the volume of freezable water in embryos when intracellular ice formation occurred. If the isothermal hold temperature was -7.5 or -10 degrees C, a 10- to 30-min holding time was required to achieve a comparable reduction in the incidence of IIF.     

Scanning electron microscopic analysis of spontaneous and UV-induced abnormal segment patterns in Chironomus samoensis (Diptera,Chironomidae)

Summary The embryonic body pattern of Chironomus samoensis, as well as other chironomids, can be altered dramatically by irradiating their eggs with ultraviolet light (UV). Anterior UV irradiation leads to the formation of double abdomen embryos whose anterior segments are replaced by posterior segments with reversed polarity. Most double abdomens are symmetrical showing a mirror image duplication of the posterior six or seven segments. However, in some cases the anterior end of the double abdomen is shorter, and comprises fewer segments, than its posterior counterpart. These asymmetries range from moderate to extreme. They involve the juxtaposition, at the plane of polarity reversal, of disparate segments. The same range of symmetrical and asymmetrical double abdomens is also formed spontaneously in an apparently mutant strain of C. samoensis. There are striking similarities between this natural variant and the Drosophila melanogaster mutant bicaudal which are also discussed with respect to models of embryonic pattern formation.     

Cryopreservation of sea urchin embryos (Paracentrotus lividus) applied to marine ecotoxicological studies

Current strategies for marine pollution monitoring are based on the integration of chemical and biological techniques. The sea urchin embryo-larval bioassays are among the biological methods most widely used worldwide. Cryopreservation of early embryos of sea urchins could provide a useful tool to overcome one of the main limitations of such bioassays, the availability of high quality biological material all year round. The present study aimed to determine the suitability of several permeant (dimethyl sulfoxide, Me₂SO; propylene glycol, PG; and ethylene glycol, EG) and non-permeant (trehalose, TRE; polyvinylpyrrolidone, PVP) cryoprotectant agents (CPAs) and their combination, for the cryopreservation of eggs and embryos of the sea urchin Paracentrotus lividus. On the basis of the CPAs toxicity, PG and EG, in combination with PVP, seem to be most suitable for the cryopreservation of P. lividus eggs and embryos. Several freezing procedures were also assayed. The most successful freezing regime consisted on cooling from 4 to −12 °C at 1 °C/min, holding for 2 min for seeding, cooling to −20 °C at 0.5 °C/min, and then cooling to −35 °C at 1 °C/min. Maximum normal larvae percentages of 41.5% and 68.5%, and maximum larval growth values of 42.9% and 60.5%, were obtained for frozen fertilized eggs and frozen blastulae, respectively.     

Reversal of cellular polarity and early cell-cell interaction in the embryos of Caenorhabditis elegans

During early embryogenesis of Caenorhabditis elegans the serial stem cell-like cleavages of the germ line cells P0-P3 generate a number of somatic founder cells with different developmental potentials. Observations on partial embryos show that in the first two of these unequal divisions in the germ line the somatic daughter cell comes to lie anterior to the new germ line cell. In the following two, however, the somatic daughter cell comes to lie posterior to the new germ line cell, suggesting a reversal of polarity in the germ line. By the use of a laser microbeam, egg fragments can be extruded from young embryos; the fragments often cleave like partial twins. Depending on whether the fragment is derived from the posterior region of the uncleaved zygote P0 or its daughter P1, the mirror image duplications that are generated are joined at their larger soma-like cells or at their smaller germ line-like cells, respectively. This result is best explained as a reversal of polarity taking place in the germ line cell P2. This notion is strengthened by the finding that partial embryos derived from the posterior region of the P2 cell in late interphase do not undergo stem cell-like (i.e., unequal) cleavages in contrast to those derived from P0 or P1. Finally, an apparent early cell-cell interaction is described which is inconsistent with the classical notion of "mosaic" nematode development: removal of the germline cell P2 results in an altered developmental pattern of its somatic sister cell EMS. A working model is presented linking reversal of polarity and cell-cell interaction and offers an explanation for the unique behavior of the EMS cell in normal development.     

Vax2 inactivation in mouse determines alteration of the eye dorsal-ventral axis, misrouting of the optic fibres and eye coloboma

Vax2 is a homeobox gene whose expression is confined to the ventral region of the prospective neural retina. Overexpression of this gene at early stages of development in Xenopus and in chicken embryos determines a ventralisation of the retina, thus suggesting its role in the molecular pathway that underlies eye development. We describe the generation and characterisation of a mouse with a targeted null mutation of the Vax2 gene. Vax2 homozygous mutant mice display incomplete closure of the optic fissure that leads to eye coloboma. This phenotype is not fully penetrant, suggesting that additional factors contribute to its generation. Vax2 inactivation determines dorsalisation of the expression of mid-late (Ephb2 and Efnb2) but not early (Pax2 and Tbx5) markers of dorsal-ventral polarity in the developing retina. Finally, Vax2 mutant mice exhibit abnormal projections of ventral retinal ganglion cells. In particular, we observed the almost complete absence of ipsilaterally projecting retinal ganglion cells axons in the optic chiasm and alteration of the retinocollicular projections. All these findings indicate that Vax2 is required for the proper closure of the optic fissure, for the establishment of a physiological asymmetry on the dorsal-ventral axis of the eye and for the formation of appropriate retinocollicular connections.     

Windbeutel is required for function and correct subcellular localization of the Drosophila patterning protein Pipe

Drosophila embryonic dorsal-ventral polarity originates in the ovarian follicle through the restriction of pipe gene expression to a ventral subpopulation of follicle cells. Pipe, a homolog of vertebrate glycosaminoglycan-modifying enzymes, directs the ventral activation of an extracellular serine proteolytic cascade which defines the ventral side of the embryo. When pipe is expressed uniformly in the follicle cell layer, a strong ventralization of the resulting embryos is observed. Here, we show that this ventralization is dependent on the other members of the dorsal group of genes controlling dorsal-ventral polarity, but not on the state of the Epidermal Growth Factor Receptor signal transduction pathway which defines egg chamber polarity. Pipe protein expressed in vertebrate tissue culture cells localizes to the endoplasmic reticulum. Strikingly, coexpression of the dorsal group gene windbeutel in those cells directs Pipe to the Golgi. Similarly, Pipe protein exhibits an altered subcellular localization in the follicle cells of females mutant for windbeutel. Thus, Windbeutel protein enables the correct subcellular distribution of Pipe to facilitate its pattern-forming activity.     

Pattern formation during early amphibian development: Embryogenesis in inverted anuran and urodele eggs

Early embryogenesis was monitored in Xenopus, Rana (anurans), and Ambystoma (urodele) eggs which were inverted at various times between fertilization and first cleavage. The pattern of cleavage furrow formation, site of involution, and extent of organogenesis were observed. In several instances, pattern formation was dramatically altered. The small/large blastomere pattern was, for example, reversed in some inverted embryos. Developmental arrest at early organogenesis usually followed pattern reversal. By employing a series of tissue transplantations, it was possible to establish that the activity of the primary embryonic organizer of inverted embryos was diminished drastically. The developmental competence of the prospective ectoderm of inverted embryos was, however, reversed. Incomplete organogenesis in inverted embryos is therefore probably due to either abnormal mesoderm formation or defective tissue interactions.     

Topology of the germ plasm and development of primordial germ cells in inverted amphibian eggs

Abstract. Inverted Xenopus eggs have reduced numbers of primordial germ cells (PGCs). The extent of the reduction varies from spawning to spawning. Histologic examination revealed that PGC counts were lowest in inverted eggs which displayed the greatest amount of shift in the vegetal mass of large yolk platelets, although the germ plasm itself always remained localized in the egg's original vegetal hemi-sphere. Even at blastulation the germ plasm continued to be localized in the egg's original vegetal hemisphere. In many cases, however, it was confined to the periphery of the embryo, which probably accounts for the reduced PGC number in some tadpoles. In other cases it may have been dispersed and therefore not detectable in histologic analyses.
Although the altered site of involution in inverted embryos did not influence PGC development, subsequent cell movement patterns apparently did. Those embryos which displayed the largest degree of pattern reversal at the tail-bud stage also exhibited the most extreme reduction in PGC numbers. A brief cold shock (4° C, 10 min) prior to first cleavage leads to a further reduction in PGC numbers in inverted embryos, probably as a result of the displace-ment of the germ plasm away from its original vegetal pole location.     

Deadlock, a novel protein of Drosophila, is required for germline maintenance, fusome morphogenesis and axial patterning in oogenesis and associates with centrosomes in the early embryo

The deadlock gene is required for a number of key developmental events in Drosophila oogenesis. Females homozygous for mutations in the deadlock gene lay few eggs and those exhibit severe patterning defects along both the anterior-posterior and dorsal-ventral axis. In this study, we analyzed eggs and ovaries from deadlock mutants and determined that deadlock is required for germline maintenance, stability of mitotic spindles, localization of patterning determinants, oocyte growth and fusome biogenesis in males and females. Deadlock encodes a novel protein which colocalizes with the oocyte nucleus at midstages of oogenesis and with the centrosomes of early embryos. Our genetic and immunohistological experiments point to a role for Deadlock in microtubule function during oogenesis.