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At least 649 records · Page 36Linked to original sources
PubMed1993

Nuclear and cytoplasmic pH increase at fertilization in Urechis caupo.

Intracellular pH (pHi) was measured in Urechis caupo (Echiura) eggs during fertilization using the pH-sensitive dye BCECF [bis(carboxyethyl)carboxyfluorescein] and fluorescence microscopy. When eggs were inseminated at pH 8, pHi began to rise 22-36 sec (n = 7) after sperm contact and reached a plateau by 3 min (2.8 +/- 1 SD; n = 14). The net increased was 0.25-0.3 pH units and the alkalinization persisted through 1 hr after insemination (after second polar body formation). Separate measurements of germinal vesicles and cytoplasm revealed that pH rose dramatically within the nuclei well before germinal vesicle breakdown (GVBD), as well as in cytoplasm. When eggs were fertilized at pH 6.9 (previously shown to inhibit proton release and egg activation despite sperm entry; see Paul, 1970, 1975; Holland et al., 1984), there was no net pH increase in either cytoplasm or germinal vesicles. When fertilization was at pH 7.45, the average pHi increase in whole eggs was 58% of that at pH 8, and 61% of the eggs activated partially or completely. These results show that the pHi rise is correlated with GVBD and egg activation.

Animals↗
PubMed1995

Parthenogenesis in Urechis caupo (Echiura). I. Persistance of functional maternal asters following activation without meiosis.

A peptide (P23) isolated from sperm acrosomal protein initiates development in eggs of the marine worm Urechis caupo (Echiura). However, in these earlier experiments the eggs were exposed continuously to P23 and did not cleave unless they were provided with a sperm centrosome. The present study reports that parthenogenetic cleavage can be induced when the eggs are exposed to P23 for 1.5 to 2 min in seawater at pH 8 followed by acidifying the seawater to pH 7 or washing off the peptide at pH 7 or 8. The mechanism of parthenogenesis induction was studied by comparing the behavior of maternal and paternal centrosomes (microtubule organizing centers), chromosomes, nuclei and nucleoli in fertilized eggs, P23-activated eggs that failed to cleave, and P23-activated eggs that cleaved, using bright-field, phase-contrast, and fluorescence microscopy following anti-tubulin and bis-benzimide staining. Parthenogenetic cleavage to mostly diploid embryos occurred in eggs that underwent germinal vesicle breakdown without polar body formation. The centrosomes used for cleavage were the persistent maternal centrosomes that did not cycle through meiosis. When cytochalasin B was used to suppress polar body formation without inhibiting the meiotic cycles, eggs did not subsequently cleave even though all four maternal centrosomes were retained. When fertilized eggs were transferred to pH 7 seawater at 1.5 to 2 min after insemination, they underwent a partial activation like the P23-activated eggs and did not form polar bodies. Subsequent cleavage was very abnormal due to the presence of multiple asters formed by the persistant active maternal centrosomes. These results show that maternal centrosomes are normally inactivated by a maternal mechanism associated with meiotic cycling.

Animals↗
PubMed1995

Propagated and nonpropagated calcium transients during egg activation in the annelid, Chaetopterus.

Transient waves of Ca2+ release cross-fertilizing deuterostome eggs from the point of sperm entry to its antipode and provide much of the activating stimulus for the egg. Based on several indirect lines of experimental evidence, it was proposed that protostome eggs are activated by a prolonged uptake of Ca2+ from the medium due to sperm-induced membrane depolarization and that this uptake then starts an activation wave similar to those in deuterostomes, except that it moves inward from the whole surface rather than through the egg from pole to pole. To test these hypotheses, we microinjected oocytes of the polychaete annelid, Chaetopterus pergamentaceus, with semisynthetic recombinant aequorins and measured light emission in response to both fertilization and artificial activation by excess K+. Both fertilization and K(+)-activation induced multiple, brief Ca2+ transients in the eggs. The first transient did not propagate, but it was followed by a series of globally propagated Ca2+ waves interspersed with additional nonpropagated pulses. The waves traversed the egg at about 30 micrometer/sec. Sequential propagated waves and nonpropagated pulses generally originated at different regions of the egg surface, except the last few, which originated in the same "pacemaker" region. These new data are consistent with the hypothesis that the activation of protostome eggs is initiated by Ca2+ waves. However, the fact that these waves propagated from pole to pole like those in deuterostome eggs refutes the notion that Ca2+ waves in activating protostome eggs move inward from the whole surface.

Animals↗
PubMed1997

Conserved anterior boundaries of Hox gene expression in the central nervous system of the leech Helobdella.

Molecular developmental studies of fly and mouse embryos have shown that the identity of individual body segments is controlled by a suite of homeobox-containing genes called the Hox cluster. To examine the conservation of this patterning mechanism in other segmented phyla, we here describe four Hox gene homologs isolated from glossiphoniid leeches of the genus Helobdella. Based on sequence similarity and phylogenetic analysis, the leech genes Lox7, Lox6, Lox20, and Lox5 are deemed to be orthologs of the Drosophila genes lab, Dfd, Scr, and Antp, respectively. Sequence similarities between Lox5 and Antp outside the homeodomain and phylogenetic reconstructions suggest that the Antennapedia family of Hox genes (as defined by Bürglin, 1994) had already expanded to include at least two discrete Antp and Ubx/abdA precursors prior to the annelid/arthropod divergence. In situ hybridization reveals that the four Lox genes described in this study are all expressed at high levels within the segmented portion of the central nervous system (CNS), with variable levels of expression in the segmental mesoderm. Little or no expression was seen in peripheral ectoderm or endoderm, or in the unsegmented head region (prostomium). Each Lox gene has a distinct anterior expression boundary within one of the four rostral segments, and the anterior-posterior (AP) order of these expression boundaries is identical to that reported for the orthologous Hox gene products in fly and mouse. This finding supports the idea that the process of AP axis differentiation is conserved among the higher metazoan phyla with respect to the regional expression of individual Hox genes along that axis. One unusual feature of leech Hox genes is the observation that some genes are only expressed during later development -- beginning at the time of terminal cell differentiation -- whereas others begin expression at a much earlier stage, and their RNA ceases to be detectable shortly after the onset of expression of the 'late' Hox genes. The functional significance of this temporal disparity is unknown, but it is noteworthy that only the two 'early' Hox genes display high levels of mesodermal expression.

Amino Acid Sequence↗
PubMed1997

The intracellular calcium increase at fertilization in Urechis caupo oocytes: activation without waves.

The intracellular Ca2+ (Cai) increase at fertilization of the marine worm Urechis caupo (Echiura) was studied with conventional and confocal epifluorescence microscopy in oocytes microinjected with calcium green dextran or dually labeled with the calcium-insensitive dye tetramethylrhodamine dextran. Calcium green fluorescence was also measured with a photomultiplier system while the oocyte membrane potential was recorded and manipulated. The results show that Cai rises simultaneously around the oocyte cortex and peaks slightly later in the nucleoplasm. The Cai rise coincides with the initiation of the fertilization potential and we conclude that it is due primarily to external Ca2+ entering through the voltage-gated Ca2+ action potential channels that open during the fertilization potential because: (1) current clamping the oocyte membrane potential to positive values in the absence of sperm produces a similar Cai increase, (2) external Ca2+ is required, (3) and the confocal images are consistent with this mechanism. External application of sperm acrosomal peptide (P23) also caused a Cai increase that was inhibited in the presence of CoCl2. Cai and pHi (measured with BCECF dextran) were manipulated in experiments employing microinjection of BAPTA (to chelate Cai), external application of NH4Cl (to increase pHi) and CoCl2 (to block Ca2+ channels), and fertilization of eggs in pH 7 seawater (Cai increase without pHi increase). The results showed that increases in both Cai and pHi are required for GVBD; neither alone is sufficient. However, although nuclear and cytoplasmic Ca2+ levels tended to parallel each other in oocytes fertilized at pH 7, and during the initial Cai response in oocytes fertilized at pH 8, there was a disproportionate fluorescence increase in the nucleoplasm of the latter prior to GVBD which could not be explained by any artifact we tested, suggesting there may be a selective increase in nuclear Ca2+ associated with GVBD. Finally, electrophysiological experiments with BAPTA-injected oocytes showed that the opening of the fertilization potential Na+ channels was Ca2+-independent, (although they did not close at the normal time). These and earlier results suggest that Urechis sperm may activate oocytes by interacting directly with the Na+ channels or associated receptors.

Ammonium Chloride↗
PubMed1998

Redox changes during fertilization and maturation of marine invertebrate eggs.

Previous studies on sea urchin eggs indicate that activation of NAD kinase is one of the earliest Ca2+-mediated events of fertilization. The subsequently produced NADP is converted to NADPH by the pentose shunt pathway, and some of this NADPH is used by an NADPH oxidase for generation of H2O2. To examine whether these changes apply generally, we have analyzed changes in pyridine nucleotide content during meiotic maturation and fertilization in eggs from four phyla. Surprisingly, fertilization-associated increases in NAD kinase were found only in echinoid eggs. The ratio of NADPH/NADP (redox ratio) increased from 1-1.6 to 2.5-6 following fertilization of echinoid and also clam eggs. However, the ratio is already >2 for unfertilized asteroid, tunicate and echiuroid eggs, and this ratio is unaffected by fertilization. We conclude that activation of NAD kinase and shifts in pyridine nucleotide metabolism and thereby cellular redox status may have roles that vary between species. In echinoids, a major role is in providing NADPH for H2O2 production, but there may be other yet unappreciated signaling functions for this change.

Animals↗
PubMed1999

MAP kinase, meiosis, and sperm centrosome suppression in Urechis caupo.

Although MAP kinase is an important regulatory enzyme in many somatic cells, almost nothing is known about its functions during meiosis, except in frog and mouse oocytes. We investigated MAPK activation and function in oocytes of the marine worm Urechis caupo that are fertilized at meiotic prophase. Activity was first detected at 4-6 min after fertilization in immunoblots with anti-active MAPK, prior to germinal vesicle breakdown (GVBD). MAPK activation did not require new protein synthesis and was dependent on the increases in both intracellular pH and intracellular Ca(2+) that normally occur during activation. When MAPK activation was inhibited with PD98059 or U0126, GVBD still occurred, but meiosis was abnormal and there was a dramatic premature enlargement of sperm asters, which normally do not appear until second polar body formation. Failure of polar body formation and premature sperm aster enlargement also occurred when MAPK activation was inhibited by an entirely different treatment which involved lowering the pH of external seawater to interrupt the normal cytoplasmic pH increase. Thus, in Urechis, active MAPK appears to be required for (1) normal meiotic divisions and (2) suppressing the paternal centrosome until after the egg completes meiosis, a general phenomenon whose mechanism has been unknown.

Animals↗
PubMed2000

Bilaterian origins: significance of new experimental observations.

Several recent laboratory observations that bear on the origin of the Bilateria are reviewed and interpreted in light of our set-aside cell theory for bilaterian origins. We first discuss new data concerning the phylogeny of bilaterian phyla. Next, we use systematic, molecular, and paleontological lines of evidence to argue that the latest common ancestor of echinoderms plus hemichordates used a maximal indirect mode of development. Furthermore, the latest common ancestor of molluscs and annelids was also indirectly developing. Finally, we discuss new data on Hox gene expression patterns which suggest that both sea urchins and polychaete annelids use Hox genes in a very similar fashion. Neither utilizes the complete Hox complex in the development of the larva per se, while the Hox complex is expressed in the set-aside cells from which the adult body plan is formed. Our current views on the ancestry of the bilaterians are summarized in phylogenetic terms, incorporating the characters discussed in this paper.

Animals↗
PubMed2001

A polychaete hunchback ortholog.

We report the first characterization of a segmentation gene homologue in the basal polychaete Capitella capitata using a pan-annelid cross-species antibody to the hunchback-like gene product. In flies, the gap segmentation gene hunchback (hb) encodes a C(2)H(2) zinc-finger transcription factor that plays a pivotal role in patterning the anterior region of the fly body plan. The hb orthologue in Capitella (Cc-hb) is expressed maternally and in all micromere and macromere cells throughout cleavage. At gastrulation, nuclear Cc-hb protein is expressed in the micromere-derived surface epithelium that undergoes epiboly and in the large vegetal blastomeres that gradually become internalized. During organogenesis, Cc-hb is expressed in the developing gut epithelium, the prostomial and pygidial epithelium, and in a subset of differentiated neurons in the adult central nervous system. Cc-hb is not expressed in the segmental precursor cells in the trunk. The Cc-hb expression domains in Capitella are similar to those reported for the leech hb orthologue (LZF2), and many of the observed differences between the annelid classes correlate with changes in life history. The lack of detectable annelid hb protein in the trunk at the time of AP pattern formation in leech and in polychaete suggests that the anterior organizing function of hb in flies originated in the arthropod or insect lineage.

Amino Acid Sequence↗
PubMed2001

The spatial and temporal expression of Ch-en, the engrailed gene in the polychaete Chaetopterus, does not support a role in body axis segmentation.

We are interested in understanding whether the annelids and arthropods shared a common segmented ancestor and have approached this question by characterizing the expression pattern of the segment polarity gene engrailed (en) in a basal annelid, the polychaete Chaetopterus. We have isolated an en gene, Ch-en, from a Chaetopterus cDNA library. Genomic Southern blotting suggests that this is the only en class gene in this animal. The predicted protein sequence of the 1.2-kb cDNA clone contains all five domains characteristic of en proteins in other taxa, including the en class homeobox. Whole-mount in situ hybridization reveals that Ch-en is expressed throughout larval life in a complex spatial and temporal pattern. The Ch-en transcript is initially detected in a small number of neurons associated with the apical organ and in the posterior portion of the prototrochophore. At later stages, Ch-en is expressed in distinct patterns in the three segmented body regions (A, B, and C) of Chaetopterus. In all segments, Ch-en is expressed in a small set of segmentally iterated cells in the CNS. In the A region, Ch-en is also expressed in a small group of mesodermal cells at the base of the chaetal sacs. In the B region, Ch-en is initially expressed broadly in the mesoderm that then resolves into one band/segment coincident with morphological segmentation. The mesodermal expression in the B region is located in the anterior region of each segment, as defined by the position of ganglia in the ventral nerve cord, and is involved in the morphogenesis of segment-specific feeding structures late in larval life. We observe banded mesodermal and ectodermal staining in an anterior-posterior sequence in the C region. We do not observe a segment polarity pattern of expression of Ch-en in the ectoderm, as is observed in arthropods.

Amino Acid Sequence↗
PubMed2000

Inactivation of Ca(2+) action potential channels by the MEK inhibitor PD98059.

In a previous study involving the inhibition of mitogen-activated protein kinase (MAPK) activation during fertilization of the marine worm Urechis caupo, we found that PD98059, but not U0126, caused multiple sperm penetrations in oocytes (Gould and Stephano, 1999, Dev. Biol. 216, 348-358). Since these oocytes are protected against polyspermy by a positive shift in membrane potential at fertilization (Gould-Somero et al., 1979, J. Cell Biol. 82, 426-440), we investigated the effects of PD98059 on the electrical properties of the oocyte membrane. PD98059, but not U0126, selectively blocked the voltage-dependent Ca(2+) channels that participate in the electrical polyspermy block. We also noted previously that PD98059 had more serious effects than U0126 on chromosome behavior during meiosis. This, too, could be explained by the effect on Ca(2+) channels, since when U0126-treated eggs were fertilized in low Ca(2+) seawater to reduce Ca(2+) uptake, similar effects were produced. These results show that PD98059 has side effects unrelated to the inhibition of MAPK activation and underscores the need for caution in interpreting the results of experiments with this widely used MEK inhibitor.

Action Potentials↗
PubMed1995

Structural comparison of cuticle and interstitial collagens from annelids living in shallow sea-water and at deep-sea hydrothermal vents.

Two types of annelid collagens of different sizes were purified, one from acetic acid extracts of the cuticle (length 2.5 microns) and the other, after pepsin digestion, from interstitial spaces of the body wall (0.3 micron). They were obtained from Alvinella pompejana, Alvinella caudata and Paralvinella grasslei collected at 2600 m depth around anoxic hydrothermal vents and from Arenicola marina and Nereis diversicolor living in shallow sea-water habitats. The length of the corresponding collagens from different species and their amino acid compositions including the hydroxylation of proline were remarkably similar. The melting point of the triple helix, however, differed between the Alvinella species (approximately 45 degrees C), Paralvinella (approximately 35 degrees C) and the shallow sea-water annelids (approximately 28 degrees C), indicating adaption to habitats with different temperatures. The cuticle collagens of the annelids possess a globular domain, which is apparently involved in oligomer formation, and show similar fragment pattern. Almost identical cross-striation patterns of segment-long-spacing segments of the interstitial collagens indicated sequence similarity, which was confirmed by partial Edman degradation of alpha-chains. These data showed almost complete identity between the two Alvinella species and a lower sequence identity with Paralvinella (approximately 95%), Arenicola (67 to 72%) and the vent vestimentiferan Riftia pachyptila (64 to 71%). The data suggest a close evolutionary relationship between these worms, despite a clear separation of habitat preference and thermal stability of the collagens.

Amino Acid Sequence↗
PubMed1994

Structural analysis of Urechis caupo hemoglobin.

The structure of Urechis caupo hemoglobin in the cyanomet state has been refined to R = 0.148 at 2.5 A resolution. Although the tertiary structure is similar to that of other vertebrate and invertebrate hemoglobins the quaternary structures of this tetramer is unique as suggested by the earlier determination of the 5.0 A resolution structure. The G and H helices of the hemoglobin are on the outside of the tetramer facing the solvent in contrast to human hemoglobin where the G and H helices form inter-subunit contacts. A substantial number of tightly bound water molecules help mediate interactions between subunits. The unusual arrangement of subunits is consistent with the general lack of co-operativity of oxygen uptake for Urechis caupo hemoglobin.

Amino Acid Sequence↗
PubMed2001

A cellular oscillator model for periodic pattern formation.

In this paper, we present a model for pattern formation in developing organisms that is based on cellular oscillators (CO). An oscillatory process within cells serves as a developmental clock whose period is tightly regulated by cell autonomous or non-autonomous mechanisms. A spatial pattern is generated as a result of an initial temporal ordering of the cell oscillators freezing into spatial order as the clocks slow down and stop at different times or phases in their cycles. We apply a CO model to vertebrate somitogenesis and show that we can reproduce the dynamics of periodic gene expression patterns observed in the pre-somitic mesoderm. We also show how varying somite lengths can be generated with the CO model. We then discuss the model in view of experimental evidence and its relevance to other instances of biological pattern formation, showing its versatility as a pattern generator.

Animals↗
PubMed1998

Paraphyletic status of Polychaeta suggested by phylogenetic analysis based on the amino acid sequences of elongation factor-1 alpha.

In order to judge whether or not Polychaeta is a paraphyletic group, I determined almost the entire amino acid sequence of elongation factor-1 alpha from thirteen polychaetes, two oligochaetes, two hirudineans, two vestimentiferans, and two molluscs. Phylogenetic analysis by the neighbor-joining (NJ) method and the maximum likelihood (ML) method indicated the monophyly of Clitellata (the oligochaetes and hirudineans). In both the NJ and ML trees, vestimentiferans and clitellates were derived from polychaetes independently. The present results strongly suggest that Polychaeta is a paraphyletic group.

Amino Acid Sequence↗