Chaetopterus Pergamentaceus Research Articles

Activin/Nodal signaling mediates dorsal\u2013ventral axis formation before third quartet formation in embryos of the annelid Chaetopterus pergamentaceus

BackgroundThe clade of protostome animals known as the Spiralia (e.g., mollusks, annelids, nemerteans and polyclad flatworms) shares a highly conserved program of early development. This includes shared arrangement of cells in the early-stage embryo and fates of descendant cells into embryonic quadrants. In spiralian embryos, a single cell in the D quadrant functions as an embryonic organizer to pattern the body axes. The precise timing of the organizing signal and its cellular identity varies among spiralians. Previous experiments in the annelid Chaetopterus pergamentaceus Cuvier, 1830 demonstrated that the D quadrant possesses an organizing role in body axes formation; however, the molecular signal and exact cellular identity of the organizer were unknown.ResultsIn this study, the timing of the signal and the specific signaling pathway that mediates organizing activity in C. pergamentaceus was investigated through short exposures to chemical inhibitors during early cleavage stages. Chemical interference of the Activin/Nodal pathway but not the BMP or MAPK pathways results in larvae that lack a detectable dorsal–ventral axis. Furthermore, these data show that the duration of organizing activity encompasses the 16 cell stage and is completed before the 32 cell stage.ConclusionsThe timing and molecular signaling pathway of the C. pergamentaceus organizer is comparable to that of another annelid, Capitella teleta, whose organizing signal is required through the 16 cell stage and localizes to micromere 2d. Since C. pergamentaceus is an early branching annelid, these data in conjunction with functional genomic investigations in C. teleta hint that the ancestral state of annelid dorsal–ventral axis patterning involved an organizing signal that occurs one to two cell divisions earlier than the organizing signal identified in mollusks, and that the signal is mediated by Activin/Nodal signaling. Our findings have significant evolutionary implications within the Spiralia, and furthermore suggest that global body patterning mechanisms may not be as conserved across bilaterians as was previously thought.

Protein tyrosine phosphatases in Chaetopterus egg activation.

Changes in protein tyrosine phosphorylation are an essential aspect of egg activation after fertilization. Such changes result from the net contributions of both tyrosine kinases and phosphatases (PTP). This study was conducted to determine what role(s) PTP may have in egg activation. We identified four novel PTP in Chaetopterus pergamentaceus oocytes, cpPTPNT6, cpPTPNT7, cpPTPR2B, and cpPTPR2A, that have significant homology to, respectively, human PTPsigma, -rho, -D2 and -BAS. The first two are cytosolic and the latter two are transmembrane. Several PTP inhibitors were tested to see if they would affect Chaetopterus pergamentaceus fertilization. Eggs treated with beta-bromo-4-hydroxyacetophenone (PTP inhibitor 1) exhibited microvillar elongation, which is a sign of cortical changes resulting from activation. Those treated with Na3VO4 underwent full parthenogenetic activation, including polar body formation and pseudocleavage and did so independently of extracellular Ca2+, which is required for the Ca2+ oscillations that initiate development after fertilization. Fluorescence microscopy identified phosphotyrosine-containing proteins in the cortex and around the nucleus of vanadate-activated eggs, whereas in fertilized eggs they were concentrated only in the cortex. Immunoblots of vanadate-activated and fertilized eggs showed tyrosine hyperphosphorylation of approximately 140 kDa protein. These results suggest that PTP most likely maintain the egg in an inactive state by dephosphorylation of proteins independent of the Ca2+ oscillations in the activation process.

Mechanisms of calcium release and sequestration in eggs of Chaetopterus pergamentaceus

Increases in the intracellular free calcium concentration are of great importance to the initiation of development in deuterostomes. Their involvement has not yet been clearly defined in protostomes. We used endogenous ligands (IP 3, cADPR, ryanodine and NAADP) and pharmacological agents (thapsigargin [Tg], thimerosal, caffeine and heparin) to study smooth endoplasmic reticulum Ca 2+ pump and release mechanisms in eggs of an annelid, Chaetopterus. Oocyte homogenates effectively sequestered Ca 2+ and released it in response to IP 3 in a concentration-dependent manner. Repeated additions of IP 3 were unable to cause further release. Heparin inhibited Ca 2+ release in response to IP 3. The homogenates also released Ca 2+ in response to thimerosal, and this release was sensitive to heparin. Two antibodies to IP 3 receptors recognized an appropriate band in Chaetopterus egg lysates. These results indicate that the oocytes possess type-1 IP 3-gated Ca 2+ channels. Neither calcium itself, nor strontium, cADPR, ryanodine, caffeine nor NAADP released appreciable Ca 2+. At low concentrations, Tg caused a slow release of Ca 2+; at higher concentrations, it elicited a rapid release. Release of Ca 2+ by Tg activated development. Since one theory of fertilization invokes the introduction of a Ca 2+ releasing soluble protein into the egg upon sperm-egg fusion, we also tested whether soluble extracts of Chaetopterus sperm could stimulate Ca 2+ release in Chaetopterus egg homogenates. There was no Ca 2+ release when the sperm extract was added to the homogenate; however, homogenates exposed to sperm extract became refractory to IP 3. Thus, Ca 2+ release at fertilization in these oocytes occurs through IP 3-gated channels.

Regulation of maturation-promoting factor by protein kinase C inChaetopterusoocytes

Summary We present the results of a variety of studies showing that activation of protein kinase C (PKC) in oocytes of Chaetopterus pergamentaceus results in germinal vesicle breakdown (GVBD). Phorbol esters and diacylglycerol can initiate a morphologically normal GVBD accompanied by a spectrum of associated biochemical processes, including increased protein phosphorylation, a shift in protein synthesis and activation of a protein kinase, maturation promoting factor (MPF). MPF activation is essential for GVBD in response to phorbol esters. In addition, inhibitors of PKC can block naturally-induced GVBD. We also present evidence that PKC can phosphorylate p34cde2, the catalytic subunit of MPF and that phosphorylation by PKC increases the histone H1 kinase activity of immunoprecipitated MPF. Immunoblot studies show that Chaetopterus oocyte p34cdc2 is not tyrosine phosphorylated prior to the initiation of GVBD, indicating that activation of MPF at GVBD in this species does not require p80cdc25, the activator of MPF at mitosis. These results suggest that PKC is an essential regulator of GVBD which can directly phosphorylate and regulate p34cdc2. Since PKC is the intracellular receptor for and is directly activated by tumor-promoters, tumor promotion might involve acceleration of the cell cycle through modification of the enzymatic activity of MPF by PKC.

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 μm/sec. Sequential propagated waves and nonpropagated pulses generally originated at different regions on 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.

Regulation of M-phase progression in Chaetopterus oocytes by protein kinase C

We have examined the presence of protein kinase C in oocytes of Chaetopterus pergamentaceus and its role in the initiation of germinal vesicle breakdown (GVBD). First, we demonstrated that the oocytes contain a phospholipid- and calcium-dependent protein kinase, protein kinase C (PKC). Since PKC is the primary intracellular receptor for phorbol esters, we tested the ability of phorbol 12,13-dibutyrate (PDBu) to induce GVBD and compared several critical events and processes involved in GVBD induced by PDBu to those induced normally (by seawater). Seawater and 100–200 n M PDBu induced chromosome condensation, spindle formation, and spindle migration over a similar time course. Both treatments induced similar alterations in the SDS-PAGE pattern of newly synthesized proteins. The synthesis of polypeptides of approximately 46 and 54 kDa increased specifically. Both treatments increased oocyte protein phosphorylation, especially of proteins of 22, 32, 46, 55, 64, and 84 kDa. Both treatments resulted in the activation of an M-phase-specific histone H1 kinase activity, which demonstrates the appearance of maturation-promoting factor. Staurosporine, a potent protein kinase C inhibitor, blocked GVBD and the activation of M-phase-specific H1 kinase, whereas HA1004, which preferentially antagonizes protein kinase A, had no effect. The results of this study demonstrate that protein kinase C can activate a wide spectrum of essential biochemical and morphological processes involved in GVBD. Further, these studies suggest that protein kinase C elicits GVBD by activating maturation-promoting factor and support the hypothesis that protein kinase C plays an essential role in oocyte maturation in this species.

Possible involvement of calmodulin in maturation and activation of Chaetopterus eggs

We report the isolation of calmodulin from oocytes of Chaetopterus pergamentaceus. The identification of this protein is based on (1) activation of beef heart cAMP phosphodiesterase, (2) heat stability, (3) sensitivity to chlorpromazine, and (4) electrophoretic mobility identical to that of porcine brain calmodulin after sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of either Ca 2+ or EGTA. We treated oocytes with chlorpromazine and W-7 to investigate the involvement of calmodulin in meiosis initiation and egg activation. Very low concentrations of chlorpromazine inhibited germinal vesicle breakdown (GVBD). This effect was shown to be dependent upon bright indirect light, since the drug was much less effective at GVBD inhibition under conditions of very low illumination. Higher concentrations of chlorpromazine and W-7 (100 μ M) inhibited GVBD and activated eggs with intact germinal vesicles as determined by fertilization envelope formation and the onset of ameboid activity. Neither egg activation nor inhibition of calmodulin stimulation of phosphodiesterase activity in vitro was affected by light. These results are consistent with a role for calmodulin in egg activation and GVBD, but suggest that chlorpromazine in bright light may prevent GVBD by some mechanism other than calmodulin inhibition.

A CYTOLOGICAL ANALYSIS OF FERTILIZATION INCHAETOPTERUS PERGAMENTACEUS

We have examined sperm-egg interaction in Chaetopterus pergamentaceus by electron microscopy. The initial contact between sperm and egg involved the membrane of the unreacted acrosome and either the tips of egg microvilli which penetrated the vitelline layer or jelly emanating from the tips of the microvilli. This resulted in an acrosome reaction and fusion between the inner acrosomal membrane and the tip of the microvillus. Sperm did not produce acrosomal processes like those of many other invertebrates, and no part of the sperm penetrated the vitelline layer until the sperm was incorporated into the fertilization cone. The fertilization cone was very small and was composed of egg microvilli. The sperm nucleus and mitochondrion were incorporated into the fertilization cone, but a recognizable sperm mitochondrion could not subsequently be seen in the egg cytoplasm. Although the axoneme of the sperm tail was present in the fertilization cone at early stages of sperm penetration, the sperm tail evidently detached in the later stages of incorporation because it could not be seen in the zygote cytoplasm after sperm incorporation. The sperm chromatin decondensed uniformly and became surrounded by a typical nuclear envelope. The results indicate that Chaetopterus provides an example of a previously undescribed model for sperm penetration of egg vestments in which the sperm needs neither to produce an acrosomal process nor to liberate vitelline layer lysins because it penetrates the vitelline layer passively after incorporation into the egg cytoplasm.

Localization of messenger RNA in the cortex ofChaetopterus eggsand early embryos

The distribution of mRNA in Chaetopterus pergamentaceus eggs was examined by in situ hybridization with poly(U) and specific cloned DNA probes. Eggs contain three distinct regions; the cortical ectoplasm, endoplasm, and a plasm released from the germinal vesicle (GV) during maturation. The ectoplasm of the mature egg showed a 15-fold enrichment in poly(A) and in histone and actin mRNAs relative to the endoplasm and the GV plasm after in situ hybridization. More than 90% of the total mass of egg poly(A) + RNA and histone and actin messages was estimated to be present in the ectoplasm. The mRNA molecules co-distributed with ectoplasmic inclusion granules during ooplasmic segregation. During the extensive cytoplasmic rearrangements which occur at the time of the first cleavage the ectoplasm was divided into animal and vegetal portions. The animal portion was segregated evenly between the AB and CD blastomeres, whereas the vegetal portion entered the polar lobe and was preferentially segregated to the CD blastomere. Histone and actin mRNA entered both the AB and CD blastomeres of the 2-cell embryo. The results demonstrate that mRNA is quantitatively localized in the cortex of the Chaetopterus egg and early embryo.

The Effects of Cytoskeleton Inhibitors on Cytoplasmic Localization in Chaetopterus pergamentaceus

We have treated fertilized and KC1-activated eggs of Chaetopterus pergamentaceus with microfilament and microtubule inhibitors to test the relationship of these cytoskeletal components to cytoplasmic localization. Low doses of cytochalasin B inhibited cleavage in fertilized egges. Such embryos underwent differentiation without cleavage, a process characterized by relocalization of the yolky endoplasm to the center of the uncleaved egg and by the formation of cilia. Similar treatment of KC1-activated eggs inhibited ciliation, but not endoplasmic relocalization. Reversible inhibition of the first cleavage resulted in equal cleavage and differentiation of a larva lacking an apical organ. Inhibition of the first two cleavages resulted in differentiation without cleavage. At all concentrations high enough to block mitosis, colchicine prevented ciliation and endoplasmic relocalization. Thus microtubule organization, but not microfilament organization, is required for ooplasmic reorganization and differentiation without cleavage.

AN ULTRASTRUCTURAL ANALYSIS OF CYTOPLASMIC LOCALIZATION INCHAETOPTERUS PERGAMENTACEUS

During early development of Chaetopterus pergamentaceus, cytoplasmic components visible by electron microscopy became segregated. The hyaloplasm, or spongy layer, composed of granular bodies surrounded by a matrix of endoplasmic reticulum and microtubules was segregated towards (1) the upper pole of the unfertilized egg, (2) the interior of the cleaving egg, (3) the superficial regions of the 16-cell stage, and (4) the ectoderm of the trochophore larva. The ectoplasm, characterized by cortical granules, surrounded the unfertilized and cleaving egg endoplasm. It was restricted to the apical end of cells at the 16-cell stage, and to the ectoderm of the trochophore larva. The endoplasm, characterized by yolk and lipid, (1) was found throughout the unfertilized egg except at the upper pole, (2) was restricted to a ring just below the cortex of the cleaving egg, (3) moved to the basal ends of blastomeres by the 16-cell stage, and (4) was limited to the endoderm of the trochophore. Granular bodies were found throughout the embryo. Mitochondria, found throughout the cytoplasm by first cleavage, were absent from the hyaloplasm of the unfertilized egg. The cytoplasm of the polar lobe was similar to that of the surrounding endoplasm. The localization of morphogenetic determinants was thus accompanied by cytoplasmic organelle localization in Chaetopterus. The results are interpreted in terms of localization mechanisms involving the embryo cortex and ground substances.

Changes in excitability of the cell membrane during 'differentiation without cleavage' in the egg of the annelid, Chaetopterus pergamentaceus.

1. The egg of the polychaete, Chaetopterus pergamentaceus, differentiates parthenogenetically without cleavage after 1 hr in a high K+ solution. The changes in the electrical properties of the membrane during differentiation have been investigated. 2. The treatment with the K solution for 40-60 min made unfertilized eggs become amoeboid cells in 4-5 hr and finally ciliated unicellular embryos in 14-16 hr. 3. In the untreated egg the action potential is Ca dependent and no Na component is found. The steady-state current-voltage relation has a marked inward rectification and shows a less marked outward rectification. 4. There are no significant changes in these properties of the cell membrane immediately after 40-60 min K treatment. 5. In the amoeboid cell stage (4-5 hr) the outward rectification increases significantly. 6. In the ciliated unicellular embryo the action potential is Ca and Na dependent. 7. The result suggests that Ca channels are present in the egg initially, K channels appear next and Na channels appear later.

THE CYTOLOGICAL EFFECTS OF PODOPHYLLIN AND PODOPHYLLOTOXIN ON THE FERTILIZED EGGS OF CHAETOPTERUS

1. Fertilized eggs of the polychaete annelid, Chaetopterus pergamentaceus, were treated with various dilutions of podophyllin and podophyllotoxin, beginning five minutes after insemination. Observations were made on the living eggs and on whole-mount cytological preparations made from samples fixed at various intervals during development. Dosages ranging from 0.1 to 0.00001 mg./ml. were tested.2. Beginning as soon as 60 seconds after the beginning of treatment with moderate to high dosages (0.1 to 0.005 mg./ml.) of podophyllin, the asters of the egg maturation figure began to fade, followed by disappearance of the spindle by about three minutes after the initiation of treatment. The nine egg chromosomes remained in the ring configuration (characteristic of the first maturation division in this form) for approximately two to five minutes longer (depending on the exact dosage), after which they were gradually enclosed in a membrane-surrounded area which appears to have been derived from the spindle substance. By this time they had taken on a somewhat vesicular appearance. The sperm chromosomes were often similarly affected by the podophyllin, although the onset of these effects was usually somewhat slower than for the eggs. Both egg and sperm chromosomes remained contained in this membrane for approximately an hour, after which they were released to lie loose in the egg cytoplasm. No further development occurred at these dosages.3. After treatments with podophyllin concentrations of 0.00005 to 0.00001 mg./ml., a semblance of normal development ensued, although the resulting larvae were usually abnormal. In egg-samples fixed 24 minutes after the beginning of treatment, there was occasional evidence of reduction in size of the asters; by 47-57 minutes after the initiation of treatment, multipolar figures were observed, as well as two entirely separate complex metaphases, often in uncleaved cytoplasm. There was frequently karyokinesis without accompanying cytokinesis. When cleavage continued to occur, it was often retarded and/or abnormal.4. Shape changes ("pear" and polar lobe stages) characteristic of the normal development of Chaetopterus eggs were observed, even after treatment with high dosages which destroyed the achromatic figure. Pseudo-polar bodies, usually without chromatin, were also noted, as well as giant polar bodies.5. The effects of podophyllotoxin were qualitatively much like those of podophyllin, but the dosage required to produce them was very much lower (0.001 mg./ml. for podophyllotoxin vs. 0.01 mg./ml. for podophyllin to completely block division, for example).6. Even short durations of exposure to either podophyllin or podophyllotoxin (10-20 minutes, as compared with the 60-minute duration routinely used in the other experiments) were sufficient to induce marked abnormality with moderate dosages.7. The effects of mitomycin C, N-dichloroacetyl DL serine (sodium) and quercetin were also tested. None of these agents was effective in producing cytological abnormality in Chaetopterus eggs, at the concentrations tested.

Heavy water inhibition of cell division: an approach to mechanism.

ABS>At concentrations higher than about 75%, deuterium oxide blocks cell division in the eggs of the sea urchin Arbacia punctulata and in those of the annelid Chaetopterus pergamentaceus. The block is reversible, although cells that have been kept is a deuterium-rich medium for long periods relative to the time for one mitotic cycle may show multipolar and irregular divisions after removal to normal water, and may subsequently cytolyze. The antimitotic action is manifested at all stages of the mitotic cycle and, to a significant extent, during cytokinesis. An important feature of the mitotic block is that it becomes effective almost immediately after the cells are placed in D2O and is quickly reversed when the cells are washed free of D2O. Whereas D2O blocks mitosis at all stages it does not destroy the achromatic apparatus, nor does it cause wholesale regression of cleavage furrows. Instead, it exerts a powerful stabilizing effect upon these structures. This appears to be related to a sharp and reversible increase in cytoplasmic viscosity evoked by immersion of nondividing cells in D2O. Examination of a number of alternative explanations of the effect leaves as the most useful working hypothesis a rigidification of the mitotic apparatus, possibly throughmore » a small increase in the strength of individual hydrogen bonds which cross-link macromolecules or small particles. For large, multiply cross-linked structures, such changes for individual bonds would produce very large cooperative effects in the direction required by the data.« less

EXAGGERATED ELEVATION OF THE FERTILIZATION MEMBRANE OF CHAETOPTERUS EGGS, RESULTING FROM COLD-TREATMENT

1. Fertilized eggs of the polychaete annelid, Chaetopterus pergamentaceus, were cold-treated for various periods of time, ranging from 150 to 720 minutes, beginning immediately after insemination. Two general methods were employed; in one, the eggs were plunged into pre-chilled, filtered aerated sea water (2-3° C.); these experiments are referred to as involving temperature shock. In the other type, the eggs were gradually chilled to approximately the above temperature. At the end of the treatment period all eggs were allowed to return gradually to room temperature.2. When eggs were cold-treated, with or without temperature shock, there was a pronounced asymmetrical exaggerated elevation of the vitelline membrane, which reached its greatest incidence about 40 minutes after the end of treatment, or shortly before the first cleavage. This exaggerated elevation continued in some cases after prolonged cold-treatment, so that the eggs were eventually denuded.3. Most of the cold-treatments used were followed by...

Nuclei of Arbacia and Chaetopterus eggs as photographed by infrared light

Photographs, taken with infrared light, of all stages of dividing Arbacia punctulata eggs show the configuration of mitotic figures and nuclei as brilliant white areas against the dark granular cytoplasmic background. The photographs are very different from those taken with visible light. The nucleus and cytoplasm of the unfertilized Chaetopterus pergamentaceus egg which are quite opaque with visible light are quite striking with infrared light.

COMPARATIVE SENSITIVITY OF SPERM AND EGGS TO ULTRAVIOLET RADIATIONS

1. The action spectrum for the retardation of division of eggs fertilized with irradiated sperm resembles the absorption of ultraviolet light by nucleoproteins.2. The action spectrum for retardation of division of irradiated eggs of the sea urchin resembles absorption by simple proteins like albumin except that at the short wave-length end there is no increase in action at λ 2,483 Å where absorption shows a definite upswing.3. The absolute amount of energy required to affect division to the same extent by affecting the sperm is very much less than that required to affect eggs.4. Other Echinoderms tested show a similar difference in susceptibility of eggs and sperm: S. franciscanus, Arbacia punctulata, Dendraster excentricus, and Pateria miniata.5. Animals other than Echinoderms tested did not show as striking a difference between susceptibility of eggs and sperm: Urechis caupo, Mactra sp., Chaetopterus pergamentaceus, and Nereis limbata.6. In the eggs listed in paragraph 5, determinations are made more difficult by the tendency for the eggs to show irregular cleavage rather than retarded cleavage as the dosage increases. Such irregular cleavage occurs in Echinoderm eggs as well but the threshold is higher.7. If both eggs and sperm of the sea urchin are irradiated the effect on the rate of division is less than the sum of the effects which would be expected on each of the gametes alone. However, the percentage of abnormal cleavage greatly increases.

Differentiation without cleavage in the egg of the annelid chaetopterus pergamentaceus

Differentiation without cleavage in the egg of the annelid chaetopterus pergamentaceus