Spatio-temporal pattern of MAP kinase activation in embryos of the ascidian Halocynthia roretzi

To understand developmental mechanisms, it is important to know when and where signaling pathways are activated. The spatio-temporal pattern of activation of mitogen-activated protein kinase (MAPK/ERK) was investigated during embryogenesis of the ascidian Halocynthia roretzi, using an antibody specific to the activated form of MAPK. During cleavage stages, activated MAPK was transiently observed in nuclei of the precursor blastomeres of endoderm, notochord, mesenchyme, brain, secondary muscle, trunk lateral cells and trunk ventral cells. These sites of MAPK activation are consistent with results of previous studies that have analyzed the embryonic induction of various tissues, and with results of inhibition of MAPK kinase (MEK) in ascidians. Activation of MAPK in notochord and mesenchyme blastomeres was observed in a short period in a single cell cycle. In contrast, in brain and secondary muscle lineages, MAPK activation spanned two or three cell cycles, and upon each cleavage, MAPK was asymmetrically activated in only one of the two daughter cells that remained brain or secondary muscle lineages. During later stages, MAPK activation was predominantly observed in the central nervous system. A conspicuous feature at this stage was that activation appeared to alternate between positive and negative along the anterior-posterior axis of the neural tube. During the tail elongation stage, MAPK was quiescent.     

Localization and roles in fertilization of sperm proteasomes in the ascidian Halocynthia roretzi

We previously reported that sperm proteasome is responsible for degradation of the ubiquitinated vitelline-coat during fertilization in the ascidian Halocynthia roretzi. Here, we report the roles in fertilization and localization in the sperm cell surface of H. roretzi sperm proteasome. An anti-proteasome antibody, as well as the proteasome inhibitors MG115 and MG132, inhibited the fertilization, indicating that the sperm proteasome functions extracellularly in ascidian fertilization. In order to further assess this issue, the sperm surface proteasome activity was labeled with a cell-impermeable labeling reagent, NHS-LC-biotin, extracted with 0.1% CHAPS, and was subjected to a pull-down assay with avidin-agarose beads. It was found that a substantial amount of sperm proteasome is exposed to the cell surface. Partition analysis with Triton X-114 also revealed that a considerable amount of the sperm proteasome activity is partitioned into a lipid layer. Localization of the proteasome activity was investigated by fluorescence microscopy with succinyl-Leu-Leu-Val-Tyr-4-methylcoumaryl-7-amide as a substrate. The sperm proteasome activity was specifically detected in the sperm head region, and it was markedly activated upon sperm activation. The membrane-associated proteasome was purified from the membrane fraction of H. roretzi sperm by affinity chromatography using an anti-20S proteasome antibody-immobilized Sepharose column. SDS-PAGE of the purified preparation showed a similar pattern of subunit composition to that of the 26S proteasome of mammalian origin. Taken together, these results indicate that H. roretzi sperm has the membrane-associated proteasome on its head, which is activated upon sperm activation, and that sperm proteasome plays an essential role in H. roretzi fertilization.     

Wnt5 is required for notochord cell intercalation in the ascidian Halocynthia roretzi

Background information. In the embryos of various animals, the body elongates after gastrulation by morphogenetic movements involving convergent extension. The Wnt/PCP (planar cell polarity) pathway plays roles in this process, particularly mediolateral polarization and intercalation of the embryonic cells. In ascidians, several factors in this pathway, including Wnt5, have been identified and found to be involved in the intercalation process of notochord cells. Results. In the present study, the role of the Wnt5 genes, Hr‐Wnt5α (Halocynthia roretzi Wnt5α) and Hr‐Wnt5β, in convergent extension was investigated in the ascidian H. roretzi by injecting antisense oligonucleotides and mRNAs into single precursor blastomeres of various tissues, including notochord, at the 64‐cell stage. Hr‐Wnt5α is expressed in developing notochord and was essential for notochord morphogenesis. Precise quantitative control of its expression level was crucial for proper cell intercalation. Overexpression of Wnt5 proteins in notochord and other tissues that surround the notochord indicated that Wnt5α plays a role within the notochord, and is unlikely to be the source of polarizing cues arising outside the notochord. Detailed mosaic analysis of the behaviour of individual notochord cells overexpressing Wnt5α indicated that a Wnt5α‐manipulated cell does not affect the behaviour of neighbouring notochord cells, suggesting that Wnt5α works in a cell‐autonomous manner. This is further supported by comparison of the results of Wnt5α and Dsh (Dishevelled) knockdown experiments. In addition, our results suggest that the Wnt/PCP pathway is also involved in mediolateral intercalation of cells of the ventral row of the nerve cord (floor plate) and the endodermal strand. Conclusion. The present study highlights the role of the Wnt5α signal in notochord convergent extension movements in ascidian embryos. Our results raise the novel possibility that Wnt5α functions in a cell‐autonomous manner in activation of the Wnt/PCP pathway to polarize the protrusive activity that drives convergent extension.     

Self‐sterility of eggs induced by exogenous and endogenous protease in the solitary ascidian,Halocynthia roretzi

The unfertilized eggs (UFE) of the solitary ascidian, Halocynthia roretzi, which are released naturally, are strictly self‐sterile. However, ovarian eggs isolated after spawning, which are expected to develop into UFE on the following day, are self‐fertile. Some exogenous proteases‐trypsin, chymotrypsin, papain and elastase‐induced self‐sterility in the self‐fertile ovarian eggs within an hour in vitro. The establishment of self‐sterility by the exogenous protease did not require the synthesis of new protein, or the participation of follicle cells. Some of the ovarian eggs were able to differentiate into self‐sterile eggs spontaneously in vitro. The protein synthesis inhibitors puromycin and cycloheximide had no effect on the spontaneous establishment of self‐sterility. However, several protease inhibitors such as leupeptin, soybean trypsin inhibitor (SBTI) and antipain, did inhibit the spontaneous establishment of self‐sterility. The possible participation of trypsin‐like protease in the establishment of self‐sterility in the ovary is discussed. Mol. Reprod. Dev. 52:99–106, 1999. © 1999 Wiley‐Liss, Inc.     

The development of three identified motor neurons in the larva of an ascidian, Halocynthia roretzi

The generation of distinct classes of motor neurons underlies the development of complex motile behavior in all animals and is well characterized in chordates. Recent molecular studies indicate that the ascidian larval central nervous system (CNS) exhibits anteroposterior regionalization similar to that seen in the vertebrate CNS. To extend the understanding about the diversity of motor neurons in the ascidian larva, we have identified the number, position, and projection of individual motor neurons in Halocynthia roretzi, using a green fluorescent protein under the control of a neuron-specific promoter. Three pairs of motor neurons, each with a distinct shape and innervation pattern, were identified along the anteroposterior axis of the neural tube: the anterior and posterior pairs extend their axons toward dorsal muscle cells, whereas the middle pair project their axons toward ventral muscle. Overexpression of a dominant-negative form of a potassium channel in these cells resulted in paralysis on the injected side, thus these cells must constitute the major population of motor neurons responsible for swimming behavior. Lim class homeobox genes have been known as candidate genes that determine subtypes of motor neurons. Therefore, the expression pattern of Hrlim, which is a Lim class homeobox gene, was examined in the motor neuron precursors. All three motor neurons expressed Hrlim at the tailbud stage, although each down-regulated Hrlim at a different time. Misexpression of Hrlim in the epidermal lineage led to ectopic expression of TuNa2, a putative voltage-gated channel gene normally expressed predominantly in the three pairs of motor neurons. Hrlim may control membrane excitability of motor neurons by regulating ion channel gene expression.     

Evidence for acrosin-like enzyme in sperm extract and its involvement in fertilization of the ascidian,halocynthia roretzi

The presence of a protease has been demonstrated in sperm of the solitary ascidian, Halocynthia roretzi, by using t-butyloxycarbonyl-L-Val-L-Pro-L-Arg-4-methylcoumaryl-7-amide (Boc-Val-Pro-Arg-MCA) and other arginyl or lysyl MCA derivatives as substrates. Several properties of the enzyme were investigated in a crude extract. The activity had a pH optimum near 8.0 and was enhanced by the addition of CaCl₂. The Km value of 87μM was determined for Boc-Val-Pro-Arg-MCA under the optimal conditions. An apparent molecular weight was estimated to be 35,000 by gel filtration. The enzyme was inhibited with diisopropyl fluorophosphate, leupeptin, antipain, p-aminobenzamidine, Val-Pro-Arg-CH₂Cl, and soybean trypsin inhibitor, but scarcely inhibited with chymostatin, elastatinal, p-chloromercuribenzoic acid, tosyl-Lys-CH₂Cl, and tosyl-Phe-CH₂Cl. Boc-Val-Pro-Arg-MCA, the most susceptible of the substrates examined, showed the most effective inhibition against fertilization of ascidian eggs. Thus, this enzyme in ascidian sperm extract has features closely similar to mammalian acrosin [EC 3.4.21.10], and we conclude that the enzyme is involved in fertilization as one of the lysins.     

Distribution of cytoplasmic determinants in unfertilized eggs of the ascidian Halocynthia roretzi

 Cytoplasmic determinants that specify the fate of endoderm, muscle and epidermis cells are known to be localized in specific areas of fertilized eggs of ascidians. The presence of such cytoplasmic determinants in unfertilized eggs was demonstrated in previous studies, but no information has yet been proved about their distribution. To investigate the distribution of cytoplasmic determinants in unfertilized eggs, we devised a method for distinguishing the polarity of unfertilized eggs using vital staining and we performed cytoplasmic-transfer experiments by fusing blastomeres and cytoplasmic fragments from various identified regions of unfertilized eggs. Cytoplasmic fragments, that contained cortical and subcortical material, from five different positions along the animal-vegetal axis were prepared, and they were fused with a4.2 (presumptive-epidermis) or A4.1 (non-epidermis) blastomeres. The ectopic development of endoderm, muscle and epidermis cells that was promoted by the transplanted cytoplasm was assessed by examining the expression of alkaline phosphatase (ALP), myosin and epidermis-specific antigen, respectively. Differentiation of endoderm and muscle was observed at higher frequencies as cytoplasmic fragments closer to the vegetal pole were transplanted. Conversely, formation of epidermis was observed at higher frequencies as cytoplasmic fragments closer to the animal pole were transplanted. The results suggest that, in cortical and subcortical regions of unfertilized ascidian eggs, endoderm and muscle determinants are widely distributed along a gradient, with maximum activity at the vegetal pole, whilst epidermis determinants are also distributed along a gradient but with maximum activity at the animal pole. Recieved: 10 June 1996 / Accepted: 12 September 1996     

Common cell-surface antigens functioning in self-recognition reactions by both somatic cells and gametes in the solitary ascidian Halocynthia roretzi

The "contact reaction" is an extremely rapid allogeneic cytotoxic reaction (ACR) mediated by hemocytes in the solitary ascidian Halocynthia roretzi. It has been proposed that regulation of the alloreactivity of hemocytes may be involved in preference for fertilization or self-sterility in this species. To identify the receptors and target ligands involved both in self-recognition by somatic cells and self-discrimination by gametes, we produced monoclonal antibodies (mAbs) that inhibit the ACR mediated by hemocytes and tested their effects on fertilization. Six different mAbs that inhibit the ACR were prepared and categorized into three groups. Although all three mAbs seemed to have the same ability to inhibit the ACR, almost constant and statistically significant inhibition (CRB1.1) and infrequent but significant inhibition (CRB2.1, and CRB3.1) of the ACR were observed in the same pairs of animals. Pretreatment of the unfertilized eggs with CRB1.1, CRB2.1, and CRB3.1, resulted in the constant and statistically significant inhibition, infrequent but significant inhibition, and no inhibition, respectively, of fertilization. Antigens recognized by CRB1.1 (CRB1.1 antigens) were detected on the cell surface of all types of hemocytes and on the vitelline coat and follicle cells of unfertilized eggs. CRB2.1 and CRB3.1 antigens were detected on the surface of certain types of hemocytes and follicle cells, but not on the vitelline coat. CRB mAbs were directed against different epitopes in the N-linked glycan on glycoproteins. These common carbohydrate antigens on somatic cells and gametes may function in some recognition processes in ACR and fertilization in H. roretzi.     

A novel G protein alpha subunit in embryo of the ascidian,Halocynthia roretzi

A cDNA clone encoding a novel G protein alpha subunit, HrGalpha(n) was isolated from the larvae of ascidian, Halocynthia roretzi. In contrast with overall amino acid identity (63%) with G protein alpha subunit of G(i) or G(o) subclass, HrGalpha(n) has a unique amino acid sequence, which lacks a residue for pertussis toxin substrate, but retains for cholera toxin substrate for ADP-ribosylation. The sequence characteristics and molecular phylogenetic analysis suggest that HrGalpha(n) defines a novel subclass within G(i) class of G protein alpha subunits. The zygotic expression of HrGalpha(n) was first detected at the 64-cell stage and observed in all blastomeres except for B7.4, B7.5 and B7.6 cells till the 110-cell stage. As progress of the developmental stages, the expression of HrGalpha(n) became restricted and was observed in the muscle, mesenchyme and a part of trunk lateral cells in tailbud embryos. With HrGalpha(n)-GFP fusion-gene construct it was showed that the genomic fragment containing 2674 bp upstream of the putative translation start site of HrGalpha(n) contained the regulatory sequence responsible for the expression in the muscle and mesenchyme cells, and that the regulatory sequence functioned also in Ciona intestinalis. Our results suggest a possible involvement of HrGalpha(n) in the signaling system regulates the cell fate during the embryogenesis of the ascidian.     

Molecular cloning and sequence analysis of an ascidian egg beta-N-acetylhexosaminidase with a potential role in fertilization

Beta-N-acetylhexosaminidase, which is found almost ubiquitously in sperm of invertebrates and vertebrates, supposedly mediates a carbohydrate-based transient sperm-egg coat binding. In ascidians and mammals, beta-hexosaminidase released at fertilization from eggs has been proposed to modify sperm receptor glycoproteins of the egg envelope, thus setting up a block to polyspermy. Previously, it was shown that in potential sperm receptor glycoproteins of the ascidian Phallusia mammillata, N-acetylglucosamine is the prevailing glycoside residue and that the egg harbors three active molecular forms of beta-hexosaminidase. In the present study, P. mammillata beta-hexosaminidase cDNA was isolated from an ovarian cDNA library and characterized. The deduced amino acid sequence showed a high similarity with other known beta-hexosaminidases; however, P. mammillata beta-hexosaminidase had a unique potential N-glycosylation site. A phylogenetic analysis suggested that P. mammillata beta-hexosaminidase developed independently after having branched off from the common ancestor gene of the chordate enzyme before two isoforms of the mammalian enzyme appeared. In situ hybridization revealed stage-specific expression of beta-hexosaminidase mRNA during oogenesis in the oocyte and in the accessory test and follicle cells. This suggests that the three egg beta-hexosaminidase forms are specific for the oocyte, test cells and follicle cells.     

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.     

Periodic appearance and disappearance of microvilli associated with cleavage cycles in the egg of the ascidian, Halocynthia roretzi

The surface of eggs of the ascidian Halocynthia roretzi, observed with SEM, is essentially smooth until immediately before cell division when numerous microvilli appear and remain during cytokinesis. As the dividing blastomeres become closely adherent, however, the microvilli disappear and the eggs recover their smooth surface. This periodic appearance-disappearance of microvilli is repeated at each cleavage cycle up to at least the 32-cell stage. During blastomere adhesion, microvilli that have appeared near the plane of the first cleavage or of the bilateral symmetry seem to fuse together across the plane to form a zipper-like complex of cytoplasmic processes, which might be responsible for attachment of the two halves of these bilaterally symmetrical embryos via the blastomeres bordering the plane of symmetry.