Signaling pathway from [Ca2+]i transients to ooplasmic segregation involves small GTPase rho in the ascidian egg

Intracellular Ca2+ transients occur at fertilization in the eggs of all animal species and are thought to be critical for the initiation of several events in egg activation. The rho family of small GTPases are known to organize and maintain the actin filament-dependent cytoskeleton, and rho is involved in the control mechanism of cytokinesis. In the ascidian Ciona savignyi, the first step of ooplasmic segregation observed just after fertilization is cortical contraction with egg deformation, mediated by the cortical actin filaments. C3 exoenzyme, a rho-specific inhibitor, did not affect the pattern of [Ca2+]i transients in the ascidian egg, but inhibited ooplasmic segregation and cytokinesis at the first cleavage. Injection of inositol 1,4,5-trisphosphate or treatment of Ca2+ ionophore induced deformation of the egg and extrusion of the first polar body, but these phenomena did not occur in the C3 exoenzyme-injected egg. These results suggest that rho proteins are involved in egg deformation, ooplasmic segregation and cytokinesis downstream of the [Ca2+]i transients.     

Role of the FGF and MEK signaling pathway in the ascidian embryo

In the ascidian embryo, a fibroblast growth factor (FGF)-like signal from presumptive endoderm blastomeres between the 32-cell and early 64-cell stages induces the formation of notochord and mesenchyme cells. However, it has not been known whether endogenous FGF signaling is involved in the process. Here it is shown that 64-cell embryos exhibit a marked increase in endogenous extracellular signal-regulated kinase (ERK/MAPK) activity. The increase in ERK activity was reduced by treatment with an FGF receptor 1 inhibitor, SU5402, and a MEK (ERK kinase/MAPKK) inhibitor, U0126. Both drugs blocked the formation of notochord and mesenchyme when embryos were treated at the 32-cell stage, but not at the 2- or 110-cell stages. The dominant-negative form of Ras also suppressed notochord and mesenchyme formation. Both inhibitors suppressed induction by exogenous basic FGF. These results suggest that the FGF signaling cascade is indeed necessary for the formation of notochord and mesenchyme cells during ascidian embryogenesis. It is also shown that FGF signaling is required for formation of the secondary notochord, secondary muscle and neural tissues, and at least ERK activity is necessary for the formation of trunk lateral cells and posterior endoderm. Therefore, FGF and MEK signaling are required for the formation of various tissues in the ascidian embryo.     

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.     

Expressed Sequence Tag Analysis of Vanadocytes in a Vanadium-Rich Ascidian, <Emphasis Type="Italic">Ascidia sydneiensis samea</Emphasis>

Some species in the family Ascidiidae accumulate vanadium at concentrations in excess of 350 mM, which corresponds to about 10⁷ times higher than that in seawater. In these species signet ring cells, with a single huge vacuole in which vanadium ion is contained, function as vanadium-accumulating cells, vanadocytes. To investigate the mechanism underlying this phenomenon, we performed an expressed sequence tag (EST) analysis of a complementary DNA library from vanadocytes of a vanadium-rich ascidian, Ascidia sydneiensis samea. We determined the nucleotide sequences of 1000 ESTs and performed a BLAST analysis against the SwissProt database. We found 93 clones of metal-related gene homologues, including the ferritin heavy subunit, hemocyanin, and metallothionein. Two ESTs, in particular, exhibited significant similarity to vanabins that have been extracted from A. sydneiensis samea blood cells as low molecular weight vanadium-binding proteins. We have named the genes encoding these ESTs vanabin3 and vanabin4. Immobilized metal ion affinity chromatography revealed that these novel vanabin homologues bind vanadium(IV) ions.     

Tail morphogenesis in the ascidian, Ciona intestinalis, requires cooperation between notochord and muscle

We present evidence that notochord and muscle differentiation are crucial for morphogenesis of the ascidian tail. We developed a novel approach for embryological manipulation of the developing larval tissues using a simple method to introduce DNA into Ciona intestinalis and the several available tissue-specific promoters. With such promoters, we misexpressed the Xenopus homeobox gene bix in notochord or muscle of Ciona embryos as a means of interfering with development of these tissues. Ciona embryos expressing bix in the notochord from the 64-cell stage develop into larvae with very short tails, in which the notochord precursors fail to intercalate and differentiate. Larvae with mosaic expression of bix have intermediate phenotypes, in which a partial notochord is formed by the precursor cells that did not receive the transgene while the precursors that express the transgene cluster together and fail to undergo any of the cell-shape changes associated with notochord differentiation. Muscle cells adjacent to differentiated notochord cells are properly patterned, while those next to the notochord precursor cells transformed by bix exhibit various patterning defects. In these embryos, the neural tube extends in the tail to form a nerve cord, while the endodermal strand fails to enter the tail region. Similarly, expression of bix in muscle progenitors impairs differentiation of muscle cells, and as a result, notochord cells fail to undergo normal extension movements. Hence, these larvae have a shorter tail, due to a block in the elongation of the notochord. Taken together, these observations suggest that tail formation in ascidian larvae requires not only signaling from notochord to muscle cells, but also a "retrograde" signal from muscle cells to notochord.     

Tunic morphogenesis in the sea peach (Halocynthia papillosa,Ascidiacea, rochordata): Cellular events,the initiation of twisted fibrous arrangements and spine formation

Summary— The adult tunic of the sea peach (Halocynthia papillosa) shows a high degree of organisation. Tunic morphogenesis was monitored from the onset of tunic secretion until juveniles reached the age of 3 months. While some characteristics of the adult tunic are still missing, like certain types of intratunical cells and striated bodies, its main features have already developed by this time. Crucial events take place at or soon after the onset of metamorphosis (stage M 0). Cuticular spines cover the external surface of the juvenile. At least two types of intratunical cells enter the tunic and the fibrous material adopts a three-dimensional twisted helicoidal architecture. The initiation of this helicoidal arrangement of fibrils directly after stage M 0 is discussed regarding accompanying developmental events. The existence of cells that penetrate the outer compartment of the tunic at the end of larval life is reported for the first time.     

Retinoic acid affects patterning along the anterior–posterior axis of the ascidian embryo

Because retinoic acid (RA) is known to affect anterior-posterior patterning in vertebrate embryos, it was questioned whether it shows similar effects in a more primitive chordate, the ascidian Halocynthia roretzi . Ascidian embryos treated with RA exhibited truncated phenotypes in a dose-dependent manner similar to the anterior truncations seen in vertebrate embryos. The most severely affected larvae possessed a round trunk without the papillae characteristic of the anterior terminal epidermis. Retinoic acid also altered the expression of HrHox-1 and Hroth in a dose-dependent manner. Expression of HrHox-1 increased, whereas expression of Hroth decreased with increasing levels of RA. In treated embryos, HrHox-1 was first expressed pan-ectodermally, then degraded in all but specific regions of the embryo. By contrast, initiation of Hroth expression was not affected, but epidermal expression was lost while expression in the neural tube narrowed toward the anterior in tail-bud embryos. These alterations in the expression of homeobox genes appear to correlate closely to the morphological defects elicited by RA treatment, suggesting broad conservation of developmental patterning mechanisms within the Phylum Chordata.     

Functional design in the evolution of embryos and larvae

A function of development is to put the right kind of cells in the right place at the right time. Other functional analyses help define what is right. As examples, functional analyses offer explanations for the unicellular bottleneck in life histories that necessitates embryos, evolutionary divergences in embryonic cell cycles, conditions permissive of loss of larval structures and consequent change in embryonic development, and the decoupled development of larval bodies and juvenile rudiments. Functional analyses also reveal the specifications required of morphogenesis, hence defining developmental phenomena to be explained.