Regulation of metamorphosis in ascidians involves NO/cGMP signaling and HSP90.

Treatment of larvae of the ascidians Boltenia villosa (Family: Pyuridae) and Cnemidocarpa finmarkiensis (Family: Styelidae) with drugs that inhibit the function of the molecular chaperone HSP90 increased the frequency of tail resorption, the primary morphogenetic event of metamorphosis. If treatment was initiated at hatching, metamorphic events subsequent to tail resorption failed to occur, indicating an ongoing role for HSP90 during morphogenesis. Removal of tails from heads of mature, but not newly hatched larvae, induced metamorphosis of the head. Decapitation experiments indicate that the capacity of tails to shorten in response to inhibition of HSP90 function requires communication with heads. To identify candidate proteins with which HSP90 may interact to regulate metamorphosis, we noted that in mammalian cells, nitric oxide synthase (NOS) interacts with HSP90 and its activity is sensitive to drugs that inhibit HSP90 function. In addition, nitric oxide (NO) signaling in the marine snail Ilyanassa obsoleta is an important regulator of metamorphosis. Inhibition of NOS activity in these ascidian larvae with L-NAME increased the frequency of metamorphosis, consistent with a putative interaction of NOS and HSP90. NOS is present in tail muscle cells, implicating them as targets for the drug treatments, consistent with the decapitation experiments. Inhibition of soluble guanylyl cyclase, the most common effector of NO signaling, also increased the frequency of metamorphosis. In contrast to treatment with anti-HSP90 drugs, metamorphosis induced with L-NAME or ODQ was complete. The results presented suggest that an HSP90-dependent, NO-based regulatory mechanism localized in tails represses ascidian metamorphosis. We discuss these results in relation to the induction of ascidian metamorphosis by several unrelated agents.

The evolution of anural larvae in molgulid ascidians.

Ascidians are urochordates, marine invertebrates with non-feeding motile chordate tadpole larvae, except in the family Molgulidae. Urodele, or tailed, Molgulids have typical ascidian chordate tadpole larvae possessing tails with muscle cells, a notochord, and a dorsal hollow nerve cord. In contrast, anural (or tail-less) Molgulids lack a tail and defining chordate features. Molecular phylogenies generated with 18S and 28S ribosomal sequences indicate that Molgulid species fall into at least four distinct clades, three of which have multiple anural members. This refined and expanded phylogeny allows careful examination of the factors that may have influenced the evolution of tail-less ascidians.

Developmental roles of nuclear complex factors released during oocyte maturation in the ascidians Halocynthia roretzi and Boltenia villosa.

The developmental roles of factors associated with the nuclear complex of Halocynthia roretzi and Boltenia villosa oocytes were investigated by cutting mature oocytes into animal and vegetal merogons before and during GVBD. Animal and vegetal merogons were cultured in sea water until the GV cytoplasm had dispersed within the cytoplasm of control oocytes and then they were cross-fertilized and scored for their ability to undergo normal development. Halocynthia oocyte fragments produced from the animal region of oocytes containing intact GVs exhibited a low frequency of polyspermy, a high frequency of fertilization and cleavage, and a high frequency of expressing an epidermal antigen, Epi-2. In contrast, merogons produced from the vegetal region of Halocynthia oocytes in which GVs were intact exhibited a high frequency of polyspermy, did not undergo cell division, and expressed a high frequency of Epi-2 expression. When vegetal fragments were produced after the dispersal of approximately 50-70% of the GV nucleoplasm, these merogons exhibited a low frequency of polyspermy, high frequencies of cell division (including the formation of epidermal layer), and in most cases expressed Epi-2. Vegetal Boltenia fragments produced during GVBD in some cases developed into larvae. These results suggest that the ascidian GV nucleoplasm may contain factors required for fertilization and cell division and that epidermal determinants reside in the oocyte cytoplasm.

Evolutionary Implications of FGF and Distal-Less Expressions During Proximal-Distal Axis Formation in the Ampulla of a Direct-Developing Ascidian, Molgula pacifica.

The present results provide the first evidence of a fibroblast growth factor (FGF) family protein in a urochordate. Anti-FGF2 immunoreactive hemoblast cells were detected at day 3 of juvenile development in a direct-developing urochordate ascidian, Molgula pacifica. The detection of FGF in hemoblast cells coincided with the appearance of distal-less protein along the proximal-distal axis of growing ampullae. Ampullae are limb-like, fluid-filled ectodermal appendages that contain hemoblast cells and have holdfast, respiratory, and immunological functions (1). Given the evolutionary conservation of the genes encoding FGF (2) their receptors (2), and distal-less (3) the present results suggest that the formation of non-homologous ascidian appendages shares genetic elements in common with proximal-distal axis formation in arthropod limbs and vertebrate limbs (4,5). The possible evolutionary implications of these findings are discussed.

p58, a Cytoskeletal Protein, Is Associated With Muscle Cell Determinants in Ascidian Eggs.

The theory that p58, a cytoskeletal protein, has an important role in ascidian muscle cell development was tested by altering normal distributions of orange-pigmented myoplasm in Boltenia villosa embryos and determining if muscle development is correlated with the presence of p58. Removal of the animal region of fertilized Boltenia eggs resulted in the redistribution of myoplasm into the anterior endoderm cells of the embryo. Despite alterations in the normal distribution of myoplasm, these embryos developed into larvae. However, when four-celled embryos that exhibited altered distributions of pigmented myoplasm were stained with NN18, an antibody that stains p58, a maximum of two blastomeres were stained, as in control embryos. Compression of Boltenia embryos at the four-celled stage caused the myoplasm to be partitioned into four blastomeres of an eight-celled embryo, instead of into two blastomeres. Compressed and cleavage-arrested eight-celled embryos developed myosin and muscle actin RNA in a maximum of four blastomeres, compared to a maximum of two blastomeres in control embryos. When compressed eight-celled embryos were stained with NN18, p58 was present in a maximum of four blastomeres. These results support the idea that the cytoskeletal protein p58 is associated with muscle cell determinants in ascidian eggs.

Direct Development in the Ascidian Molgula retortiformis (Verrill, 1871).

The cellular features of the ascidian Molgula retortiformis (Verrill, 1871), a direct developing species, were investigated with the aid of transmission electron microscopy, histochemistry, and immunocytochemistry. Developmental comparisons between direct and indirect developing ascidians will further our understanding of how developmental processes evolve. M. retortiformis eggs are surrounded by a follicular envelope comprising a layer of outer follicle cells attached to an acellular chorion. The cytoplasm of M. retortiformis eggs contains large quantities of yolk and glycogen. Immediately after hatching, at day 2.5 of development, the cells constituting a juvenile exhibited similar ultrastructural features, except that the larger, deeper cells contained more yolk and glycogen than the epidermal cells. Differentiated muscle cells were absent in newly hatched M. retortiformis juveniles, and acetylcholinesterase (AChE) activity was not detected. Immunocytochemistry experiments using a vertebrate intermediate filament antibody (NN18) support the idea that the failure of newly hatched M. retortiformis juveniles to develop muscle cells may be due to the absence of a factor localized in the egg myoplasm. This paper concludes with a discussion of the "substrate hypothesis" and the evolution of ascidian direct development.

Evolutionary modifications of morphogenetic mechanisms and alternate life history strategies in ascidians.

Comparative embryological studies using anural and urodele ascidians provide an experimental system to study interactions between developmental and evolutionary mechanisms that produce alternate life history strategies. In this paper, cellular features of anural morphogenesis in Molgula pacifica are compared to morphogenesis in species that develop tailed (urodele) larvae and other anural molgulid species. The results of these studies are discussed with regard to possible mechanisms responsible for the evolution of anural morphogenesis and the ecological consequences of anural development. Early developmental processes including ooplasmic segregation, cleavage patterns, and the site and timing of gastrulation were similar in M. pacifica compared to urodele embryos and embryos produced by other anural species. The limited extent of invagination caused by large, yolky cells that restricted vegetal pole cell movements in M. pacifica gastrulae contrasted with the extensive movements of vegetal pole cells that accompanied invagination in M. provisionalis embryos and the embryos of four urodele species. The modified mode of gastrulation exhibited by M. pacifica embryos is likely due to the relatively high yolk content of their eggs. The developmental fates of muscle and epidermal progenitor cells in M. pacifica embryos were altered compared to urodele embryos. Ultrastructural studies and acetylcholinesterase histochemistry experiments indicate that muscle progenitor cells have lost their potential to develop muscle cell features. This loss in myogenic potential suggests that muscle progenitor cells were re-programmed to die. However, this possibility was not supported by the results of ultrastructural studies. A second possibility is discussed in that muscle progenitor cells may have been re-specified to differentiate into adult cells after metamorphosis. Evidence is presented suggesting that the timing mechanism responsible for controlling the onset of metamorphosis, first evident by the outgrowth of epidermal ampullae, was modified in M. pacifica. This paper concludes with a discussion of how anural morphogenesis altered the ancestoral urodele life cycle and the possible ecological benefits of these evolutionary alterations.

Effects of chronic ethanol and diet treatment on urinary folate excretion and development of folate deficiency in the rat.

Because the folate deficiency of chronic alcoholism has been proposed to result from ethanol-induced effects on metabolism or urinary excretion of folate, the present study was designed to evaluate the role of chronic ethanol-induced urinary folate loss on folate homeostasis in the rat. Male Sprague-Dawley rats were fed nutritionally sufficient liquid diets for 12 wk with or without ethanol, folate and sulfonamide. Urinary folate excretion was increased in ethanol-fed rats consuming folate-containing diets, but not in rats fed folate deficient diets. Consumption of folate-deficient diets led to a rapid decrease in urinary folate excretion, suggesting renal adaptation to conserve folate. Tissue and plasma levels of folate were mostly unaffected by ethanol ingestion in rats fed folate-containing diets. Ethanol treatment did not consistently enhance tissue folate depletion in rats fed folate-deficient diets. The results suggest that in rats consuming diets containing high levels of folate, chronic ethanol ingestion increased urinary folate excretion, but not to a sufficient magnitude to consistently affect folate homeostasis.

Polarization of ooplasmic segregation and dorsal-ventral axis determination in ascidian embryos.

During ooplasmic segregation in ascidians, the myoplasm moves from its original location in the periphery of the unfertilized egg to the vegetal pole of the zygote. The vegetal cap of myoplasm marks the future site of gastrulation and the dorsal side of the embryo. The purpose of this investigation was to determine the mechanism for polarizing the myoplasm during ooplasmic segregation. To test the possibility that the myoplasm moves toward the sperm and that the vegetal pole is the exclusive site of sperm entry, we examined fertilization in egg fragments of the ascidians Styela plicata and Ciona savignyi. Similar frequencies of fertilization were exhibited by various egg fragments, including animal and vegetal fragments or multiple fragments prepared from the same egg. These results indicate that sperm do not enter the egg exclusively at the vegetal pole. Experiments with egg fragments and constricted eggs, combined with chalk marking of the animal pole, demonstrated that after fertilization the myoplasm segregates parallel to the animal-vegetal axis, usually toward the vegetal end of the cell. Activation of primary oocytes with the Ca2+ ionophore A23187 caused the myoplasm to segregate independently of the animal-vegetal axis. This confirms previous experiments in which eggs aligned along a glass fiber coated with A23187 segregated their myoplasm toward the fiber (W.R. Jeffery, 1982, Science 216, 545-547) and suggests that the intrinsic cue for polarization is a release of sequestered Ca2+ at fertilization. Therefore, it appears that ooplasmic segregation and the dorsal-ventral axis are polarized by maternal factors distributed in a concentration gradient along the animal-vegetal axis of the ascidian egg.

Development of myoplasm-enriched ascidian embryos.

The fertilized ascidian egg is thought to be comprised of distinct regions of tissue-specific cytoplasmic determinants. This idea was tested by bisecting fertilized eggs into egg fragments and culturing them until the unoperated controls developed into larvae. Fertilized eggs were bisected using a microsurgical method in which part of the uncleaved zygote was extruded through a hole made in the follicular envelope and the cytoplasmic bridge between the two egg regions was severed. One egg fragment contained all of the egg myoplasm (termed myoplasm-enriched or ME fragment), while the other fragment lacked myoplasm. ME fragments consisting of 40-50% of the total egg volume in many cases cleaved normally and developed into larvae. In a few cases, ME larvae initiated metamorphosis and developed into normal juveniles. Triton-extraction of ME embryos and larvae showed that the myoplasm was redistributed into nonmuscle lineage cells at each stage of development. Despite the redistribution of myoplasm into many of the endoderm cells situated in the head region of ME larvae, the expression of the muscle-specific enzyme acetylcholinesterase (AchE) and a muscle-specific antigen (Mu-2) was restricted to the tail muscle cells. The endoderm cells situated in the head region of ME larvae expressed an endoderm-specific enzyme alkaline phosphatase (AP) as in the controls. Furthermore, cleavage-arrested four- and eight-cell ME embryos expressed AchE activity in the expected number of blastomeres. When a greater quantity of myoplasm was redistributed into cells that normally do not express AchE activity by producing 10-30% ME embryos, in a few cases more than the expected number of blastomeres expressed AchE activity. In conclusion, the main finding of the present investigation, based on the development of ME fragments comprising 40-50% of the total egg volume, is that ascidian embryos are capable of regulative development.

Development of a muscle actin specified by maternal and zygotic mRNA in ascidian embryos.

In this investigation, we characterize the embryonic and adult actins and describe the embryonic expression of a muscle actin in the ascidian Styela. Two-dimensional polyacrylamide gel electrophoresis showed that embryos, tadpole larvae, and adult organs contain three major and two minor isoforms of actin. Two of the major isoforms, which are present in the mantle, branchial sac, alimentary tract, and gonads of adults and in eggs, embryos, and heads and tails of tadpoles, are likely to be cytoplasmic actins. The third major isoform, which was enriched in the mantle and branchial sac of adults and localized primarily in the tails of tadpoles, is a muscle actin. The muscle actin isoform was not detected in eggs and early embryos. Radioactivity incorporation studies showed that the cytoplasmic actins were synthesized throughout early development, but muscle actin synthesis was first detected between the 16- and 64-cell stages, 2-3 hr after fertilization. Two lines of evidence indicate that embryonic muscle actin synthesis is directed in part by maternal mRNA. First, poly(A)+ RNA isolated from unfertilized eggs directed the synthesis of muscle actin in an mRNA-dependent reticulocyte lysate. Second, muscle actin was synthesized in anucleate egg fragments. Arguments are also presented that muscle actin synthesis is not directed exclusively by maternal mRNA. It is concluded that embryonic and adult Styela exhibit actin heterogeneity, that one of the actin isoforms is a muscle actin, and that the muscle actin is synthesized during embryogenesis under the direction of maternal and zygotic mRNA.

Alkaline phosphatase expression in ascidian egg fragments and andromerogons.

Alkaline phosphatase (AP) activity is expressed in the endodermal cell lineage of ascidian embryos beginning at gastrulation. AP expression is resistant to levels of actinomycin D which completely suppress the appearance of other tissue-specific enzyme and morphological markers including acetylcholinesterase (AchE), a larval muscle enzyme whose expression requires embryonic transcription. The resistance of AP expression to actinomycin D has led to the proposal that AP may be expressed independent of embryonic transcription by the translational activation of maternal AP mRNA. To test this hypothesis, AP expression was examined in fragments of unfertilized and fertilized Styela plicata eggs by histochemical methods. As expected, nucleate fragments from fertilized eggs developed into larvae which exhibited AP activity in their endodermal cells and AchE activity in their muscle cells. In contrast, anucleate fragments from fertilized eggs, cultured until controls reached the larval stage, did not develop AP or AchE activity. The lack of AP activity was unrelated to the absence of cleavage or to the ooplasmic composition of the anucleate fragments. Anucleate fragments from unfertilized eggs were also AP negative, unless they were inseminated, after which they often developed to the larval stage as andromerogons and exhibited AP activity in their endodermal cells. The development of endodermal AP in andromerogons suggests that the factors responsible for AP expression are not localized in or attached to the maternal nucleus. In summary, the results suggest that AP expression requires nuclear events and is not determined exclusively by maternal cytoplasmic factors such as preformed mRNA.

Synthesis of putative microtubule-associated proteins by mouse blastocysts during early outgrowth in vitro.

The outgrowth of mouse trophoblast in culture provides a simplified model system analogous in certain ways to blastocyst implantation in vivo. Day-four blastocysts cultured for 3 days in vitro undergo extensive changes in cell shape and motility which are likely to involve the complex cytoskeletal system of the trophoblast cells. To explore the biochemical basis of these changes, one set of cytoskeletal proteins, the microtubule-associated proteins (MAPs), was studied. Day 4 blastocysts were labeled with [35S]methionine and blastocyst outgrowths, after 3 days in culture from the blastocyst stage, were labeled with [3H]methionine. Labeled embryos were disrupted and the soluble supernatants were pooled, and newly synthesized proteins from the two stages were coassembled with taxol-stabilized brain microtubule polymer enriched for MAP-binding sites. Double-labeled coassembly proteins (putative MAPS) were then released from the microtubule polymer by treatment with 0.35 M NaCl and analyzed by one-dimensional polyacrylamide gel electrophoresis. 3H/35S dpm ratios were determined for individual protein bands to compare the relative synthesis rates for day 4 blastocyst and day 3 outgrowth MAPs. In spite of the extensive changes in cell shape and motility associated with blastocyst outgrowth, a common set of putative MAPs characterizes the two stages investigated, including several in the size range of tau factors. No synthesis of high molecular weight MAPs comparable with MAP 1 or MAP 2 from brain was detected. The synthesis rates of individual MAPs relative to each other remain constant over this period and are likely coordinated with total protein and tubulin synthesis.