Developmental autonomy of presumptive notochord cells in partial embryos of an ascidian

Summary

Ultrastructural features of larval notochord cell differentiation, sheath (membrane leaflets and filaments) and vacuoles of intracellular colloid, were found in some cells of certain partial embryos of the ascidian, Ciona intestinalis. As expected from established lineage fate maps, mature quarter-embryos developing from microsurgically isolated anterior-vegetal blastomeres (A4.1 pair) at the 8-cell stage had some cells with the notochord features. Such cells, however, also occurred in quarter-embryos resulting from the posterior-vegetal blastomere pair (B4.1) and in partial embryos derived from the B5.1 cell pair isolated at the next cleavage of the B4.1 blastomeres. These findings confirm a prediction of additional notochord cell fates from a recent revision of the ascidian lineage map based on cell marking with microinjected horseradish peroxidase. Partial embryos obtained from other lineages of the 8- and 16-cell stages did not develop notochord cells.     

Tyrosine and the synthesis of melanin in embryonic cells fromAmbystoma mexicanum

Summary Explants comprising about 15 cells were dissected from various regions of the blastula ofAmbystoma mexicanum and cultured in Barth's medium. By addition of L-tyrosine to the culture medium it was possible to induce melanin synthesis in three different cells types: undifferentiated embryonic cells, mesenchyme cells and nerve cells. Tyrosine was found to act as an inductor in a very low concentration (1 M). It is suggested that tyrosine serves both as an inductor and as a substrate for melanin synthesis in the amphibian larva.     

Development of pigment cells in the brain of ascidian tadpole larvae: insights into the origins of vertebrate pigment cells.

In vertebrates, melanins produced in specialized pigment cells are required for visual acuity, camouflage, sexual display and protection from ultra violet (UV) radiation. There are three pigment cell types that are classified based on their distinct embryonic origins. Retinal pigment epithelium (RPE) cells originate from the outer layer of the optic cup. Pigment cells of the pineal organ are formed from the developing diencephalon. Melanocytes are derived from the neural crest unique to vertebrate embryos. Some of these pigment cells also play roles that are independent of the activity of tyrosinase, the key melanogenesis enzyme, or melanin: production of substrate(s) for catecholamine synthesis, maintenance of endolymph composition in the cochlea, maintenance of photoreceptor cells in the retina and retinoid metabolism essential for the visual cycle. To deduce the evolutionary origins of vertebrate pigment cells and a possible archetypal genetic circuitry, which may have been modified and utilized to generate multiple pigment cell types, comparison of developmental mechanisms of pigment cells between vertebrates and closely related invertebrate ascidians are proposed to provide useful information. The tadpole-type larva of ascidians possesses two melanin-containing pigment cells, termed the otolith and ocellus pigment cells, in the brain that are believed to be required for photo- and geotactic responses during swimming. In this review, current knowledge on the development of the two ascidian pigment cells is summarized, i.e. complete cell lineage, structure and expression of genes encoding two melanogenesis enzymes, and molecular developmental mechanisms involving BMP-CHORDIN antagonism, and possible evolutionary relationships between ascidian and vertebrate pigment cells are discussed.     

Autonomy of acetylcholinesterase differentiation in muscle lineage cells of ascidian embryos

The two muscle lineage blastomeres were removed surgically from Ciona intestinalis embryos at the eight-cell stage and allowed to develop in isolation. Acetylcholinesterase, an enzyme that occurs only in muscle cells of the developing larva, was detected histochemically in progeny cells of these isolated blastomers. Acetylcholinesterase differentiation in muscle lineage cells is not, therefore, dependent on inductive interactions with embryonic tissues derived from other eight-cell stage blastomeres.     

Defects in cAMP-pathway may initiate carcinogenesis in dividing nerve cells: A review

The mechanisms of carcinogenesis in nervous tissues are not well understood. It is now established that adenosine 3,,5-cyclic monophosphate (cAMP)-pathway plays a crucial role in initiating differentiation in transformed and embryonic cells of neuronal and glial origin. Therefore, we propose that defects in the cAMP-pathway may initiate the first phase of carcinogenesis (immortalization). Subsequent genetic abnormalities in oncogenes, anti-oncogenes or other cellular genes individually or in combination may lead to transformation (cancer phenotype). This hypothesis is derived from the fact that an elevation of the cAMP level in murine NB cells induces terminal differentiation in many of these cells in spite of the fact that they are highly aneuploid. Additional changes in cAMP-regulated genes responsible for initiating differentiation may make these cells resistant to cAMP or may make the cAMP-effect on differentiation reversible. Indeed, cAMP-resistant cells exist in NB cell populations, and the cAMP-effect on differentiation is reversible in glioma cells. Identification of genes that initiate, promote and maintain terminal differentiation and those which prevent differentiation following elevation of cAMP in NB cells may increase our understanding of the mechanisms of carcinogenesis. This review illustrates the following: (a) historical background leading to the discovery of cAMP as an inducer of differentiation in nerve cells; (b) identification of potential sites in cAMP-pathway that may play a crucial role in initiating the first phase of carcinogenesis (immortalization) and potential gene targets in immortalized cells whose alterations may cause neoplastic transformation of nerve cells. It is interesting to note that the cAMP pathway remains responsive to an elevated cAMP level in inducing differentiation in NB cells in spite of chromosomal anomalies and genetic changes associated with the maintenance of a cancer phenotype.     

Role of TGF-beta in the retinoic acid-induced inhibition of proliferation and melanin synthesis in chick retinal pigment epithelial cells in vitro

The purpose of this study was to investigate the effects of all-trans retinoic acid (RA) on the induction of transforming growth factor-beta (TGF-beta) that is concerned with the proliferation and melanin synthesis of chick retinal pigment epithelial (RPE) cells in vitro. Chick RPE cells were cultured in the presence or absence of RA and anti-TGF-beta antibody for 7 days. The effects of RA and pan-specific TGF-beta antibody on RPE cell proliferation were assessed by counting the number of cells, and their effects on melanin synthesis were evaluated by measuring the melanin content of the cells. TGF-beta activity in the culture supernatant of RPE cells was measured using CCL-64 cells. RA significantly inhibited RPE cell proliferation and increased melanin synthesis. The addition of pan-specific TGF-beta antibody to the culture blocked the inhibition of RPE cell proliferation and the increased melanin synthesis. RA induced TGF-beta production in the culture supernatant of RPE cells. These findings indicate that RA regulates the proliferation and melanin synthesis of RPE cells via induction of TGF-beta.     

(beta)-catenin mediates the specification of endoderm cells in ascidian embryos

In the present study, we addressed the role of (beta)-catenin in the specification of embryonic cells of the ascidians Ciona intestinalis and C. savignyi and obtained the following results: (1) During cleavages, (beta)-catenin accumulated in the nuclei of vegetal blastomeres, suggesting that it plays a role in the specification of endoderm. (2) Mis- and/or overexpression of (beta)-catenin induced the development of an endoderm-specific alkaline phosphatase (AP) in presumptive notochord cells and epidermis cells without affecting differentiation of primary lineage muscle cells. (3) Downregulation of (beta)-catenin induced by the overexpression of cadherin resulted in the suppression of endoderm cell differentiation. This suppression was compensated for by the differentiation of extra epidermis cells. (4) Specification of notochord cells did not take place in the absence of endoderm differentiation. Both the overexpression of (beta)-catenin in presumptive notochord cells and the downregulation of (beta)-catenin in presumptive endoderm cells led to the suppression of Brachyury gene expression, resulting in the failure of notochord specification. These results suggest that the accumulation of (beta)-catenin in the nuclei of endoderm progenitor cells is the first step in the process of ascidian endoderm specification.     

Differentiation of histospecific ultrastructural features in cells of cleavage-arrested early ascidian embryos

Summary Ultrastructural features of histospecific differentiation were found in early cleavage stage ascidian embryos treated with cytochalasin B and held thereby in cleavagearrest until hatching time. Markers characteristic of tissue differentiation during normal embryonic and larval stages ofCiona intestinalis were expressed in muscle and two brain cell lineages of cleavage-arrested whole embryos and in epidermal and notochordal cell lineages of cleavage-arrested partial embryos. These features were muscle myofilaments and myofibrils, melanosomes of the brain pigment cells, cilium-derived structures present in a proprioceptive brain cell, extracellular test material of epidermal cell origin, and the sheath filaments, membrane leaflets, and vacuolar colloid associated with notochord cells. All of these ultrastructural markers of differentiation were blocked in their development by treatment of gastrula stage embryos with actinomycin D, an inhibitor of RNA synthesis, and presumably result from the expression of new gene activity. At the time of cleavage-arrest the five cell lineages studies still contained two or more unsegregated lineage pathways. Subsequent developmental autonomy within the lineages is consistent with the hypothesis of segregation during early development of functionally independent gene regulatory factors.     

Skeletogenic potential of induced secondary mesenchyme cells derived from the presumptive ectoderm in echinoid embryos

 During the normal development of echinoids, an animal cap consisting of 8 mesomeres in a 16-cell stage embryo differentiates exclusively into ectoderm. Micromeres in an embryo at the same stage differentiate into primary mesenchyme cells (PMC) and coelomic pouch constituents. An animal cap and a quartet of micromeres were isolated from a 16-cell stage embryo and recombined to make a chimeric embryo devoid of presumptive endoderm and secondary mesenchyme cells (SMC). The PMC in the chimeric embryo were completely removed at the mesenchyme blastula stage. The PMC-depleted chimeric embryos formed an archenteron derived from the mesomeres. Some secondary mesenchyme-like cells (induced SMC) were released from the archenteron tip. A considerable fraction of the induced SMC formed the typical mesenchyme pattern after migrating into the vegetal region, synthesized skeletogenic mesenchyme cell-surface protein (msp130) and produced the larval skeleton. These findings indicate that induced SMC derived from the presumptive ectoderm have the same nature as natural SMC in both the timing of their release and their skeletogenic potential expressed in the absence of PMC. Received: 14 November 1996 / Accepted: 30 December 1996     

The classical pathway of melanogenesis is not essential for melanin synthesis in the adult retinal pigment epithelium

Premelanosomes are presumed to be essential for melanogenesis in melanocytes and pre-natal retinal pigment epithelium (RPE) cells. We analysed melanin synthesis in adenoviral-transduced tyrosinase-gene-expressing amelanotic RPE (ARPE) 19 cells to determine whether premelanosome formation is needed for post-natal melanogenesis. The synthesis of melanogenic proteins and melanin granules was investigated by immunocytochemistry and light and electron microscopy. The occurrence of tyrosinase was analysed ultrastructurally by dihydroxyphenylalanine histochemistry. The viability of transduced cell cultures was examined via MTT assay. We found active tyrosinase in small granule-like vesicles throughout the cytoplasm and in the endoplasmic reticulum and nuclear membrane. Tyrosinase was also associated with multi-vesicular and multi-lamellar organelles. Typical premelanosomes, structural protein PMEL17, tyrosinase-related protein 1 and classic melanosomal stages I–IV were not detected. Instead, melanogenesis took place inside multi-vesicular and multi-lamellar bodies of unknown origin. Viability was not affected up to 10 days after transduction. We thus demonstrate a pathway of melanin formation lacking typical hallmarks of melanogenesis.     

Co-regulation of melanin precursors and tyrosinase in human pigment cells: roles of cysteine and glutathione.

Glutathione (GSH) and cysteine (CysH) have both been implicated in the biogenesis of the pheomelanin precursor 5-S-cysteinyldopa (5-S-CD). However, recent studies have shown that only CysH is transported across the membrane of isolated melanosomes, and that the positive regulation of CysH in pigment cells leads to an increased production of 5-S-CD. In the present study, the question was examined as to whether melanin precursors and tyrosinase could be coregulated by cellular thiols. To address this issue, the levels of CysH and GSH were varied in normal melanocytes and melanoma cells using buthionine sulfoximine (BSO), an inhibitor of GSH biosynthesis. Treatment with 50-100 microM BSO decreased GSH levels to less than 10% of control, and increased CysH levels between two- and five-fold in both cell types. Concomitant with this, an increase in the ratio of 5-S-CD to DOPA and a decrease in the pigment content of the cells were observed. The decrease in cell pigmentation was associated with strong decreases in tyrosine hydroxylase activity and 14C-melanin production. Only melanoma cells showed a modified tyrosinase isozyme pattern on Western immunoblots in response to BSO, while the mRNA expression of tyrosinase and TRP-1 were unchanged in both cell types. These results suggest that the balance between CysH and GSH, which is partly determined by the rate of utilization of CysH for GSH biosynthesis, regulates not only the levels of 5-S-CD and DOPA but also the melanogenic activity of pigment cells. Since DOPA functions as a cofactor in the monophenolase reaction of tyrosinase, it is proposed that the ratio of 5-S-CD to DOPA may be an important factor in the regulation of tyrosinase activity in situ.     

Muscle lineage cytoplasm can change the developmental expression in epidermal lineage cells of ascidian embryos

Cytoplasm from muscle lineage blastomeres of an ascidian embryo can cause cells of a nonmuscle lineage to produce larval tail muscle acetylcholinesterase. Muscle cytoplasm was partitioned microsurgically into epidermal lineage blastomeres at the eight-cell stage. Posterior half-embryos (the two B3 cells) of Ascidia nigra were obtained first by separating the anterior and posterior blastomere pairs at the four-cell stage. At third cleavage, the two B3 cells divide into an ectodermal cell pair that gives rise solely to epidermal tissues, and a mesodermal-endodermal blastomere pair from which the tail muscle cells are derived. When the ectodermal and mesendodermal blastomere pairs were isolated from one another by microsurgery and reared as partial embryos, only cells originating from the mesendodermal blastomeres produced a histochemical acetylcholinesterase reaction. Immediately after cleavage of the isolated B3 cells into ectodermal and mesendodermal cell pairs, the cleavage furrows could be made to disappear by pressing firmly on the mesendodermal cells with a microneedle. Repeated up and down pressure with the microneedle at a new position across the mesendodermal cells caused furrows to reestablish in the new position, thereby incorporating mesodermal cytoplasm and increasing the size of the ectodermal cells. The cytoplasmically altered ectodermal blastomere pairs, which became detached from the mesendodermal cells by this microsurgical procedure, continued to divide and were reared to “larval” stages. One-third of these epidermal partial larvae produced patches of cells containing acetylcholinesterase. These results lend further support to the theory that choice of particular differentiation pathways (embryonic determination) in ascidian embryos is mediated by segregation of specific egg cytoplasmic determinants.     

Determinative properties of muscle lineages in ascidian embryos

Blastomeres removed from early cleavage stage ascidian embryos and reared to 'maturity' as partial embryos often elaborate tissue-specific features typical of their constituent cell lineages. We used this property to study recent corrections of the ascidian larval muscle lineage and to compare the ways in which different lineages give rise to muscle. Our evaluation of muscle differentiation was based on histochemical localization and quantitative radiometric measurement of a muscle-specific acetylcholinesterase activity, and the development of myofilaments and myofibrils as observed by electron microscopy. Although the posterior-vegetal blastomeres (B4.1 pair) of the 8-cell embryo have long been believed to be the sole precursors of larval muscle, recent studies using horseradish peroxidase to mark cell lineages have shown that small numbers of muscle cells originate from the anterior-vegetal (A4.1) and posterior-animal (b4.2) blastomeres of this stage. Fully differentiated muscle expression in isolated partial embryos of A4.1-derived cells requires an association with cells from other lineages whereas muscle from B4.1 blastomeres develops autonomously. Clear differences also occurred in the time acetylcholinesterase activity was first detected in partial embryos from these two sources. Isolated b4.2 cells failed to show any muscle development even in combination with anterior-animal cells (a4.2) and are presumably even more dependent on normal cell interactions and associations. Others have noted an additional distinction between the different sources of muscle: muscle cells from non-B4.1 lineages occur exclusively in the distal part of the tail, while the B4.1 descendants contribute those cells in the proximal and middle regions. During the course of ascidian larval evolution tail muscle probably had two origins: the primary lineage (B4.1) whose fate was set rigidly at early cleavage stages and secondarily evolved lineages which arose later by recruitment of cells from other tissues resulting in increased tail length. In contrast to the B4.1 lineage, muscle development in the secondary lineages is controlled less rigidly by processes that depend on cell interactions.     

A genome-wide survey of genes for enzymes involved in pigment synthesis in an ascidian, Ciona intestinalis

The draft genome sequence and a large quantity of EST and cDNA information are now available for the ascidian Ciona intestinalis. In the present study, genes involved in pigment synthesis pathways were identified in the decoded genome of Ciona, and information about these genes was obtained from available EST and cDNA sequences. It was found that the Ciona genome contains orthologous genes for each enzyme of the melanin, pteridine, ommochrome, papiliochrome, and heme synthesis pathways. Several appear as independent duplications in the Ciona genome. Because cDNA clones for all but two of these genes have already been isolated by the cDNA project, C. intestinalis will provide an experimental system to explore molecular mechanisms underlying color patterns, through future genome-wide studies.     

Determination and regulation in the pigment cell lineage of the ascidian embryo

The brain of the ascidian larva comprises two pigment cells, termed the ocellus melanocyte and the otolith melanocyte. Cell lineage analysis has shown that the two bilateral pigment lineage cells (a-line blastomeres) in the animal hemisphere give rise to these melanocytes in a complementary manner. The results of the present investigation suggest that the specification of the fate of pigment cells proceeds in two distinct steps. First, the determination of pigment lineage cells requires an inductive interaction from the vegetal blastomeres of the A-line. Cell dissociation experiments demonstrated that the inductive interaction is completed by the midgastrula stage. However, the two bilaterally positioned cells destined to become the pigment cells in the first step are still equipotent at this stage in that they can give rise to either the ocellus or otolith. Thus, they constitute what is termed an "equivalence group." In the second step, the individual fates of the two cells that compose the equivalence group are determined. Namely, one cell develops into an ocellus and the other cell develops into an otolith. Photoablation of one of the pigment precursor cells at various stages indicated that the second step of determination occurs at the midtailbud stage. It is suggested that the cue to choose one of the alternative developmental pathways may be positional information that exists along the anteroposterior axis. The second step of determination is thought to be mediated by a hierarchical interaction. In the absence of this interaction, melanocyte specification proceeds along the dominant pathway that results in the differentiation of an ocellus.     

Localization of melanin synthesis within the pigment cell: Determination by a combination of electron microscopic autoradiography and topographic planimetry

Summary Localization of melanin synthesis within the pigment cells of the Cloudman S-91 mouse melanoma was determined by means of a combination of high resolution autoradiography and topographic planimetry. Initial melanin biosynthesis occurred predominantly in the endoplasmic reticulum and associated ribonucleoprotein particles of the melanocytes. By measuring a number of cell organelles and employing the index of relative specific localization it could be shown that the nucleus and mitochondria are of little or no importance in the process of melanogenesis.This investigation has been supported by the following research grants: CA 06548 CB, NIH, PHS and an Institutional Grant from the American Cancer Society (to H. M. H.); CA-05887, NIH, PHS (to A. S. Z.); M-00388 and NB-00782, NIH, PHS (to J. F. H.). One of us (H. M. H.) holds a Research Cancer Development Award (5-K 3-GM-2634) of the National Institute of General Medical Sciences, Public Health Service.We are grateful to Mr. Ronald Abler for his help with the topographic measurements; to Dr. Erhard Haus for help and advice; to Mr. J. Thornby and Mr. A. P. Basu for assistance with the statistical aspects of this study; and to Mrs. Lenore Mottaz, Miss Bernice Uittenbogaard, and Mrs. Judith Strong for careful technical assistance.