Tail Muscle Cells Research Articles

Mechanism of an Evolutionary Change in Muscle Cell Differentiation in Ascidians with Different Modes of Development

We have investigated the mechanism of an evolutionary change in ascidian muscle cell differentiation. The ascidiansMolgula oculataandMolgula occultaare closely related species with different modes of development.M. oculataembryos develop into conventional tadpole larvae with a tail containing striated muscle cells, whereasM. occultaembryos develop into tailless larvae with undifferentiated vestigial muscle cells. The muscle actin geneMocuMA1was isolated from anM. oculatagenomic library.MocuMA1is a single-copy, larval-type muscle actin gene which appears to lack introns. However, the 5′ upstream region ofMocuMA1is sufficient to drive expression of alacZfusion construct in the larval muscle cells, implying that it is a functional gene.MocuMA1mRNA first appears in the prospective muscle cells ofM. oculataembryos during gastrulation, and transcripts continue to be present throughout embryogenesis. Muscle actin mRNA was not detected duringM. occultaembryogenesis, although the same probe was capable of detecting muscle actin mRNA in more distantly related ascidian species with tail muscle cells. Interspecific hybrids produced by fertilizingM. occultaeggs withM. oculatasperm recover the ability to express muscle actin mRNA in the vestigial muscle cells, suggesting thattrans-acting factors responsible for muscle actin gene expression are conserved inM. occulta.The presence of thesetrans-acting factors was confirmed by showing that theMocuMA1/lacZfusion construct is expressed in the vestigial muscle cells ofM. occultalarvae. The orthologous larval muscle actin genesMoccMA1aandMoccMA1bwere isolated from aM. occultagenomic library. The coding regions of these genes contain deletions, insertions, and codon substitutions that would make their products nonfunctional. Although the 5′ upstream regions of theM. occultamuscle actin genes also contain numerous changes, expression ofMoccMA1a/lacZandMoccMA1b/lacZfusion constructs showed that they both retain specific promoter activity, although it is reduced inMoccMA1b.The results suggest that the regression of muscle cell differentiation is mediated by changes in the structure of muscle actin genes rather than in thetrans-acting regulatory factors required for their expression.

Expression of an Msx homeobox gene in ascidians: insights into the archetypal chordate expression pattern.

The Msx homeobox genes are expressed in complex patterns during vertebrate development in conjunction with inductive tissue interactions. As a means of understanding the archetypal role of Msx genes in chordates, we have isolated and characterized an Msx gene in ascidians, protochordates with a relatively simple body plan. The Mocu Msx-a and McMsx-a genes, isolated from the ascidians Molgula oculata and Molgula citrina, respectively, have homeodomains that place them in the msh-like subclass of Msx genes. Therefore, the Molgula Msx-a genes are most closely related to the msh genes previously identified in a number of invertebrates. Southern blot analysis suggests that there are one or two copies of the Msx-a gene in the Molgula genome. Northern blot and RNase protection analysis indicate that Msx-a transcripts are restricted to the developmental stages of the life cycle. In situ hybridization showed that Msx-a mRNA first appears just before gastrulation in the mesoderm (presumptive notochord and muscle) and ectoderm (neural plate) cells. Transcript levels decline in mesoderm cells after the completion of gastrulation, but are enhanced in the folding neural plate during neurulation. Later, Msx-a mRNA is also expressed in the posterior ectoderm and in a subset of the tail muscle cells. The ectoderm and mesoderm cells that express Msx-a are undergoing morphogenetic movements during gastrulation, neurulation, and tail formation. Msx-a expression ceases after these cells stop migrating. The ascidian M. citrina, in which adult tissues and organs begin to develop precociously in the larva, was used to study Msx-a expression during adult development. Msx-a transcripts are expressed in the heart primordium and the rudiments of the ampullae, epidermal protrusions with diverse functions in the juvenile. The heart and ampullae develop in regions where mesenchyme cells interact with endodermal or epidermal epithelia. A comparison of the expression patterns of the Molgula genes with those of their vertebrate congeners suggests that the archetypal roles of the Msx genes may be in morphogenetic movements during embryogenesis and in mesenchymal-epithelial interactions during organogenesis.

Heterochronic expression of an adult muscle actin gene during ascidian larval development

Adultation is a heterochronic mode of development in which adult tissues and organs differentiate precociously during the larval phase. We have investigated the expression of an adult muscle actin gene during adultation in the ascidian Molgula citrina. Ascidians contain multiple muscle actin genes which are expressed in the larva, the adult, or during both phases of the life cycle. In ascidian species with conventional larval development, the larval mesenchyme cells, which are believed to be progenitors of the adult mesoderm, remain undifferentiated and do not express the muscle actin genes. In M. citrina, the mesenchyme cells differentiate precociously during larval development, suggesting a role in adultation. An adult muscle actin gene from M. citrina was obtained by screening a mantle cDNA library with a probe containing the coding region of SpMA1, a Styela plicata adult muscle actin gene. The screen yielded a cDNA clone designated McMA1, which contained virtually the complete coding and 3' noncoding regions of a muscle actin gene. The deduced McMA1 and SpMA1 proteins exhibit 97% identity in amino acid sequence and may be encoded by homologous genes. The McMA1 gene is expressed in juveniles and adults, but not in larval tail muscle cells, suggesting that it is an adult muscle actin gene. In situ hybridization with a 3' non-coding region probe was used to determine whether the McMA1 gene is expressed during adultation in M. citrina. McMA1 mRNA was first detected exclusively in the mesenchyme cells during the late tailbud stage and continued to accumulate in these cells during their migration into the future body cavity and heart primordium in the hatched larva. The McMA1 transcripts persisted in mesenchyme cells after larval metamorphosis. It is concluded that an adult muscle actin gene shows a heterochronic shift of expression into the larval phase during adultation in M. citrina.

Novel genes expressed differentially in ascidians with alternate modes of development

We have used a subtractive procedure to isolate cDNA clones encoding genes expressed differentially in ascidian species with alternate modes of development. The ascidians used in this study were Molgula occulta, which develops a tailed (urodele) larva, and Molgula occulta, which develops a tailless (anural) larva. Two of the identified clones, Uro-2 and Uro-11, are described. Southern blots show that the Uro-2 and Uro-11 genes are present in both species, but the corresponding mRNAs are expressed preferentially in the urodele species. In situ hybridization showed that Uro-2 and Uro-11 transcripts accumulate in small oocytes during oogenesis. The maternal Uro-2 and Uro-11 transcripts were distributed throughout the oocyte cytoplasm. Transcript concentrations declined during vitellogenesis, but mature eggs still contain detectable levels of Uro-2 and Uro-11 mRNA. After fertilization, the maternal Uro-2 and Uro-11 transcripts were localized in the ectoplasm of uncleaved zygotes and mostly entered the ectoderm cells during cleavage. The Uro-2 gene appears to produce only maternal transcripts. In contrast, the Uro-11 gene may also produce zygotic transcripts, which accumulate between gastrulation and neurulation in posterior epidermis, neural and tail muscle cells. Zygotic expression of the Uro-11 gene was not detected in embryos of the anural species. The deduced amino acid sequences of the Uro-2 and Uro-11 cDNAs suggest that they encode novel basic proteins with distinctive structural features. The predicted Uro-2 protein contain, a leucine zipper motif, suggesting that it may dimerize with another protein. The predicted Uro-11 protein contains a nuclear localization signal, a region with similarity to part of the DNA-binding motif in the bacterial histone-like HU and IHF proteins, 12 repeats of the proposed DNA-binding motif S(T)PXX, and a potential zinc finger of the C6 or C6H2 class, suggesting that it may be a DNA-binding protein. The Uro-2 and Uro-11 proteins are candidates for regulatory factors involved in the evolutionary transition from urodele to anural development.

An ankryin-like protein in ascidian eggs and its role in the evolution of direct development

An erythrocyte anti-ankryin antibody was used to investigate the presence and distribution of ankryin in eggs and embryos of ascidian species with different modes of development. In eggs of the indirect developer Ascidia ceratodes, anti-ankryin reacted with a 210 kDa polypeptide which has an electrophoretic mobility similar to the vertebrate ankryins. Immunofluorescence microscopy showed that the ankryin-like protein is co-distributed with the myoplasm throughout development. It is restricted to a thin layer under the plasma membrane in unfertilised eggs, undergoes ooplasmic segregation to the posterior pole of the zygote after fertilisation, and is distributed to the tail muscle cells during cleavage and embryogenesis. After gastrulation and neurulation, lower levels of the ankryin-like protein, presumably of zygotic origin, were observed in brain cells and in the apical margin of epidermal cells. The ankryin-like protein was also localised in the myoplasm in eggs and embryos of another indirect developing species, Halocynthia roretzi. The ankryin-like protein may link the cytoskeleton with the plasma membrane in ascidian eggs, as it does in vertebrate erythrocytes. In contrast to A. ceratodes and H. roretzi, which are members of the families Ascidiidae and Pyuridae respectively, the pattern of ankryin-like protein expression was changed in five species in the family Molgulidae. These molgulid ascidians exhibit either indirect or direct development, and eggs of the direct developing species have lost or modified the myoplasm. The ankryin-like protein was present in young oocytes but failed to persist during oogenesis and disappeared in mature eggs and embryos of these molgulid species. The change in ankryin-like protein expression may be a preadaptation for loss of the myoplasm and the evolution of direct development.

Multiple actin genes encoding the same α-muscle isoform are expressed during ascidian development

Ascidian embryos develop rapidly into tadpole larvae containing striated tail muscle cells. We have isolated and characterized five actin cDNA clones from a Styela clava tailbud-stage library. The nucleotide sequences of these clones and genomic Southern blot analysis indicate that they represent at least four different muscle actin genes, which are designated ScTb1, ScTb24, ScTb30, and ScTb12/34. The derived protein sequences of these genes indicate that they encode the same α-muscle actin which has features related to each of the three different classes of vertebrate α-muscle actins. Northern and in situ hybridization with probes prepared from the 3′ untranslated region (UTR) of several of the ScTb clones showed that these muscle actin genes are expressed in different temporal and spatial patterns during development. ScTb1 was detected in eggs, embryos, and adults, ScTb24 and ScTb12/34 were detected in embryos and adults, and ScTb30 was detected only in embryos. The maternal transcripts disappeared shortly after fertilization and zygotic mRNAs were first detected during gastrulation and continued to accumulate during subsequent tail muscle differentiation. ScTb30 mRNA, which is expressed in the embryo, peaks during the tailbud stage and is present at low levels in the tadpole larva. In contrast, ScTb1, ScTb24, and ScTb12/34 mRNAs, which are expressed in embryos and adults, peak during the late tailbud stage and are present in substantial quantities in the larva. The ScTb24 gene was detected only in tail muscle cells, whereas the ScTb30 gene was detected in embryonic tail muscle, mesenchyme, epidermal, and neural cells. The ScTb24 mRNA also accumulates primarily in vascular tissue in the branchial sac and mantle of adults. The existence of a gene family encoding the same α-muscle actin isoform is unique among the chordates and may function to maximize muscle actin production during the rapid differentiation phase of ascidian larval muscle cells.

A gastrulation center in the ascidian egg

A gastrulation center is described in ascidian eggs. Extensive cytoplasmic rearrangements occur in ascidian eggs between fertilization and first cleavage. During ooplasmic segregation, a specific cytoskeletal domain (the myoplasm) is translocated first to the vegetal pole (VP) and then to the posterior region of the zygote. A few hours later, gastrulation is initiated by invagination of endoderm cells in the VP region of the 110-cell embryo. After the completion of gastrulation, the embryonic axis is formed, which includes induction of the nervous system, morphogenesis of the larval tail and differentiation of tail muscle cells. Microsurgical deletion or ultraviolet (UV) irradiation of the VP region during the first phase of myoplasmic segregation prevents gastrulation, nervous system induction and tail formation, without affecting muscle cell differentiation. Similar manipulations of unfertilized eggs or uncleaved zygotes after the second phase of segregation have no effect on development, suggesting that a gastrulation center is established by transient localization of myoplasm in the VP region. The function of the gastrulation center was investigated by comparing protein synthesis in normal and UV-irradiated embryos. About 5% of 433 labelled polypeptides detected in 2D gels were affected by UV irradiation. The most prominent protein is a 30 kDa cytoskeletal component (p30), whose synthesis is abolished by UV irradiation. p30 synthesis peaks during gastrulation, is affected by the same UV dose and has the same UV-sensitivity period as gastrulation. However, p30 is not a UV-sensitive target because it is absent during ooplasmic segregation, the UV-sensitivity period. Moreover, the UV target has the absorption maximum of a nucleic acid rather than a protein. Cell-free translation studies indicate that p30 is encoded by a maternal mRNA. UV irradiation inhibits the ability of this transcript to direct p30 synthesis, indicating that p30 mRNA is a UV-sensitive target. The gasturlation center may function by sequestration or activation of maternal mRNAs encoding proteins that function during embryogenesis.

Ultrastructural and histochemical study of anural development in the ascidian Molgula pacifica (Huntsman)

Tadpole development is eliminated in the life cycle of the ascidian Molgula pacifica. The elimination of a tailed larva is termed anural development, in contrast to urodele development which is exhibited by most ascidian species. In the present study, transmission electron microscopy and histochemistry were used to gain a better understanding of anural development in M. pacifica. The fine structure of M. pacifica oocytes and fertilized eggs was similar to urodele oocytes and eggs, except that a perivitelline space and test cells were absent. M. pacifica embryos exhibited the typical cleavage pattern of urodele embryos. Gastrulation was initiated at the vegetal pole, as in urodeles, and occurred at the same time as in two urodele species (Molgula manhattensis and Pyura haustor). However, changes in cell shapes and cell movements of the vegetal pole cells that participate in gastrulation were highly modified compared to commonly studied ascidians. The changes in shapes and movements of the vegetal pole cells were minimal and resulted in embryos having a very small archenteron and blastopore. The presence of large, yolky cells in the interior of the embryo likely restricted vegetal cell movements. Two ultrastructurally distinct types of epidermal cells were evident at the gastrula stage. When gastrulae were manually dechorionated from their surrounding mucous-follicular envelope layers, the embryos were already surrounded by a thin tunic. When day 1 juveniles in the process of hatching were sectioned along the anterior-posterior axis, regional differences in cell types were evident. Differentiated muscle cells in the posterior region were not evident. Day 1 M. pacifica juveniles, anural-developing M. provisionalis juveniles and tadpoles from three urodele species were tested for their abilities to express AchE activity. The highest levels of AchE activity were detected in the larval tail muscle cells of urodeles, low levels of activity were detected in the posterior region of M. provisionalis juveniles, whereas M. pacifica juveniles did not exhibit AchE activity. The results are discussed in terms of evolutionary mechanisms responsible for anural development in ascidians.

An evolutionary change in the muscle lineage of an anural ascidian embryo is restored by interspecific hybridization with a urodele ascidian

Anural ascidians do not develop into a conventional tailed larva with differentiated muscle cells, however, embryos of some anural ascidian species retain the ability to express acetylcholinesterase (AChE) in a vestigial muscle cell lineage. This study examines the number of AChE-positive cells that develop in the anural ascidian Molgula occulta relative to that in the closely related urodele (tailed) species, Molgula oculata. Histochemical assays showed that M. oculata embryos develop 36 to 38 AChE-positive cells, consistent with the number of tail muscle cells expressed in other urodele ascidians. In contrast, M. occulta embryos develop a mean of only 20 AChE-positive cells in their vestigial muscle lineage. Cleavage-arrested embryos of the anural species express AChE only in B-line blastomeres, showing that the vestigial muscle lineage cells are derived from the primary muscle lineage. Less than the expected number of AChE-positive B-line cells develop in cleavage-arrested anural embryos, however, implying that the allocation of primary muscle lineage cells is decreased. Eggs of the anural species can be fertilized with sperm of the urodele species resulting in the development of some larvae that contain a short tail and/or a brain melanocyte, specific features of urodele larvae. The typical urodele number of AChE-positive cells is restored in some of these hybrid embryos. Both primary and secondary muscle lineages are restored because cleavage-arrested hybrid embryos develop more AChE-positive cells in the B-line blastomeres and supernumerary AChE-positive cells in the A-line blastomeres. Hybrid embryos that develop the urodele complement of AChE-positive cells also form a tail and/or a brain melanocyte showing that restoration of muscle lineage cells is coupled to the development of other urodele features. AChE expression occurred in anural embryos with disorganized or dissociated blastomeres, indicating that AChE expression is determined autonomously. It is concluded that an evolutionary change in the allocation of larval muscle lineage cells occurs during development of the anural ascidian M. occulta which can be restored by interspecific hybridization with the urodele ascidian M. oculata.

Identification of a cytoskeletal protein localized in the myoplasm of ascidian eggs: localization is modified during anural development

The myoplasm of ascidian eggs is a localized cytoskeletal domain that is segregated to presumptive larval tail muscle cells during embryonic development. We have identified a cytoskeletal protein recognized by a vertebrate neurofilament monoclonal antibody (NN18) which is concentrated in the myoplasm in eggs and embryos of a variety of ascidian species. The NN18 antigen is localized in the periphery of unfertilized eggs, segregates with the myoplasm after fertilization, and enters the larval tail muscle cells during embryonic development. Western blots of one-dimensional and two-dimensional gels showed that the major component recognized by NN18 antibody is a 58 x 10(3) Mr protein (p58), which exists in at least three different isoforms. The enrichment of p58 in the Triton X-100-insoluble fraction of eggs and its reticular staining pattern in eggs and embryos suggests that it is a cytoskeletal protein. In subsequent experiments, p58 was used as a marker to determine whether changes in the myoplasm occur in eggs of anural ascidian species, i.e. those exhibiting a life cycle lacking tadpole larvae with differentiated muscle cells. Although p58 was localized in the myoplasm in eggs of four urodele ascidian species that develop into swimming tadpole larvae, this protein was distributed uniformly in eggs of three anural ascidian species. The eggs of two of these anural species contained the actin lamina, another component of the myoplasm, whereas the third anural species lacked the actin lamina. There was no detectible localization of p58 after fertilization or segregation into muscle lineage cells during cleavage of anural eggs. NN18 antigen was uniformly distributed in pre-vitellogenic oocytes and then localized in the perinuclear zone during vitellogenesis of urodele and anural ascidians. Subsequently, NN18 antigen was concentrated in the peripheral cytoplasm of post-vitellogenic oocytes and mature eggs of urodele, but not anural, ascidians. It is concluded that the myoplasm of ascidian eggs contains an intermediate filament-like cytoskeletal network which is missing in anural species that have modified or eliminated the tadpole larva.

Temporal and spatial expression of a cytoskeletal actin gene in the ascidian Styela clava

We have cloned and characterized the temporal and spatial expression of ScCA15, a cDNA clone encoding an actin gene in the ascidian Styela clava. The partial nucleotide and derived amino acid sequences of this singlecopy gene suggest that it is a cytoskeletal actin. Northern analysis shows that ScCA15 corresponds to a 1.8-kb mRNA that is transcribed during oogenesis, during embryonic development, and in the adult. In situ hybridization shows that maternal ScCA15 mRNA is distributed uniformly in the cytoplasm of the oocyte and unfertilized egg. During the period of ooplasmic segregation following fertilization, however, ScCA15 mRNA appears to be translocated into the ectoplasm, a specialized cytoplasmic region of the egg. During the early cleavages, the ectoplasmic transcripts are partitioned to ectodermal cells in the animal hemisphere, which are precursors of the epidermis and nervous system of the larva. Maternal ScCA15 mRNA is degraded just before gastrulation and replaced by zygotic transcripts which begin to accumulate between the neurula and mid-tailbud stages. Zygotic ScCA15 mRNA accumulates primarily in the epidermal and neural cells, although lower levels of these transcripts may also be present in tail muscle cells. These results show that two mechanisms are used to concentrate ScCA15 mRNA in the ectodermal cells during development: 1) localization and differential segregation of maternal transcripts and 2) specific expression of the ScCA15 gene. ScCA15 mRNA is detected by in situ hybridization in the testes, ovaries, alimentary tract, and endostyle of adults. In the testes, ScCA15 mRNA is present in developing sperm, whereas in the ovary, these transcripts are present in the germinal epithelium and developing oocytes. In the alimentary tract, ScCA15 mRNA is confined to the gastric epithelium of the esophagus, stomach, and intestine. Since the ScCA15 gene is expressed in embryonic and adult tissues that are undergoing rapid cell division, this actin is likely to function in some aspect of cell proliferation.

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.

Differential expression of the Ca 2+-binding protein parvalbumin during myogenesis in Xenopus laevis

We have used immunocytological techniques to examine the developmental expression of the Ca 2+-binding protein parvalbumin in Xenopus laevis embryos. Western blot experiments show that at least three different forms of parvalbumin are expressed during embryogenesis; the tadpole tail expresses one form, adult brain expresses another, mylohyoid muscle expresses both, and gastrocnemius and sartorius muscles express these two plus a third form. Parvalbumin (PV) is first detectable by immunofluorescence at stages 24–25 of development, a time when myotomal muscles are differentiating and contractile activity occurs spontaneously in embryos. At metamorphosis, PV is expressed in developing limb muscles. While the majority of skeletal muscle fibers express high levels of PV in both embryos and adults, a second fiber type has no detectable PV. The arrangement of PV-containing fibers is stereotyped in each muscle group examined. Histochemical staining of tadpole muscles indicate that PV-containing fibers correspond to fast-twitch skeletal muscles, whereas those without PV correspond to slow-twitch muscles. During tail resorption at metamorphosis, PV appears to be extruded from dying tail muscle cells and taken up by phagocytic cells.

Identification of proteins and mRNAs in isolated yellow crescents of ascidian eggs

The yellow crescent or myoplasm is a localized cytoplasmic region in eggs of the ascidian Styela that is partitioned to the larval tail muscle and mesenchyme cells during embryonic development. To determine whether the myoplasm contains a specific subset of maternal macromolecules, its abundant proteins and mRNAs were identified and compared to those present in the remainder of the egg. This was accomplished by exploiting a newly developed method for the mass fractionation of yellow crescents which is based on the presence of a unique cytoskeletal domain in the yellow crescent region. The fractionation yields a yellow crescent fraction (YCF) representing the myoplasm and a supernatant fraction representing the nonmyoplasmic regions. The YCF comprises structures which contain the characteristic myoplasmic organelles and about 10% of the protein, 8% of the RNA, and 1-3% of the poly(A) of whole eggs. Two-dimensional gel electrophoresis indicated that the YCF contains 15 polypeptides that are undetectable in the supernatant fraction and 43 polypeptides that are significantly depleted in the latter fraction. The proteins restricted to the YCF are both of cytoskeletal and noncytoskeletal origin. In vitro translation of RNA in a message-dependent lysate and analysis of [35S]methionine-labelled polypeptide products by two-dimensional gel electrophoresis did not reveal qualitative differences between the YCF and the supernatant fraction. Furthermore, the mRNAs coding for two polypeptides that were localized in the myoplasm were not restricted to the YCF. The results suggest that qualitative differences in proteins but not in prevalent mRNAs exist between the yellow crescent and the other cytoplasmic regions of Styela eggs.

Tracing of cell lineage in embryonic development of Phallusia mammillata (ascidia) by vital staining of mitochondria

Vital staining of mitochondria with a fluorescent dye 3,3′-diethyloxacarbocyanine was used to follow cell lineage in embryos of Phallusia mammillata. The results agree in general with the plan established by Conklin in 1905. Strong fluorescence migrated after fertilization similarly to the pigment of the “yellow crescent” in Styela. Later, fluorescence segregated into muscle cell primordia, but not into mesenchyme cells. An animal hemisphere cell, b 8.17 also exhibited strong fluorescence and joined a group of muscle primordia, very likely becoming a muscle cell itself. In the tadpole, all the tail muscle cells were fluorescent. Fluorescence was also noticed in nerve cell primordia of the vegetal hemisphere, particularly in the cell A 8.16 whose descendants appeared to become part of the sensory vesicle which was strongly fluorescent in the tadpole. The usefulness of this type of vital staining in following cell lineage of colorless embryos is stressed.

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.

Budding and the Reproductive Cycle of Distaplia

The complete developmental cycle, sexual and asexual, is described for several species of the ascidian genus, Distaplia. Part I. The cycle of D. rosea is described, from the active larva to the establishment of the first blastozooid generation. Of the four or five buds produced from the larval probud, only one develops, the rest remain abortive. Some larvae are often retained in the colony after the dissolution of the parent zooid and give rise to blastozooids (Text-figs. 1-3). The general morphology of mature zooids is described for D. bermudensis, and their arrangement into typical systems in the colony. Zooids are at first functional males, while the brood-pouch develops later as the first egg in the ovary approaches maturity. The brood-pouch is essentially a loop of the oviduct and does not involve the atrial wall (Text-figs. 4-5). In the gross development of the bud up to the time it becomes functional, the originally spherical bud elongates relatively along its antero-posterior axis but otherwise does not exhibit differential growth. After the functional state is reached, differential growth of the atrial siphon commences and eventually transforms the small circular siphon into the relatively enormous atrial aperture of the mature zooid (Text-figs. 4 and 6). Gonads develop as hermaphrodite structures in buds of D. rosea, D. bermudensis, D. occidentalis, and D. clavata, but as either ovaries or testes alone in D. magnilarva (Text-fig. 7). Part 2. Tadpole larvae of Distaplia vary greatly in size according to the species, but not in organization, the cell number of purely larval structures such as adhesive organs and sensory organs being constant (Text-fig. 8). Cleavage of the egg, in spite of large size and yolk content, is typical of ascidians in general, and shows bilateral patterning comparable with that of Styela and Ciona (Text-fig. 9). There is great growth in the development from the egg to the tadpole stage, though the full number of cells of both the notochord and tail muscle is reached at an early stage, about 40 in the case of the notochord (as in all other ascidians) and over 2,000 tail-muscle cells (the largest number known). In the later development of the tadpole larva, the anterior region bearing the adhesive organ and the ampullae becomes progressively constricted off until it remains attached only by a vascular stolon. The probud is separated from the abdomen at a precise stage in the whole development and subdivides before tadpole development is complete (Text-figs. 10-12). The sensory vesicle contains a typical unicellular otolith and an ocellus consisting of optic pigment cup, layer of retinal cells, and three unicellular lens cells. In the tail the intracellular notochordal vacuoles run together to form a continuous fluid cylinder with the chordal cells forming a limiting sheath. Myofibrillae are continuous from cell to cell in the lateral interfaces of the ectoplasmic regions (Text-fig. 13). Part 3. Contrary to all previous reports, buds are produced by adult zooids and are not confined to the tadpole larva, but they are produced at a different phase of the life-cycle of the individual zooid. After sexual reproduction is complete, the zooids atrophy, their tissues autolysing and becoming phagocytosed in part. All of the zooid degenerates with the exception of the epicardium and surrounding epidermis. This structure actually undergoes extensive elongation, at the close of which the posterior end of the pericardium and enclosing epidermis constricts off as one of two probuds (Text-figs. 14-15). This is essentially the same process, apart from the stage with which it is associated, as occurs in the tadpole larva. In both cases the probud elongates and constricts into two or more definitive buds (Text-figs. 16-17), the smallest of which fail to develop. The formation of hermaphrodite or unisexual gonads depends primarily on the mass of the presumptive gonad tissue segregated at a very early critical period in bud morphogenesis (Text-fig. 18). The process of bud formation is compared with that of the allied genus Colella, and an interpretation very different from that of Brien is presented (Text-fig. 20).