Early specification of ascidian larval motor neurons

In the tadpole larvae of the ascidian Halocynthia roretzi, six motor neurons, Moto-A, -B, and -C (a pair of each), are localized proximal to the caudal neural tube and show distinct morphology and innervation patterns. To gain insights into early mechanisms underlying differentiation of individual motor neurons, we have isolated an ascidian homologue of Islet, a LIM type homeobox gene. Earliest expression of Islet was detected in a pair of bilateral blastomeres on the dorsal edge of the late gastrula. At the neurula stage, this expression began to disappear and more posterior cells started to express Islet. Compared to expression of a series of motor neuron genes, it was confirmed that early Islet-positive blastomeres are the common precursors of Moto-A and -B, and late Islet-positive cells in the posterior neural tube are the precursors of Moto-C. Overexpression of Islet induced ectopic expression of motor neuron markers, suggesting that Islet is capable of regulating motor neuron differentiation. Since early expression of Islet colocalizes with that of HrBMPb, the ascidian homologue of BMP2/4, we tested a role of BMP in specification of the motor neuron fate. Overexpression of HrBMPb led to expansion of Lim and Islet expression toward the central area of the neural plate, and microinjection of mRNA coding for a dominant-negative BMP receptor weakened the expression of these genes. Our results suggest that determination of the ascidian motor neuron fate takes place at late gastrula stage and local BMP signaling may play a role in this step.     

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.     

The BMP signaling pathway is required together with the FGF pathway for notochord induction in the ascidian embryo

The 40 notochord cells of the ascidian tadpole invariably arise from two different lineages: the primary (A-line) and the secondary (B-line) lineages. It has been shown that the primary notochord cells are induced by presumptive endoderm blastomeres between the 24-cell and the 64-cell stage. Signaling through the fibroblast growth factor (FGF) pathway is required for this induction. We have investigated the role of the bone morphogenetic protein (BMP) pathway in ascidian notochord formation. HrBMPb (the ascidian BMP2/4 homologue) is expressed in the anterior endoderm at the 44-cell stage before the completion of notochord induction. The BMP antagonist Hrchordin is expressed in a complementary manner in all surrounding blastomeres and appears to be a positive target of the BMP pathway. Unexpectedly, chordin overexpression reduced formation of both primary and secondary notochord. Conversely, primary notochord precursors isolated prior to induction formed notochord in presence of BMP-4 protein. While bFGF protein had a similar activity, notochord precursors showed a different time window of competence to respond to BMP-4 and bFGF. Our data are consistent with bFGF acting from the 24-cell stage, while BMP-4 acts during the 44-cell stage. However, active FGF signaling was also required for induction by BMP-4. In the secondary lineage, notochord specification also required two inducing signals: an FGF signal from anterior and posterior endoderm from the 24-cell stage and a BMP signal from anterior endoderm during the 44-cell stage.     

Induction of ascidian peripheral neuron by vegetal blastomeres.

Ascidian tadpole larvae have a similar dorsal tubular nervous system as vertebrates. The induction of brain formation from a4.2-derived (a-line) cells requires signals from the A4.1-derived (A-line) cells. However, little is known about the mechanism underlying the development of the larval peripheral nervous system due to the lack of a suitable molecular marker. Gelsolin, an actin-binding protein, is specifically expressed in epidermal sensory neurons (ESNs) that mainly constitute the entire peripheral nervous system of the ascidian young tadpoles. Here, we address the role of cell interactions in the specification of ESNs using immunostaining with an anti-gelsolin antibody. Animal half (a4.2- and b4.2-derived) embryos did not give rise to any gelsolin-positive neurons, indicating that differentiation of ESNs requires signals from vegetal cells. Cell isolation experiments showed that A4.1 blastomeres induce gelsolin-positive neurons from a-line cells but not from b4.2-derived (b-line) cells. On the other hand, B4.1 blastomeres induce gelsolin-positive neurons both from b-line cells and a-line cells. This is in sharp contrast to the specification of brain cells which is not affected by the ablation of B4.1-derived (B-line) cells. Furthermore, basic fibroblast growth factor (bFGF) induced ESNs from the a-line cells and b-line cells in the absence of vegetal cells. Their competence to form ESNs was lost between the 110-cell stage and the neurula stage. Our results suggested that the specification of the a-line cells and b-line cells into ESNs is controlled by distinct inducing signals from the anterior and posterior vegetal blastomeres. ESNs in the trunk appear to be derived from the a8.26 blastomeres aligning on the edge of presumptive neural region where ascidian homologue of Pax3 is expressed. These findings highlight the close similarity of ascidian ESNs development with that of vertebrate placode and neural crest.     

Chordin and noggin promote organizing centers of forebrain development in the mouse

In this study we investigate the roles of the organizer factors chordin and noggin, which are dedicated antagonists of the bone morphogenetic proteins (BMPs), in formation of the mammalian head. The mouse chordin and noggin genes (Chrd and Nog) are expressed in the organizer (the node) and its mesendodermal derivatives, including the prechordal plate, an organizing center for rostral development. They are also expressed at lower levels in and around the anterior neural ridge, another rostral organizing center. To elucidate roles of Chrd and Nog that are masked by the severe phenotype and early lethality of the double null, we have characterized embryos of the genotype Chrd(-/-);Nog(+/-). These animals display partially penetrant neonatal lethality, with defects restricted to the head. The variable phenotypes include cyclopia, holoprosencephaly, and rostral truncations of the brain and craniofacial skeleton. In situ hybridization reveals a loss of SHH expression and signaling by the prechordal plate, and a decrease in FGF8 expression and signaling by the anterior neural ridge at the five-somite stage. Defective Chrd(-/-);Nog(+/-) embryos exhibit reduced cell proliferation in the rostral neuroepithelium at 10 somites, followed by increased cell death 1 day later. Because these phenotypes result from reduced levels of BMP antagonists, we hypothesized that they are due to increased BMP activity. Ectopic application of BMP2 to wild-type cephalic explants results in decreased FGF8 and SHH expression in rostral tissue, suggesting that the decreased expression of FGF8 and SHH observed in vivo is due to ectopic BMP activity. Cephalic explants isolated from Chrd;Nog double mutant embryos show an increased sensitivity to ectopic BMP protein, further supporting the hypothesis that these mutants are deficient in BMP antagonism. These results indicate that the BMP antagonists chordin and noggin promote the inductive and trophic activities of rostral organizing centers in early development of the mammalian head.     

Notch signaling is involved in nervous system formation in ascidian embryos

Notch signaling plays crucial roles during embryogenesis in various metazoans. HrNotch, a Notch homologue in the ascidian Halocynthia roretzi, has been previously cloned, and its expression pattern suggests that HrNotch signaling is involved in nervous system formation. To determine the function of HrNotch signaling, in the present study we examined the effects of the constitutively activated forms of HrNotch. Overexpression resulted in larvae with defects in neural tube closure and brain vesicle formation. In embryos expressing the activated HrNotch, the expression of a neural marker gene, HrETR-1, was enhanced and expanded in the central nervous system, although ectopic expression decreased during the tailbud stage. The activated HrNotch also suppressed the formation of the adhesive organ (palps) and the peripheral nervous system, which consists of ciliary mechanosensory neurons, whereas it promoted epidermal differentiation. The suppression and promotion of the formation of these respective cell types were confirmed by examination of the expression of relevant tissue-specific markers. We also cloned Hrdelta, an ascidian homologue of DSL family genes, which encode ligands for which Notch acts as a receptor. The expression of Hrdelta was observed in the precursors of palps and peripheral neurons in addition to the CNS. These results suggest that Notch signaling is important for ascidian nervous system formation and that it affects the fate choice between palps and epidermis and between peripheral neurons and epidermis within the neurogenic regions of the surface ectoderm by suppressing the formations of palps and peripheral neurons and promoting epidermal differentiation.     

The ascidian homolog of the vertebrate homeobox gene Rx is essential for ocellus development and function

The tadpole larvae prosencephalon of the ascidian Ciona intestinalis contains a single large ventricle, along the inner walls of which lie two sensory organs: the otolith (a gravity-sensing organ) and the ocellus (a photo-sensing organ composed of a single cup-shaped pigment cell, about 20 photoreceptor cells, and three lens cells). Comparison has been drawn between the morphology and physiology of photoreceptor cells in the ascidian ocellus and the vertebrate eye. The development of vertebrate and invertebrate eyes requires the activity of several conserved genes and it is regulated by precise expression patterns and cell fate decisions common to several species. We have isolated a Ciona homeobox gene (Ci-Rx) that belongs to the paired-like class of homeobox genes. Rx genes have been identified from a variety of organisms and have been demonstrated to have a role in vertebrate eye formation. Ci-Rx is expressed in the anterior neural plate in the middle tailbud stage and subsequently in the larval stage in the sensory vesicle around the ocellus. Loss of Ci-Rx function leads to an ocellus-less phenotype that shows a loss of photosensitive swimming behavior, suggesting the important role played by Ci-Rx in basal chordate photoreceptor cell differentiation and ocellus formation. Furthermore, studies on Ci-Rx regulatory elements electroporated into Ciona embryos using LacZ or GFP as reporter genes indicate the presence of Ci-Rx in pigment cells, photoreceptors, and neurons surrounding the sensory vesicle. In Ci-Rx knocked-down larvae, neither basal swimming activity nor shadow responses develop. Thus, Rx has a role not only in pigment cells and photoreceptor formation but also in the correct development of the neuronal circuit that controls larval photosensitivity and swimming behavior. The results suggest that a Ci-Rx "retinal" territory exists, which consists of pigment cells, photoreceptors, and neurons involved in transducing the photoreceptor signals.     

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.     

Mesoderm patterning and somite formation during node regression: differential effects of chordin and noggin.

In Xenopus, one of the properties defining Spemann's organizer is its ability to dorsalise the mesoderm. When placed ajacent to prospective lateral/ventral mesoderm (blood, mesenchyme), the organizer causes these cells to adopt a more axial/dorsal fate (muscle). It seems likely that a similar property patterns the primitive streak of higher vertebrate embryos, but this has not yet been demonstrated clearly. Using quail/chick chimaeras and a panel of molecular markers, we show that Hensen's node (the amniote organizer) can induce posterior primitive streak (prospective lateral plate) to form somites (but not notochord) at the early neurula stage. We tested two BMP antagonists, noggin and chordin (both of which are expressed in the organizer), for their ability to generate somites and intermediate mesoderm from posterior streak, and find that noggin, but not chordin, can do this. Conversely, earlier in development, chordin can induce an ectopic primitive streak much more effectively than noggin, while neither BMP antagonist can induce neural tissue from extraembryonic epiblast. Neurulation is accompanied by regression of the node, which brings the prospective somite territory into a region expressing BMP-2, -4 and -7. One function of noggin at this stage may be to protect the prospective somite cells from the inhibitory action of BMPs. Our results suggest that the two BMP antagonists, noggin and chordin, may serve different functions during early stages of amniote development.     

Ultraviolet irradiation during ooplasmic segregation prevents gastrulation, sensory cell induction, and axis formation in the ascidian embryo

The effect of ultraviolet (uv) light on embryonic development was examined in the ascidian Styela clava. uv irradiation (3.0 x 10(-3) J mm-2) of the entire surface of fertilized eggs during ooplasmic segregation prevented gastrulation, sensory cell induction, and embryonic axis formation. The uv-irradiated embryos completed ooplasmic segregation and cleaved normally, but vegetal blastomeres did not invaginate at the beginning of gastrulation, sensory cells in the larval brain did not develop tyrosinase or melanin pigment, and the larval tail did not develop. Endoderm, epidermis, and muscle cells differentiated in the uv-irradiated embryos, however, as evidenced by expression of endodermal alkaline phosphatase (AP), an epidermal-specific antigen, and alpha-actin, myosin heavy chain, and acetylcholinesterase (AChE) in muscle cells. Higher doses of uv light (6.0-9.0 x 10(-3) J mm-2) suppressed expression of the epidermal antigen and muscle cell markers, whereas the development of endodermal AP was insensitive. Irradiation at various times between fertilization and the 16-cell stage revealed that gastrulation, sensory cell differentiation, and axis formation are sensitive to uv light only during ooplasmic segregation. Irradiation of restricted regions of the zygote during ooplasmic segregation showed that the uv-sensitive components are localized in the vegetal hemisphere. The absorption characteristics of the uv-sensitive components suggest that they are nucleic acids. The results show that uv-sensitive components that specify gastrulation, sensory cell induction, and embryonic axis formation are localized in the vegetal hemisphere of Styela eggs.     

An FGF signal from endoderm and localized factors in the posterior-vegetal egg cytoplasm pattern the mesodermal tissues in the ascidian embryo

The major mesodermal tissues of ascidian larvae are muscle, notochord and mesenchyme. They are derived from the marginal zone surrounding the endoderm area in the vegetal hemisphere. Muscle fate is specified by localized ooplasmic determinants, whereas specification of notochord and mesenchyme requires inducing signals from endoderm at the 32-cell stage. In the present study, we demonstrated that all endoderm precursors were able to induce formation of notochord and mesenchyme cells in presumptive notochord and mesenchyme blastomeres, respectively, indicating that the type of tissue induced depends on differences in the responsiveness of the signal-receiving blastomeres. Basic fibroblast growth factor (bFGF), but not activin A, induced formation of mesenchyme cells as well as notochord cells. Treatment of mesenchyme-muscle precursors isolated from early 32-cell embryos with bFGF promoted mesenchyme fate and suppressed muscle fate, which is a default fate assigned by the posterior-vegetal cytoplasm (PVC) of the eggs. The sensitivity of the mesenchyme precursors to bFGF reached a maximum at the 32-cell stage, and the time required for effective induction of mesenchyme cells was only 10 minutes, features similar to those of notochord induction. These results support the idea that the distinct tissue types, notochord and mesenchyme, are induced by the same signaling molecule originating from endoderm precursors. We also demonstrated that the PVC causes the difference in the responsiveness of notochord and mesenchyme precursor blastomeres. Removal of the PVC resulted in loss of mesenchyme and in ectopic notochord formation. In contrast, transplantation of the PVC led to ectopic formation of mesenchyme cells and loss of notochord. Thus, in normal development, notochord is induced by an FGF-like signal in the anterior margin of the vegetal hemisphere, where PVC is absent, and mesenchyme is induced by an FGF-like signal in the posterior margin, where PVC is present. The whole picture of mesodermal patterning in ascidian embryos is now known. We also discuss the importance of FGF induced asymmetric divisions, of notochord and mesenchyme precursor blastomeres at the 64-cell stage.     

Vestigial Brain Melanocyte Development During Embryogenesis of an Anural Ascidian

Anural development was examined in the ascidian Bostrichobranchus digonas using specific markers for differentiated urodele ascidian larval cells and tissues. In this ovoviviparous anural ascidian, eggs, embryos and developing juveniles were present in the gonads, brood sacs, and atrial cavity, respectively. Morphological studies indicated that B. digonas embryos do not develop into tailed larvae with an extended notochord and differentiated muscle cells. In addition, these embryos lack detectable expression of the muscle-specific markers acetylcholinesterase, alpha actin, and myosin heavy chain. In striking contrast to other anural ascidian embryos, however, B. digonas embryos can develop tyrosinase in several melanocyte precursor cells and eventually form a brain pigment cell. The melanocyte does not become part of a definitive brain sensory organ (otolith) and subsequently disappears during metamorphosis. A period of tyrosinase expression was also observed following metamorphosis in which many tyrosinase-positive cells appear in the body of the developing juvenile. The results demonstrate that different urodele features can be uncoupled during the evolution of anural development. The development of a vestigial brain melanocyte also suggests that B. digonas evolved from a urodele ancestor rather than from another anural ascidian lacking a brain pigment cell.     

Nuclear plasticity and timing mechanisms of the initiation of alkaline phosphatase expression in cytoplasm-transferred blastomeres of ascidians

Egg cytoplasm containing endoderm determinants was transferred to presumptive-muscle or presumptive-epidermis blastomeres isolated from cleavage-stage embryos of the ascidian Halocynthia roretzi. We investigated three aspects of the expression of endoderm-specific alkaline phosphatase (ALP) activity. First, we examined whether ectopic ALP expression, an indication of ectopic endoderm formation, was promoted in cytoplasm-transferred blastomeres isolated at late-cleavage stage. The results showed that the cell fate was converted by the introduced cytoplasm, even in recipient blastomeres in which the cell fate was already restricted to muscle or epidermis, and in those where expression of the muscle- or epidermis-specific genes was already initiated. Next, we examined the formation of endoderm and other tissue in embryos by double staining for ALP and muscle- or epidermis-specific marker. Regions positive for ALP and positive for muscle or epidermis marker were mutually exclusive. These results suggested that muscle- or epidermis-specific genes that were already expressed in the recipient blastomeres were down-regulated in ectopically forming endoderm cells. This is evidence for nuclear plasticity during ascidian embryogenesis. In the last series of experiments, we investigated the timing of the appearance of ALP activity in cytoplasm-transferred embryos. In the partial embryos that were derived from various combination of recipient blastomeres and donor cytoplasm obtained from various staged eggs and embryos, the timing seemed to coincide with the time that starts when cell fusion for cytoplasmic transfer was done. Therefore, the clock that determines the timing of the initiation of ALP expression is likely to start at the moment of cell fusion. Several possible hypotheses for the timing mechanism are discussed.     

Synergistic action of HNF-3 and Brachyury in the notochord differentiation of ascidian embryos.

In vertebrate embryos, the class I subtype forkhead domain gene HNF-3 is essential for the formation of the endoderm, notochord and overlying ventral neural tube. In ascidian embryos, Brachyury is involved in the formation of the notochord. Although the results of previous studies imply a role of HNF-3 in notochord differentiation in ascidian embryos, no experiments have been carried out to address this issue directly. Therefore the present study examined the developmental role of HNF-3 in ascidian notochord differentiation. When embryos were injected with a low dose of HNF-3 mRNA, their tails were shortened and when embryos were injected with a high dose of HNF-3 mRNA, which was enough to inhibit differentiation of epidermis and muscle, no obvious ectopic differentiation of endoderm or notochord cells was observed. However, co-injection of HNF-3 mRNA along with Brachyury mRNA resulted in ectopic differentiation of notochord cells in the animal hemisphere, suggesting that HNF-3 acts synergistically with Brachyury in ascidian notochord differentiation. Notochord differentiation of the A-line precursor cells depends on inducing signal(s) from endodermal cells, which can be mimicked by bFGF treatment. Treatment of notochord precursor cells isolated from the 32-cell stage embryoswith bFGF resulted in upregulation of both the HNF-3 and Brachyury genes.     

Xerl, a novel CNS-specific secretory protein, establishes the boundary between neural plate and neural crest.

A novel gene, Xerl, has been found as a CNS-specific gene encoding a secretory protein. In order to clarify a function of Xerl, we first examined Xerl-expressing areas during early development. Comparison with XlSox-2-positive neural plate and ADAM13-positive neural crest showed that Xerl expression was limited within the neural plate area. Microinjection of Xerl mRNA into 2- or 4-cell stage embryos indicated that Xerl overexpression caused the regional expansion of XlSox-2- and NCAM-positive neural plate, which was concomitant with the outer shift of ADAM13-positive region. The Xerl injection resulted in incomplete neural closure because of the local overproduction of the neuroepithelium. In contrast, loss of function analysis of Xerl indicated that Xerl inhibition caused the ectopic differentiation of neural crest cells. In the conjugation experiment using chordin-injected animal caps, Xerl promoted chordin-induced XlSox-2 expression, whereas Xerl inhibition caused ADAM13expression even in the injection with a high dose of chordin. Animal cap assays also showed that Xerl expression was induced by chordin. In the functional analysis using truncated forms of Xerl, Xerl deltaL (lacking LNS domain) worked as a dominant negative form that induced the overproduction of neural crest cells. These results suggest that Xerl is involved in the boundary formation of the neural plate through exclusion of neural crest cell differentiation.