Metamorphosis in solitary ascidians
Summary
Embryonic and postembryonic development in ascidians have been studied for over a century, but it is only in the last 10 years that the complex molecular network involved in coordinating postlarval development and metamorphosis has started to emerge. In most ascidians, the transition from the larval to the sessile juvenile/adult stage, or metamorphosis, requires a combination of environmental and endogenous signals and is characterized by coordinated global morphogenetic changes that are initiated by the adhesion of the larvae. Cloney was the first to describe cellular events of ascidians' metamorphosis in 1978 and only recently elements of the molecular regulation of this crucial developmental step have been revealed. This review aims to present a thorough view of this crucial developmental step by combining recent molecular data to the already established cellular events. genesis 53:34–47, 2015. © 2014 Wiley Periodicals, Inc.
INTRODUCTION
Animal metamorphosis, the process of an organism undergoing physical transformation, has fascinated biologists for years. It refers to the profound changes at the morphological, physiological, biochemical, behavioral, and ecological levels that happen in a coordinated way during a brief period following the postembryonic developmental stage of an animal (Bishop et al., 2006). This developmental transition phase is characterized by various cellular mechanisms that are not unique to metamorphosis, such as contraction, cell migration, specific tissues' growth and differentiation or, on the contrary, cell death (Davidson et al., 2003). All these cellular events are highly coordinated in space and time by a complex combination of signal transduction pathways and gene networks (Rutherford et al., 2007).
Metamorphic events can be largely divided into two groups; events that destroy tissues/organs from the previous stage and events for constructing tissues/organs for the next stage. Because the destructive and constructive events occur in different subsets of tissues and organs, the mechanisms that initiate and process these changes may be different. Indeed, a mystery of animal metamorphosis is how completely different events are conducted simultaneously in a body. Uncovering the underlying mechanisms that are responsible for the initiation of metamorphic events is a necessary step for understanding the evolution of these processes in animals.
Among animals that undergo metamorphosis, most marine invertebrates have a bi‐phasic life cycle with pelagic larval and benthic adult phases (Jackson et al., 2002). Compared to other marine organisms, ascidians are an excellent model system to study the complex process of metamorphosis, as the cellular composition of larvae and juveniles, and tissue rearrangements during this transition, are well documented (Hirano and Nishida, 2000; Hotta et al., 2007; Lemaire et al., 2008; Nakamura et al., 2012; Satoh, 2003). We present this review to describe the current knowledge of our understanding in initiation and coordination of metamorphic events in different ascidian species.
Ascidians have a biphasic life history with two distinct body plans. After a rapid embryogenesis, the nonfeeding (lecithotropic) larvae swim for a few hours and then metamorphose into a juvenile that is a sessile filter feeder. During the swimming period, larvae prepare for the onset of metamorphosis, which usually begins with adhesion. Ascidian larvae have a set of protrusions named adhesive papillae at their anterior‐most part of their body (Fig. 1A). Once adhesion occurs, the larvae initiate metamorphic events. Larvae that have reached the intrinsic conditions that allow successful metamorphic changes are called competent. The acquisition of metamorphic competence during the larval period has been shown to occur in response to a wide variety of external and endogenous signals (Cloney, 1982; Davidson and Swalla, 2002; Jackson et al., 2002). Ascidian larvae do not start metamorphosis even after they acquire the ability or competence to do so and this is a difference of ascidian metamorphosis compared to that in other organisms like insects and amphibians, in which the maturation of the intrinsic condition (hormone balance for ex.) predominantly determines the initiation of metamorphosis. This characteristic of ascidian metamorphosis may be a countermeasure for their change to sessile life style. If ascidians initiate metamorphosis before they arrive to their final destination, they can no longer move to a better location because they become tail‐less during metamorphosis. This untimely metamorphosis could force animals to live in a nonappropriate environment for their survival, which could result in inefficient growth and reproduction. To avoid this risk, ascidian larvae may have developed the regulation to start metamorphosis strictly after adhesion.
Metamorphosis of the ascidian Ciona intestinalis. (A) swimming larva and schematic representation of tissues organization at this stage. Pa, papilla; Po, preoral lobe; SV, sensory vesicle. (B) A juvenile soon after metamorphosis and schematic representation of this stage. The preoral lobe of larva is elongated and becomes transparent to be an ampulla (Am). Adult organs, such as endostyle (ES) and gill (Gi) start to develop in the trunk. Tail is retracted towards the trunk (RT). (Right) Schematic representation.
Adult ascidians superficially show little resemblance to the chordate body plan, but they do have some characteristics that are shared with other chordates like gill slits in the pharynx (Ogasawara et al., 1999a,b).
METAMORPHIC EVENTS OF ASCIDIANS
The metamorphosis of solitary ascidians can be divided into two phases: a rapid adhesion reaction followed by a prolonged period of juvenile differentiation. Metamorphosis involves numerous rapid morphogenetic movements and physiological changes.
Tissues of ascidian larvae are largely classified into three groups in relation to their status at postmetamorphic stages (Cloney, 1982); (1) tissues that exclusively function in the larval stage (transitory larval organs or TLO), (2) tissues that function in both larval and adult stages (larval‐juvenile organs/tissues or LJO), and (3) tissues that exclusively function in the adult stage (prospective juvenile organs or PJO). TLOs and LJOs are functional in the larval body, while PJOs are premature and functionless in the larval body. Although tail structures are fully differentiated within the larvae, most of these tissues/organs are classified into TLO and they degenerate during metamorphosis by the event named tail regression (Cloney, 1978; Jeffery and Swalla, 1997; Satoh, 1994). Two exceptions have to be noted (Fig. 1B): (1) the primordial germ cells (PGCs): they are embedded in the endoderm of the tail and they migrate toward the trunk during tail regression (Shirae‐Kurabayashi et al., 2006); (2) the cells that belong to the ventral endodermal strand, which migrate toward the trunk during the swimming period before the tail regression (Nakazawa et al., 2013). Most LJOs and PJOs are present in the trunk that develops during metamorphosis to compose the main part of the postmetamorphic body. The distinction of TLO, LJO, and PJO is not always clear. For example, although the epidermis is classified into LJO, the fate is completely different between the trunk and tail epidermis. The trunk epidermis is indeed LJO that remains after metamorphosis to constitute juvenile epidermis, while the tail epidermis is regressed with other tail tissues and degenerates during metamorphosis. Curiously, the cell fate of the trunk and tail epidermis diverges following the 8–16 cell stage (Pasini et al., 2006), suggesting the possibility that their postmetamorphic fate is predetermined during cleavage stages. The central nervous system (CNS) is classified into TLO; however, a recent study has shown that larval CNS contributes to the formation of the adult CNS after metamorphosis, suggesting that CNS can be classified into LJO (Horie et al., 2011).
The above classification of tissues is useful to understand ascidian metamorphosis because these groups show different fates during metamorphosis. Ascidians achieve metamorphosis by losing TLOs while maintaining or developing LJOs and PJOs. Cloney was the first to describe ten cellular events as the basis of ascidians' metamorphosis in 1978: (1) secretion of adhesives by the papillae or epidermis of the trunk, this is a prerequisite event for metamorphosis because it is required for attachment to substrate (Table 1) (2) reversion and retraction of papillae, (3) regression of the tail, (4) loss of the outer cuticle layer of the larval tunic, (5) retraction of the sensory vesicle, (6) phagocytosis of visceral ganglion, sensory organs, and cells of the axial complex, are the events for losing larval tissues and therefore the events occur in the TLOs (Table 1), (7) emigration of blood cells or pigmented cells, occurs through the epidermis into the tunic (Cloney and Grimm, 1970), (8) rotation of visceral organs through an arc of about 90°, expansion of the branchial basket, and elongation of the oozooids of juveniles, (9) expansion, elongation, or reciprocation of ampullae, reorientation of test vesicles and expansion of the tunic, and (10) release of organ rudiments from an arrested state of development, are the events for constructing adult tissues/organs (Table 1). There are variations of metamorphic events among species, but the above classification is basically shared among many ascidians (Davidson et al., 2003).
| Events described in Cloney et al., (1982) and their characteristics | Effects in sj or trf mutants | Classification in Nakayama‐Ishimura et al. (2009) |
|---|---|---|
| Events required for the initiation of metamorphosis | ||
| Secretion of adhesives | cellulose sensitive | Not done because secretion of adhesivesis accomplished during larval stage. |
| Events for losing larval tissues (destructive events) | ||
| Tail regression | trf‐dependent | Group 3 |
| Sensory vesicle retraction | trf‐dependent | Group 3 |
| Loss of larval tunic | ND* | |
| Phagocytosis of visceral ganglion and sensory organs | ND* | |
| Papillae eversion or retraction | cellulose sensitive, trf‐dependent | Group 2 |
| Events for constructing adult tissues | ||
| Emigration of blood and pigmented cells | ND* | |
| Rotation of body axis | cellulose sensitive, trf‐independent | Group 1 |
| Development of adult organs | trf‐independent | Group 4 |
| Development of ampullae | trf‐independent | Group 4 |
| *ND, not done. |
Molecular Mechanisms Underlying Ascidian Metamorphosis
This detailed description of cell events and changes in tissue morphology has not been backed up by a detailed image of the molecular events at the origin of this transformation.
In this review, we will discuss recent molecular data and try to integrate them in the previous descriptions of the cellular events of the ascidians metamorphosis for a global and comprehensive vision of metamorphosis regulation in solitary ascidians (Figs. 2 and 3).
Apoptosis start at the tip of the tail in metamorphic larva. Superposition of TUNEL and DAPI labeling (apoptotic nuclei appear in green, all nuclei of the tadpole in blue).
Synopsis of molecular and cellular events at the onset of metamorphosis in different species of solitary ascidians. Sequential numbers refer to the order of events. Gr.: Group according to classification in Nakayama‐Ishimura et al. (2009) and Table 1.
Tail regression
Tail regression mechanisms
The first and most dramatic metamorphic event in ascidians is the regression of the larval tail. Two main mechanisms (subdivided in five forms, see Satoh, 1992 for details) have been proposed to explain tail regression in ascidians. The first that involves the contractile properties of the epithelial layer, which could induce the first phases of tail shortening was observed in Aplousobranchiata (Distaplia occidentalis, Aplidium constellatum, Diplosoma macdonaldi), in Phlebobranchiata (Ecteinascidia turbiniata, C. intestinalis, Ascidia callosa, Corella willmeriana) and in one Stodilobranchiata (the colonial ascidian Botryllus schlosseri). The second is observed in Stodilobranchiata (Boltenia villosa, Herdmania curvata, Styela gibbsii, Molgula mahattensis, Molgula occidentalis, Polycitor mutabilis)), and is based on contractile properties developed by notochord cells that would trigger tail shortening (reviewed by Cloney, 1978). These observations gave rise to the terminology tail absorption, which suggest that the cells that compose the tail ingress in the trunk. Metamorphosis also includes apoptotic events and there are striking examples of this throughout the animal kingdom (Jacobson, 1997; Vaux and Korsmeyer, 1999). In accordance with this, we and others clearly showed that most of the tail cell types die by programmed cell death during this key metamorphic event, and because many cells are eliminated, it is more accurate to use the term “tail regression” (Chambon et al., 2002; Jeffery, 2002; Tarallo and Sordino, 2004) (Fig. 2).
Using transmitted electron microscopy (TEM), we clearly defined that programmed cell death presents all the morphological features of apoptosis as they were described by Kerr et al., (1974). Moreover, using TUNEL and Western Blot approaches, we highlighted biochemical features of apoptotic programmed cell death, also called programmed cell death type I (PCD I), which are DNA cleavage and activation of a caspase‐like 3 (Chambon et al., 2002). In contrast, we never observed cell engulfment that is the cell corpse clearance mechanism, the last step of apoptotic cell death. Apoptosis affects almost all the cell types that compose the tail, tunic cells, epidermal cells, notochord cells, tail muscle cells (Chambon et al., 2002), and the CNS (Tarallo and Sordino, 2004). The only exception is the endodermal strand in which no cell death was observed, compatible with the recent finding that these cells migrate toward the trunk prior to tail regression (Nakazawa et al., 2013).
The most remarkable feature of this apoptotic tail regression is the posterior origin and propagation. We observed that apoptosis started at the tail tip (Fig. 2) and through sequential TUNEL pictures we showed that it continued up to the tail base (Chambon et al., 2002). This apoptotic cell death, affect the majority, if not all, epidermal cells of the tail, muscle cells, and at least the most posterior notochord cells (Chambon et al., 2002) In other words, one of the main mechanisms of tail regression in Ciona intestinalis is cell death which is tightly controlled and even predetermined in time and space. Interestingly the same posterior “tip of the tail” origin was observed as the initiation spot of apoptosis in two other species of ascidians, Molgula occidentalis and Asicidia ceratodes, and for the latter one, the same posterior to anterior apoptotic propagation as the one observed in C. intestinalis (Jeffery, 2002). This advance in our understanding of tail regression by an essential contribution of apoptotic mechanisms should not put aside the previous cellular descriptions of tail contraction. Indeed, these two phenomena are not mutually exclusive and our unpublished and preliminary time lapse microscopy observations suggest their cooccurence.
The phylogenetic position of ascidians could contribute to gain insight in how molecular mechanisms of programmed cell death evolved from invertebrates to vertebrates. By comparing the anural ascidian Molgula occulta and its urodele sister species Molgula oculata, W. R. Jeffery observed that diversity of apoptosic programs may explain differences in their larval form. He proposed that modulation in apoptotic patterns could be an evolutionary force in the multiple anural development emergencies of ascidians (Jeffery, 2002).
- What is the nature of the initiation signal and how is it restricted to the tip of the tail where apoptosis starts?
- How is apoptosis propagated in a posterior to anterior manner?
Although the biochemical signal for initiation of apoptotic‐dependent tail regression has not been identified, gene profiling approaches have provided some clues on gene networks involved in apoptosis, as well as in the irreversible mechanisms of larval adhesion to the substratum mediated by the adhesive papillae of the larval anterior trunk (Nakayama et al., 2001, 2002; Woods et al., 2004).
Initiation of apoptotic‐dependent tail regression
As discussed above, the onset of metamorphosis comes after the acquisition of competence and is correlated with the definitive adhesion of the larvae to the substratum. The acquisition of metamorphic competence during the larval period has been shown to occur in response to a wide variety of external and endogenous signals (Cloney, 1982; Davidson and Swalla, 2002; Jackson et al., 2002). For example, before adhesion, larvae change their response to light and gravity, and prefer to settle on dark or shaded surfaces (review by Satoh, 1994). It has also been proposed that competent larvae may respond to bacterial adhesion signals (Davidson and Swalla, 2002; Roberts et al. 2007). In Stolidobranch ascidians, an empirical way to study competency is KCl addition; incompetent larvae do not respond to this treatment. However, after competency is reached, then the tail will retract immediately after being treated with 10 mM KCl and the larvae will settle. It is not known how the added KCl induces adhesion, but it can be used to correlate competency with gene expression (Roberts et al., 2007). The period when competency is reached is highly correlated to the expression of MASP (mannose‐binding‐lectin activated serine protease) in the anterior of the larvae (Roberts et al., 2007), suggesting that the innate immune system is being activated (Davidson and Swalla, 2002). These results suggest that some ascidians tadpoles may be using bacterial cues to settle, but more experiments are necessary to see if the innate immune system activation is necessary and sufficient to trigger metamorphosis. Finally, upon adhesion, three phases are successively observed: larvae with active tail, immobile larvae and tail regression. The molecular pathway(s) controlling tail regression initiation and propagation are intriguing as they link “early” and anterior adhesion‐driven signals to “late” and posterior apoptosis‐triggering mechanisms, with the latter progressing in a posterior to anterior wave (Fig. 3).
Around adhesion: Signaling at the papillae and CNS.
The papillae‐mediated larvae adhesion is the requisite step for tail regression, and it has been reported that tail regression is totally abolished in papilla‐cut larva, in opposition with other metamorphic events as body axis rotation or ampulla formation (Nakayama‐Ishimura et al., 2009).
EGF pathway at the papillae: In Herdmania curvata larvae, at the time of adhesion, the EGF‐like molecule, Hemps, is secreted by a group of cells termed the papillae associated tissue (PAT) cells, just anterior to the papillae (Degnan et al., 1997; Eri et al., 1999). Further experiments suggested that this signaling pathway controls the onset of adhesion (Degnan et al., 1997; Eri et al., 1999). Some elements of this pathway have been found in other species:
The PAT cells have been shown to migrate through the anterior tunic towards the external medium, in Boltenia villosa, pleading for their role in mediating external cues and triggering Hemps‐dependent adhesion (Davidson et al., 2001)
In this same species, and by a differential screen, Davidson and Swalla showed that an actor of the developmental EGF signaling in Drosophila, cornichon, is expressed in the anterior papillary region of larvae at the acquisition of competence (Davidson and Swalla, 2001).
Finally, by differential screening in Ciona intestinalis, Nakayama et al. identified Ci‐meta1, with a predicted amino acid sequence containing numerous repeats of EGF‐like and calcium‐binding EGF‐like domains, and whose transcript is expressed strongly at the region of the papillae at the moment of adhesion (Nakayama et al., 2001).
The above results underline the relevance of a papillae‐localized EGF‐emanating signaling pathway, at the interface between external cues and the onset of adhesion, which invariably precedes metamorphic events.
ERK kinase at the papillae: A second major molecular actor that is activated specifically at the papillae, around the period of Ciona intestinalis larvae adhesion, is the extracellular signal‐regulated kinase (ERK). More importantly, chemical inhibition of this ERK activation abolishes initiation of apoptosis in metamorphic larvae, supporting the fact that this localized activation is essential for initiation of the tail regression (Chambon et al., 2007).
Thrombospondin family at the papillae and base of trunk: Two differential screens done in Ciona intestinalis by the Satoh laboratory (Nakayama et al., 2001) and in Boltenia villosa by Davidson and Swalla (Davidson and Swalla, 2002) respectively, provide us with an unexpected common actor: the thrombospondin family. The corresponding gene was shown to be expressed at the moment of adhesion, and localized at the papillae, central dorsal region of the trunk and at the frontier between trunk and tail (or neck region). Its predicted protein sequence contains repeats that classify it to thrombospondins, a family of “matricellular” proteins, extracellular proteins that can bind to several ligands (Resovi et al., 2014). These proteins have been described as being able to regulate cellular phenotype during tissue genesis and/or repair, by interacting with numerous molecules as cytokines, growth factors, matrix components, membrane receptors and extracellular proteases (Adams, 2001). Interestingly, the thrombospondin is expressed in the neck region at a time when JNK activation was reported in Ciona (Chambon et al., 2007), and thrombospondin is known as an activator of the JNK pathway in endothelial cells (Jimenez et al., 2001), that lead to a caspase dependent apoptosis (Microchnik et al., 2008). Although practically challenging, it would be very interesting to investigate the role of this gene, by a “loss of function strategy”. Is its expression a consequence of the molecular pathway preparing larvae for metamorphosis or does it itself participate actively in it?
JNK and β‐adrenergic receptor mediated signaling at the CNS: The potential role of the CNS in metamorphosis was previously raised in a review and analysis of ascidian metamorphosis in 1978 by R. A. Cloney. He proposed a preponderant role for the larval nervous system and sensory organs in selecting sites for adhesion and in the onset of metamorphosis (Cloney, 1978, 1982). Moreover, since the CNS links the papillae to the brain vesicle to the tip of the tail, it is hypothesized that the nervous system, and conduction and diffusion of one or more secreted factors are likely to be involved in metamorphosis (Takamura, 1998; reviewed by Satoh, 1994).
We observed specific activation of the c‐Jun N‐terminal kinase (JNK) MAP kinase in the CNS (posterior part of the sensory vesicle, neck region, visceral ganglion and nerve cord) starting during the swimming period of the Ciona intestinalis larvae and getting more intense just at the adhesion phase (Chambon et al., 2007; A. Karaiskou unpublished data). Moreover, the chemical inhibition of JNK abolished initiation of apoptosis in metamorphic larvae, supporting the possibility that this activation is a key component of the tail regression triggering pathway (Chambon et al., 2007). The MAPK implication in triggering apoptosis in Ciona was confirmed and extended by Tarallo and Sordino (2004) who carefully analyzed the involvement of the MAP kinase pathway in the various apoptotic events occurring in the larval trunk and brain.
Furthermore, it has been reported that addition of noradrenaline or adrenaline to cultured Ciona savignyi and Ciona intestinalis larvae, promoted tail regression, without adhesion (Kimura et al., 2003). The same authors showed a specific expression of the β1‐adrenergic receptor at the adhesive papillae, around the brain vesicle and in fibers running along the notochord, localizations that coincide with the CNS (Kimura et al., 2003). This data gives us the following order of events: adhesion with the adhesive papillae may cause noradrenaline and/or adrenaline release from the most anterior CNS and this signal could be propagated through the trunk and tail CNS to promote tail regression.
Apart from a role in the initiation of molecular pathways triggering tail regression, the above mentioned CNS‐located events offer the intriguing possibility of a role as the link between the adhesion (trunk‐located) and the regression initiation (tip of the tail‐located), through the CNS and the nerve cord.
From the above it is obvious that the MAPK pathways are key factors that could link papillae, CNS and tail regression and it was observed that their chemical inhibition abolishes tail regression. These facts led us to the setup of a differential microarray approach in Ciona intestinalis, with chemical inhibitors of either ERK or JNK, which gave the opportunity to identify subsequent gene networks controlled by these MAP kinases (Chambon et al., 2007). Approximately fifty genes presenting a down‐ or up‐regulation were identified in each case (Chambon et al., 2007). Interestingly, some of these genes were previously reported as involved in metamorphosis of Ciona or other ascidian species (Davidson and Swalla, 2002; Nakayama et al., 2001, 2002; Woods et al., 2004).
Initiation of regression: Molecular events at the tail:
A few hours after adhesion, apoptosis‐dependent tail regression initiates at the tip of the tail (Fig. 2). Recent data has provided molecular candidates playing important roles in this process.
ERK and JNK genes network and innate immunity genes: Firstly, there is obvious candidate genes identified by the ERK or JNK inhibition screening (see above, Chambon et al., 2007). Differential gene expression was observed seven hours after hatching, which theoretically coincides with the onset of networks active at the moment on tail regression initiation. A set of genes were those encoding protein precursors, with both extracellular and intracellular domains, i.e., secreted proteins. Among them there is Ci‐sushi and Ci‐Sccpb, two genes under JNK control, with expression patterns respectively all along the tail or at the tip of the tail. Interestingly, functional experiments were conducted and initiation of apoptosis was prevented in a Ci‐sushi gene silencing context (Chambon et al., 2007). This result reinforces the interest of such differential screens and opens up the possibility of identifying further important tail regression actors downstream of JNK and ERK.
Another aspect concerning these genes (Sushi, Sccpb), that contain sushi domains (or complement control protein modules—CCP), is that they belong to a larger group of invertebrate innate immunity genes, already revealed as up‐regulated at the moment of adhesion and in young juveniles in Boltenia villosa (Davidson and Swalla, 2002). For example, in Boltenia villosa, compounds that inhibited the complement pathway allowed adhesion of the larvae, but they did not undergo organ rotation and further development (Roberts et al., 2007). Genes coding for complement signaling, and selectins or proteins with von Willebrand factor a (VWa) domains have come up in both the above mentioned screens in notable and surprisingly high rates compared to more expected “developmental” signaling molecules. It would be of high interest to focus on the roles of this family of genes at the moment when larvae initiate tail regression: do they keep their canonical immune response roles or participate in this phase of deconstruction/reconstruction mainly by their ability to control adhesion and migration pathways (see also discussion in Davidson and Swalla, 2002)?
Nitric oxide (NO): Nitric oxide (NO), a physiological messenger produced by the enzyme NO synthase (NOS), is known to be involved in metamorphosis (Heyland and Moroz, 2006) and in apoptosis (Chung et al., 2001). In ascidians, depending on the species, NO has contradictory effects. For example, in Herdmania momus, NO acts as a positive regulator to induce metamorphosis (Ueda and Degnan, 2013). On the contrary it has been reported that increasing or decreasing NO levels resulted in a delay or acceleration of tail regression in Ciona intestinalis and in Boltenia villosa (Bishop et al., 2001; Comes et al., 2007). Furthermore, in Ciona intestinalis a correlation was proposed between NO levels and caspase‐3 activity, placing the NO pathway as another possible modulator of the apoptosis in the tail (Comes et al., 2007). Although NO pathway contribution needs further analysis, there is a potentially interesting link between the above mentioned innate immunity genes group and this pathway: various forms of programmed cell death can be initiated by injury, stress, or exposure to pathogens and in many of these pathways hormones or NO have key mediator roles (Heyland and Moroz, 2006; Rutherford et al., 2007).
In summary, extracellular signals drive competent larvae to adhesion via the papillae, where a local activation of EGF and ERK pathways seems to play an important role; this signaling could communicate with the CNS‐located JNK and β‐adrenergic pathways' activation and possibly result in a modulation of gene expression in different parts and/or tissues of the tail to drive initiation of tail regression (Fig. 3). An interesting thought would be that this module corresponds to the humoral factor conducted and diffused by the CNS, hypothesized by Cloney (Cloney, 1978).
Late events:
On the contrary to the above data, on molecular cues playing different roles in adhesion and in the major larval tissue deconstructing event (tail regression), little is known on molecules involved in other major deconstructing or rearrangement metamorphic events. One interesting example of such events is the major reorganization of the CNS, always quoted among the major metamorphic events as “phagocytosis of visceral ganglion and sensory organ” (Cloney, 1978). Although recent research has provided us with a detailed picture of the cellular events going on (maintenance of almost all the larval CNS except the nerve cord, disappearance of most of the larval neurons, differentiation of some larval ependymal cells into adult neurons, in Ciona intestinalis, Horie et al., 2011), further investigation is needed in order to understand the molecular pathways involved. One of the mechanisms that has been reported is apoptotic cell death, for at least part of the Ciona intestinalis larval CNS (Tarallo and Sordino, 2004). Furthermore, in terms of formation of adult structures, it would be very interesting to further analyze the process by which larval ependymal cells contribute to the formation of the adult CNS (Sasakura et al., 2012a). Finally, almost no data exist on molecular regulation of other major constructing events, as emigration of blood cells, rotation of visceral organs, or expansion of the branchial basket.
In conclusion, recent studies focusing on the molecular features of metamorphosis in different species of solitary ascidians, often using gene profiling approaches and differential mRNA screening, have emerged (Chambon et al., 2007; Davidson and Swalla, 2002; Nakayama et al., 2001, 2002; Woods et al., 2004,). These studies reveal important aspects implicating diverse signaling pathways going from innate immunity genes to the MAPK family. The future challenge will be to investigate finely tuned coordination between different pathways and cell fates.
Metamorphic pathways
In C. intestinalis, mutants that exhibit metamorphosis defects gave new insights in how the different pathways could coordinate the metamorphic events in ascidians, and this is discussed in the following section. As mentioned above, larvae of ascidians start a set of metamorphic events soon after they attach to substrates with anterior adhesive papillae. The quick response after adhesion may be a countermeasure against their nonfeeding style at the larval stage: ascidian larvae swim with a limited amount of nutrients stored in eggs and therefore they need to convert their body quickly to start feeding. Since ascidian metamorphosis takes place in a short period, several events proceed in parallel. Metamorphosing larvae destruct their tails, and at the same time they construct adult tissues and organs. Metamorphic events are caused in a coordinated manner: none of them start prior to adhesion, and all of them start in the regular order after adhesion. For example, retraction of papillae and tail regression occur quickly after adhesion, and body axis rotation and growth of adult organs follow. How do these metamorphic events coordinately take place? A simple explanation is that stimuli provided by adhesion initiate all metamorphic events simultaneously through a single and same pathway. An alternative explanation is that several different pathways are included in the metamorphosis of ascidians, and the initiative cue of metamorphosis, that is the stimuli of adhesion, starts them at once. The latter includes two further hypotheses; one is that each metamorphic pathway initiates a set of nonrelated metamorphic events, and the other is that a metamorphic pathway initiates a set of related metamorphic events. The recent mutant analyses gave us a clue to deduce which hypothesis is likely to be true in ascidian metamorphosis.
Mutants of Ciona intestinalis showing defects in the process of metamorphosis
The techniques of mutants isolation were recently established in two ascidians Ciona intestinalis and Ciona savignyi (Nakatani et al., 1999; Sasakura et al., 2005). In Ciona intestinalis, two methods have been reported; one is isolation of spontaneous mutants maintained in wild populations (Nakayama‐Ishimura et al., 2009; Sordino et al., 2008), and the other is transposon‐mediated insertional mutagenesis (Sasakura et al., 2005, 2012b). By these methods, two mutants were so far discovered showing abnormal initiation of some metamorphic events without occurrence of another set of them. Their detailed phenotypes are described below.
Swimming juvenile (sj): sj is a mutant isolated by transposon‐mediated mutagenesis. sj mutant larvae autonomously initiate a part of metamorphic events, namely retraction of papillae and body axis rotation (Fig. 4A). The term “autonomously” means that sj larvae do not require the stimuli of adhesion for initiating papillae retraction and body axis rotation. The resultant larvae have the trunk structure similar to metamorphosed juveniles, while their tails remain and continue strokes for swimming. Because adhesive papillae are lost from sj larvae prior to adhesion, sj mutant larvae seldom adhere to substrate. However, sj larvae that manage to achieve adhesion can complete metamorphosis to be normal juvenile and subsequent growth until reproductive stage. The causative gene of sj is Ci‐CesA, the gene encoding cellulose synthase (Nakashima et al., 2004). Tunicates, including ascidians, share the characteristic that they produce cellulose and can utilize it (Hirose et al., 1999; Sagane et al., 2010). Ascidian cellulose is present in the tunic, the outer cuticular layer surrounding their body to protect them from predators. In sj mutants, cellulose is lost from the tunic (Sasakura et al., 2005). The phenotypes of sj mutant suggest that cellulose and/or cellulose synthase is responsible for the proper process of metamorphosis. In addition, sj mutants at the adult stage cannot sustain adhesion and they are easily stripped off from substrates. Their tunic is jelly‐like and thus it is too soft to protect the body. Massive algal growth is seen on the surface of tunic of sj mutant adults compared to wild type animals. These phenotypes suggest that ascidian cellulose is necessary for establishing and maintaining the sessile life style at the postmetamorphic stage.
Mutants showing phenotypes during metamorphosis. (A) A swimming juvenile (sj) mutant larva. The larva loses papillae (arrowhead) and starts body axis rotation by developing endostyle, while its tail remains complete and continues swimming. (B) A tail regression failed (trf) mutant larva. After adhesion, this mutant larva starts ampulla formation and adult organs growth, while tail regression and sensory vesicle retraction do not take place.
Tail regression failed (trf): Larvae of trf mutant have normal body structure and initiate metamorphosis by adhesion as in the case of wild‐type larvae (Nakayama‐Ishimura et al., 2009). trf mutant larvae undergo body axis rotation, development of ampullae and adult organs, but the remaining events, such as retraction of papillae, tail regression and sensory vesicle retraction, are not triggered. The resultant trf mutant juveniles have tails that continue strokes, while their trunk has developed adult organs including gill slit, endostyle, heart, body wall muscles and ampullae (Fig. 4B). Most of these organs become functional in the trunk of trf mutants, as can be seen by the contraction of muscular structures, heartbeat and waving of cilia of gill slits. Apoptosis in the tail does not occur in trf mutant, and this is supposed to be a cause of the failure in the tail regression in this mutant (see the above sections: Chambon et al., 2002). trf is a spontaneous mutant line isolated from a wild population, and the causative gene has not been isolated.
Ascidian metamorphosis is conducted by plural pathways
A shared characteristic of sj and trf mutants is that a part of metamorphic events occur while other events are not started. This suggests that ascidian metamorphic events can be subdivided into several groups whose initiation is controlled by different mechanisms. Phenotypes of sj mutants divide metamorphic events into two groups; events that are triggered autonomously in the sj mutant larvae (cellulose‐sensitive events) and events that are not triggered in the sj mutant larvae (Table 1, middle column). Phenotypes of trf mutants also divide metamorphic events into two; events that are triggered and that are not triggered in trf mutant larvae. Because trf mutants require adhesion for initiating metamorphic changes at the trunk, initiation of the metamorphic events that are not triggered in trf mutants is abnormal in this mutant and these events are named “trf‐dependent events”, while events triggered in trf mutants are named “trf‐independent events” (Table 1, middle column). By the association of these phenotypes, a model has been proposed that divides metamorphic events into four groups (Table 1, right column; Nakayama‐Ishimura et al., 2009). Group 1 comprises a cellulose‐sensitive and trf‐independent event, which includes body axis rotation. Group 2 is a cellulose‐sensitive and trf‐dependent event, which includes papillae retraction. Group 3 comprises cellulose‐independent and trf‐dependent events, including sensory vesicle retraction and tail regression. Group 4 comprises cellulose‐independent and trf‐independent events, including ampullae formation and adult organ growth. This classification does not deal with all of the 10 metamorphic events because of the limitation of observation, but the relationship of these events would be important to uncover by future studies (Fig. 3).
The above grouping was done according to mutant phenotypes. However, there is a strong correlation between classification of the metamorphic events and that of larval tissues/organs into TLO, LJO, and PJO. For instance, groups 2 and 3 events are those observed in the tissues that exclusively function in the larvae; these events are for destructing larval tissues and named “destructive events”. All of trf‐dependent events are included in the destructive events and therefore they occur in TLO. Group 4 includes events occurring in the tissues constructing adult organs. These events require cell proliferation and are named “cell division‐requiring events”. These classifications suggest that metamorphic events responsible for a similar process are initiated by the same pathway.
The order of the progression of metamorphic events has plasticity, although it seems rigid in the normal metamorphosis as mentioned above. The plasticity is revealed by the fact that sj mutant larvae that finish the body axis rotation and papillae retraction can complete metamorphosis to be normal juveniles after the mutant larvae somehow achieved adhesion or are administrated with chemicals that induce metamorphosis. In trf mutant, papillae retraction occurs when the mutant larvae are cultured for a long period. Therefore there may be compensative pathways that can complete metamorphosis even in the irregular order. A crosstalk between pathways for each groups of metamorphic events has been suggested (Nakayama‐Ishimura et al., 2009), and this crosstalk may constitute the compensative pathways.
Difference of the larval trunk among ascidian species: A discussion based on the knowledge from mutant analyses
The presence of multiple pathways in ascidian metamorphosis leads us to the idea that small changes in a part of the pathways could contribute to the different larval or juvenile structure among ascidians. The degree of development of adult organ primordia in the larval trunk differs among ascidian species. In general, the state of differentiation of the digestive and circulatory system is more advanced in larvae of compound ascidians than in solitary ascidians, and some compound ascidians have well‐developed visceral organs (Fig. 5 and Cloney, 1978). The well‐developed larval trunks seen in the compound ascidians can be generated by an evolutionary process referred to as adultation (Jeffery and Swalla, 1992). The observation of sj and trf mutants has suggested that changes in a small subset of genes can create functional adult organs in the larval trunk. Particularly, phenotypes of trf mutants show that mutation in a single gene can create functional adult organs in the swimming larval body of a solitary ascidian (Nakayama‐Ishimura et al., 2009), that is more similar to the situation of the compound ascidians. Therefore, functions of genes that are involved in the regulation of adult organs growth may have been changed during divergence of ascidian species, and the genetic modifications may have caused adultation in compound ascidians. Characterization of the genes affecting adult organ growth, such as the causative gene of trf, should contribute to the validation of this hypothesis, about the evolutionary history of ascidian metamorphosis.
Larvae of solitary and compound ascidians. (A) A larva of the solitary ascidian Herdmania momus. (B) A larva of the solitary ascidian Styela plicata. (C) A larva of the compound ascidian Botrylloides sp. (D) A larva of the compound ascidian Distaplia sp.
CONCLUSION
Metamorphosis in ascidians results in a dramatic modification of their body plan, transforming them in a few hours from swimming larvae to sessile adults. This profound rearrangement is characterized by a variety of cellular events, finely orchestrated in time and space, by a combination of signal transduction pathways. In this review, we have presented the current knowledge of our understanding of metamorphic events in different ascidian species.
Larvae Acquisition of Metamorphic Competence and Subsequent Adhesion are the Initiation Point of Metamorphosis
The main actors described at this section are summarized in Figure 3. The adhesive organ, or papilla, seems to be a hot point for mediating external adhesion signals (via Hemps, an EGF repeats protein) and onset of at least one of the events of metamorphosis, tail regression, via activation of a yet to unravel ERK pathway. Around adhesion time, in the CNS at the close vicinity of the papillae, two different sets of signaling molecules get activated: JNK and the β1‐adrenergic receptor network. Since JNK activation was also observed through the nerve cord, a tempting hypothesis is that this activation through the CNS could serve as the link between the anterior events at the time of adhesion and the posterior ones, regulating the initiation of tail regression. It would be very important to explore this hypothesis.
The First and Most Spectacular Metamorphic Event is Tail Regression, Mainly Due to a Polarized Apoptotic Cell Death
Tail regression in Ciona intestinalis occurs mainly by apoptosis, which is tightly controlled and even predetermined in time and space, starting always at the tip of the tail and propagating in a posterior to anterior manner and this is a unique feature among Chordate organisms. This invariant pattern of apoptosis observed during tail regression, makes ascidians an excellent “genetic tool” to investigate the apoptotic molecular mechanisms during development, as in C. elegans, the reference model in the apoptosis research field (S. Brenner, Nobel Prize acceptance speech, 2003). This is even more relevant since C. intestinalis genome exhibits a cell death signaling machinery that shares many central participants with vertebrates (Takada et al., 2005, Terajima et al., 2003; Weill, 2005) and the biochemical cell death machinery is close to that found in mammals (Baghdiguian et al., 2007). Moreover the ongoing sequencing of different species of ascidians coupled with the emerging genetic tools would contribute to identify new specific regulatory pathways of cell death.
From a molecular point of view, it has been shown that treatment of Ciona intestinalis larvae with either ERK or JNK inhibitors, around adhesion time, inhibited apoptotic tail regression. A differential screening provided two lists, one with genes modulated downstream of ERK, and the second one downstream of JNK (Chambon et al., 2007). These lists revealed one interesting family of genes, already identified by a differential screening at the onset of metamorphosis in Boltenia villosa, genes of the invertebrate innate immunity family. Focusing on the precise roles of these genes in metamorphosis, understanding how these immunity genes interfere with postembryonic development would be an exciting future direction in ascidian research.
Decisions Between Survival and Cell Death
One of the emerging issues in ascidian metamorphosis research, is the compelling fact that groups of cells will either commit to apoptosis, while surrounded by tissues that survive (as is the case of part of the trunk CNS) or in the opposite, will survive, even though belonging initially to the body part destined to destruction, the tail (as is the case of the endodermal strand and the PGCs). Furthermore, these two latter tissues, achieve survival, at least partially, by a posterior to anterior migration, which takes place prior the onset of metamorphic events for the endodermal strand, or during tail regression for the PGCs. For the later one, apoptotic cells in ascidians tail could act as a driving force for their migration towards the trunk as it was reported in drosophila during dorsal closure or in mouse during neural tube closure (review in Suzanne and Steller, 2013). Detailed analysis of the molecular mechanisms involved will be important for understanding the way by which a chordate organism coordinates cell fate decisions.
The Posterior to Anterior Wave of Events
Another intriguing observation emanates from the above mentioned data: the apoptotic‐driven tail regression is finely orchestrated to a posterior to anterior wave, as are the two cell migrations that “protect” the endodermal strand and the PGCs from cell death. What is the fine mechanism orchestrating the sequential “destruction” of a large part of the tail tissues and what kind of signal triggers and drives the posterior to anterior cell migration? Is there a determinant? If yes, how was it created? If no, could it be a cell to cell communication, meaning that apoptotic cells communicate an apoptotic destiny to the ones at a more anterior position? And how is this directional signaling connected to the apoptosis initiation signaling and the adhesion one? One hypothesis that deserves exploration would be that the posterior part of the tail of ascidian embryos contains the signaling source as it expresses several secreting molecules such as FGF and Wnt, starting at earlier developmental stages (Ikuta et al., 2010; Treen et al., 2014).
What Metamorphosis Can Teach Us About Tunicates Evolution
From an evolution point of view, it is supposed that the ancestors of tunicates were free‐swimming animals like larvaceans (Wada, 1998). This means that the ancestor of ascidians had functional adult organs in a tadpole‐like body, like those of trf mutants, and ascidians restricted the growth of adult organs during the tadpole stage after acquisition of the life style of adhesion via metamorphosis. Comparison of the regulation mechanisms of the ascidian and larvacean focusing on the genes involved in the metamorphic events, especially those which regulate adult organ growth, is important to understand the evolutionary history of tunicates.
All the above intriguing questions could be the starting point of future investigations. Studying ascidian metamorphosis is not only acquiring essential knowledge about the evolution of this process in animals, but can also provide a simple model for the analysis and comprehension of challenging fundamental questions on how cells commit to different fates (cell death versus survival, migration, and differentiation) and most importantly how these processes are coordinated in a Chordate.
ACKNOWLEDGMENTS
This study was supported by: Emergence‐UPMC‐2012 Research Program and “André Picard Network” to JPC and AK, Grants‐in‐Aid for Scientific Research from the Japan Society for the Promotion of Science (JSPS) and Ministry of Education, Culture, Sports, Science and Technology (MEXT) to YS. YS was supported by the Toray Science and Technology Grant and by grants from the National Bioresource Project (YS).
