Molgula tectiformis is a new ascidian species recently described by Nishikawa (1991). In Otsuchi Bay, Iwate, Japan, they are easily obtainable from cages for culturing scallops. We report here that M. tectiformis is another example of a direct developer: their embryonic development is lacking the tadpole larva. The fertilized egg is orange and about 150 μm in diameter. At 18°C, the egg cleaves at about 20 min intervals and gastrulation occurs about 5 hr after fertilization. In contrast to conventionally-developing ascidians, M. tectiformis does not form a tadpole larva. Immediately before hatching, three stolons or ampullae begin to extend from the tailless embryo. After hatching the stolons mediate the attachment of the juvenile body to the substratum. Histochemistry for tissue-specific enzyme activity did not detect muscle-specific acetylcholinesterase, endoderm-specific alkaline phosphatase, and pigment cell-specific tyrosinase. In addition, in situ hybridization could not prove the presence of muscle actin gene transcripts in the embryo. These results suggest that these larval tissues do not differentiate in M. tectiformis embryos. Because M. tectiformis is common and gravid year-around in Otsuchi Bay, this direct developer provides the opportunity for further analysis of molecular changes during evolution that cause an alternative mode of development.
In addition to the usual developmental processes involved in forming a tailed (tadpole or urodele) larva, a dozen or so ascidian species, exhibit other modes of embryonic development or the formation of tailless (anural) larvae (Lacaze-Duthiers, 1874; Berrill, 1931; Millar, 1954; Whittaker, 1979; Swalla and Jeffery, 1990, 1992; Bates and Mallett, 1991; Bates, 1995). These tailless (or anural) species belong to the families Molgulidae and Styelidae and include Molgula bleizi, M. occulta, M. arenata, M. pacifica, M. provisionalis, M. robusta, M. retortiformis, Bostrichobranchus pilularis, B. digonas, Eugyra arenosa, Pelonaia corrugata and Polycarpa tinctor (reviewed by Jeffery and Swalla, 1990, 1992; Bates, 1993). Anural species may be subdivided into those that exhibit indirect (incomplete) or direct (complete) development. In species with indirect anural development, such as M. arenata and M. occulta, a tailless, slug-like larva hatches from the chorion before metamorphosis (Whittaker, 1979; Swalla and Jeffery, 1990). Species with direct anural development, such as M. provisionalis and M. pacifica (Bates and Mallett, 1991; Bates, 1995), have completely eliminated the larval stage. In these species, hatching occurs after the beginning of metamorphosis, which is initiated within the chorion. A recent molecular phylogenetic analysis suggests that anural species have been derived at least five different times from ancestors with urodele larvae (Hadfield et al., 1995).
The tadpole larva has a head containing a dorsal brain and pigmented sensory organs (otolith and ocellus) and a tail containing a notochord, spinal cord, and striated muscle cells. These chordate features are lacking in anural larvae, although undifferentiated precursor cells are frequently observed (Whittaker, 1979; Swalla and Jeffery, 1990). Therefore, ascidian species with these two modes of development provide an opportunity to study molecular mechanisms involved in an evolutionary change in development (Swalla and Jeffery, 1990, 1996; Swalla et al., 1993; Satoh and Jeffery, 1995; Kusakabe et al., 1996).
No ascidian species with anural development have been previously reported from the coast of Japan. Recently, an ascidian species belonging to the family Molgulidae has appeared in large numbers off the coast of northern Japan. This new species has been described by Nishikawa (1991) as Molgula tectiformis. Here we report that M. tectiformis develops without a larval stage and is another example of an anural (direct) developer.
MATERIALS AND METHODS
Molgula tectiformis (Nishikawa, 1991) was collected in Otsuchi Bay near the Otsuchi Marine Research Center, Ocean Research Institute, University of Tokyo, Otsuchi, Iwate, Japan. A large number of M. tectiformis are found at a depth of 5~10 m in this location. The animals are attached to fixed substrates, such as cages for culturing scallops and ropes.
Gametes can be obtained by controlling the light and dark periods (cf. Satoh, 1994). The animals spawn by light stimulation after long exposure of light followed by dark adaptation. In this study, however, we obtained gametes by dissecting the gonad. After exposure to seawater, oocytes matured by breaking down the germinal vesicle. Eggs were fertilized by mixing them with a suspension of non-self sperm. Fertilized eggs were cultured in filtered seawater at about 18°C. At this temperature, first cleavage took place about 1 hr after insemination, and the embryos divided at approximately 20 min intervals. Juveniles hatched out of the chorion about 13 hr after insemination. Photomicrographs of M. tectiformis embryos and juveniles were taken through a Zeiss Axioplan microscope.
M. tectiformis embryos at appropriate stages and juveniles were collected by low-speed centrifugation and fixed for histochemistry and in situ hybridization. Embryos and larvae of Ciona savignyi or Molgula occidentalis, which form conventional tadpole larvae, were used as controls.
Histochemistry for enzymatic activity specific to larval tissues
Acetylcholinesterase (AChE): AChE is a marker of muscle cell differentiation in ascidian embryos (cf. Satoh, 1994). Histochemical detection of AChE was carried out according to the method described by Karnovsky and Roots (1964) after specimens were fixed for 10 min in 5% formalin seawater at room temperature.
Alkaline phosphatase (AP): AP is predominantly expressed in endoderm cells of ascidian tadpole larvae, and is a marker of endoderm differentiation (cf. Satoh, 1994). AP activity was detected histochemically as described by Whittaker and Meedel (1989).
Tyrosinase: Tyrosinase (dopa oxidase) is a key enzyme for the production of melanin. In ascidian embryos, tyrosinase is produced by the otolith and ocellus pigment cell lineages (cf. Satoh, 1994). Tyrosinase activity was detected according to the method described by Whittaker (1973).
In situ hybridization
An antisense RNA probe containing most of the coding region of the MocuMA1 gene, which hybridizes to muscle actin mRNA from a variety of ascidian species (Kusakabe et al., 1996), was used to determine whether M. tectiformis embryos express muscle actin. The procedures used for MocuMA1 probe preparation and in situ hybridization of sectioned embryos were as described previously (Kusakabe et al., 1996).
Embryogenesis of M. tectiformis
Figure 1 shows the embryonic development of M. tectiformis. The egg was orange in color and about 150 μm in diameter (Fig. 1A). It is surrounded by numerous, large test cells which vigorously interact with the surface of the embryo during development. M. tectiformis is self-fertile: eggs fertilized with sperm of the same individual develop normally. In this study, however, we followed embryogenesis of eggs fertilized with sperm of another individual. Fertilization initiates ooplasmic segregation. After ooplasmic segregation, the animal hemisphere became transparent whereas the vegetal hemisphere became orange-brown (compare Fig. 1B with 1A). The transparent, animal cytoplasm was segregated mainly into the epidermis of the embryo, while the orange-brown vegetal cytoplasm was partitioned mainly into the endoderm of the embryo (Fig. 1). Whether the myoplasm, an egg cytoplasmic region containing tail-muscle cell determinants (cf. Satoh, 1994), was present in M. tectiformis eggs could not be determined.
As in the case of ascidians with urodele larvae, cleavage of M. tectiformis eggs was bilaterally symmetrical. The first and second cleavages were meridional, dividing the egg into two (Fig. 1B) or four cells (Fig. 1C), usually of equal sizes. The third cleavage was latitudinal, and it usually produced animal and vegetal cells of unequal sizes. In 8-cell embryos, the four animal cells were transparent and small, whereas the four vegetal cells were intensely in orange color and large (Fig. 1D). Therefore, the future dorsal (vegetal)-ventral (animal) axis could be discerned by the position of colored blastomeres at the 8-cell stage. At the fourth cleavage of ascidian eggs, the division pattern of the animal tier cells is reversed relative to the vegetal tier cells (cf. Satoh, 1994). This division pattern reversal was also observed at the fourth cleavage in M. tectiformis embryos (Fig. 1E). The fifth cleavage occurred about 20 min later to form a 32-cell embryo. The subsequent cleavages increased the number of embryonic cells, although precise division pattern could not been followed (Fig. 1F, G). Gastrulation occurred at about 4.5 hr after fertilization (18°C). At first, the vegetal side of the embryo flattened (Fig. 1G). Then the vegetal cells invaginated (Fig. 1H). Gastrulation appeared to be accompanied by epibolic movement of the animal cells. At about 11 hr after insemination and after the completion of gastrulation, a transparent stripe became visible on the posterior-vegetal side of the embryo. This transparent stripe may correspond to the position of the neural tube, which forms on the dorsal side of ascidian embryos during neurulation (cf. Satoh, 1994). The morphogenetic processes of gastrulation and neurulation were completed within a few hours.
Later during embryogenesis, the outer surface of the embryo became transparent, indicating the formation of the epidermis (Fig. 1J). No other conspicuous changes in the embryonic morphology were evident during later phases of embryogenesis. About 13 hr after fertilization, M. tectiformis embryos hatched from the chorion. A portion of the chorion became thin, a hole was eventually formed in this region, and the juvenile passed through the hole in the chorion (Fig. 1J). The hatched larva was ovoid. Three transparent ampullae appeared while the embryo was still in the chorion (Fig. 1J, K). Following hatching and settlement, they form a firm attachment to the substratum (Fig. 1L).
Therefore, M. tectiformis does not develop a conventional tadpole larva. Because juvenile formation took place during late embryogenesis, this species is also a direct developer. Other direct-developing ascidian species are B. digonas (Swalla and Jeffery, 1992) and M. pacifica (Young et al., 1988; Bates and Mallett, 1991): there was no tail region with apparent differentiation of the notochord and muscle (Fig. 1). No otolith and ocellus pigment cells were observable in the embryo (Fig. 1). In addition, no larval adhesive organ was evident (Fig. 1).
After hatching, the formation of young adults proceeded rapidly (Fig. 2). Juveniles soon lost the ampullae. About 3 or 4 days after hatching, the development of various internal organs was evident in the young adult (Fig. 2B).
Muscle cell differentiation in M. tectiformis embryos
During indirect anural development in M. occulta, neither the larval muscle actin nor the myosin heavy chain genes are expressed but the muscle-lineage cells develop the muscle-specific enzyme AChE (Jeffery and Swalla, 1991; Kusakabe et al., 1996). As described above, M. tectiformis is a direct developer, suggesting that differentiation of muscle cells is suppressed. We examined this issue by detection of AChE by histochemistry and muscle actin gene expression by in situ hybridization.
As shown in Fig. 3A, the control C. savignyi larva developed AChE activity in the tail muscle cells. However, AChE activity was not detected in M. tectiformis embryos (Fig. 3B). This was confirmed by examining more than 30 embryos at different stages of later embryogenesis (at 10 and 12 hr of development). AChE activity, however, was detected in the body-wall muscle cells of juveniles 4 days after hatching (data not shown). The results show that M. tectiformis embryos do not express AChE.
Sections of M. tectiformis embryos at 12 hr of development were hybridized in situ with the antisense MocuMA1 probe to determine whether muscle actin is expressed. The MocuMA1 probe, which contains most of the coding region of an M. oculata larval-muscle actin gene, has been shown to detect muscle actin transcripts in urodele ascidian embryos (Kusakabe et al., 1996). As shown in Fig. 4A, the MocuMA1 probe detected transcripts in tail muscle cells of M. occidentalis, the urodele developer used as a control in our experiments. In contrast, muscle actin transcripts could not be detected in M. tectiformis embryos, although small cells visible in the posterior region may be vestigial-muscle cell precursors (Fig. 4B). The results suggest that M. tectiformis embryos do not express muscle actin.
In summary, the lack of AChE and muscle actin expression suggests that M. tectiformis embryos lack differentiated muscle cells.
Development of alkaline phosphatase in M. tectiformis embryos
Alkaline phosphatase (AP) activity is used to monitor endoderm differentiation in ascidian embryos (cf. Satoh, 1994). We examined development of AP activity in M. tectiformis embryos. As a control, C. savignyi larvae clearly showed the AP activity in endodermal cells (Fig. 3D). However, AP activity was not detected in M. tectiformis embryos and newly hatched juveniles (Fig. 3C), although AP activity became detectable in juveniles 4 days after hatching (Fig. 3D). The results suggest that endoderm cell differentiation has also been modified in M. tectiformis embryos.
Development of tyrosinase activity in M. tectiformis embryos
Ascidian tadpole larvae usually develop two pigment cells in the brain vesicle: one is the otolith and the other is the ocellus (cf. Satoh, 1994). The stage at which these pigment cells differentiate varies with species (cf. Satoh, 1994). During embryogenesis of most direct developers, pigment cells do not differentiate (cf. Jeffery and Swalla, 1990). The direct developer B. digonas, which develops one or two pigment cells (Swalla and Jeffery, 1992), is a notable exception. As described above, no pigment cells were formed in M. tectiformis embryos. We performed histochemistry to determine whether tyrosinase expression is also lacking in M. tectiformis embryos. Although C. savignyi embryos showed tyrosinase activity in two pigment cells (Fig. 3E), none of the M. tectiformis embryos examined (more than 30 specimens) developed enzymatic activity (Fig. 3F). The results show that the melanin synthesis pathway has been eliminated in M. tectiformis embryos.
In this investigation, we have shown that M. tectiformis is an anural (direct) developer. Therefore, similar to other anural species, M. tectiformis does not exhibit a tadpole larva. Despite this modification in the conventional mode of embryogenesis, fertilized M. tectiformis eggs undergo ooplasmic segregation and cleavage similarly to those of urodele ascidian species, and they gastrulate and form a neural tube. After neurulation, however, they do not differentiate a pigmented neural sensory organ or exhibit the morphogenetic movements resulting in notochord and tail development. In addition, M. tectiformis embryos do not express AP, suggesting a deficiency in endoderm differentiation. Similar to a few other anural species, including M. bleizi, M. pacifica, M. retortiformis, and B. digonas (Berrill, 1931; Bates and Mallett, 1991; Swalla and Jeffery, 1992; Bates, 1995), M. tectiformis embryos begin metamorphosis prior to hatching. In most urodele ascidian species, metamorphosis, as marked by the extension of the ampullae, is initiated after hatching (cf. Satoh, 1994). Thus, there appears to be two major differences in development between M. tectiformis and conventional urodele ascidian embryos. The first difference, which is shared with all other anural species, is that M. tectiformis embryos lack the urodele neural sensory and tail structures. The second difference is that metamorphosis begins precociously before hatching. The form of anural development characteristic of M. tectiformis may have evolved through a combination of elimination and heterochronic acceleration of developmental processes.
The anural species that has been most thoroughly investigated is M. occulta, an indirect developer (Swalla and Jeffery, 1990, 1996; Jeffery and Swalla, 1991, 1992; Kusakabe et al., 1996). Although no tail appears during embryogenesis, M. occulta embryos contain vestigial notochord and muscle cells, which fail to undergo terminal differentiation. The vestigial muscle cells of M. occulta embryos express AChE but they do not contain muscle actin transcripts (Swalla and Jeffery, 1990; Kusakabe et al., 1996). AChE is also expressed in the vestigial muscle cells of another anural indirect developer, M. arenata (Whittaker, 1979). A recent study showed that lack of muscle actin expression in M. occulta was caused by structural deficiencies in the coding regions rather than the regulatory machinery of the larval muscle actin genes (Kusakabe et al., 1996). In M. tectiformis embryos, we have been unable to detect either the expression of muscle actin or AChE during embryonic development, suggesting that the program of muscle differentiation has degenerated even further than in M. occulta and M. arenata. It will be interesting to determine if larval muscle actin genes are still present in the M. tectiformis genome.
The extent to which urodele features are modified differs among anural ascidian species. All known anural species, with the notable exception of B. digonas (Swalla and Jeffery, 1992), have lost the pigmented neural sensory organ (Berrill, 1931; Jeffery and Swalla, 1990). The lack of pigment cell differentiation stems from a deficiency in the melanin synthesis pathway, as shown by the absence of tyrosinase activity in M. occulta embryos (Swalla and Jeffery, 1990). In the present study, we have shown that tyrosinase activity and pigmented neural sensory cells are also absent in M. tectiformis embryos. Differences in AChE expression have also been reported in anural ascidian species. The indirect anural developers M. arenata (Whittaker, 1979) and M. occulta (Jeffery and Swalla, 1991) express AChE, whereas the activity of this enzyme is reduced to very low levels or absent in the anural direct developers M. provisionalis, M. pacifica, and B. digonas (Bates and Mallett, 1991; Swalla and Jeffery, 1992; Bates, 1995). These comparisons suggest that loss of AChE expression is correlated with the direct mode of anural development, consistent with the suggestion that these anural species are older, in an evolutionary sense, than species with indirect anural development (Bates, 1993).
There is one feature of anural development we have observed in M. tectiformis eggs and embryos that is distinct from other anural species: the fact that they are surrounded by large and active test cells. It has been shown previously that M. pacifica and M. occulta embryos either lack test cells or contain reduced numbers of smaller test cells (Young et al., 1988; Bates and Mallett, 1991). The test cells have a secretory function in tunic synthesis and assembly during larval development (cf. Satoh, 1994). The reason why M. tectiformis has retained the test cells while other anural ascidian species have eliminated them is unclear, but may be related to fundamental differences in the structure of the larval tunic.
Many anural ascidian species, including M. arenata, M. occulta, B. pilularis, and B. digonas, are restricted to rather homogeneous habitats at the ocean bottom, where they lie partially buried in sand or mud. Berrill (1931) suggested that these anural species evolved from urodele ancestors because there was no selective advantage for a larval dispersal stage in these habitats. More recently, Hadfield et al. (1995) proposed that the tadpole larva may be under negative selective pressure in patchy bottom habitats because extensive larval dispersal would interfere with settling and feeding of the rapidly-developing juveniles. It is difficult to adapt either hypothesis to M. tectiformis, which are attached to firm substrates in Otsuchi Bay. Other anural ascidian species, including M. bleizi, M. pacifica, and M. provisionalis (Berrill, 1931; Young et al., 1988; Bates and Mallett, 1991; Bates, 1995), are also attached to substrates rather than buried in sand or mud flats. It is possible that anural developers initially evolved in homogeneous bottom habitats, then speciated and invaded the shoreline, as first suggested by Berrill (1931). If this is the case, the presumed bottom-dwelling ancestor of M. tectiformis must have become extinct because no other anural Molgula species have been reported in the coastal waters of northern Japan.
Anural species have been useful as models for elucidating the molecular basis of urodele ascidian development, the evolution of developmental mechanisms, and the regulatory genes that may have operated in the ancestral chordate (Jeffery and Swalla, 1992; Swalla et al., 1993; Satoh and Jeffery, 1995; Kusakabe et al., 1996; Swalla and Jeffery, 1996). The Japanese ascidian M. tectiformis has several features that are unique among anural species, including year round availability of gametes, relatively large eggs and embryos, and existence of colored cytoplasmic regions. These features will be important assets in future studies of the mechanisms of anural development.
We wish to thank Dr. Hiroshi Wada for his suggestion on M. tectiformis embryogenesis. We also thank to all staff members of the Otsuchi Marine Research Center, Ocean Research Institute, University of Tokyo for their hospitality. This research was supported by Grants-in-Aid for Specially Promoted Research (No. 07102012) and International Scientific Research (06044120) from the Ministry of Education, Science, Sports and Culture of Japan to N.S. and NSF (IBN-94-17799) and NIH (HD-13970) to W.R.J.