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

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

Expression of a muscle determinant gene, macho-1, in the anural ascidian Molgula tectiformis

In anural (tailless) ascidian species, functional embryonic muscle is not formed. In urodele (tailed) ascidians, macho-1 functions as a maternally supplied factor for embryonic muscle formation. The failure of embryonic muscle development in anural ascidians may be due to the suppression of macho-1 expression. In this paper, however, we report the expression of macho-1 in embryos of an anural ascidian, Molgula tectiformis. Although M. tectiformis has lost the developmental potential to form functional embryonic muscle, macho-1 was expressed in a very similar manner as in urodele ascidians. This result, together with those of previous studies, strongly suggests that in M. tectiformis the upstream genetic cascade responsible for muscle formation is intact, while the downstream cascade including the expression of muscle structural genes is severely affected.Electronic Supplementary Material Supplementary material is available for this article at     

Programmed cell death in the ascidian embryo: modulation by FoxA5 and Manx and roles in the evolution of larval development

Programmed cell death (PCD) has been discounted in the ascidian embryo because the descendants of every embryonic cell appear to be present in the tadpole larva. Here we show that apoptotic PCD is initiated in the epidermis and central nervous system (CNS) but not in the endoderm, mesenchyme, muscle, and notochord cells during embryogenesis in molgulid ascidians. However, the affected cells do not actually die until the beginning of metamorphosis. Although specific patterns of PCD were different in distantly related ascidian species, the results suggest that removal of CNS cells by apoptosis is a urchordate feature predating the origin of the vertebrates. Certain molgulid ascidian species have evolved an anural (tailless) larva in which notochord cells fail to undergo the morphogenetic movements culminating in tail development. These anural species include Molgula occulta, the sister species of the urodele (tailed) species Molgula oculata. We show that PCD in the notochord cell lineage precedes the arrest of tail development in M. occulta and other independently evolved anural species. The notochord cells are rescued from PCD and a tail develops in hybrid embryos produced by fertilizing M. occulta eggs with M. oculata sperm, implying that apoptosis is controlled zygotically. Antisense inhibition experiments show that zygotic expression of the FoxA5 and Manx genes is required to prevent notochord PCD in urodele species and hybrids with restored tails. The results provide the first indication of PCD in the ascidian embryo and suggest that apoptosis modulated by FoxA5 and Manx is involved in notochord and tail regression during anural development. Differences in PCD that occur between ascidian species suggest that diversity in programming apoptosis may explain differences in larval form.     

Interspecific hybridization between an anural and urodele ascidian: differential expression of urodele features suggests multiple mechanisms control anural development

Anural development in the ascidian Molgula occulta was examined using tissue-specific markers and interspecific hybridization. Unlike most ascidians, which develop into a swimming tadpole larva (urodele development), M. occulta eggs develop into a tailless slug-like larva (anural development) which metamorphoses into an adult. M. occulta embryos show conventional early cleavage patterns, gastrulation, and neurulation, but then diverge from the urodele developmental mode during larval morphogenesis. M. occulta larvae do not contain a pigmented sensory cell in their brain or form a tail with differentiated notochord and muscle cells. As shown by in situ hybridization with cloned probes and analysis of in vitro translation products, M. occulta embryos do not accumulate high levels of alpha actin or myosin heavy chain mRNA. In contrast, acetylcholinesterase is expressed in muscle lineage cells, indicating that various muscle cell features are differentially suppressed. M. occulta embryos also lack tyrosinase activity, suggesting that suppression of brain pigment cell differentiation occurs at an early step in development. M. occulta eggs fertilized with sperm from Molgula oculata (a closely related urodele species) develop into hybrid larvae exhibiting some of the missing urodele features. Some hybrid embryos develop tyrosinase activity and differentiate a brain pigment cell and a short row of notochord cells, and form a short tail. These urodele features appeared together or separately in different hybrid embryos suggesting that they develop by independent mechanisms. In contrast, alpha actin and myosin heavy chain mRNA accumulation was not enhanced in hybrid embryos. These results suggest that multiple mechanisms control anural development.     

Evolution of the ascidian anural larva: evidence from embryos and molecules.

Most ascidians pass through a tadpole (urodele) larval stage, although some species have derived a tailless (anural) larva. New insights into the evolution of anural larvae in the Roscovita clade of molgulid ascidians were obtained from studing embryonic development of the transitional anural species Molgula bleizi and from phylogenetic analysis based on muscle and cytoskeletal actin gene sequences. By observing in vitro fertilized eggs, we found that M. bleizi, previously described as a typical anural developer, actually forms a short immotile tail during embryogenesis. The short tail contains notochord lineage cells, which undergo abbreviated morphogenetic movements but eventually arrest in development. Molgula bleizi tail muscle lineage cells produce the muscle enzyme acetylcholinesterase (AChE) but do not express muscle actin genes. The presence of a short tail, a vestigial notochord, and AChE-positive muscle cells suggest that M. bleizi is a recently derived anural species. An M. bleizi larval muscle actin gene (MbMA1) was isolated, sequenced, and shown to be a pseudogene based on critical deletions in its coding region that would result in a nonfunctional actin protein. The mutations in MbMA1 are distinct from and have evolved independent of the larval muscle actin pseudogenes MoccMA1a and MoccMA1b in Molgula occulta, another anural developer in the Roscovita clade. Pseudogene formation explains the absence of muscle actin mRNA in M. bleizi embryos. The 3' untranslated region of an M. bleizi cytoskeletal actin gene was also isolated and sequenced. Phylogenetic trees reconstructed using muscle and cytoskeletal actin sequences suggest that the anural developer M. bleizi evolved prior to the divergence of the urodele developer Molgula oculata and the anural developer M. occulta in the Roscovita clade. Since M. bleizi lives attached to hard substrata in the tidal zone, whereas M. oculata and M. occulta live buried in subtidal sand flats, our results suggest that the anural larva evolved at least twice in the Roscovita clade of molgulid ascidians as an adaptation to different habitats.     

Ecological Consequences of Altering the Timing Mechanism for Metamorphosis in Anural Ascidians

SYNOPSIS. In the present study the timing of metamorphosis inan anural ascidian, Molgula pacifica, was compared to metamorphosisin a urodele species Boltenia villosa. Metamorphosis in M. pacificawas triggered at a fixed time in development (32–36 hoursafter fertilization), just prior to hatching. In contrast, metamorphosiswas triggered in B. villosa after the hatched larvae respondedto substrate cues. The timing of metamorphosisin B. villosawas often delayed for up to four days, whereas delays in M.pacifica were not observed. An antibody, termed Epi-3, was foundto cross-react exclusively with epidermal cells in both species.The binding of FITC-labelled Epi-3 was very low prior to metamorphosisand then it increased dramatically after metamorphosis was triggered.The cytoplasm of ampulla tip cells and the tunic immediatelysurrounding each ampulla showed the highest levels of Epi-3fluorescence. The histological and ultrastructural featuresof the ampulla cells suggest that Epi-3 antibody recognizesgranules localized in the apical cytoplasm. How the evolution of an internal "clock" mechanism responsiblefor initiating metamorphosis may be beneficial to anural speciesis discussed. One possibility is that the anural type of timingmechanism reduces mortality rates during this critical phaseof its life cycle.     

Ultraviolet irradiation of eggs and blastomere isolation experiments suggest that gastrulation in the direct developing ascidian, Molgula pacifica, requires localized cytoplasmic determinants in the egg and cell signaling beginning at the two-cell stage

Gastrulation in the maximum direct developing ascidian Molgula pacifica is highly modified compared with commonly studied "model" ascidians in that endoderm cells situated in the vegetal pole region do not undergo typical invagination and due to the absence of a typical blastopore the involution of mesoderm cells is highly modified. At the gastrula stage, embryos are comprised of a central cluster of large yolky cells that are surrounded by a single layer of ectoderm cells in which there is only a slight indication of an inward movement of cells at the vegetal pole. As a consequence, these embryos do not form an archenteron. In the present study, ultraviolet (UV) irradiation of fertilized eggs tested the possibility that cortical cytoplasmic factors are required for gastrulation, and blastomere isolation experiments tested the possibility that cell signaling beginning at the two-cell stage may be required for the development of the gastrula. Irradiation of unoriented fertilized eggs with UV light resulted in late cleavage stage embryos that failed to undergo gastrulation. When blastomeres were isolated from two-cell embryos, they developed into late cleavage stage embryos; however, they did not undergo gastrulation and subsequently develop into juveniles. These results suggest that cytoplasmic factors required for gastrulation are localized in the egg cortex, but in contrast to previously studied indirect developers, these factors are not exclusively localized in the vegetal pole region at the first stage of ooplasmic segregation. Furthermore, the inability of embryos derived from blastomeres isolated at the two-cell stage to undergo gastrulation and develop into juveniles suggests that important cell signaling begins as early as the two-cell stage in M. pacifica. These results are discussed in terms of the evolution of maximum direct development in ascidians.     

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.     

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

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

Multiple origins of anural development in ascidians inferred from rDNA sequences

Ascidians exhibit two different modes of development. A tadpole larva is formed during urodele development, whereas the larval phase is modified or absent during anural development. Anural development is restricted to a small number of species in one or possibly two ascidian families and is probably derived from ancestors with urodele development. Anural and urodele ascidians constitute a model system in which to study the evolution of development, but the phylogeny of anural development has not been resolved. Classification based on larval characters suggests that anural species are monophyletic, whereas classification according to adult morphology suggests they are polyphyletic. In the present study, we have inferred the origin of anural development using rDNA sequences. The central region of 18S rDNA and the hypervariable D2 loop of 28S rDNA were amplified from the genomic DNA of anural and urodele ascidian species by the polymerase chain reaction and sequenced. Phylogenetic trees inferred from 18S rDNA sequences of 21 species placed anural developers into two discrete groups corresponding to the Styelidae and Molgulidae, suggesting that anural development evolved independently in these families. Furthermore, the 18S rDNA trees inferred at least four independent origins of anural development in the family Molgulidae. Phylogenetic trees inferred from the D2 loop sequences of 13 molgulid species confirmed the 18S rDNA phylogeny. Anural development appears to have evolved rapidly because some anural species are placed as closely related sister groups to urodele species. The phylogeny inferred from rDNA sequences is consistent with molgulid systematics according to adult morphology and supports the polyphyletic origin of anural development in ascidians. Correspondence to: W.R. Jeffery     

Development of vestigial tail muscle acetylcholinesterase in embryos of an anural ascidian species.

1. The ascidian Molgula arenata produces an anural larva lacking a tail and other structural features of typical urodele larvae in the family Molglidae, yet its embryos developed a histochemically detectable acetylcholinesterase in the tail muscle rudiment. Development of the myoblasts seemed to fail during the neurula stage. 2. Larval enzyme activity occurred at a mean of 5--6% of the level found in the urodele species Molgula occidentalis and Molgula manhattensis, as measured by scanning integrating microdensitometry of the histochemical reaction product. Some anural larvae had as much as 20% of the enzyme activity in urodele larvae. 3. This example of vestigial expression in the absence of other urodele larval features further illustrates the autonomy of a histospecific enzyme development thought to be controlled by an egg cytoplasmic determinant. Partial suppression of the determinant might be the cause of this diminished expression. 4. Two other anural molgulid species, Molgula occulta and Bostrichobranchus pilularis, did not have vestigial larval enzyme and possibly have lost the determinant completely.     

Factors necessary for restoring an evolutionary change in an anural ascidian embryo.

The ascidian Molgula oculata has a tailed (or urodele) larva, whereas Molgula occulta develops directly via a tailless (or anural) embryo. Interspecific hybrid embryos produced by fertilizing M. occulta eggs with M. oculata sperm (M. occulta x M. oculata hybrids) can develop urodele larval structures, including a brain pigment cell and a short tail containing a small notochord. Development of larval features differs in individual M. occulta clutches: some eggs develop into hybrids with both a brain pigment cell and a tail, some into hybrids with either a brain pigment cell or a tail, and others into hybrids without urodele features. The expression of a 58-kDa protein (p58), which is present in eggs and embryos of urodele ascidians but lacking in those of most anural species, also varies in expression between different clutches of M. occulta eggs. Western blot and immunofluorescence studies show that p58 expression is correlated with the ability of hybrid embryos to express urodele features. For example, clutches of M. occulta eggs containing relatively high levels of p58 produce many hybrids with both a brain pigment cell and a tail. Differential expression of p58 occurs during oogenesis in M. occulta individuals: p58 is found at similar levels in previtellogenic oocytes, but in some animals it disappears during vitellogenesis, while in others it persists throughout oogenesis and is present in mature eggs. When M. occulta eggs are extracted with Triton X-100, p58 remains in the detergent-insoluble fraction, suggesting that it is associated with the cytoskeleton. In most unfertilized M. occulta eggs, p58 is uniformly distributed, but after fertilization it is localized in the uncleaved zygote and then concentrated in embryonic ectoderm, notochord, and muscle lineage cells. Despite containing high levels of p58, gynogenetic hybrid embryos, produced by fertilizing M. occulta eggs with uv-irradiated M. oculata sperm, develop into hybrids without a brain pigment cell or a tail. The results suggest that both a functional paternal genome and p58 are necessary for restoration of larval features in M. occulta x M. oculata hybrids. The cytoskeletal complex containing p58 may mediate the localization of key maternal factors in the egg or may be involved in cellular interactions during embryogenesis which are responsible for development of urodele cell fates.     

The forkhead gene FH1 is involved in evolutionary modification of the ascidian tadpole larva.

The forkhead gene FH1 encodes a HNF-3beta protein required for gastrulation and development of chordate features in the ascidian tadpole larva. Although most ascidian species develop via a tadpole larva, the conventional larva has regressed into an anural (tailless) larva in some species. Molgula oculata (the tailed species) exhibits a tadpole larva with chordate features (a dorsal neural sensory organ or otolith, a notochord, striated muscle cells, and a tail), whereas its sister species Molgula occulta (the tailless species) has evolved an anural larva, which has lost these features. Here we examine the role of FH1 in modifying the larval body plan in the tailless species. We also examine FH1 function in tailless speciesxtailed species hybrids, in which the otolith, notochord, and tail are restored. The FH1 gene is expressed primarily in the presumptive endoderm and notochord cells during gastrulation, neurulation, and larval axis formation in both species and hybrids. In the tailless species, FH1 expression is down-regulated after neurulation in concert with arrested otolith, notochord, and tail development. The FH1 expression pattern characteristic of the tailed species is restored in hybrid embryos prior to the development of chordate larval features. Antisense oligodeoxynucleotides (ODNs) shown previously to disrupt FH1 function were used to compare the developmental roles of this gene in both species and hybrids. As described previously, antisense FH1 ODNs inhibited endoderm invagination during gastrulation, notochord extension, and larval tail formation in the tailed species. Antisense FH1 ODNs also affected gastrulation in the tailless species, although the effects were less severe than in the tailed species, and an anural larva was formed. In hybrid embryos, antisense FH1 ODNs blocked restoration of the otolith, notochord, and tail, reverting the larva back to the anural state. The results suggest that changes in FH1 expression are involved in re-organizing the tadpole larva during the evolution of anural development.     

Evolution and development of brain sensory organs in molgulid ascidians

The ascidian tadpole larva has two brain sensory organs containing melanocytes: the otolith, a gravity receptor, and the ocellus, part of a photoreceptor. One or both of these sensory organs are absent in molgulid ascidians. We show here that developmental changes leading to the loss of sensory pigment cells occur by different mechanisms in closely related molgulid species. Sensory pigment cells are formed through a bilateral determination pathway in which two or more precursor cells are specified as an equivalence group on each side of the embryo. The precursor cells subsequently converge at the midline after neurulation and undergo cell interactions that decide the fates of the otolith and ocellus. Molgula occidentalis and M. oculata, which exhibit a tadpole larva with an otolith but lacking an ocellus, have conserved the bilateral pigment cell determination pathway. Programmed cell death (PCD) is superimposed on this pathway late in development to eliminate the ocellus precursor and supernumerary pigment cells, which do not differentiate into either an otolith or ocellus. In contrast to molgulids with tadpole larvae, no pigment cell precursors are specified on either side of the M. occulta embryo, which forms a tailless (anural) larva lacking both sensory organs, suggesting that the bilateral pigment cell determination pathway has been lost. The bilateral pigment cell determination pathway and superimposed PCD can be restored in hybrids obtained by fertilizing M. occulta eggs with M. oculata sperm, indicating control by a zygotic process. We conclude that PCD plays an important role in the evolution and development of brain sensory organs in molgulid ascidians.     

Analysis of ascidian <Emphasis Type="Italic">Not</Emphasis> genes highlights their evolutionarily conserved and derived features of structure and expression in development

The ascidian larva is often regarded as an organism close to the ancestral form of chordates, while it is generally accepted that the Spemanns organizer is absent from ascidian embryos. Not is one of the genes expressed in the organizer to execute functions in vertebrate embryos. To address the extent of conservation of Not gene expression among ascidians and vertebrates, we examined the structure and developmental expression of Not of the two distantly related ascidian species, Halocynthia and Ciona. Putative ascidian Not proteins were noted by the absence of one of the two motifs conserved among Not proteins of sea urchin and vertebrates. Analysis by in situ hybridization revealed that Not gene expression of ascidians could be categorized into three types: expression likely to be conserved between ascidians and vertebrates, that probably unique to ascidians, and that specific to ascidian species. Expression of ascidian Not in the posterior end of the tail as well as the notochord and a small part of the anterior neural tube at the tailbud stage is reminiscent of the expression of the vertebrate counterparts in the tailbud, which is regarded as a continuation of the organizer and the pineal gland, respectively. The expression of Not in the epidermis precursors during cleavage stage may be unique to ascidians. In the light of the present findings, evolutionary aspects of Not genes are discussed.Electronic Supplementary Material Supplementary material is available for this article at Edited by N. Satoh     

污损性海鞘的生态特点研究展望

海鞘生长快,繁殖迅速,能产生大量在短时间内附着的幼虫,是海洋污损生物群落中的重要成员,对海上人工设施会产生严重危害。污损性海鞘主要由悉尼海鞘(Ascidia sydneiensis)、史氏菊海鞘(Botryllus schlosseri)、米氏小叶鞘(Diplosoma listerianum)、柄瘤海鞘(Styela clava)、红贺海鞘(Herdmania momus)等9科29属103种组成,其中在太平洋海域64种、印度洋23种、大西洋44种,而北冰洋海域仅3种;另外,其附着污损具有明显的地域性和季节特点,并与深度有关。今后应加强污损性海鞘的生态调查和分类研究,阐明深海及两极海域附着污损特点,揭示幼虫附着变态过程的分子调控机理,完善幼虫采集培养技术,以期更好地掌握海鞘生物学特性与生态特点,丰富和发展海洋生态学内容,并为海洋污损生物的防除奠定基础,促进海洋经济产业的发展。     

Maternal information and localized maternal mRNAs in eggs and early embryos of the ascidian Halocynthia roretzi

Summary

The mosaic behavior of blastomeres isolated from ascidian embryos has been taken as evidence that localized ooplasmic factors (cytoplasmic determinants) specify tissue precursor cells during embryogenesis. Experiments involving the transfer of egg cytoplasm have revealed the presence and localization of various kinds of cytoplasmic determinants in eggs of Halocynthia roretzi. Three cell fates, epidermis, muscle and endoderm, are fixed by cytoplasmic determinants. The three kinds of tissue determinants move in different directions during ooplasmic segregation. Prior to the onset of the first cleavage the three kinds of determinants reside in egg regions that correspond to the future fate map of the embryo and then they are differentially partitioned into specific blastomeres. In addition to tissue-specific determinants, there is evidence suggesting that ascidian eggs contain localized cytoplasmic factors that are responsible for controlling the cleavage pattern and morphogenetic movements. Transplantation of posterior-vegetal egg cytoplasm to an anterior-vegetal position causes a reversal of the anterior-posterior polarity of the cleavage pattern. Localized cytoplasmic factors in the posterior-vegetal region are involved in the generation of a unique cleavage pattern. When vegetal pole cytoplasm is transplanted to the animal pole or equatorial position of the egg, ectopic gastrulation occurs at the site of transplantation. This finding supports the idea that vegetal pole cytoplasm specifies the site of gastrulation. Recently, we started a cDNA project to analyze maternal mRNAs. An arrayed cDNA library of fertilized eggs of H. roretzi was constructed, and more than 2000 clones have been partially sequenced so far. To estimate the proportion of the maternal mRNAs that are localized in the egg and embryo, 150 randomly selected clones were examined by in situ hybridization. We found eight mRNAs that are localized in the eight-cell embryo, of which three were localized to the myoplasm (a specific region of the egg cytoplasm that is partitioned into muscle-lineage blastomeres) of the egg, and then to the postplasm of cleavage-stage embryos. These results indicate that the proportion of localized messages is much higher than we expected. These localized maternal messages may be involved in the regulation of various developmental processes.