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The twist gene is known to be involved in mesoderm formation in two of the three clades of bilaterally symmetrical animals: viz. deuterostomes (such as vertebrates) and ecdysozoans (such as arthropods and nematodes). There are currently no data on the spatiotemporal expression of this gene in the third clade, the lophotrochozoans (such as mollusks and annelids). To approach the question of mesoderm homology across bilaterians, we decided to analyze orthologs of this gene in the gastropod mollusk Patella vulgata that belongs to the lophotrochozoans. We present here the cloning, characterization, and phylogenetic analysis of a Patella twist ortholog, Pv-twi, and determine the early spatiotemporal expression pattern of this gene. Pv-twi expression was found in the trochophore larva in a subset of the ectomesoderm, one of the two sources of mesoderm in Patella. These data support the idea that twist genes were ancestrally involved in mesoderm differentiation. The absence of Pv-twi in the second mesodermal source, the endomesoderm, suggests that also other genes must be involved in lophotrochozoan mesoderm differentiation. It therefore remains a question if the mesoderm of all bilaterians is homologous.

The orthodenticle/ otx and orthopedia/ otp classes of homeobox gene families have been identified in all three major classes of bilaterians: deuterostomes, lophotrochozoans, and ecdysozoans. Otx genes have been studied extensively and play a role in the development of anterior neural structures. Otp genes have been found to be involved in nervous system development in mouse and Drosophila. To date, no members of these genes are known in molluscs. We cloned orthologs of orthodenticle/ otx and orthopedia/ otpfrom the gastropod Patella vulgata, and designated them Pv-otx and Pv-otprespectively. Our analysis of the spatio-temporal expression pattern of otx and otp orthologs during P. vulgata embryogenesis leads to the following conclusions. First, Pv-otx is expressed in and around the stomodaeum and our analysis thus supports the previously suggested conservation of the protostome and deuterostome larval mouth regions. Second, we find that Pv-otp is involved in the development of the larval apical sensory organ, suggesting a conserved role for this gene family in nervous system development. A similar conserved role in nervous system development has been proposed for orthodenticle/otx genes and we suggest that part of the cells expressing Pv-otx are involved in the development of the anterior nervous system. Last, we postulate that otx genes were ancestrally involved in the development of ciliary bands in bilaterians.

According to the dorsoventral axis-inversion theory, protostomes (such as insects, snails and worms) are organized upside-down by comparison with deuterostomes (vertebrates), in which case their respective ventrally (belly-side) and dorsally (back-side) located nervous systems, as well as their midline regions, should all be derived from a common ancestor. Here we provide experimental evidence for such homology by showing that an orthologue of hedgehog, an important gene in midline patterning in vertebrates, is expressed along the belly of the larva of the limpet Patella vulgata. This finding supports the existence of a similar mechanism for the development of the midline of the nervous system in protostomes and deuterostomes.

The engrailed gene is well known from its role in segmentation and central nervous system development in a variety of species. In molluscs, however, engrailed is involved in shell formation. So far, it seemed that engrailed had been co-opted uniquely for this particular process in molluscs. Here, we show that, in the gastropod mollusc Patella vulgata, an engrailed ortholog is expressed in the edge of the embryonic shell and in the anlage of the apical sensory organ. Surprisingly, a dpp-BMP2/4 ortholog is expressed in cells of the ectoderm surrounding, but not overlapping, the engrailed-expressing shell-forming cells. It is also expressed in the anlage of the eyes. Earlier it was shown that a compartment boundary exists between the cells of the embryonic shell and the adjacent ectoderm. We conclude that engrailed and dpp are most likely involved in setting up a compartment boundary between these cells, very similar to the situation in, for example, the developing wing imaginal disc in Drosophila. We suggest that engrailed became involved in shell formation because of its ancestral role, which is to set up compartment boundaries between embryonic domains.

Snail genes have been found to play a role in mesoderm formation in two of the three clades of bilaterians, deuterostomes (comprising the chordates) and ecdysozoans (comprising the arthropods). No clear data are available on the role these genes play in development of the mesoderm in the third clade, that of lophotrochozoans (comprising annelids and molluscs). We identified two new members of the snail gene family in the gastropod mollusc Patella vulgata. Phylogenetic analysis showed that the two genes clearly belong to the snail sub-family. Their expression patterns do not indicate a role during early mesoderm formation. In fact, contrary to expectations, the snail genes of Patella were mostly expressed in the ectoderm. In view of the location of their expression sites, we suggest that these genes could be involved in regulating epithelial-mesenchymal transitions (EMT) and cell motility, as has recently been demonstrated for snail genes in vertebrates. This may well correspond to the ancestral function of these genes. The results are discussed in the light of the evolutionary origin of the mesoderm. Electronic supplementary material to this paper can be obtained by using the Springer LINK server located at http://dx.doi.org/10.1007/s00427-002-0228-1.

In the mollusc Patella vulgata a cDNA clone named Esther 32 (E32) was found to be expressed in a specific spatio-temporal pattern. DNA sequence analysis showed that E32 represents a putative RNA-binding protein containing a KH domain. In early trochophore larvae, expression of E32 was found in all cells except the already differentiated trochoblasts and the apical tuft cells. Later on in development, expression was also abolished in the presumptive shell gland and restricted to specific areas, among which were the head and foot anlage. This suggests that E32 is involved in maintaining cells in an undifferentiated state via a post-transcriptional mechanism.

Trochoblasts are the first cells to differentiate during the development of spiralian embryos. Differentiation is accompanied by a cell division arrest. In embryos of the limpet Patella vulgata, the participation of cell cycle-regulating factors in trochoblast arrest was analysed as a first step to unravel its cause. We determined the cell cycle phase in which the trochoblasts are arrested by analysing the subcellular locations of mitotic cyclins. The results show that the trochoblasts are most likely arrested in the G2 phase. This was supported by measurement of the DNA content in trochoblast nuclei after the last division. Trochoblasts complete their final division at the sixth mitotic cycle. This mitotic cycle resembles the first postblastoderm cell cycle of Drosophila, in which mitotic activity is controlled by expression of the string gene. As failure of string expression results in cell cycle arrest in the G2 phase, negative regulation of a Patella string homolog could be responsible for trochoblast arrest. Although Stl messengers disappeared from trochoblasts during their final division, expression was observed again 20 min later. Messengers remained present in all trochoblasts at low levels during further development. Thus, expression of the stringlike gene allows the cell cycle arrest of these cells, whereas in Drosophila cells arrested in division lack string messengers.

Embryos of molluscs, annelids, nemerteans and platyhelminthes show remarkable intra- and interphyletic resemblances and differences in mesentoblast, dorso-ventral axis and trochoblast specification. These variations have been used to investigate their evolutionary relationship. In molluscs and annelids a heterochronic shift parallels evolutionary relations based on adult characters. Nemerteans and platyhelminthes lack trochal cells and differ in the specification of the mesodermal precursor cell. Nemerteans also differ fundamentally with respect to axis specification related to the first cleavage. Therefore, close phylogenetic relations exist between molluscs and annelids, whereas nemerteans and platyhelminthes are only remotely related with each other and with molluscs and annelids.

Trochoblasts of the mollusc Patella vulgata differentiate early in development into ciliated cells due to a cell intrinsic developmental capacity. The third cleavage appears to be decisive for their specification. Permanent inhibition of cleavage after the third cleavage, as induced by a continuous incubation with the drug Cytochalasin B, had no effect on trochoblast-specific gene expression later in development. However, permanent inhibition of cleavage before third cleavage abolished trochoblast differentiation including trochoblast-specific gene expression. Inhibition of just the third cleavage itself was sufficient to repress trochoblast-specific gene expression. In contrast, inhibiting the second or fourth cleavage did not effect trochoblast-specific gene expression. Correct formation of micromeres at the third cleavage is required to obtain trochoblast-specific gene expression as was shown in experiments in which the formation of micromeres during third cleavage was suppressed by pressure. In addition, centrifugation before or during the third cleavage disturbs trochoblast-specific gene expression, whereas centrifugation after the third cleavage does not affect trochoblast-specific gene expression. Thus, during the third cleavage a decisive step in the determination of developmental fate of trochoblasts takes place, likely resulting in a segregation of activating and inhibitory determinants. Trochoblast-specific markers in Patella were only expressed in embryos that were division arrested after third cleavage. This suggests that a segregation of differentiation potentials has to take place before trochoblast differentiation markers are expressed. The restriction of differentiation potentials in the cleaving stage embryo is thus required to enable trochoblast-specific gene expression.

As a first step in analyzing the function of a cdc25 homolog during the embryonic development of Patella vulgata (Pv), genomic clones encoding these stringlike proteins (Stl) were isolated and characterized. These clones belong to four groups which are derived from different regions of the Pv genome. As the sequences of Stl genes from two of these groups are almost identical, we suggest that these genes represent copies of the same gene. The Stl3 gene, which has been analyzed in detail, consists of four exons separated by three introns. Its sequence encodes a 250-amino-acid protein with a calculated weight of 28 kDa. The Stl protein contains regions conserved in all other cdc25 proteins. Stl messengers are not stored maternally in Pv oocytes and Stl transcription only starts after the first embryonic cleavages.

During early embryogenesis of the mollusc Patella vulgata the alpha-tubulin-4 gene is specifically expressed in the differentiation process of a particular cell type, the trochoblasts. The 5' region of the gene has been analysed for elements that are required for the regulation of the cell-type-specific activation of the gene. In a functional assay, seven elements that play a role in the spatiotemporal activation of the gene were detected in the promoter sequence. They are not required all together at the same time. A core of two elements, together with two or three auxiliary elements, are sufficient in controlling trochoblast-specific expression of the gene in the Patella embryo. One of the core elements is always required, without it, the gene is not expressed at all. The other core element acts as a positive element in trochoblasts and as a negative element in non-trochoblasts and thus is involved in directing the cell-type specificity of expression. The seven elements act cooperatively, and at least some of them can substitute for each other. Sequence comparison revealed that six of the seven fragments contain the sequence element GTTAA, including one of the core elements. Both core elements seem to play a crucial role in the expression of the alpha-tubulin-4 gene during the differentiation process of trochoblasts in the Patella embryo. A model for the regulation of the cell-type specificity of the gene during trochoblast differentiation is proposed.

As the first five cleavages of the Patella vulgata embryo are synchronous, they are well suited to determine the mRNA level of cyclin A and B genes in an embryo. During the third and fourth cleavage cycle the quantity of A and B mRNA is regulated in a cell-cycle-dependent way, reaching a high level between cleavages and a lower level just after mitosis. This implies that transcription of the cyclin genes occurs before the overall transcription increases directly after the fifth cleavage. During the first cleavages cyclin A and B mRNA is localized in distinct parts of the cytoplasm. Between two successive cell devisions it is found as a crescent-shaped domain at the peripheral side of the nucleus. At cytokinesis it is present between two separating nuclei and at newly formed cell membranes. At the fifth cleavage this localization disappears. Changes in the expression pattern of cyclin A and B may be expected after the fifth cleavage, when the first cells become arrested in cell division and differentiate. The mechanism causing cell division arrest of these primary trochoblasts is still unknown. Cell division arrest caused by the absence of cyclin A and/or B mRNA could be conditional for further differentiation. However, a decrease in cyclin A and B mRNA level in the trochoblasts is not detectable until 4 h after their last division. Later in development no cyclin A and B mRNA can be detected in these cells, whereas cyclin A and B mRNA is present in other cells of the embryo. Thus, the absence of cyclin A and B mRNA in primary trochoblasts, and in the later differentiating secondary and accessory trochoblasts is not obligatory for cell division arrest or cell differentiation.

In this study we show that the onset of embryonic transcription in the marine snail Patella vulgata coincides with the start of the sixth cleavage, when the cell-cycle elongates and divisions become asynchronous. Changes in mRNA content before and after onset of transcription were initially demonstrated by in vitro translation of isolated mRNA from different stages. Before the sixth cleavage, three major mRNAs encoding proteins of 36, 50 and 52 kDa were present. These proteins probably correspond to cyclin A and B and ribonucleotide reductase. After this stage, three major proteins with molecular weights of 36.5, 52.5 and 53 kDa were found after in vitro translation. Via hybrid selected translation and differential screening cDNAs corresponding to the 52.5 and 53 kDa proteins were cloned. The encoded proteins resemble tubulins from other animals to a high extent (between 96.5 and 93.1% identity for α-tubulin and 97.9 and 75.9% for β-tubulin). The 36.5 kDa protein is the previously described actin. Both tubulins were expressed at or shortly after the first asynchronous division after the fifth cleavage.

The expression of alpha- and beta-tubulin genes during the early development of the marine mollusk Patella vulgata has been investigated. From the 32-cell stage onwards, an enhanced expression of both alpha- and beta-tubulin mRNAs was detected in the primary trochoblasts. After one additional cleavage, these cells become cleavage-arrested and then form cilia. They are the first cells to differentiate during Patella development. Later, alpha- and beta-tubulin mRNA is also found in the accessory and secondary trochoblasts. Together these three cell-lines form the prototroch, the ciliated locomotory organ of the trochophore larva. The early and abundant expression of tubulin genes precede and accompany cilia formation in the trochoblasts and provides us with an excellent molecular differentiation marker for these cells. Apart from the trochoblasts, tubulin gene expression was also found in other cells at some stages. At the 88-cell stage, elevated tubulin mRNA levels were found around the large nucleus of the mesodermal stem cell 4d. In later stages, tubulin gene expression was detected in the cells that form the flagella of the apical tuft and in the refractive bodies. An alpha-tubulin gene was isolated and characterized. A lacZ fusion gene under control of the 5' upstream region of this tubulin gene was microinjected into embryos at the two-cell stage. The reporter gene product was only detected in the three trochoblast cell-lines at the same time as tubulin genes were expressed in these cells. Reporter gene product was not detected in any other cells. Thus, this 5' upstream region of this alpha-tubulin gene contains all the elements required for the correct spatiotemporal pattern of expression.

The suc1 protein is a cell cycle regulator whose precise function remains to be elucidated. The suc1 cDNA of the mollusk Patella vulgata was cloned and sequenced. It encodes a 9 kD protein showing a strong similarity with its human counterparts and to a lesser extend with its yeast counterparts. The expression of suc1 in maturing oocytes was shown to be tightly cell cycle-regulated. The abundance of the suc1 transcripts is high in prophase- and metaphase-arrested oocytes but drops dramatically upon exit from M-phase, after fertilization. The microinjection of suc1 synthetic messengers into embryonic blastomeres delayed the cell cycle clock, thus disrupting the perfect cell cycle synchrony exhibited by the blastomeres of early Patella embryos. Interestingly, this suc1 delaying effect was significantly reversed when cyclin B messengers were co-injected with suc1 messengers. These results show that Patella embryos offer a quite valuable model to study cell cycle regulation. Moreover, they support the existence of a negative control exerted by suc1 on the cell cycle traverse in a higher eukaryote.

The metaphasic block of Patella vulgata oocytes depends on protein synthesis as an emetine treatment triggers metaphase/anaphase transition and leads to the sequential disappearance of cyclin A and B. Both cyclins are stable in metaphase-arrested oocytes which indicates that inhibition of protein synthesis activates cyclin proteolysis. The use of extracts prepared from metaphase-arrested oocytes and from emetine-treated oocytes fully confirms these in vivo findings. Considering previous observations about the regulation of protein synthesis throughout the cell cycle, we propose the involvement of a transient inhibition of translation in the activation of cyclin proteolysis and in exit from the M-phase arrest.

The actin gene family of the marine molluscPatella vulgata was chosen as a model system to study the regulation of genes expressed during early development in molluscs. Using a hamster actin cDNA clone as a probe, we isolated nine actin cDNA clones from trochophore larvae. The total nucleic acid sequence of three of these clones has been determined. Each clone contains the whole protein encoding region. The deduced amino acid sequences resemble actin proteins from other species to a high extent. The nucleotide sequence from the 3'UTR (UnTranslated Region) and 5'UTR from all nine clones has been resolved. In this way we could identify four different subtypes. Southern blots with genomic DNA were probed with different 3'UTR's corresponding to each subtype to determine the genomic organization. One 3'UTR detected one band probably corresponding with one gene. Another 3'UTR detected one or two genes and the third 3'UTR between two and four genes. Northern blots were used to detect the presence of actin mRNA during different stages of development. In the mature oocyte, actin mRNA is present in low amounts. The level of actin mRNA starts to rise steadily from 8 h after fertilization (88-cell stage) onwards. The level of the different subtype mRNAs, as specified by their 3'UTR rises at different developmental stages and to various extents. This indicates that the expression of each type is regulated independently and in relation to the developmental stage of the embryo.

We have cloned and sequenced the cDNAs encoding Patella vulgata cyclins A and B. The cDNA clones contain an open reading frame of 426 and 408 amino acids respectively, which present similarity with cyclins from other species. Cyclin A and B RNAs are present as polyadenylated and non-polyadenylated RNA in prophase oocytes and are completely polyadenylated in metaphase I. During the first cleavages after fertilization the level of cyclin A and B mRNAs is high and drops when the free swimming stage is reached. Using p13suc1-Sepharose bead precipitation we demonstrate that cyclin synthesis is triggered during maturation and that inhibition of protein synthesis makes the cyclins disappear rapidly from the metaphase I oocytes, which shift to interphase condition. By microinjecting antisense oligonucleotides into metaphase I oocytes, we demonstrate that in vivo ablation of cyclin A and B messengers together gives the same result, whereas microinjection of only one oligonucleotide does not show any effect.

To study the transforming capacity of the two fastidious adenoviruses 40 and 41 (Ad40 and Ad41), we have cloned the left terminal regions of their genomes. Transfection with plasmids containing these regions leads to transformation of primary baby rat kidney (BRK) cells. Cell lines derived from transformed cells show an intermediate transformed phenotype. The left terminal region was present in at least 50 copies in cells transformed by Ad41 and in 2-3 copies in Ad40-transformed cells. The integration patterns of the plasmids containing the left terminal region of Ad41 were similar in three different cell lines.

A detailed procedure is described for the preparation of hybrid hybridomas, that produce bispecific antibodies. This is achieved by fusing two hybridoma cell lines that are phenotypically distinct (HAT(s)/neo(r) and HAT(r)/neo(s)) and thereby allow for the selection of the appropriate hybrid cells. HATs mutants were obtained from one of the two fusion partners by 8-azaguanine treatment; these mutant phenotypes were found in an unexpected high frequency. For the introduction of the dominant neo(r) marker gene in one of the HAT(s) fusion partners, a retroviral vector was used in order to obtain a high efficiency of gene transfer. Our method was very effective in the production of hybrid hybridomas, so-called quadromas. The detection of bispecific antibodies was based on simultaneous binding by one antibody of two different antigens, or on the presence of two different H chain isotypes in this molecule.

To determine whether differences in structure and organization of the transforming regions of Ad40 and Ad41 could explain the fastidious growth of these viruses, we have sequenced these regions and analyzed the structure of the corresponding mRNAs. These regions are 85% homologous to each other and exhibit 52% of homology to the analogous Ad5 region. Like the other adenoviruses, the Ad40 and Ad41 transforming regions contain two transcriptional units, E1a and E1b. Differences and similarities in the strategic sequences of these transcription units and Ad5 regulatory elements are discussed. Northern blotting and S1-nuclease analysis of RNA isolated from transformed cells reveal that in Ad40-transformed cells region E1a is transcribed into three partially overlapping mRNAs, while no transcription of region E1b can be detected. In Ad41-transformed cells only one E1a mRNA is found, comparable to the larger Ad40 E1a mRNA. In the Ad41 E1b region a single mRNA species is synthesized, which is comparable to the E1b messenger found in cells transformed by other adenovirus serotypes. Comparison of the large proteins encoded by the Ad40 and Ad41 E1a regions with the corresponding Ad5 E1a protein shows a relatively low homology in three conserved regions. These results are discussed in relation to the transforming capacity and the fastidious growth of Ad40 and Ad41 in conventional cell lines.

In order to establish whether the poor growth of the two fastidious adenoviruses types 40 and 41 (Ad40 and Ad41) in HeLa cells is due to a reduced trans-activation by the early region 1A (E1A), we have determined the trans-activating effect of this region on the expression of the chloramphenicol acetyltransferase (CAT) gene controlled by the Ad2 E4 promoter. Cotransfection of HeLa cells with plasmids containing the E1A regions of Ad5, Ad40, and Ad41, respectively, and the CAT gene controlled by the Ad2 E4 promoter showed that activation of the E4 promoter by the E1A regions of Ad40 and Ad41 depends on the same sequence elements of the E4 promoter as activation by the Ad5 E1A gene products. The level of activation, however, is significantly lower. This might partly explain the reduced growth in HeLa cells of the two viruses.

The adenovirus (Ad) single-stranded DNA binding protein (DBP) is a multifunctional protein. It is thought to consist of two domains, the amino-terminal domain involved in host-range determination and the carboxyl-terminal domain functioning in DNA replication and DNA binding. We have determined the nucleotide sequences of the DBP genes of Ad40 and Ad41, two human adenoviral serotypes that differ significantly from other adenoviruses. Regions of structural and functional importance in the corresponding proteins could be identified by comparison of the amino acid sequences with those of other known DBPs. In addition, the nucleotide sequences of the DBP early promoters, of the 23K protease genes, and of parts of the hexon and 100K protein genes have been determined. It can be deduced from the nucleotide sequences, that the Ad40 and Ad41 DBPs are relatively small (473 and 474 amino acids (a.a.), respectively, versus 529 a.a. for the Ad5 DBP). This is caused by the presence of very small amino-terminal domains of 119 a.a. (Ad40) and 120 a.a. (Ad41), as compared to 173 a.a. for the corresponding Ad5 domain. Only a few amino acids in this domain have been conserved in all known DBPs. The carboxyl-terminal domains show a higher degree of sequence conservation. In this domain, four strongly conserved regions can be identified, one of which might form a metal-binding site. The 23K proteases of both Ad40 and Ad41 show a strong homology to the Ad2 and Ad5 proteins, with the exception of the carboxyl-terminal end of the proteins. The 23K protease gene of Ad41 has an open reading frame that extends beyond the polyadenylation signal, in contrast to the Ad40 gene that ends well in front of the signal.

The DNAs of the two fastidious adenovirus species 40 and 41 (Ad40 and Ad41) have been studied by restriction endonuclease cleavage, CsCl density gradient centrifugation, and liquid-phase hybridization. For Ad40 DNA, cleavage maps were constructed for the restriction endonucleases EcoRI, SalI, ClaI, BstEII, BclII, and PvuI. The EcoRI, SalI, ClaI, and XhoI maps could be established for Ad41 DNA. The buoyant density in CsCl of DNAs of the fastidious species is 1.711 g/cm3 corresponding to a G + C content of 52%. The homology of the DNAs from Ad40 and Ad41 proved to be 62-69%. These results are consistent with previous reports on antigenic and DNA restriction enzyme analysis in which the classification into two species was proposed (J. C. De Jong, R. Wigand, A. H. Kidd, G. Wadell, J. G. Kapsenberg, C. J. Muzerie, A. G. Wermenbol, and R.-G. Fritzlaff, J. Med. Virol. 11, 215-231 (1983), and I. Uhnoo, G. Wadell, L. Svensson, and M. Johansson, Dev. Biol. Stand. 53, 311-318 (1983].