André E. van Loon

A lophotrochozoan twist gene is expressed in the ectomesoderm of the gastropod mollusk Patella vulgata.

SUMMARY 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.

Evolutionary biology: Hedgehog crosses the snail's midline

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.

Characterisation of two snail genes in the gastropod mollusc Patella vulgata. Implications for understanding the ancestral function of the snail-related genes in Bilateria

Abstract.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 Patellavulgata. 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.

The genes encoding the DNA binding protein and the 23K protease of adenovirus types 40 and 41.

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.

Cell-specific gene regulation in early molluscan development

Summary In order to understand the processes that regulate cell-specific gene expression in early molluscan development, we studied the expression of tubulin genes in embryos of Patella vulgata (Gastropoda, Mollusca). Tubulin genes are first expressed at the 32-cell stage, which is also the stage at which the embryonic transcription starts. At this stage expression of tubulin genes is observed exclusively in two cells in each quadrant, the trochoblasts. These cells, after one additional division, will differentiate into ciliated and cleavage arrested cells. Later they will form part of the prototroch, the locomotory organ of the free-swimming trochophore larva. A trochoblast-specific α-tubulin gene was cloned and its 5′ upstream region was fused to the lac-Z reporter gene. After micro-injection into 2-cell stage embryos, the expression of this fusion gene appears to be restricted to the trochoblasts at the right time. Mutagenic analysis showed that two elements located between −108 and −42 were essential ...

Onset of transcription in Patella vulgata coincides with cell cycle elongation and expression of tubulin genes

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.

Spatio-temporal expression of a gene encoding a putative RNA-binding protein during the early larval development of the mollusc Patella vulgata

Abstract. 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.