Wim J.A.G. Dictus

Cell-lineage and clonal-contribution map of the trochophore larva of Patella vulgata (Mollusca)

Molluscan development is characterised by its extremely regular cleavage pattern. In numerous molluscs the fate of various early-cleavage stage blastomeres has been determined and fate maps have been constructed. On the basis of similarities between these fate maps, a generalised molluscan cell-lineage map has been constructed. Recently, the validity of this map has been challenged. In this study, the cell-lineage of the first-, second-, and third-quartet micromeres and third-generation macromeres of the equally-cleaving gastropod mollusc Patella vulgata was studied by fluorescent cell-lineage tracer injection followed by epifluorescence microscopy and confocal laser scanning microscopy. For the first time, a complete cell-lineage map, in the form of a clonal-contribution map of the trochophore, has been constructed with the use of fluorescent cell-lineage tracers. This map both agrees and differs in a number of respects with the generalised cell-lineage map of molluscs. The most important deviation is that the micromere 2d, formerly referred to as the first somatoblast, is not the only cell that forms the foot and shell gland in Patella.

Development of the musculature in the limpet Patella (Mollusca, Patellogastropoda).

Abstractu2002Whole-mount technique using fluorescent-labelled phalloidin for actin staining and confocal laser scanning microscopy as well as semi-thin serial sectioning, scanning and transmission electron microscopy were applied to investigate the ontogeny of the various muscular systems during larval development in the limpets Patella vulgata L. and P. caerulea L. In contrast to earlier studies, which described a single or two larval shell muscles, the pretorsional trochophore-like larva shows no less than four different muscle systems, namely the asymmetrical main head/foot larval retractor muscle, an accessory larval retractor with distinct insertion area, a circular prototroch/velar system, and a plexus-like pedal muscle system. In both Patella species only posttorsional larvae are able to retract into the shell and to close the aperture by means of the operculum. Shortly after torsion the two adult shell muscles originate independently in lateral positions, starting with two fine muscle fibres which insert at the operculum and laterally at the shell. During late larval development the main larval retractor and the accessory larval retractor become reduced and the velar muscle system is shed. In contrast, the paired adult shell muscles and the pedal muscle plexus increase in volume, and a new mantle musculature, the tentacular muscle system, and the buccal musculature arise. Because the adult shell muscles are entirely independent from the various larval muscular systems, several current hypotheses on the ontogeny and phylogeny of the early gastropod muscle system have to be reconsidered.

The development of the musculature in the limpet Patella with implications on its role in the process of ontogenetic torsion

Summary Scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) following application of a fluorescent dye for filamenteous actin revealed four distinct muscle systems in the pretorsional larvae of Patella, i.e., the velum ring, the pedal plexus and the main and accessory larval retractors. After torsion, two adult (i.e., left and right) shell muscles arise independently from all larval muscles. In addition, the tentacular as well as the adult mantle and buccal musculature are formed during subsequent development. Both larval retractors and the velum ring are lost during or shortly after metamorphosis, while the pedal plexus, left and right (adult) shell muscles, tentacular, mantle and the buccal musculature remain functional in the adult animal. These findings, together with observations of living larvae, strongly support the hypothesis that muscular and hydraulic activity are primarily responsible for the process of ontogenetic torsion. Shell formation in Patella caerulea includ...

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.

Translational Control of the Xenopus laevisConnexin-41 5′-Untranslated Region by Three Upstream Open Reading Frames

The Xenopus laevis Connexin-41 (Cx41) mRNA contains three upstream open reading frames (uORFs) in the 5′-untranslated region (UTR). We analyzed the translation efficiency of constructs containing the Cx41 5′-UTR linked to the green fluorescent protein reporter after injection of transcripts into one-cell stageXenopus embryos. The translational efficiency of the wild-type Cx41 5′-UTR was only 2% compared with that of the β-globin 5′-UTR. Mutation of each of the three uAUGs into AAG codons enhanced translation 82-, 9-, and 4-fold compared with the wild-type Cx41 5′-UTR. Based on these increased translation efficiencies, the percentages of ribosomes that recognized the uAUGs were calculated. Only 0.03% of the ribosomes that entered at the cap structure scanned the entire 5′-UTR and translated the main ORF. The results indicate that all uAUGs are recognized by the majority of the scanning ribosomes and that the three uAUGs strongly modulate translation efficiency inXenopus laevis embryos. Based on these data, a model of ribosomal flow along the mRNA is postulated. We conclude that the three uORFs may play an important role in the regulation of Cx41 expression.

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.

Organiser role of the stem cell of the mesoderm in prototroch patterning in Patella vulgata (Mollusca, Gastropoda)

During early development of the gastropod mollusc Patella vulgata, the stem cell of the mesoderm (3D-macromere) is induced. As a result of this induction, the embryo becomes dorsoventrally organised. At about the same time in development, ciliated cells, so-called trochoblasts, are formed. Later in development, some trochoblasts deciliate and, together with the ciliated trochoblast, form the dorsoventrally organised prototroch, the locomotory organ of the larva. In order to study the role of the 3D-macromere in the specification of trochoblasts and in the induction of the dorsoventral organisation of the prototroch, induction of 3D has been prevented in various ways. it is shown that preventing 3D-induction results in the formation of a radially symmetrical prototroch. The trochoblasts of all four quadrants developed like corresponding trochoblasts of the A-quadrant. Somewhere between 30 and 120 min after fifth cleavage the 3D-macromere induces the formation of specific trochoblasts and organises the dorsoventral pattern of the prototroch. Besides a role of the 3D-macromere, a role of other cells has been demonstrated in the conditionally specified deciliation of trochoblasts.

Translational control of the Xenopus laevis Connexin41 5′UTR by three upstream open reading frames

The Xenopus laevis Connexin-41 (Cx41) mRNA contains three upstream open reading frames (uORFs) in the 5′-untranslated region (UTR). We analyzed the translation efficiency of constructs containing the Cx41 5′-UTR linked to the green fluorescent protein reporter after injection of transcripts into one-cell stageXenopus embryos. The translational efficiency of the wild-type Cx41 5′-UTR was only 2% compared with that of the β-globin 5′-UTR. Mutation of each of the three uAUGs into AAG codons enhanced translation 82-, 9-, and 4-fold compared with the wild-type Cx41 5′-UTR. Based on these increased translation efficiencies, the percentages of ribosomes that recognized the uAUGs were calculated. Only 0.03% of the ribosomes that entered at the cap structure scanned the entire 5′-UTR and translated the main ORF. The results indicate that all uAUGs are recognized by the majority of the scanning ribosomes and that the three uAUGs strongly modulate translation efficiency inXenopus laevis embryos. Based on these data, a model of ribosomal flow along the mRNA is postulated. We conclude that the three uORFs may play an important role in the regulation of Cx41 expression.

Cell-lineage analysis of the prototroch of the gastropod molluscPatella vulgata shows conditional specification of some trochoblasts

Embryos of many spirally cleaving species possess a characteristic cell type, the trochoblasts. These cells differentiate early in development into ciliated cells and give rise to the prototroch, the locomotory organ of the trochophore larva. As a necessary prelude to the investigation of the mechanisms that are responsible for specification of trochoblasts in the equally cleaving gastropod molluscPatella vulgata, the cell-lineage of the prototroch was studied. This was done by microinjection of the cell-lineage tracer lucifer yellow-dextran in trochoblasts and by scanning electron microscopical analysis of formation of the prototroch. The results show that trochoblasts that form the prototroch are of different clonal origin and that the four quadrants of the embryo have an unequal contribution to the prototroch. Since the four quadrants of the equally cleaving embryo are initially equipotent, some trochoblasts must become conditionally specified. Other trochoblasts seem to become autonomously specified. After initial ciliation some trochoblasts become deciliated and for some cells the choice between a larval and an adult cell fate is conditionally specified. Cell-lineage analysis demonstrates that the various autonomously and conditionally specified trochoblasts are organised according to the dorsoventral axis of the embryo. Possible mechanisms that can account for the conditional specification of trochoblasts — including a role for the 3D macromere, which forms the primary mesoderm and is responsible for the formation of the dorsoventral axis of the embryo — are discussed.