Gisèle Van De Vyver

Primary cultures from the marine sponge Xestospongia muta (Petrosiidae, Haplosclerida)

In the context of the investigations on the origin and in vitro production of bioactive compounds, primary cultures were developed from ectosomal and choanosomal cell suspensions from the sponge Xestospongia muta. Dissociated cells aggregated and reorganized into a striking reticulated network of cells, typical for X. muta. Moreover, in some cultures an isotropic reticulation of small spicules, very similar to that found in the ectosome of adult sponges, was observed. Phytohaemagglutinin promoted aggregation and the reorganization of the cells. HPLC analyses revealed that straight-chain acetylenic compounds were recovered from short-term cultures and that they were synthesized during culture. Heterotrophic bacteria were assumed to be involved in the process. Together our results established that X. muta would be an excellent experimental model to study, in laboratory conditions, the differentiation of the skeleton and the in vitro biosynthesis of straight-chain acetylenic compounds.

Ultrastructural study of differentiation processes during aggregation of purified sponge archaeocytes

Archaeocytes from the spongeEphydatia fluviatilis were dissociated and then isolated on Ficoll density gradients. Their aggregation and reconstitution processes were studied by transmission electron microscopy to determine their capabilities for differentiation.Archaeocyte aggregates follow a well defined sequence of differentiation to generate the characteristic structures of a sponge. Pinacoderm is the first structure to be regenerated and appears progressively at the surface of the 12 h aggregates. Pinacocytes which have differentiated in archaeocyte aggregates are identical to native ones except that the nucleolus remains in most cells. The choanocytes appear only after 24 h by a two step process. First, small cells (choanoblasts) are formed from archaeocytes by mitosis. These cells then transform into fully differentiated choanocytes possessing collars and flagella. The early choanocyte chambers are small, irregular and randomly dispersed in the aggregates. Finally, collencytes and sclerocytes begin to appear just before the aggregates spread on the substrate.The differentiation of a suspension of pure archaeocytes is a unique model system to study sponge cell differentiation and has allowed us to demonstrate that archaeocytes isolated from developed sponges maintain the capacity to differentiate even though this capacity is not usually expressed.

Temporal and spatial expression of EmH-3, a homeobox-containing gene isolated from the freshwater sponge Ephydatia muelleri

Homeoboxes have been particularly valuable in identifying genes involved in development. This prompted us to look for homeobox-containing genes in sponges, the most primitive metazoans, and to explore the potential role of these genes in their development. Using the reverse transcription polymerase reaction (RT-PCR), we analyzed the expression of EmH-3 homeobox-containing gene at different stages of development, and in different cell-type populations. The patterns of EmH-3 expression show that this gene is expressed differentially in the course of development and in a cell-type specific manner. The level of transcripts increases from undetectable levels in resting gemmules to higher levels at the moment of hatching and throughout the sponges life. EmH-3 is strongly expressed in the pluripotent archaeocytes, whether isolated from fully differentiated sponges (adult archaeocytes) or from HU-treated sponges (embryonic archaeocytes). Conversely, in differentiated cells such as pinacocytes and choanocytes, EmH-3 expression is very weak and similar to that found in the resting gemmules. On the other hand, another freshwater sponge homeobox-containing gene, prox1 from Ephydatia fluviatilis is expressed almost at the same level at all stages of development and in all the investigated cell populations. Together, these results suggest that EmH-3 plays a role in cell determination and/or differentiation. In particular EmH-3 would determine which archaeocytes will multiply and undergo differentiation and which ones will remain undifferentiated.

Retinoic acid down-regulates the expression of EmH-3 homeobox-containing gene in the freshwater sponge Ephydatia muelleri

The effects of retinoic acid (RA), a common morphogen and gene expression regulator in vertebrates, were studied in the freshwater sponge Ephydatia muelleri, both on morphogenesis and on the expression of EmH-3 homeobox-containing gene. At 0.3 microM, RA had no noticeable influence on sponge development, slightly up-regulating EmH-3 expression. In contrast, in sponges reared in 10, 8 microM and to a lesser extent 2 microM RA, there was a strong down-regulation of EmH-3 expression after hatching. This induced modifications in cell composition and morphology, greatly disturbing normal development. Archaeocytes kept the features found in newly hatched sponges while choanocytes and a functional aquiferous system were completely absent. The inhibition of morphogenesis and down-regulation of EmH-3 expression were reversible when sponges were no longer subjected to RA. After RA removal, EmH-3 expression returned to the high values found in untreated sponges, archaeocytes differentiated into choanocytes and sponges achieved a normal development. These results clearly show that, in freshwater sponges, the most primitive metazoan, RA may also act as a morphogen, regulating the expression of a homeobox-containing gene. They demonstrate that the expression of EmH-3 is necessary for the differentiation of archaeocytes into choanocytes and hence for the formation of a complete functional aquiferous system.

Conservation and phylogeny of a novel family of non-Hox genes of the Antp class in demospongiae (Porifera)

A survey across the most basal animal phylum, the Porifera, for the presence of homeobox-containing genes led to the isolation of 24 partial or complete homeobox sequences from 21 sponge species distributed in 15 families and 6 orders of Demospongiae. All the new sequences shared a high identity/similarity with EmH-3 (Ephydatia muelleri), a non-Hox gene from the Antp class. The Demox sequences, EmH-3, and related homeodomains formed a well-supported clade with no true affinity with any known bilaterian family, including the Tlx/Hox11 family, suggesting that the EmH-3 family of genes, comprising 31 members, represents a novel family of non-Hox genes, called the Demox family, widespread among Demospongiae. The presence of the Tlx/Hox11 specific signature in the Demox family and common regulatory elements suggested that the Demox and Tlx/Hox11 families are closely related. In the phylogenetic analyses, freshwater Haplosclerida appeared as monophyletic, and Haplosclerida and Halichondrida as polyphyletic, with a clade comprising Agelas species and Axinella corrugata. As for their expression, high levels of Demox transcripts were found in adult tissues. Our data add to the number of published poriferan homeobox sequences and provide independent confirmation of the current Demospongiae phylogenies.

In situ manifestations of nonself recognition between incrusting sponges

The present study investigates the morphological aspects of nonself recognition between three incrusting sponge species living in contact.