Cilia and Hedgehog: When and how was their marriage solemnized?

Primary cilia are essential for Hedgehog (Hh) signaling in mammals, and this requirement appears to be conserved in other vertebrates as well. Here, I review recent work that has scrutinized the evolution of the link between the Hh pathway and cilia, discuss what we have learnt from these studies and speculate on how this fascinating problem can be further explored.     

Continuing dispersal of freshwater triclads (Platyhelminthes; Turbellaria) in Britain with particular reference to lakes

1. A survey of lakes in Anglesey and Snowdonia, Wales, in the summer of 1997 recorded changes in the triclad fauna which had occurred since earlier studies in the 1950s and in 1973. Two native species, Polycelis tenuis and Dugesia polychroa, and two immigrants, Planaria torva and especially Dugesia tigrina, have increased their range. The triclad fauna has remained unchanged with time in eleven out of sixteen lakes in Snowdonia, in contrast to only one out of fourteen lakes on Anglesey. This supports the hypothesis produced in the 1950s, that the triclad fauna of the ion-poor, unproductive lakes in Snowdonia would change little with time, being maintained by ecological interactions, whereas the ion-rich, productive lakes on Anglesey, from which native Dugesia spp. and Dendrocoelum lacteum are historically absent, would increase in triclad diversity. 2. An examination of national records for the distribution of D. tigrina and Pl. torva indicated their continuing dispersal in both still and running waters in mainland Britain, with the former species having the greater frequency of occurrence. 3. The reasons for this may be that D. tigrina is (a) more easily dispersed, (b) a more opportunistic, catholic feeder, (c) a more vigorous competitor, (d) able to reproduce asexually and (e) able to prey to some extent on other triclad species, as shown in the laboratory but not yet confirmed in the field. The absence of D. tigrina and Pl. torva from unproductive lakes may be explained in terms of low temperature and feeding mode, and a low standing crop of gastropods, respectively. 4. Seven surveys (1961–1997) of the triclad fauna (six species) of Colemere, England, revealed that D. tigrina was confined to one small area of the littoral zone until the mid-1980s, after which it spread fairly rapidly to occupy the entire shore by 1997. Its expansion has been to the detriment of all the native species, particularly P. tenuis, except Dd. lacteum. The possible extinction of Pl. torva may be linked to a numerical reduction in snails, particularly Potamopyrgus jenkinsi. 5. Dugesia tigrina has an adverse effect on the native triclad fauna in lakes, particularly when only a few species are present. This has been explained in terms of the availability of a wider variety of food. The presence of a larger number of triclad species denies the immigrant food items, particularly crustaceans and snails, the food refuges of Dd. lacteum and Dugesia spp., respectively. However, the long-term study of Colemere has shown that more diverse triclad faunas may also be vulnerable to this immigrant. The effect of Pl. torva on native triclads remains uncertain.     

An in situ hybridization protocol for planarian embryos: monitoring myosin heavy chain gene expression

The monitoring of gene expression is fundamental for understanding developmental biology. Here we report a successful experimental protocol for in situ hybridization in both whole-mount and sectioned planarian embryos. Conventional in situ hybridization techniques in developmental biology are used on whole-mount preparations. However, given that the inherent lack of external morphological markers in planarian embryos hinders the proper interpretation of gene expression data in whole-mount preparations, here we used sectioned material. We discuss the advantages of sectioned versus whole-mount preparations, namely, better probe penetration, improved tissue preservation, and the possibility to interpret gene expression in relation to internal morphological markers such as the epidermis, the embryonic and definitive pharynges, and the gastrodermis. Optimal fixatives and embedding methods for sectioning are also discussed. A. Cardona and J. Fernández have contributed equally to this work.     

Expression of hsp90 mediates cytoprotective effects in the gastrodermis of planarians

Heat shock proteins (HSPs) play a crucial role in the protection of cells. In the present study, we have identified an hsp90-related gene (Djhsp90) encoding a cytosolic form of HSP90 that is primarily expressed in gastrodermis of the planarian Dugesia japonica. Djhsp90 becomes significantly induced after traumatic amputation or other stress stimuli, such as exposure to X-ray or ultraviolet radiations, heat shock, or prolonged starvation. When Djhsp90 is silenced by ribonucleic acid interference (RNAi), planarians dramatically decrease in size, becoming unable to eat, and die in a few weeks. Our results indicate that this gene plays an essential cytoprotective role in the gastrodermis of planarians and suggest that this chaperone can be involved in autophagic processes that are activated by this tissue.     

Characterization of innexin gene expression and functional roles of gap-junctional communication in planarian regeneration

Planaria possess remarkable powers of regeneration. After bisection, one blastema regenerates a head, while the other forms a tail. The ability of previously-adjacent cells to adopt radically different fates could be due to long-range signaling allowing determination of position relative to, and the identity of, remaining tissue. However, this process is not understood at the molecular level. Following the hypothesis that gap-junctional communication (GJC) may underlie this signaling, we cloned and characterized the expression of the Innexin gene family during planarian regeneration. Planarian innexins fall into 3 groups according to both sequence and expression. The concordance between expression-based and phylogenetic grouping suggests diversification of 3 ancestral innexin genes into the large family of planarian innexins. Innexin expression was detected throughout the animal, as well as specifically in regeneration blastemas, consistent with a role in long-range signaling relevant to specification of blastema positional identity. Exposure to a GJC-blocking reagent which does not distinguish among gap junctions composed of different Innexin proteins (is not subject to compensation or redundancy) often resulted in bipolar (2-headed) animals. Taken together, the expression data and the respecification of the posterior blastema to an anteriorized fate by GJC loss-of-function suggest that innexin-based GJC mediates instructive signaling during regeneration.     

Planarian pharynx regeneration in regenerating tail fragments monitored with cell-specific monoclonal antibodies

 The special morphological features of freshwater planarians make them an attractive and informative model for studying the processes of regeneration and pattern formation. In this work, we investigate pattern formation and maturation of the planarian pharynx during regeneration in tail fragments. Using three monoclonal antibodies (TCAV-1, TF-26 and TMUS-13) specific for epithelial, secretory and muscle cells, respectively, we followed the sequence and timing of differentiation and maturation of these three cell types within the regenerating pharynx. Two of these monoclonal antibodies, TCAV-1 and TMUS-13, also labelled morphologically immature cells that appear to be committed to the differentiation pathway leading to their respective adult cell types. Our results show that the cells forming the new pharynx come from undifferentiated cells through proliferation and differentiation processes rather than from differentiated cells of the old stump. We describe three stages of pharynx regeneration according to the immunoreactivity shown: (1) no immunoreactivity, corresponding to the accumulation of undifferentiated cells that form the pharynx primordium; (2) immunoreactivity to TCAV-1 and TMUS-13, corresponding to the re-building of the pharynx; and (3) immunoreactivity to TF-26, corresponding to a fully mature and functional pharynx. The sequence of differentiation of these three cell types suggests that the pharynx grows by intercalation of new undifferentiated cells coming from the parenchyma between the older pharyngeal cells, in agreement with existing models of pharynx regeneration. Finally, our results suggest an intercalary model for pharynx epithelial cell renewal. Received: 30 September 1996 / Accepted: 6 December 1996     

No news on the flatworm front! Nitric oxide synthase in parasitic and free-living flatworms

The free radical nitric oxide (NO) has emerged as a simple and unique signalling molecule that can serve as neurotransmitter, paracrine substance or hormone. NO is a gas, formed by various neuronal cells, both centrally and peripherally. NO regulates cyclic GMP synthesis. The production of NO can be detected using the NADPH diaphorase (NADPH-d) histochemical stain for nitric oxide synthase (NOS). NOS was detected in two parasitic flatworms, Diphyllobothrium dendriticum and Hymenolepis diminuta, and two free-living flatworms, Planaria torva and Girardia tigrina. The staining for NOS was very strong in the nervous system of both parasitic worms. The main nerve cords, the transverse ring commmissures, nerves in association with the musculature, especially the cirrus musculature and sensory nerve endings showed NADPH-d staining. The NADPH-d staining in the free-living flatworms was much weaker. Still NOS activity was found in the neuropile of the brain and in association with the pharynx musculature. The demonstration of NOS in flatworms, indicates that NO is an old signal molecule in evolutionary terms. This revised version was published online in July 2006 with corrections to the Cover Date.     

Stem cells are differentially regulated during development, regeneration and homeostasis in flatworms

The flatworm stem cell system is exceptional within the animal kingdom, as totipotent stem cells (neoblasts) are the only dividing cells within the organism. In contrast to most organisms, piwi-like gene expression in flatworms is extended from germ cells to somatic stem cells. We describe the isolation and characterization of the piwi homologue macpiwi in the flatworm Macrostomum lignano. We use in situ hybridization, antibody staining and RNA interference to study macpiwi expression and function in adults, during postembryonic development, regeneration and upon starvation. We found novelties regarding piwi function and observed differences to current piwi functions in flatworms. First, macpiwi was essential for the maintenance of somatic stem cells in adult animals. A knock-down of macpiwi led to a complete elimination of stem cells and death of the animals. Second, the regulation of stem cells was different in adults and regenerates compared to postembryonic development. Third, sexual reproduction of M. lignano allowed to follow germline formation during postembryonic development, regeneration, and starvation. Fourth, piwi expression in hatchlings further supports an embryonic formation of the germline in M. lignano. Our findings address new questions in flatworm stem cell research and provide a basis for comparison with higher organisms.     

Schistosoma mansoni: histochemical analysis of the postacetabular gland secretion of cercariae

Contents of the funduses and ducts of the postacetabular glands of Schistosoma mansoni cercariae, the secreted deposits, and the surface film were compared by their histochemical reactions. Techniques for carbohydrate-containing substances, neutral and acid mucosubstance, proteins and amino acids, and enzymes were used. The secretion reacted differently before (within the glands) and after (in secreted deposits) emission.Before emission, the postacetabular gland contents reacted as a neutral mucosubstance containing periodate-engendered and periodate-reactive aldehydes rich in vic-glycols or their substituted amines, probably hexoses other than glucose, such as fucose or galactose. No reactions of significance were observed for acid groups or for glycogen or lipids. In this state, the secretion is termed mucigen.After emission, the secretion stained not only as mucigen, but also as acid mucosubstance, apparently sialomucin. After emission, it is termed mucin.It is probable that acid radicals were present in mucigen but were masked stearically by the presence of adjacent neutral radicals or basic proteins. The surface film reacted as both a neutral and an acid mucosubstance. Evidence suggested that the film itself was neutral and that the reaction for acid mucosubstance was from an overlay of mucin secreted from the postacetabular glands.Proteins and amino acids, especially arginine, and some tyrosine and tryptophan were indicated in mucigen and in mucin by the histochemical tests. There was no histochemical evidence of enzymes. Secretion of the postacetabular glands is concluded from histochemical reactions, as from earlier chromatographic data (Stirewalt and Evans 1960), to be a carbohydrate-protein-lipid complex.