Dentro de la etapa de periodo no electoral

5.1.3.1.Transport

In comparison to mammals, very little is known about ALP functions in non- vertebrate taxa. Transport-related functions can be inferred in a variety of groups. In the model polychaete Platynereis dumerilii, ALP activity is a marker of nephridia throughout development (Hasse et al., 2010). Alkaline phosphatase activity was also detected in the nephridia of the oligochaete Enchytraeus japonensis (Myohara, 2004). Similar to the mammalian small intestine, the silkworm larval gut expresses both a membrane-bound and a soluble form of alkaline phosphatase, and insect ALPs are also active in salivary glands (Eguchi, 1995). In addition to vertebrates and insects, gut expression has been observed in diverse animals including annelids (Myohara, 2004; Myohara et al., 1999; Kitamura and Shimizu, 2000), larval echinoderms (Hsiao and Fujii, 1963) and invertebrate chordates (Zhang and Wang, 2001).

5.1.3.2.Biomineralisation

The extent of alkaline phosphatase involvement in invertebrate biomineralisation systems is far from clear. Unlike vertebrates, most invertebrate groups with hard shells or skeletons use some form of calcium carbonate as their main skeletal mineral. Thus, the Pi supplier explanation for the role of ALPs does not apply. The “disinhibitor” hypothesis, however, still has plausibility. Simkiss (1964) found PPi to be a potent crystallisation inhibitor during inorganic CaCO3 precipitation. He found the same about Pi, leading him to doubt the usefulness of ALPs in removing crystallisation inhibitors, but he also speculates that converting PPi to Pi may make it easier to remove by additional mechanisms. Furthermore, other roles of ALP activity besides a direct involvement in mineral precipitation, such as regulation of transport to the mineralisation site and modification of the extracellular matrix, could be relevant in carbonate-based mineralisation systems. Data regarding alkaline phosphatases in invertebrate mineralisation are mostly limited to histochemical assays in skeletogenic tissues. Numerous reports of alkaline phosphatase activity in the mantle epithelium exist in bivalve and gastropod molluscs (Bevelander and Benzer, 1948; Bevelander, 1952; Durning, 1957; Beedham, 1958; Saleuddin, 1967; Timmermans, 1969; Ganagarajah and Saleuddin, 1972; Gaume et al., 2011). Developmental studies of ALP activity in larval and juvenile molluscs also report its presence in shell-forming tissues (Bidwell et al., 1990; Marxen et al., 2003; Hohagen and Jackson, 2013). Some of their results, such as the appearance of ALP activity well before shell mineralisation and its spatial association with the periostracum in Marxen et al.’s study, may suggest that it is more involved in the production and maturation of the organic components of the shell than in mineralisation itself.

69 In crustaceans, alkaline phosphatase activity is present in the epidermis under the cuticle and increases before moulting (Travis, 1955; 1957). ALP activity is present around calcified granules in the hepatopancreas (Travis, 1957; Dias Corrêa Junior et al., 2003), which serve as calcium storage sites during moults. One of the few functional studies of invertebrate alkaline phosphatases in a biomineralisation context was done by Chockalingham (1971). In this study, inhibition of crab alkaline phosphatase by levamisole apparently decreased carapace calcification, but true quantitative results were not reported.

An interesting result comes from Domart-Coulon et al. (2001), who cultured scleractinian coral cells and observed increased ALP activity shortly before the culture began to precipitate aragonite. In culture conditions that delayed aragonite precipitation, the ALP peak was also correspondingly delayed. Earlier, Hayes and Goreau (1977) reported widespread presence of alkaline phosphatase on epidermal cell membranes of coral planulae and speculated on the role of phosphatases in the regulation of calcification via Pi.

Reports conflict in echinoderms, which can have calcareous endoskeletons in both larval and adult stages. Histochemical analysis suggested high ALP activity in the skeleton-forming primary mesenchyme cells of sea urchin larvae (Hsiao and Fujii, 1963). However, Donachy et al. (1990) reported that ALP activity in the regenerating arms of a starfish increased long before ossicle formation, and it was localised to the coelomic epithelium rather than the skeleton-forming mesodermal cells. In a recent genome-wide analysis of gene regulation in Strongylocentrotus purpuratus primary mesenchyme cells (PMCs), the cells responsible for larval spicule formation in sea urchins, Rafiq et al. (2014) report no alkaline phosphatases among hundreds of genes with PMC-biased expression. Five predicted phosphatases are among the 420 genes this study found to be differentially expressed in PMCs compared to the rest of the embryo, but the supplementary information listing these genes suggests that none of them are ALPs.

5.1.3.3.Stemness and regeneration

The evidence for ALP involvement in invertebrate regeneration and stem cells is likewise mixed. Stem cell-related activity for ALPs has been claimed in ascidians, non-model hydrozoans and parasitic crustaceans, (Shukalyuk et al., 2005; Akhmadieva et al., 2007; Isaeva et al., 2008; 2011; Jeffery, 2014), although the stemness of the cells in question is not always well-established. In the case of hydrozoans, where most cells of the body may have stem cell-like properties (Bosch et al., 2010) and display ALP activity in both intact and regenerating animals (Lentz and Barnett, 1962), it could be difficult to assess the relationship between alkaline phosphatases and stemness. Although early reports describe ALP activity in planarian neoblasts (Osborne and Miller, 1963), modern studies are lacking except for the occasional ALP hidden in the supplements of a transcriptome study (among genes responsive to smed-prep RNAi in Kao et al., 2013). In the oligochaete

70 annelid Enchytraeus japonensis, neoblasts, regeneration blastema, gonads and posterior growth zone are conspicuously devoid of ALP activity (Myohara, 2004).

5.1.4.Aims:

 To describe the spatial and temporal pattern of ALP activity in the mature and regenerating opercular filament

o Are opercular ALPs active in the regenerating epidermis, which is

proliferative and also secretes cuticle?

o In particular, is ALP activity associated with opercular plate mineralisation?

 To test whether opercular ALPs can be inhibited by levamisole, and whether inhibition causes regeneration and/or calcification defects

 To characterise the protein sequences of opercular ALPs and place them in the context of metazoan ALP diversity

5.2.Methods

General alkaline phosphatase staining protocol is described in section 2.4.1. Levamisole experiments are described in 2.4.2-3, and sequence collection and phylogenetic analysis are described in 2.4.4-5.

5.3.Results