Implicancias pastorales del reconocimiento del joven como lugar

2.4.1.Staining for alkaline phosphatase activity

Alkaline phosphatase activity in mature and regenerating opercular filaments was detected using nitro-blue tetrazolium/5-bromo-4-chloro-3'-indoylphosphate (NBT/BCIP, Roche, cat # 11-681-451-001) as a chromogenic substrate. Anterior ends of worms were fixed in 4% PFA at 4°C overnight or 1 h at room temperature, then rinsed three times in PBS. Opercular filaments were dissected and moved into PBT. A number of specimens were further dissected into portions as described for BrdU immunohistochemistry, except that cups ≥ 3 dpo were cut into left and right halves rather than proximal and distal. At this point, in some experiments, the specimens were digested with a 1:100 dilution of Proteinase K at 37°C (15 min for regenerates and 30 min for unoperated specimens), then washed for 3 x 5 min in PBT and post-fixed for 1 h in 4% PFA, followed by three more 5 min PBT washes. Before staining, specimens were washed twice (5 min each) in AP buffer (100 mM Tris, 100 mM NaCl, 50 mM MgCl2 and 0.1% Tween-20). Staining was carried out under a dark box without agitation in AP buffer with 7.5 µl/ml NBT/BCIP stock (18.75 mg/ml NBT and 9.4 mg/ml BCIP, toluidine salt, in 67% DMSO). Development of the stain was monitored approximately every 15 minutes. Development took between 30-80 minutes, with a typical staining reaction taking 45-50 min. The reaction was stopped with two changes of PBT, and stained specimens were post-fixed for an hour in 4% PFA (or overnight at 4°C), followed by 3 x 5 min PBT washes. Specimens were documented and stored as described for the BrdU and PH3 immunohistochemistry procedures above.

2.4.2.Testing for inhibition by levamisole

To test whether the alkaline phosphatase activity in the opercular filament is sensitive to inhibition by levamisole, three samples of 6 dpo specimens (n = 7 each) were stained as described above (without proteinase digestion), but either deionised water (control) or levamisole (2 mM or 10 mM) was added to their staining buffer. All specimens in this experiment were stained for 50 minutes for comparability.

2.4.3. In vivo effects of levamisole exposure

In order to test whether inhibition of opercular alkaline phosphatase activity has an effect on regeneration and/or calcification, live, regenerating worms were exposed to levamisole. Four experiments were done in total. In all experiments, worms were exposed to levamisole for 24 hours beginning approximately 2 dpo (see below for details). Levamisole stock (500 mM) was prepared in deionised water, and an appropriate amount of dH2O was added to control dishes at the same time as levamisole was added to the experimental dishes. After 24 hours, the inhibitor was removed with five changes of clean FSW and control worms were given a

21 change of FSW. Regenerates in experiments 2-4 were photographed under a dissecting microscope at every data collection time. Differences between the experiments are summarised in Table 2.1 below.

Table 2.1. Summary of in vivo levamisole exposure experiments. Hpo = hours post-operation.

Experiment Treatments (n) Treatment _started Data collected Times of data _collection

1 _{100 µM (7), 1 mM (8)} Ctrl (9), 10 µM (10), 48 hpo Regeneration stage, Presence/absence of calcification, malformations, health status 48 hpo, 3 dpo, 6 dpo 2 Ctrl (10) 2 mM (10), _{10 mM (11)} 46 hpo Regeneration stage, presence/absence of calcification, malformations, health status 46 hpo, 70 hpo, 6 dpo 3 Ctrl (18), 2 mM (19) 54 hpo Regeneration stage, verbal description of calcification, malformations, health status 54 hpo, 78 hpo, 6 dpo 4 Ctrl (18), 2 mM (18) 55 hpo Regeneration stage, calcification stage, malformations, health status 55 hpo, 79 hpo, then daily until 6

dpo 5 Ctrl (20), 1 mM (20) 55 hpo Regeneration stage, calcification stage, malformations, health status 55 hpo, 79 hpo, then daily until 6

dpo

Regeneration stages for experiments 1-3 correspond to the early stages of regeneration described in section 3.4.1.1. Stages up to cup were distinguished, but more advanced regenerates were pooled together. In experiment 4, all morphogenetic stages were distinguished, while pigmentation (which appears after all morphogenetic landmarks are present) was only recorded as a general presence/absence.

During opercular plate calcification, the appearance, growth and merging of mineralised “tiles” is a continuous process and discrete stages are difficult to define. The process was nevertheless roughly partitioned by the size of tiles (“small” or “large”), number (0, ≤ 5, ≤ 10, > 10) and their degree of clustering (disjointed tiles, disjointed clusters of tiles, partial crust, complete crust).

2.4.4.Manual assembly of

S. lamarcki alkaline phosphatase

sequences

Three novel alkaline phosphatase protein sequences containing complete ALP domains (NCBI conserved domain cd00016) were manually assembled at the

22 protein level from the raw RNA-seq reads derived from unoperated and regenerating opercular filaments. First, the reads were queried with human placental alkaline phosphatase (NP_001623.3) and small “seed” contigs belonging to four different conserved regions were established by alignment to the human protein and the S. lamarcki alkaline phosphatase detected in the larval transcriptome of Kenny and Shimeld (2012). These seeds were extended through iterated TBLASTN searches against the raw reads until no more partially overlapping reads were found or stop codons were reached. Stop codons were only accepted when they fell outside the conserved alkaline phosphatase domain, and were supported by at least three reads. On a few occasions, TBLASTN yielded no new reads; in these cases BLASTN using the most terminal reads from the previous round was tried. In cases of polymorphism, the most common variant was used unless multiple variants had equal numbers of supporting reads, in which case an arbitrary decision was made. Nucleotide contigs were also produced from the reads used to build the protein sequences (polymorphisms were resolved to match the assembled protein sequence).

2.4.5.Phylogenetic analysis of metazoan alkaline phosphatases

2.4.5.1.Sequences

Alkaline phosphatase sequences were collected from 15 other metazoan species and used to build an alignment of a total of 69 alkaline phosphatase domains. Species were chosen to represent a manageable but broad sample of the animal kingdom, and species with published genome sequences were chosen in order to cover the diversity of ALP sequences in each major lineage. Alkaline phosphatases are strongly conserved, and BLAST searches with any metazoan ALP yield the same hits, but in all searches, human placental alkaline phosphatase was used for consistency. Details of the sequences collected, source databases and corrections done to the published sequences can be found in Table A2. Key taxathat did not yield any recognisable alkaline phosphatases were sponges, ctenophores and choanoflagellates, which had been intended as a non-metazoan outgroup. Databases searched for these taxa were the NCBI nr and EST databases with taxon restricted to Porifera, Ctenophora or Choanoflagellata, respectively; the Amphimedon queenslandica, Oscarella carmela and Mnemiopsis leidyi genome assemblies (available at http://metazoa.ensembl.org/Amphimedon_queenslandica/ Info/Index, http://www.compagen.org/blast.html, and http://research.nhgri.nih.gov/ mnemiopsis/, respectively), and EST and transcriptome data for O. carmela and M. leidyi available from the above databases.

2.4.5.2.Alignment and phylogenetic analysis

Alkaline phosphatase domains were aligned in Jalview 2.8 (Waterhouse et al., 2008) using MAFFT 6 (Katoh and Toh, 2010). Poorly aligned regions were re- aligned by MUSCLE (Edgar, 2004) in MEGA 5 (Tamura et al., 2011) and/or edited manually in MEGA. Neighbour-joining trees were constructed in MEGA using the

23 default model, with pairwise deletion of gaps. Maximum likelihood trees were constructed using the PhyML 3.0 (Guindon et al., 2010) web service at the South of France bioinformatics platform (http://www.atgc-montpellier.fr/phyml) with the WAG + I + Γ model selected by all three information criteria in Modelgenerator 0.85 (Keane et al., 2006). In PhyML, subtree pruning and regrafting was used as the tree search algorithm. Both NJ and ML trees were tested for robustness with 500 bootstrap replicates.