Biology

The broad area of interest in this study is to give an insight into the evolution of body plans and left-right specifications of bilateria. Number of animals shows internal and external features with asymmetry. It is believed that in vertebrates the asymmetry utilizes the signaling molecule Nodal. In this study an attempt has been made to study the effect of Nodal and Pitx expression in non-deuterostomes group.  The method used to test the effect of expression of Nodal and Pitx was by introducing a chemical inhibitor on the Nodal pathway at the molecular level. The study hypothesizes on the suggestion that asymmetrical expression of Pitx might be an ancestral feature and also that nodal was present in the common ancestor of all bilaterians asymmetrically.

Nodal and Pitx (one of the targets of Nodal signaling) was isolated in two species of snails. To gain insight into the left-right asymmetry in gastropods they investigated previously described genes critical for chirality in other organisms. They subsequently characterized cDNAs encoding proteins related to nodal and Pitx in both the species. In order to identify if nodal is present in other lophotrochozoans, they traced orthologs of nodal in annelid, Capitella Species I. In addition, a single potential orthologs of Pitx was identified in both snail species. Sexually matured L.gigantea was collected from Los Angeles and breeding population of B.glabrata was maintained in freshwater tanks. To investigate the function of Nodal signaling in snails, they introduced a chemical inhibitor called SB-431542, which specifically interferes with type I receptors, inhibiting only the activity of Nodal signaling prior to the juvenile stage. The drug was introduced to two species, Lottia Gigantea and Biomphalaria Glabrata, at various developmental stages. Nodal inhibition was performed by introducing 1 DMSO and SB 431542 at a concentration of 5 or 10uM and also treated with Rapamycin at a concentration of 10uM. The entire drug treated and control embryos were kept in the dark during the entire test period. The percentage of abnormalities was studied depending on the concentration and the timing of the drug treatment.

The results indicated that Pitx is downstream from nodal in snails, just as in deuterostomes. Snails, unlike in deuterostomes, nodal does not appear to regulate its own expression. The results also indicate that nodal expression in snails is asymmetrical from the onset, whereas in vertebrates it is initially symmetrical and depends on the nodal signaling to achieve the asymmetric pattern. Thus, suggesting that nodal was present in the common ancestor of bilaterians. The data also supports the fact that nodal and Pitx are expressed on the right side in bilaterians.

The data presented does justify the authors conclusion. To test the effects of the drug on the Nodal pathway at the molecular level, the specimens were divided into test and control group. Although, there was not much variation in the Nodal expression in both the groups (the control was negative), it was noticed that in 30 of the treated embryos the level of expression of Pitx was greatly reduced and in 9.5 of treated embryos asymmetric expression was undetectable. This shows that the drug was effective only on the expression of Pitx and the Nodal expression was unaffected by the drug. The data presented completely justifies that there was no variation in nodal expression in snail at molecular level and that asymmetry in snails is from the outset, whereas in vertebrates nodal expression is initially symmetric and depends on the regulated feedback of nodal signaling to achieve an asymmetric pattern. Previous studies suggest that some mechanisms contributing to asymmetry are shared between distant phyla. This study brings a closer linkage. Although Pitx orthologs have been identified in non- deuterostomes, Pitx in these species has not been reported in asymmetrical expression patterns. The result of this study suggests that nodal was present in the common ancestors of al bilaterians. There are evidences that the last common ancestor of all snails had a dextral body, and then this study contributes to the fact that snail expressed both nodal and Pitx on the right side combined with the fact that in sea urchins also the nodal and Pitx are expressed on the right side.Number of studies have been conducted to prove the molecular pathway that leads to asymmetry utilizing signaling molecule nodal in vertebrates, but this study focuses on the nodal orthologs of Ecdysozoa (flies and nematods) and Lophotrochozoa (snails and annelids). This is the first report for a nodal ortholog in a non-deuterostomes group. The study also focuses on the ancestral feature of bilateral in non-deuterostomes group. One of the important strength of the study was data gathered from induction of the drug to detect the activity of Nodal expression. The fact that the entire experiment was done in a controlled environment adds to the strength of the study. The introduction of drug and effects further supported the hypothesis.

Although, there has been no major limitations in this study as the drug was introduced during all the developmental stages, but further studies are required to determine how nodal expression is regulated. We still need to understand the actual symmetry breaking event in snails. Also, this study does not provide insights into the developmental control of complex animal morphologies such as shell coiling.

The drug treatment results along with other analysis of expression of nodal and Pitx provide support to the contention that signaling plays an important role in left-right asymmetry in snails. The study also suggests that the asymmetrical activity of the Nodal pathway could lead to unequal formation of shell producing cells. The reduction of nodal signaling causes randomization of asymmetry in vertebrates, but interestingly in snails it causes a lack of asymmetry.