BACKGROUND  

The taxon Xenacoelomorpha has been a key, for a long time, in discussions of the origin and diversification of bilateral animals (our relatives). The fact that they have been classified as the sister group to the remaining bilateral groups, not without controversy, has prompted many groups to investigate their morphological and molecular characters. In a sense, it has been clear that they can be a good proxy for what an “early” bilaterian might have looked like (Riutort and Baguña and Riutort, 2004[1]).

The group shows an amazing diversity of organ systems architectures. Whether we pay attention to the nervous system, the gut, the gonads or the muscles, the wide variety of morphological arrangements is astounding. This, in itself, justifies the use of these animals in any comparative analysis aiming at understanding how organs change, and diversify, over evolutionary time. The fact that during the last few years, genomes and transcriptomes of several members of the Xenacoelomorpha have entered public databases provides us with the unique opportunity of correlating changes in organ architecture with specific changes in gene expression, whether in space or time. However the main drawback we are facing in the field is the need of well-developed, sensitive, detection protocols, for transcripts and proteins alike. This, in part, has been tackled in a few laboratories (the community working with xenacoelomorphs is still very small).

  Historically, the detection of protein expression profiles was the first problem to be tackled in our, Developmental Biology, laboratory. The use of pan-bilaterian antibodies (neurotransmitters) or chemical reagents (phalloidin) was instrumental in describing the wide range of organ architectures in the acoelomorphs. From commercial antibodies to taxon-specific antibodies, all collaborated in giving us a general description of morphologies and, thus, were used as complementary tools in tracing phylogenetic relationships. Muscle and nervous systems were described for many species, whether in embryos or juveniles and adult animals. From the very beginning it became clear that what was needed was to understand how these diversity of architectures were regulated at the gene or genome level. Needless to say, there was a pressing need for developing in situ hybridization protocols, with one or multiple RNA probes.

Others and we started developing in situ protocols in the early 2000’s. In our laboratory, the first in situs were done using Hox genes, prompted by the idea that these genes would be instrumental in understanding how a “first” bilaterian might have organized their major body axis. Our initial efforts (done by the student Eduardo Moreno) were greatly facilitated by the expertise and guidance of Andreas Hejnol (then at Mark Martindale’s laboratory, Hawaii), who was also detecting the expression of Hox genes in the species Convolutriloba longifissura (we were using the European Symsagittifera roscoffensis).  This were single gene, colorimetric, in situs but were giving us the first views of how regulatory genes were used in an acoel. The procedure (see below) was quite informative but missed the possibility of unravelling comparative aspects of gene expression. This would need the introduction of double-fluorescent in situ hybridization protocols. The first contact with the procedure, and the detection methods, came in a visit one member of the group (P Martinez) did to the laboratory of William McGinnis (UCSD) who had developed multiplex protocols for Drosophila embryos. Later on, in Barcelona, with the help of the students Marta Chiodin, first, and Elena Perea-Atienza, later, we developed a mature protocol specifically geared at detecting expression in acoels (see, also, below). This new method was instrumental in detecting the different domains of expression for all members of the so-called neurogenic, bHLH, genes in acoel juveniles.

The mature protocols to which we have referred above are described in our Methods Chapter. However, a few tips on how they were adapted to the xenacoelomorphs, with the problematics associated to the use of these particular animals are explained below.

PREPARING ACOELS FOR IMMUNOCHEMISTRY AND IN SITU HYBRIDIZATION  

One of the problems with studying xenacoelomorphs is that they are, most species, quite rare and also difficult, or impossible, to culture. In fact, out of more than 400 species known, only two are being cultured in the lab with some success, Isodiametra pulchra and Hofstenia miamia (the only species for which RNAi methods have been developed). Besides the cultured species, only a few more are amenable due to the great quantity of specimens that can be obtained at specific locations. One is Convolutriloba, which normally grows, and in great numbers, in aquarium of tropical species, and the other is Symsagittifera roscoffensis, a species that can be found in some locations (particularly in Brittany, France) in great numbers at low tide. In particular periods of the year (mostly winter) this last species lays many embryos (cocoons), a process stimulated by the transfer of gravid animals to the laboratories (perhaps induced by stress).

In our laboratory we rely on the use of S. roscoffensis animals and their embryos or juveniles. Embryos are laid in groups (around 15-20) inside a capsule, which is called a cocoon. The embryos develop almost synchronously within a cocoon and with good temporal synchronicity across several cocoon lay at a particular period in the lab. This provides us with a rough estimate of their developmental time, or, at least, a window of time. After about one week the juveniles will hatch from the cocoon and will start swimming. At this stage they are small miniatures of the adult and have most of the organ primordial in place. This is our most used developmental time for ascertaining the expression patterns of regulatory genes.

Cocoons are problematic. They are covered by thick membrane that is difficult to remove. Moreover, to gain access to the embryo, one has to remove a second membrane attached to the growing embryo. These two membranes have been equated (jokingly) to rubber, because the difficulty of removing them or even to perforate (a reason why no microinjection protocols have, so far, being established in these animals).  In fact, the problem of removing the two external membranes is so severe that immunochemistry protocols have been never effective. This problem is, somehow, alleviated in the in situ hybridization analysis, since the incubation reagents used in the protocols are effective in breaking/removing the membranes to a good extend. This has allowed us to perform in situ protocols in embryos but not those for immunochemistry.  Some alternatives, as the use of enzymes and/or tweezers have been used in our laboratory but without a consistent success.

Acoel worms move by gliding, using cilia, which are embedded in a layer of mucous, secreted by many glands around the body. The presence of the mucous secretions has been problematic in developing immunochemistry methods since they tend to give the animals with sticky properties, including the unspecific absorption of antibodies and RNAs. For that particular reason we introduced a step of cysteine treatment before the immunochemistry protocol. However this treatment turned out to be too aggressive as was breaking our samples and we decided to eliminate it and increase the permeabilization and incubation times instead. Another handicap that we had to overtake was the fluorescence caused by the symbiotic algae, which is present in our reference species (especially in the adults) and can lead to strong background. We resolve it adding a previous step of photobleaching, as explained in the chapter, in which the time had to be very careful adjusted in order to not damage the samples.

INTERPRETATION OF DATA  

While the immunochemistry and in situ protocols, once established, may look like any other used in animals, each group (clade) has particularities that affect to the interpretation of patterns. This is particularly accentuated in the case of in situ hybridization, where RNA probes tend to give (in adult animals) a lower resolution picture of the spatial domain than antibodies (normally sharper images are obtained). In the acoelomorphs, an additional problem of interpretation is given by the histological structure of many tissues. This is due to the fact that most of the organisms are filled with a mass of so-called parenchymal cells, which are highly interdigitated and many times ramified. To complicate matters, the nuclei of epithelial, muscular and nervous systems are also intermingled in the most external part of the animal. This pattern of cell arrangements tends to give a fuzzy look to the patterns of genes expressed there. Moreover, in many acoelomorphs, the digestive system adopts the form of a syncytial mass, without internal cell borders. The “soupy” appearance of this structure makes a bit difficult to delineate specific, sharp, domains of expression. In other bilaterians, in general, tissues are better delineated, with tissue borders well delimited. The detection of these structures is easier, since the domains that occupy are clearly separated from the rest. A good resolution of this problem can be obtained by the combined use of in situ hybridization and immunochemistry. The use of specific gene probes and antibodies against specific tissue markers should help us in resolving the finer aspects of the expression of genes, whether in time or in space. Nevertheless, for studying in more detail the organization of the cell types and structures, and for more accurate interpretation of our results, it is imperative to compare this molecular detection approaches with high resolution electron transmitted microscopy images, a job carried out by our student Brenda Gavilán (and not further discussed in our chapter). This approach helps us to have a detailed reference to interpret better the information given by the detection methods.

FUTURIBLES  

With the technologies that we outlined in our Chapter, we should be able to tackle very specific problems in development of acoelomorphs. The use of similar protocols in nemertodermatids (and Xenoturbella; M. Elphick personal communication) is also encouraging. In the near future an emphasis will have to be put in developing better methodologies in embryos, since it is though the analysis of embryo patterns that we can trace the development (and, in the long term, the evolution) of cell lineages and tissues. This is a great challenge, mostly in a field in which the number of practitioners is still small and where the access to different species is, most of the time, quite difficult. Needless to say, the addition of culture techniques and functional gene analysis methodologies would open the field to a thorough identification of gene networks, a necessary tool to understand the mechanisms that have controlled the origin and diversification of all Bilateria. In addition to those experiments geared to understand developing mechanisms, a new challenge is using the hybridization technologies to understand physiological processes, such as regenerative capacities of cellular functions. The laboratory of Prof. Sprecher (Fribourg, CH) has introduced methodologies to combine expression of genes/proteins in combination with functional tests, opening new avenues in the study of neural systems. Moreover, his group is also using the approaches of single cell sequencing to identify cell types in the acoels. Needless to say, the identification of cell types will rely on the parallel use of inmuno or in situ detection methods.

REFERENCES

1. Baguñà J, and Riutort M. The dawn of bilaterian animals: the case of acoelomorph flatworms. Bioessays. 2004;26:1046–57. doi:10.1002/bies.20113.

AUTHORS:  BRENDA GAVILÁN (UNIVERSITAT BARCELONA), ELENA PEREA-ATIENZA (UNIVERSITAT BARCELONA), SIMON G. SPRECHER (UNIVERSITY FRIBOURG) AND PEDRO MARTINEZ (UNIVERSITAT BARCELONA)