p1: We have seen that because of its morphological, developmental and
p1: genomic characteristics, and because of its key phylogenetic position,
p1: amphioxus is a good model to understand the evolution of chordates.
p1: However, this is not sufficient for an organism to be a good model.
p1: You need to also have access to several other things.
p1: First, you need to have access to biological material and in this video
p1: we will see how to keep and use amphioxus in the lab.
p1: That is essential knowledge for an organism to become a model species.
p1: For example, if you like to work on deve lopment, you need to have access to embryos,
p1: and in this video we will see how to obtain them from amphioxus.
p1: By the end of this chapter you should be able to describe the general steps for how
p1: amphioxus embryos can be obtained in the lab and manipulated
p1: and to explain what tools are available for amphioxus developmental biology.
p1: In the case of cephalochordates, five different species are used
p1: in the laboratory to study embryogenesis.
p1: Among them, four belong to the genus Branchiostoma. So the American species
p1: Branchiostoma floridae, the Asian species Branchiostoma japonicum,
p1: and Branchiostoma belcheri.
p1: And the European species Branchiostoma lanceolatum.
p1: The other species is Asymmetron lucayanum from the Bahamas.
p1: As we said in the 1st chapter, the first live embryos of amphioxus were obtained
p1: from Linda and Nick Holland in Florida from the Branchiostoma floridae species
p1: and for many years, the embryos of this species were the only ones available
p1: for European and American researchers.
p1: This species is collected in Tampa Bay at different locations, and the animals
p1: live at shallow depths.
p1: Collection of ripe animals is done during the breeding or spawning season,
p1: which ranges from late spring to late summer
p1: and the animals then are brought to the lab and kept under the light.
p1: The animals can then be induced to spawn at sunset by turning off the light
p1: or by applying a mild electric shock.
p1: However, only specimens that would spawn that particular night in the location they
p1: were collected from can be induced to spawn.
p1: In other words, you cannot know if your animal will spawn on a given night.
p1: Males and females are separated and single individuals are kept in plastic cups
p1: with a small volume of seawater.
p1: Once the animals have released their gametes, the oocytes that can be several
p1: thousand per female and sperm are mixed in Petri dishes for in vitro fertilization.
p1: The synchronous embryos then can be manipulated
p1: in Petri dishes filled with seawater.
p1: As it was a big financial investment and time-consuming for European researchers
p1: to work with the American species, they decided at the beginning of the 2000s
p1: to try to obtain embryos from the European species, Branchiostoma lanceolatum.
p1: This species was described as abundant in the Mediterranean coast close to
p1: the marine station of Banyuls-sur-mer.
p1: Thus several European teams decided to collect animals at that location and try to
p1: define the timing of the spawning season there, and to obtain spawning in the lab.
p1: Animal collection has to be undertaken using a dredge
p1: since some amphioxus live at 5-10 metres of depth.
p1: Then the sand is sieved on the boat or in the harbour.
p1: The researchers that did the first try in Banyuls-sur-mer could define that the
p1: spawning season ranges from May to July.
p1: They tried the electric shock method to induce the release of gametes.
p1: But even if it worked fine, the embryos were not developing correctly.
p1: Finally a temperature shift was successfully used to induce gamete release
p1: in the Branchiostoma lanceolatum species.
p1: How does it work? Ripe animals are collected and kept at 17-18°C
p1: for a couple of weeks with the day- night cycle close to the natural one.
p1: Then the day before the researchers want the animals to spawn
p1: they place them at 23° in the afternoon.
p1: The desired spawning day, the animals are separated in the afternoon
p1: ﻿﻿in plastic cups with a small volume of seawater.
p1: ﻿﻿The light is shut down at sunset, and then animals usually
p1: spawns one or two hours later.
p1: This allows researchers to obtain embryos in any desired day of the spawning season.
p1: The day-night cycle can also be shifted if researchers want spawning at a
p1: ﻿﻿different hour of the day.
p1: ﻿The Branchiostoma lanceolatum species can also be kept efficiently in aquaria
p1: with artificial sea water, allowing inland labs to work with this species.
p1: In Asia, there are two Branchiostoma species that are used for research:
p1: Branchiostoma belcheri and Branchiostoma japonicum.
p1: And they too have a breeding season that ranges from the end of the spring
p1: to the end of the summer.
p1: For a long time the spawning in the lab was not controlled for these species,
p1: but now the temperature shift method developed for Branchiostoma lanceolatum
p1: has been successfully used for this species too.
p1: Additionally, long term culture in the laboratory over several years,
p1: the completion of the full life cycle,
p1: and obtaining embryos in laboratory outside the natural breeding season
p1: is now possible for Branchiostoma belcheri.
p1: In the genus Asymmetron, animals of the Asymmetron lucayanum species
p1: collected near Bimini in the Bahamas have been efficiently used for spawning
p1: in the Holland laboratory.
p1: These species still have a breeding period during the year in fall and spring
p1: when the temperature of the water is moderate and the animals tend to spawn
p1: 0, 1, or 2 days before the new moon.
p1: This species is very important for our understanding of features present
p1: in the common ancestor of cephalochordates, as this genus is the first to diverge in
p1: this lineage before the Branchiostoma and Epigonichthys split.
p1: However, the use of these species for developmental biology studies
p1: is quite recent and still limited.
p1: The larva of these species die after several days of development in the lab,
p1: making the study of metamorphosis impossible.
p1: Besides having access to the embryos of a given species, if you want to use
p1: an animal as a model for evo-devo study, you need also to develop some analytical
p1: tools. Nowadays, genomic and transcriptomic data are powerful resources
p1: to ask questions about genomic evolution, but also to understand more the genetic
p1: control of development. The first amphioxus genome to be sequenced is the
p1: Branchiostoma floridae genome, which is available since 2008.
p1: Then the Asian species was sequenced in 2012 and finally the genome of the
p1: European species Branchiostoma lanceolatum together with many epigenomic
p1: data was published in 2018. For these three Branchiostoma species
p1: and for the Asymmetron lucayanum, ref erence transcriptomes are also available.
p1: Another important tool to study the control of embryonic development is
p1: the possibility to modify gene expression.
p1: In the three Branchiostoma species, microinjection techniques have been
p1: developed in order to introduce nucleotidic and/or proteic molecules into the embryo.
p1: These injections are undertaken in the egg before fertilization.
p1: To express a given gene, messengerRNA injection is efficient in these species.
p1: To knock down/knock out a given gene, available techniques differ by species.
p1: In Branchiostoma floridae,
p1: down regulation of a gene by morpholino injection has been successfully used.﻿
p1: In Branchiostoma belcheri, direct mutagenesis using the TALEN technique
p1: that stands for Transcription Activator-Like Effector Nucleases has been also proven
p1: successful. In Branchiostoma lanceolatum, the morpholino injection is not efficient
p1: and so several laboratories are trying to implement the CRISPR-Cas9 technique
p1: in this model. Transient transgenesis is available in Branchiostoma species using
p1: injection of linearized plasmids alone or in combination with a Tol2 transposase.
p1: In both cases, the embryos are mosaic and this technique has mainly been used to
p1: study the function of putative enhancer regions with the help of reporter genes
p1: like LacZ or GFP. Moreover, more classical tools are also available for developmental
p1: studies in amphioxus, as for example the gene expression analysis by whole-mount
p1: in situ hybridization, or the localization of proteins by immunolabeling,
p1: the modification of cell signalling pathways or specific protein activities
p1: through pharmacological treatments.
p1: Some characteristics that make the amphioxus a good model for embryogenesis
p1: studies is the fact that many embryos can be obtained from a single female.
p1: They develop externally and rapidly and this allows us to perform pharmacological
p1: studies by using a small amount of the molecule of interest.
p1: Moreover, the embryos are small and transparent, which allows for easy imaging
p1: as you will see during the practical course.
p1: In this chapter, we have seen how to maintain amphioxus in the lab and how
p1: to obtain amphioxus embryos. As you have seen, this is not an easy model organism,
p1: and for most of the species, the embryos are available only in short periods
p1: of the year when the animals have gametes in the field. This is the case
p1: for many animal models outside the most conventional ones. However, animals do
p1: not only become models for their ease of use, but above all for the possibility
p1: they offer to answer biological questions. I hope we have convinced you about
p1: amphioxus, and you will see either in Roscoff or in the next chapter
p1: what specific questions it has already allowed us to answer.