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