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What are sila camps why would a marine fish contain a sugar that makes up the exoskeleton of insects? How do you sharks sense fish?
Why do we care about lamprey immune systems dr.?
Chris and Amaya from Bri studies these questions using comparative genomics this research will improve our understanding
Of marine ecology as well as immunology and holds implications for both the medical and bio technical fields. This is radio by
Hey everyone I am Jeff Lauder a 2nd year student in the QSB program
I'm Genevieve first year in the QSB program, and I'm Morgan and I'm a second year student here in the QSB program
And we're here with dr.
Chris Amamiya from the
Benaroya Research Institute
Is really broad far-reaching research exploring everything from amia logical systems of lampreys to the way sharks can sense fish

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And how we can use some of these model organisms to study immunology in general. Thank you for being here dr. Amiya well
Thank you for having me
So audio work that you described today in your seminar to the QSB Department and program was focused on
Lamprey and the coelacanth so why are these organisms interesting? Why do we study them a lot of people?
I think are familiar with my operators actually widely distributed even in, California
But people don't even know about them so Ken tell us just a little bit of background of lampreys
Why we're interested in them and kind of how you got into researching them
Yes, so lampreys are very basal
Representatives of the vertebrates they are
Together with the hagfishes at the base of the of the living vertebrates and
It's a long story of how I actually got interested in working with lampreys in the first place

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This was back when I was in the Boston University School of Medicine
I was in the center for human genetics there, but working on
Hox genes and
and
In order to do the work we had developed when I was still opposed
At the Lawrence Livermore Lab we had developed
Methods for large insert cloning which were necessary for the Human Genome Project and in particular we're developing these
bacterial artificial chromosomes cloning systems
For cloning these pieces of DNA that are 100 to 150 or 200 kilobases in length
as it turns out
Back, then there was no
Way to actually sequence genomes that like we have today
so we had to clone out all the pieces that we were interested in and in particular the Hox genes and
my collaborator at the time
Frank Ruddle who was at Yale University?

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Who's who at the you know who was a pioneer in doing Mouse transgenesis and for studying Hox genes?
Figured out that it would be a great opportunity to combine
Our our skill sets as it were and to study these Hawks genes
What were the Hox genes how were they comprised in these basal vertebrates?
What were their sister egg you Latorre elements all these kinds of things and in order to do that we actually had to have these
these bacterial artificial chromosomes libraries
so, that's
It was a match
Made by necessity. I guess and and
he is
Frank died a couple of years ago
But this had actually led to a couple of grant proposals actually three three that were funded to study
To use these bacterial artificial chromosomes do the requisite sequencing and Frank was an expert at making?

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transgenic mice and so the idea was to take these long stretches of DNA to
to insert into these pieces of DNA
reporters such as Lac Z reporters
and
Then to introduce these into the mice to see where these things were going to be expressed if they had the same sort of control
that for example the mouse Hox genes had and
That eventually
Led to some some really interesting findings of course we expanded these
Many other organisms as well, but the ability to take these pieces of DNA to alter them
using genetic engineering techniques
At that time in the yeast now done in bacteria and then to take those and put those into the mice and express those
That was really a key to doing the kind of evo-devo that eventually a lot of people had
Ended up doing so that's that was my first foray into studying lampreys and it like I said it
Occurred because we were studying Hox genes

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And and bacterial artificial chromosomes, and so just a brief injection here for people who are familiar
Just a quick reminder of what Hox genes are and why we study them how they're implicated
Right so the Hox genes when people study evolution it had always been
to this very day people are very interested in Hox genes because they contain a stretch of
Of DNA
And amino acids about 60 amino acids that are encoded by one hundred one hundred and eighty base pairs
that encode this thing called the homeobox which is a
piece of
as part of the protein that can physically bind to certain stretches of DNA and they serve as
basically
transcription factors so most of these Hawks genes, and they're the the human genome has
45 or 46 of these

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Hawks genes, they're they're intimately involved in regulating many other processes so early in development
These Hawks genes are turned on
Once once they're turned on they do regulate other genes in this cascade so they're very important from from a developmental standpoint and so
Frank Ruddle who's a very prominent?
developmental biologist who had discovered the Hox genes in in mice
Along with his colleagues in Drosophila
He had a very large interest in understanding how these Hawks genes could contribute to evolutionary divergence
So that this is the reason why we actually pursued the Hox genes in particular in the lampreys?
Actually, you brought up a good point you brought back the lamprey. I'm curious
What do you use this model organism know before use it for auctions, but what are you doing with it currently, so?

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Right so, that's a good question. We act we have many many projects that have since taken off on the lamprey
Because we had these bacterial artificial chromosomes
reagents
we were able to
to collaborate also with some other folks
namely
max Cooper who was at the time was at University of Alabama at Birmingham and
Max and
his postdoc at the time Zeb Panzer were very interested in trying to understand the immune system of the lamprey and
I should point out that the immune system of the lamprey
it was shown several years ago at least 40 years ago that when you immunize lampreys you can generate a robust a
Fairly robust immune response the immune response was similar to what you found in us or in a mouse
And one of the hallmarks was a melodica memory
so

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Several years ago about 40 years ago experiments were done using basically taking red blood cells a certain type of red blood cell
For these these type o blood cells as it turns out you could immunize with these human type O blood cells
These lampreys and the lampreys would generate this nice robust immune response, and it was a very repeatable kind of immune response
But nobody was able to ever isolate
Immunoglobulins which are the hallmarks of an adaptive immune system?
so to
Cut a tip to make a long story short max had had a way to
to immunize these these lampreys and to isolate certain immune populations of cells and using a transcriptome approach and
by altering our
Null hypothesis as it were we were able to
Isolate what we thought were molecules that would be good kin for being

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surrogates for the immunoglobulins or the antibodies and these were molecules that had these leucine-rich repeat
motifs
And that eventually led us to to clone out these these genes from these bacterial artificial chromosomes
From the libraries that we had and show that these
Using a combination of approaches show immunologically that these were in fact
involved in
immune recognition and that the glose locus itself undergoes these massive
Rearrangements at the local level to generate these diversities to generate the diversity. That is necessary for
Combating all these these antigens that are that are being
That the organism actually sees throughout the lifetime so like you were saying before
So Darwin organisms all have t-cell receptors, and then the lamprey sore our out-group

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Don't have these types of methods and but they still can do an adaptive immune response
So they instead use these leucine rich
proteins like you were saying and you were calling these ones the
Variable several lymphocyte receptors yeah, right so there are a lot of black boxes here. We don't know
We have some ideas how the rearrangement occurs
But clearly they're doing it in a completely different way than
the
Rearrangements that are necessary for generating the diversity for the immunoglobulin sort of t-cell receptors this we know
but
We think that
During the development of their respective cell types
And they also have t-cells that make this variable lymphocyte receptor
So b-cells empty cells so they have both lineages that that we have

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But the ones that are in the the B, cell type cells we call VL
Are B's the ones that are in the T. Cell? Type are the VL Ras and?
but nonetheless
There are certain kinds of transcription factors that are associated with the development of these respective lineages
Which as it turns out are kind of similar to what you see in the in T. Cell development?
We there are a lot of questions that we're currently trying to ask
in terms of
The the origins of the VL RS
You know the development of the VL ARS etc. There's a lot of questions in fact the VL ARS
We know based on transcriptome and some other kinds of data
That they're also expressed very early in embryonic development
Which seems kind of strange that these genes are being expressed and and?

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Presumably utilized even before there's an immune system, so this is well before an immune system or any kind of tissue for immune recognition
They are expressing these genes and we can find them in places that are actually that would suggest that they're necessary for embryonic development
And this is all unpublished work, but you know I've been talking about it for a while so
It's not so it's not like
A secret or anything yeah, so I mean just broadly speaking. I mean this seems like is there opportunities for looking into
The lamprey munis problems versus human immune response
And this is kind of helping us understand more of either the human immune response by studying this kind of alternative system
Yeah, I think what the in the selling point for when we actually
Did get our in our NIH grant to study this is that this is a large?
multi gene family
That is being utilized
for immunity

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Clearly because we can we can immunize and we can get these immune responses we can even find
we can clone out the specificities against some of these antigens but
It's also being utilized in another clearly in another fashion
Like I said it probably is involved in early development because when you knock out the VL ours early in development as it turns out
You you abrogate normal development of the lampreys, so it's a good example of a gene family
That's being utilized for at least a couple of different purposes
And yes, we're pursuing this
Are there any parallels to the immune system in human?
That awaits further investigation, I think
Different point you're saying there might be you know different laboratories there like maybe a
System that we don't know of our network that is deciding this or it could it be an epigenetic

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More environmental cue is there something else that we're not looking into that we might that might give us a cue of what's really going
On yeah, I think that's a really good question
The the only answer I can say is that?
When we study their their genes that are expressed in the earliest
When they're expressed early in development
and we we sequence the the repertoire the repertoire is very limited and
So it seems that the they're not undergoing as much rearrangement that there's some sort of predetermined
Specificities that are necessary for the early
Developmental function and it has been our thought that perhaps
The because these things are receptors that they're they're serving some role in
in
In cell lineage tracing or something to this effect
Where whereby?

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It's requiring some innate signal in order to to
Contribute to that early development, so it's it could be it's some sort of developmental
Like a cell adhesion molecule or something like that
but
Where like I said we're still pursuing this can this this line of investigation
There's a lot of really interesting implications there. Yeah, I didn't even know lampreys have something resembling B cells
something
So what is chitin and why do we care about it like what makes it so important?
Well chitin is is really simple. It's like cellulose
Cellulose is number one polymer
in the biosphere it's made by plants and
It's basically a repeating unit of glucose well chitin is similar in that
It's a glucose derivative. It's called n acetyl glucosamine, and it's found

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very
Prominently in the natural world it's the second most
prevalent
polymer in the natural world and it is the most prevalent polymer in in the aquatic environment and so
In terms of just the sheer amount of it from an ecological standpoint, it's
it's
The fact that you're finding
chitin turnover and about
anywhere from 10 to 20 Giga tons
Is is spectacular because if you don't yeah?
If you didn't turn over this chitin if you didn't have and the only way you're going to turn it over is by using
Effectively using these chitin or lytic enzymes which are found in bacteria if you didn't have these things
The ocean would be one very thick slurry
Okay, so it's an important. It's an important molecule for a lot of reasons and you know

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of course it's found on horny exoskeletons of
All these arthropods as well as in fungi. It was first family right right you know so
in fact one of the diagnostic things that they look for in the clinical lab for when you have a
Fungal infection of the lung as you look for chitin. You know it's very simple si
So we use those same kind of simple assays to study chitin in
in the organisms for which we found chitin sensations, which were absolutely surprising because
And and we're I'm talking about the fishes and the amphibians these these were not thought to have
and
if you go online
I mean almost every source will tell you that there's no chitin in any of the vertebrates and in fact in the literature
It's it's considered
Dogma that chitin is not in any of the vertebrates
So this was kind of surprising finding and once I didn't read you

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But just to touch on that so what is the fish that you first kind of discovered this chitin in and how did you?
Again get to that point of studying this fish. Yeah, so it wasn't we didn't necessarily use a
Chitin per se what we did was we were able to show when we looked for
Genes that were actually missing in the mammals
But they were found in the in the fishes we found a handful of actually about 50 of these genes
and
As it turns out one of these genes which was an unknown gene in the NCBI
As it turns out this gene was most closely related to chitin synthesis kuttan synthase is the final step in chitin
Biosynthesis. It's a kind of a complicated
Process, but it is the final step and so
and because it was so highly related to the

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Invertebrate chitin synthesis from Drosophila or from shrimp or or what have you?
It was really quite surprising, and and we had two good. They're there for figure out whether or not these chitin synthesis
Synthesis were making chitin in these vertebrates, and as it turns out they are and so it's our
Our mission now to figure out what what they're doing you know it's certainly not making horny exoskeletons then
Because based on the way these the chitin is actually distributed
It's not they're not in these really hard structures most of it is actually seems to be in
some sort of a gelatinous
Kind of structure we find it in the fishes and the amphibians we do not find them in the the so-called amniotes
Which are the reptiles and?

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including birds and the mammals
But we do find them in some tetrapods we find them in in things like amphibians and and axolotls and frogs and
so
Chitin per se based on the physical characteristics of chitin
We find well it's known that chitin is less soluble in aquatic
solvents and and you can take chitin and
For example you can buy chitin from from Sigma's these shrimp shells and you can try to solubilize them
They don't really go into water very well, and you have to basically treat them in a certain way with certain with acids or bases
To get them into water, and then they'll come out of water really easily if you're not careful, so
We're not
It's it's really a mystery to us

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Why it is that the chitin is even in these organisms, and how they're actually existing and maybe that hydrogel that is forming
Clearly therein in some sort of mucus form new coid form
is protective you know we know that chitin is is a
Can serve as an antibiotic?
We know that chitin as you say is less soluble, but it also serves as as
as
filters for example in in
There's this structure called a pair of trophic membrane in
Drosophila and and many other invertebrates it actually serves as a filter to protect the organism from toxic wastes
In their gut for example
We don't know I mean this is like really really new stuff
But based on what we've seen in the distribution of the chitin especially in the integument which is the skin?
Of fishes where we've done most of the work
We can we can see that. It's it's forming this this very

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Mucus like
structure which may be involved in in the extracellular matrix, which is absolutely critical for cell-to-cell communication
So specifically, what fishes what organisms are you?
Studying in this case and finding the chitin in so did you talk you mentioned the coelacanth? Yeah, so so again
we've been lucky because we found in every case where we look and
Found chitin synthesis in their genome we find the presence of chitin
And we've we've lots of these examples lots of fishes have chitin
And they they express them in the same way we find them in the gut we find them in the skin etc in the eye
but
Most of the work experimental work has been done on the zebrafish because
Zebrafish you can get embryos and you can do a lot of experimental biology and a lot of them
There are lots of lines available that have then for which

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You have fluorescent markers in these lines, so they mix it makes it a lot easier to actually study
If you're studying for example we think chitin is involved we see it early in development in the endoderm
So there are some endoderm markers of fluorescent lines
For the endoderm that we can get ahold of and we can do the requisite experiment for example
We can knock down the chitin stand face genes and see what happens to these cells for example
Those are the kinds of experiments that are that are very basic, but they can tell us a lot about
The potential function of what kind might actually be doing you just kind of talked about
Specifically knocking out chitin genes. Can you go over specifically what happens when you knock out those genes?
So what are we are we seeing in these?
Zebrafish or see the cancer there. It is, but you're using when you actually do those gene knockouts
Yeah, so we can't do any experiments with the coelacanth yes. Yeah, because coelacanths are in danger, and they're also quite large

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and so those experiments, I mean we were really really lucky to be able to see sequence of coelacanth you know because you know the
Just getting material. It's on the the
appendix of the sides--
Endangered species
In any case
when we knock out the chitin synthesis
using either this CRISPR technology
Or
using
morpholino x' or
chitin synthesis inhibitors
Such as these pesticides that have been developed to
Prevent insects from forming a
One of these horny exoskeletons
We see some
the embryos really don't develop normally you have all kinds of effects on the eye on the axis on the
on the skin and

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It doesn't form a gut normally
Kind of effect wouldn't have only hi like what do you think it's actually affecting the eye?
Development anyway, what's going down there? Yeah? That's that's a really good question, and we really haven't pursued it as as
As thoroughly as we should
This is one of those areas that we don't have really a lot of expertise on the eye
So we have to collaborate with someone on this
But the places where chitin is found or the place where chitin is found in the eye is
Surprising so we see it
In all these all the eyes of the fit every single fish that we've looked at as well as amphibians
such as Excel Otto and
The place where chitin is found is in particular
On the rods and the cones, there's a structure called an outer segment now the outer segment in
these photoreceptor cells

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are
these
structures
are
some ciliated structure at least
evolutionarily, and they have all these like Christi in them and in between all these little layers are
The options and there are many different options right every
Each photoreceptor has its expressing a different opsin gene, so options are
these
proteins that are involved
in
Seeing light in us particular wavelength because they're there
when they are
Exposed to light in a particular wavelength then they can resonate and that eventually
fires that particular photoreceptor
to
Generate a signal right so of so if there's a photon in the right

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Wavelength whether it be for the rods or the cones you get a signal and ultimately
Of course that signal goes to the optic nerve and the constitution of the color the perception of a color is what actually?
Allows also the spatial
resolution the topographical map right and
So what it's actually doing in the outer segments is anyone's guess and of course
Why it should be the case that
Knocking down the chitin in these will give rise to
Basically a defect in I formation is anyone's guess, but I'd like to move to the opposite end of the animal
In developing in developing fishes you'll have fecal pellet
I don't have a lot of chitin in it under these pellets kind of continue does it create an envelope
During development and do they ever stop

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After the first fecal drop or do they continue throughout development, and they continue throughout the fish's life. Yes
Oh, that's a good question, and I didn't I probably didn't
Specify
What we actually see I don't think the chitin is actually sloughing off
I think it's part of the in the tube at the at the at the anus
However, it's clear to me that it's in enveloping this fecal pellet and we know
that of course
We know that when they drop the fecal pellets that they don't have chitin except for the chitin. That's in the fecal matter
Itself okay the the sheath is actually it stays on the fish. I think but clearly
that
purpose of actually
So as you being ecologists and whatnot?
Fecal pellets are actually are really really important for the ocean they formed the marine
marine snow

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Not zebrafish cuz they're freshwater, but then
We have evidence that most or of the ones that we've looked at the marine organisms then the mean marine fishes they have
Chitin that surrounds the fecal pellet now why that's important is that?
people have done these studies of
fecal pellets, and if the fecal pellet doesn't
sink
Then it won't go down to the bottom of the ocean of course you as you know that the the fecal pellets are really
Important for for the biomass in these really deep zones, so they has to it has to sink
And I suspect that the chitin is involved in compaction of these fecal pellets
Yeah, no exactly so you know when when you look at when you look at these schools of fishes
And you see the fecal pellets like herring and things they all drop to the bottom right so they have to be more compact than
than the
surrounding environment I

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Personally want to thank you for your time here in the lab of radio bio
You know this is a great discussion, so thanks for taking part in this well
I just it's always fun to talk about science. You know it's my favorite subject. Mmm. Thanks guys
This is radio bio signing off
Radio bio is supported by the quantitative and systems biology graduate group at the University of California Merced
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