Hello.
My name is Didier Stainier.
I'm a director at the Max Planck Institute for Heart and Lung Research
in Bad Nauheim, Germany.
And today, in this third part, we're going to be talking about the phenomenon of genetic
compensation in the context of vascular development in the zebrafish embryo.
Historically, gene function in zebrafish has been studied initially through a forward genetic
approach, namely... mutagenizing the genome -- introducing mutations randomly -- and then
doing phenotypic screening.
More recently, morpholinos -- antisense oligos -- have been used to knockdown gene function.
And then, more recently, just a few years ago, using the zinc finger nucleases as well
as TALENs and the CRISPR/Cas9 technology, mutations have been introduced in specific
genes to study not only the genes that were studied previously using morpholinos, but
also other genes.
And the studies that I'll be telling about today were essentially inspired by the fact
that looking at specific genes that have been studied both using morpholinos, as well as
reverse genetic techniques, it became apparent that in many cases the phenotypes induced
by mutations were much milder than those induced by the morpholinos technology.
And so, essentially... just to give you a little background, morpholinos are, as you
can see here, modified oligos, they are highly stable, they bind RNA, and they're used to
blocked either translation or splicing.
And so the question, then, is why mutant phenotypes are in fact often milder than antisense phenotypes...
So, mutant phenotypes versus antisense phenotypes...
I will also be using the word morphant for morpholino-induced, so we'll be talking about
mutant versus morphant phenotype, and mutants are often referred to as knockouts and antisense
as knockdowns.
So, let's start by some history of the antisense approach, antisense technology.
And... more than thirty years ago, people working in developmental biology were using
or started using antisense RNA to essentially block in function.
This was birthed in the context of frog embryo as well as the fly embryo.
But this period was fairly short-lived, probably because people were concerned about off-target
effects, and so people moved to, now, overexpressing either wild-type or dominant-negative versions
of genes or proteins.
And, essentially... for example, in this case, dominant-negative activin receptor certainly
was known and... to interfere with other proteins besides the activin receptor, and yet approaches
such as this gave us important insights into developmental processes, and so it's important
to realize that, while no reagent is perfect, certainly these reagents can be used to make
important insights into biological processes.
And so, in a zebrafish, this antisense, using the morpholinos, was introduced in 2000, at
the same time as it was introduced in the frog.
And a few years later, a number of guidelines were written up essentially to try and...
as best as people could, essentially control for these morpholino studies and specifically
trying to avoid and recognize off-target effects.
And so, essentially, with this in mind and, as I said, as the various reverse genetics
techniques became available, people started seeing, essentially, important differences
between the mutation-induced phenotypes and the morpholino- or antisense-induced phenotypes.
And this was further emphasized by a larger study from Nathan Lawon's lab, where they
essentially looked at a large number of genes and, again, found a poor correlation between
morpholino-induced and mutant phenotypes in zebrafish.
And so, essentially, this is not specific, I should mention, to the zebrafish field.
In fact, if you will now look at antisense work in the mouse... is now using transgenesis
to drive antisense trans... transcripts, essentially, again, you see more severe phenotypes from
the antisense approach than from the mutation approach.
And so it's clearly a question that spans beyond, or goes beyond, just using morpholinos
and probably applies to all antisense work.
So, we decided to revisit this issue in detail, and we picked this gene called Egfl7 for a
number of reasons, but mostly because, in three different settings, using the antisense
approach, this gene had been implicated in the... playing an important role in
vascular development.
This was in zebrafish, in frog, as well as in human endothelial cells.
And yet, in the mouse mutant, there was no phenotype, not discernible phenotype.
Just to give you a little background about this gene, in encodes an ECM protein, it's
expressed mostly by endothelial cells, and it's apparently expressed by tumor cells in
human cancers, and for this reason was a good drug target, and Genentech, in fact, had a
clinical trial for one of the humanized monoclonal antibody against this protein.
So, as I mentioned, in zebrafish, where the first work was done on this gene, when you
knock down Egfl7 using morpholinos, you see severe defects in a number of processes including
vascular tube formation, and you also get pericardial edema, indicative of a failing heart.
A similar phenotype was seen in the frog embryo, again using an antisense approach, and also
in human ES cells, human endothelial cells.
And, as I mentioned, in the contrast to these studies, to these findings, the mouse mutant
was in fact phenotypically normal.
Now, this was a little complicated, initially, by the fact that there is an... in the Egfl7,
as you can see here, there is a microRNA, a microRNA-126, embedded in this locus.
And this microRNA is also expressed in endothelial cells, and so in fact the original mutant
that was made deleted this microRNA as well, and this led to the appearance of vascular
phenotypes, when, in fact, when specific knockouts, specific mutations were made either in the
microRNA or the Egfl7 gene itself, it was realized that, in fact, the microRNA was the
one responsible for the phenotype seen in the original mutant.
So, essentially, the bottom line is that we have severe phenotypes using antisense technology
in the fish, frog, and in human cells, but no phenotype in the mouse.
And so we went on to make, using, now, TALEN technology, an Egfl7 mutant, and we identified
a number of mutant alleles and focused on two alleles: one is the delta-3, which removes
a proline, another one is the delta-4 that leads to a premature stop codon.
And this is the main allele, mutant allele that we'll be using for the rest of this study.
And we used high-resolution melt analysis to develop the very rigorous and reproducible
genotyping protocol, which, as you'll see, is essential for the studies I'll be telling
you about.
So, the... much like the mouse mutant, in fact, the zebrafish mutant shows a very mild
if any phenotypes; only about 5% of the mutants show this transient hemorrhage that you can
see, here, in the head of the mutant, but... essentially, looking at both the trunk as
well as the head vasculature, essentially one sees no discernible or no major phenotype.
We also made and analyzed maternal zygotic mutants and, again, these mutants, these maternal
zygotic mutants did not show a more severe phenotype than just the zygotic mutants.
So, essentially, as I said, we now have a situation where the mutants in zebrafish,
much like the mutants in the mouse, show very mild vascular defects, if any; only about
5% of mutants showed this defect.
But, as you can see here, again, from the original data using the antisense technology,
we have both severe vascular defects, vascular tube defects, that is, so, defects in vasculogenesis,
as well as defects in angiogenesis that leads to the sprouting of new vessels.
So, essentially, we have... as had been seen/observed for several other genes, now... we have profound
phenotypic differences between the Egfl7 mutants and the Egfl7 morphants, and one can think
about a number of simple or trivial explanations why that would be.
It's possible that the mutant allele we had made was a hypomorph, and it's also possible
that the morpholino that had been used for these studies were inducing off-target effects,
and this is the phenotypes that, essentially, people had been looking at.
It's also possible that there was a more interesting observation, and so we decided to look further
and try and understand the discrepancy between the mutant and the morphant phenotype.
So, essentially, the first question then is, is the mutation a null allele?
And you might think this is a simple question to answer, but in fact the genome has come
up with many different ways to bypass mutations, especially mutations that lead to stop codons,
premature stop codons in the 5-prime end of the gene.
So, for example, it's been observed now, several times, that downstream ATGs, or even non-ATG
codons can be used for the initiation of translation.
We can... we've also observed, and I'll show you in a minute, exon skipping.
And then, in terms of secreted proteins like Egfl7, certainly one can also imagine scenarios
where unconventional secretion pathways are used for truncated proteins, for example.
Let me show you an example of exon skipping, again, that's been observed several times
in the field.
And, in this case, we made mutations in exon 2 and, as you can see, again, here, these
are now two different mutations -- there's a mutation 1 and mutation 2 -- and in the
mutant you can see, essentially... they're right here... that there's a smaller band
that's also present in the heterozygote, and this band comes from the skipping of exon 2,
as you can see there.
So, is it... essentially, as I said, many ways by there... by which the genome can circumvent
what looks to be severe lesions.
So, in terms of our lesion, our delta-4 mutation, specifically, what we did is first look at
the RNA levels, as shown here, and you can see there's a reduction in the delta-4 allele
compared to wild-type or the delta-3 allele, so there's an about 50 percent reduction in
the transcript level, possibly through nonsense-mediated decay.
If you look at the protein... we expressed both the wild-type and the mutant protein
in cells, and, as it is a secreted protein, you can see that most of the wild-type protein
is present in the medium; if you look at the mutant protein, you can see there's a reduction
in the level of expression, but you can see very little protein, in fact, secreted.
So, these two data together suggest that this allele, this delta-4 allele that we had generated,
could in fact be a severe allele.
How about the second question -- what about off-target effects caused by morpholinos?
Now, in order to do this, we're going to be injecting the morpholino into this mutant
allele that we made.
And essentially the reasoning here is that, if this allele, this mutant allele, is null,
the morpholinos... any additional phenotypes that's seen from morpholino injection, should...
should by definition be an off-target effect.
So, essentially, before we do that, before we inject this morpholino into the... the
Egfl7 mutants, we want first to introduce a myc tag in the Egfl7 locus, again, by gene
editing following cleavage by TALENs, and this is to allow us to look at the efficiency
of the morpholino at different doses.
And so by Western blot analysis, then, after injecting one nanogram of this morpholino
into these transgenic embryos, one can see, in fact, there's about an 80% reduction of
protein levels, Egfl7 protein levels, using one nanogram of morpholino.
We chose this small dose of 1 nanogram because if you inject higher doses, as you can see
here, you'd essentially induce the expression of p53, which has been reported to be indicative
of an off-target effect from morpholino injections, and so, essentially, one nanogram does not
cause p53 induction, but two will, and so we stuck with one nanogram.
So essentially, now, we are ready for the experiment, so we're going to be injecting
this Egfl7 morpholino into Egfl7 mutants in the following manner.
We're going to be crossing hets, injecting one nanogram of the morpholino and then taking
32 affected embryos and genotype them.
And so let's sli... first look at the various scenarios that... and the outcomes of what
one would predict.
So, essentially, if the mutant allele is not null, then the mutant embryo should be more
sensitive than the wild type to the morpholino injections.
Let's say, for example, there's 20% gene function left, you, you know, inject the morpholino
at one nanogram, the mutant embryo should be more sensitive.
If the mutant is null and the morpholino phenotype is due to off-target effects, then essentially
the genotype of the embryo should not matter; the mutant and wild-type embryos should be
equally sensitive to the morpholino injections.
However, if the mutant is null and the morpholino phenotype is not due to off-target effects,
then the mutant embryo should be less sensitive than the wild type to the morpholino injections.
And this is exactly, in fact, what we saw.
So, we... as I said, genotyped 32 affected embryos, and we would expect, through Mendelian
segregation, 8 of them to be mutants, but in fact we only found 3 of them, here, shown
in these red curves, and so this indicates that, in fact, the Egfl7 mutants are less
sensitive, they're somewhat protective... protected to the Egfl7 morpholino.
And so, in fact, these are the data on our different experiments.
In the control experiment, you can see Mendelian segregation of the various genotypes, but
when you inject the morpholino you can see that, out of 32 injected embryos, fewer than
8 of them are in fact showing a phenotype.
And you can of course, then, look at this retrospectively -- after you've genotyped
the embryos, go back to the pictures that you took -- and this is, for example, here,
a wild-type embryo that was injected with one nanogram of the morpholino.
In this case, a mutant embryo… you can see that the mutant embryo
does not show any angiogenesis phenotype.
We're looking, here, at the formation of these vessels, here, in the trunk, that form angiogenesis,
the so-called intersomitic vessels, and you can see a clear phenotype in the wild type
but not in the mutant.
So essentially, we have this situation where the mutants do not show a severe phenotype,
but if you inhibit translation you see a severe phenotype.
This is by, now, using these morpholino antisense.
What about, now, if you inhibit transcription?
What will you see?
And the way we did this -- inhibiting transcription -- was to take advantage, again, of a recently
developed technique called CRISPR interference, and we're using, now, a dead version of CAS9,
one that doesn't have nuclease activity, to block transcription.
And so, here are the experiments, first to show that in fact we can block transcription
to about a 50% level, and so here are the guides used against both the template and
non-template strands.
And, in fact, when you use this approach, you can phenocopy the morpholino-induced phenotypes,
or the morphant-like phenotypes.
So, again, these are, again, control and two experimental, and you can see defects in the
intersomitic vessels, as shown here.
So, what we have is a situation where the mutants don't show a phenotype, but if you
use a morpholino to block translation, or you use this CRISPR interference to block
transcription, you see a severe vascular defects.
So, essentially, the hypothesis then became one of gene compensation, and to use a classical
example, one from the muscular dystrophy field, when mouse mutants were made for the dystrophin
gene, the utrophin gene was up regulated, and so one needs to make the double mutant
to see the kind of phenotype that Duchenne patients exhibit.
And so the hypothesis, then, in our case was that there was in fact the activation of a
network, a compensatory network, that would buffer against deleterious mutations, and
this compensation was present in mutant embryos but not in morphants or in
CRISPRi-injected embryos.
And so we used, then, proteomics and transcriptomics to test this hypothesis, and let's go... look
first at the proteomics, so we're comparing wild type, mutant, and morphants.
And what we found is, essentially, a single protein... this is now comparing mutant to
wild-type... we found a single protein, Emilin3a, that's upregulated in the mutant compared
to the wild type, but interestingly this Emilin3a... 3a was not significantly upregulated in the
morphant, compared to the wild type.
Looking at the RNA levels, we found, in fact, not only Emilin3a, but other family members,
including Emilin3b and Emilin2a, and you can see, again, that they are upregulated in the
mutant compared to the wild type, but not in the morphant.
Similarly, when we use CRISPRi, we did not see... we did not see upregulation of these
genes, Emilin3a... 3a, 3b, and 2a.
What are these Emilin genes?
We know, in fact, that, like Egfl7, Emilins are negative regulators of elastogenesis and
one of the main and functional domain of Egfl7, shown here in yellow, is in fact an EMI domain,
and this name, EMI, comes in fact from the Emilin genes.
Now, on these genes, the upregulation of these Emilin genes is in fact important functionally,
and as it... can it explain, in fact, the lack of phenotypes in Egfl7 mutants?
And the way we addressed this question is by essentially making Egfl7 morphants and
then rescuing them with wild-type as well as mutant Egfl7, as well as Emilin2 and Emilin3.
And, as you can see here, again, this is now the number of effect... in green, we're looking
at the phenotypically normal embryos, which are a few, when you inject the Egfl7 morpholino.
When you come in with Egfl7 RNA for rescue, you can see that this frequent number...
frequency increases.
If you use a mutant version of Egfl7, you fail to rescue and, again, much like wild-type
Egfl7, you can partially rescue the Egfl7 morphant phenotype by using these Emilin2
and Emilin3 genes.
This uhh... compensation phenomenon that we observed in zebrafish, this difference between
knockout and knockdown, or morphant and mutant... mutant and morphant embryos, is also observed
in yeast.
There was a recent study from Orion Weiner's lab, where they looked at the bem1 gene.
If you look at the mutation in bem1, as you can see here, essentially... that causes a
very minor phenotype, but now if you use optogenetics to drive this protein away from its site of
action, then you see, now, more severe phenotypes, including cell cycle arrest and cell lysis.
So, to summarize and essentially provide some outlook on these studies, clearly there are
some morpholinos that phenocopy mutations, at least at the morphological level, for example,
cardiac troponin T, that we used extensively in Part 2 to block contraction of the heart.
There are other morpholinos that do not phenocopy mutations, so there are possible... a number
of possible explanations, including the fact that mutant alleles could be hypomorphic and
that certainly could be the case for many mutations that were induced in the 5-prime
end of genes.
There are going to be morpholinos that, in fact, do cause a number of off-target effects,
even if used at a relatively low dose.
And then, in some cases, for example, as we just observed with Egfl7's, we can have compensation
in the mutants but not in the morphants.
What about morpholinos?
How, now, with this in mind, and with the ability to essentially, then, mutate any gene
using TALEN or CRISPR/Cas9... how should we think about using morpholinos?
And the argument would be that, in fact, to find a morpholino that causes no off-target
effects, and probably the best way to do this is to find a dose and a sequence of morpholino
that has no effect in the corresponding null mutant embryos, or maybe, even better yet,
in embryos that are lacking the morpholino binding site.
Since we do see differences between the morphant and mutant phenotypes, a question of course
arises as to which of these phenotypes is the real phenotype, and which tool to use.
And we would argue that, of course, both of these tools, the mutation approach as well
as the morpholino or antisense approach, should be used; even if they give you different answers,
both of these answers could in fact be correct, especially if the antisense reagent has been
validated previously.
Now, the zebrafish is particularly well suited to do this kind of work, that is, to compare
mutant and morphant phenotypes, and one way we are thinking of using it is essentially
to identify members of the network.
So, for example, in this context, the context of the work I just described, you might think
of Emilins as being part of a network with Egfl7.
And, Emilin could in fact... because it can at least partially compensate for the lack
of Egfl7, it can be by definition seen as a modifier gene.
And so, essentially, the idea now is to take the genes implicated in vascular development
or vascular biology and see if the morphants and mutants for these genes show different
phenotypes, and, if they do, can we identify the compensating genes and thereby identify
the modifier genes?
Now, mechanistically, there are a number, of course, of interesting questions including,
what are the mechanisms of this transcriptional upregulation?
So, in the Egfl7 mutants, how does Emilin transcription get upregulated?
What is the trigger for this upregulation?
What are the mechanisms between the trigger itself and this transcriptional upregulation?
And so this is sort of some of the ongoing work now in the lab, and let me just give
you a few slides... show you a few slides about what we're doing, not only in zebrafish
but also in mammalian cells.
We now have a number... identified a number of genes where essentially we see this RNA
level upregulation in the paralog, the non-mutated paralog.
And, in four of these cases, we've also seen that the heterozygous embryos, as well as
the mutant embryos, show in fact this increase in mRNA levels.
So, for example, I've told you about, in this case... earlier, about Egfl7 and the upregulation by...
of Emilin.
We've also looked at vegfa mutants and we see upregulation of vegfa-b.
If you looked at the heterozygous embryo, again, for example, looking at Egfl7, right
here, you can see that the heterozygous embryos show an intermediate level of upregulation
compared to the mutants, and of course here is the wild type, and similarly vegfa also...
heterozygous embryos also show this intermediate level of upregulation.
Now, of course, we've also identified and seen... observed genes that, when mutated,
do not cause the upregulation of the paralog, and some of these cases are shown here.
And so, with that, I'll thank the.. and acknowledge the people who have been driving these projects,
including the original paper, Rossi and Kontarakis et al, and also Mohamed's work, who is now
working to essentially look further into this phenomenon of compensation using zebrafish
mutants, but also using mammalian cell lines, where we observe similar compensation of phenomenon,
trying to see if we can get it to mechanisms, again, not only to identify the trigger, but
also the mechanism between the trigger and the transcriptional upregulation.
And I also want to thank the funding bodies who are supporting this work.
Thank you.
