So I've discussed the shadow enhancers as a mechanism of transcriptional precision
ensuring robust expression in natural populations
and more precise, sharper patterns of gene activation.
A second mechanism of transcriptional precision that I'd like to discuss today
is paused RNA polymerase.
And paused PolII was identified initially performing whole-genome ChIP binding assays
using antibodies against RNA polymerase II.
And of course this was done on a whole genome scale
but what I'm showing you here is just one example of paused polymerase.
This is for our old friend the Sog gene which is activated by
low levels of the dorsal gradient in the lateral neurogenic ectoderm.
Again, Sog is kept off the ventral mesoderm by the snail repressor.
What you see here is the binding of RNA polymerase II to the promoter region of this Sog gene
before it's switched on in the early embryo.
So I refer to this as paused polymerase.
We know that this polymerase is fully activated
and that is indicated by as assay called a nuclear run-on assay
which I'm not going to discuss in any particular detail.
But the assay not only identified where polymerase is bound,
but where, specifically, activated polymerase is found in the genome.
So we know that this polymerase: It has begun transcription.
It has escaped from the promoter,
melted the DNA, made a short nascent transcript of about 30-50 nucleotides,
and then arrests 30-50 nucleotides downstream of the Sog transcription start site.
So prior to Sog activation, this gene is ready to go.
Polymerase is bound, and engaged, and activated.
And this situation is not just true for Sog,
but most of the genes that control embryonic development
show paused polymerase prior to the activation.
So these are the Hox genes,
genes encoding different signaling pathways
such as Notch signaling, Wnt and Hedgehog signaling.
These types of genes tend to contain paused polymerase in the Drosophila embryo.
And the question is why. What does paused polymerase do for gene activation?
And a hint came from quantitative in situ hybridization assays
done by a graduate student in my lab Allister Boettiger.
And what Allister did was to look very carefully at the first nascent transcripts,
at the moment of activation, produced by genes that contained paused polymerase
or lacked paused polymerase.
And here you're seeing the early activation of two different genes
activated by low levels of the Dorsal gradient; Sog in green and Thisbe in red.
You can see they're both turned on in the lateral regions that will form the neurogenic ectoderm
however Sog contains paused polymerase whereas Thisbe does not.
Thisbe lacks paused polymerase at its promoter prior to activation.
And so what you see in the case of Sog which contains the paused polymerase,
is that, within a few minutes of the first detection of the first nascent transcripts,
over 90% of the nuclei, which will eventually express the gene, show transcription.
And so a very synchronous pattern of gene activation.
In contrast, in the case of Thisbe, which lacks the paused polymerase,
you can see that only about 50% of the nuclei show nascent transcripts
within the first several minutes of gene activation.
And it will take another 20-30 minutes before this pattern fills out
and becomes uniform as seen for Sog.
So paused polymerase fosters rapid and synchronous patterns of gene activation.
And this synchrony is a property of the core promoter.
And some evidence for that is shown here.
So if you look at the bottom panel, you see the early activation of a gene called Pannier.
Pannier is switched on in the dorsal ectoderm of the early embryo.
Its initial transcription is highly erratic or stochastic.
Only about 30% of the nuclei which will eventually express the gene
show nascent transcripts during the early phase of its activation.
Now what you are looking at in these top panels are the same Pannier transgenes
except that the core promoter of Pannier, which lacks paused polymerase,
has been replaced by two paused promoters; either Snail in the middle or Tail-up.
Ok? So, we're switching the non-paused Pannier promoter on the bottom
to the paused paused Snail or Tail-up promoters but otherwise the transgene is kept the same.
And what you observe is much more synchronous patterns of gene activation
with the paused Snail promoter or the paused Tail-up promoter
as compared to the natural Pannier promoter which comes on in a highly erratic fashion.
So it is the minimal promoter, in this case just 100 nucleotides
centered around the transcription start site that is sufficient
for pausing of the polymerase and producing synchronous patterns of gene activation.
Now, it turns out that many of the most critical embryonic patterning genes
contain both paused polymerase and shadow enhancers.
And I want to show you evidence that this combination
of paused polymerase and shadow enhancer is a potent one-two punch
for rapid, synchronous, robust, precise patterns of gene expression
that also produce very high rates of RNA synthesis.
And I'm going to come back to our old friend the Snail gene to illustrate this point.
So Snail, like a number of key patterning genes in the early embryo,
contains both paused polymerase and a shadow enhancer;
two enhancers for the early pattern of gene activation.
And what you see in panel A is a high magnification view of the Snail expression pattern.
I've mentioned how Snail is activated by high levels of the dorsal gradient.
It shows very sharp limits of gene expression that delineate the boundary between
the mesoderm in red and the lateral ectoderm, the neurogenic ectoderm
which will produce the nervous system of the fly.
And I want to be very clear about this boundary. It is a critical boundary in the early embryo.
The mesoderm cells (the red expressing cells) will invaginate during gastrulation
to form internal tissues and muscles.
In contrast, just on the other side of the Snail border,
these lateral cells will remain initially on the surface of the embryo,
forming surface ectoderm which will eventually give rise to the nervous system
and to the nerve core of the adult fly.
So very different fates across that border.
Sharp borders are often the sites of active cell signaling
and in this case, there's Notch signaling across the border.
The Snail expressing cells send a Notch signal
to the row of cells just on the other side of the border.
That row of cells receives the Notch signal
and activates a regulatory gene called Sim which is important for
establishing the ventral midline of the nervous system
and the midline is important for the patterning of the entire nervous system
similar to the role of the floor plate in the vertebrate neural tube.
So I want to discuss the formation of that critical, sharp Snail border,
that site of signaling and activation of Sim
within the ventral most regions of the neurogenic ectoderm.
Now, the sharp border depends on mutual repression
between neurogenic repressors present in lateral regions of the embryo
and Snail itself which functions, as I mentioned, as a transcriptional repressor.
So you have repressors fighting each other to sharpen that border
and you'll see this in more detail in a moment.
On the right you see the consequences of removing the Snail repressor.
So this is a mutant embryo that fails to make the Snail repressor.
The Snail RNA you can see is unevenly distributed. There's no longer a sharp border.
Snail expression is being rapidly shut down
and neurogenic repressors that are normally restricted to lateral regions
are now de-repressed throughout the embryo,
converting what should become the mesoderm into extra lateral neurogenic ectoderm.
This process can be seen in a little more detail in this slide.
So here you see the refinement of the lateral limits of the Snail expression pattern.
When Snail first comes on the borders are rather indistinct
but in a period of just 30 to 40 minutes,
they are quickly sharpened to form that razor sharp, not straight but sharp boundary
between the future mesoderm and the neurogenic ectoderm.
The sharpening of the Snail expression pattern, as I mentioned,
depends on mutual repression between Snail and lateral neurogenic repressors such as Vnd.
Vnd is not the only repressor involved in this process
of sharpening the Snail border but it's one of them.
So the idea is that Snail gets turned on.
It represses Vnd, restricts Vnd to the lateral neurogenic ectoderm
and then Vnd returns the favor by feeding back on Snail, repressing it to sharpen that border.
Now this is a classical bistable threshold switch
with Snail and Vnd duking it out to form that sharp border.
It gives a sharp border but it's a dangerous mechanism
because it depends on Snail being very quickly expressed
in order to achieve critical, threshold levels in the early embryo
to keep these neurogenic repressors out of the mesoderm
and restricted to the lateral neurogenic ectoderm.
If you don't make enough Snail at the right place and the right time,
then the mesoderm is not formed and it's transformed into neurogenic ectoderm.
So to get a feeling for just how fast is the rate of Snail synthesis
in this bistable threshold switch and the sharpening of the Snail border,
Allister developed a quantitative in situ hybridization assay.
And I'm not going to take you through all the hideous details here
but basically he takes a fluorescent probe, hybridizes to staged embryos,
uses a variety of deconvolution methods to identify individual hybridization dots
and we have evidence that each dot represents a single Snail mRNA.
And then he counts the dots to get a feeling for the number of Snail products
present in a given cell at a given stage in development.
On the right here you see a massaged image containing
4 consecutive nuclei across the Snail border.
Now, the embryo is still a syncytium.
The cell membranes have not completely formed between the nuclei
so what I'm calling a cell is the nucleus plus the most closely associated cytoplasm.
And you can see that the two cells to the left have
high levels of the Snail hybridization dots (of Snail mRNAs).
The nuclei or cells on the right basically lack Snail transcripts
and there's the sharp border.
You'll also see that the hybridization dots tend to be asymmetrically distributed
within the cell with more dots on the apical side of the cell than in the basal cytoplasm.
And this is true for many of the mRNAs encoded by developmental patterning genes
in the early fly embryo. They tend to show this apical localization.
So the next slide shows some of the actual numbers.
So what you're looking at here are
each dot is counting the Snail mRNAs in a given cell at a given stage
and so at the beginning of nuclear cleavage cycle 13,
there is an average of 50 Snail mRNAs per cell.
And then this number rises to 120 mRNAs per cell
during the 15 minute window that is the interphase period of nuclear cleavage cycle 13.
Then the cell divides and of course you go from an average of 120 mRNAs per cell
down to 60 mRNAs per cell at the beginning of nuclear cleavage cycle 14
and then during the next 25 minutes this number rises to its steady state level
from 60 to an average of 180 mRNAs per cell over a period of about 25 minutes.
This is a very fast rate of synthesis and that's summarized on the next slide.
So the steady state, as I mentioned, is 180 Snail mRNAs per cell
and this was reached in a period of about 25 minutes
during the first half of nuclear cleavage cycle 14.
That is a rate of synthesis of 20 mRNAs per minute
assuming a half-life of 10 minutes and we think that's an over-estimate.
The actual half-life for the Snail mRNA may be more like 7 to 8 minutes.
But even assuming a half-life of 10 minutes, that means that one Snail mRNA
is synthesized every 5 to 10 seconds per allele.
and that is a very fast rate of synthesis. That's pushing the theoretical limit.
The limit of transcription rates in the early Drosophila embryo is 1 RNA per 4 to 5 seconds.
That number is based on the rate of polymerase elongation (about 20 nucleotides per second).
That was measured by John Liss some years ago
and we have evidence that it's completely consistent with the rate of 20 nucleotides per second
and it's only possible to load one polymerase at the promoter
every 4 to 5 seconds because the polymerase is very large and bulky
and you can only load one every 80 to 100 nucleotides.
So that's your rate of synthesis.
One transcript every 4 to 5 seconds due to the rate of polymerase elongation
and the size of the polymerase and the ability to load polymerase onto a crowded promoter.
You've got to think about bumper to bumper traffic on the freeway
trying to all go at the speed limit.
And so we were within a factor of 2 of the theoretical limit
of the rate of synthesis of Snail mRNA
and as I said, that's important to ensure that Snail represses the neurogenic repressors
during this mutual fight that sharpens the Snail border.
So you always get that right by getting a very fast rate of synthesis
and this is fostered by both shadow enhancer and paused polymerase.
So I just want to finish this part of my talk by emphasizing the importance
of paused polymerase in giving rapid and synchronous patterns of gene activation.
Paused polymerase is also associated with genes that are
subject to very rapid rates of synthesis as seen for Snail.