My name is Joe Gall.
I'm at the Carnegie Institution in Baltimore
in the Department of Embryology,
and I'm going to talk today
about in situ hybridization.
In situ hybridization is the technique
by which a labeled, single-stranded
RNA or DNA molecule in solution
is hybridized to immobilized
single-stranded RNA or DNA in a tissue
or tissue section.
And by having a tag on the molecule in solution,
eventually the hybrid is visible
in the tissue section
as viewed through a microscope.
This technique of in situ hybridization
was first carried out successfully
in my laboratory in 1968.
The origin of in situ hybridization,
at least as an intellectual phenomenon,
derives from experiments
that had been carried out much earlier,
mainly around 1950,
by two investigators named Coons and Kaplan,
and what Coons and Kaplan had done
was to develop the technique of immunocytochemistry,
a technique by which
a fluorescent antibody
was made to bind to the antigen
immobilized in a tissue section,
so that one could detect the position of
specific protein molecules in cells
by their fluorescence.
And shown here is a modern image.
This is a nuclear body from a salamander oocyte.
Here, shows immunostaining
with an antibody against the protein Coilin.
Here's immunostaining with an antibody
against an Sm protein.
This is a slide which I made more recently,
using the Coons and Kaplan technique.
The point, however, is that
they had shown that it was possible to detect
specific protein molecules in tissues,
and it occurred to me that it would be wonderful
if one could detect specific nucleic acids in tissues.
Actually, I was thinking less about nucleic acids
as I was thinking about genes,
because I had been studying the giant chromosomes
of salamanders, shown here,
or the giant chromosomes of Drosophila,
shown here,
and in these chromosomes
one can see bands
that were pretty much regarded as genes
by people working with them at that time.
We now know this is a little bit more complicated than that,
but it seemed that we had here a tissue
- a chromosome -
where specific genes
- specific nucleic acids...
it should be possible to do something comparable to the
immunofluorescent staining technique
to detect specific genes in these tissues.
So, actually,
I kept this in my mind for quite a long time,
and it became possible to think about doing this kind of thing
in the early 1960s,
when investigators discovered that
one could take double-stranded DNA,
denature it into its single strands,
and immobilize those strands
on a nitrocellulose filter.
This was done by a couple of investigators
named Gillespie and Spiegelman,
and what they showed was that,
having the denatured DNA on a filter,
one could then hybridize
radioactive RNA to it
and actually determine, quantitatively,
the amount of a particular gene sequence on the filter.
So, I thought,
"Well, if they can do it on a filter
you ought to be able to do it on a slide".
So, I tried denaturing the DNA on a slide,
that is, in cells - nuclei -
that were squashed out onto a slide,
and I found that I could denature the DNA quite adequately,
without damaging the tissue,
but then when I tried to hybridize
a radioactive RNA to it,
I got no signal.
In this case,
I was looking for the radioactivity by autoradiography,
that is by covering the tissue
with a thin layer of photographic emulsion
and looking for silver grains in the emulsion
caused by the radioactivity of the probe.
As I said, nothing happened,
and I more or less gave up on that technique.
That was in the early to mid 1960s.
Now, a break came, quite accidentally,
from studies that I and others
had been carrying out
on salamander oocytes and frog oocytes,
and what we found was that
in the giant oocyte nucleus,
which is shown here,
derived from this large oocyte here
- the oocyte is the egg of the frog -
inside of this giant nucleus
are of course the chromosomes,
but in addition we found that
there were several hundred to a thousand nucleoli,
these round bodies here shown on this particular slide.
Now, the interesting thing about these nucleoli
that we discovered was that
they contained DNA,
and it was specifically the genes
that code for ribosomal RNA
that were in these nucleoli.
This was a most unusual situation
to have DNA outside of the chromosomes,
but it was quite clear from work
that had been carried out in my laboratory,
as well as in the laboratory of Don Brown
and of Oscar Miller,
that there was extrachromosomal DNA
outside in these nucleoli.
And I studied the origin of that DNA
and what I found was that
it actually was synthesized at one specific time
in the early history of the formation of the oocyte,
and one could see in these early cells
- early nuclei -
that there was a cap of DNA here...
and you can see the cap over here,
you can see the chromosomes here
and down here you can see the chromosomes in this nucleus...
so, in these two nuclei
you have the chromosomes,
but then you have this huge cap of
extra DNA which in fact
was the ribosomal genes
that had been somehow...
had somehow come out of the chromosome
and amplified to give you
this one specific sequence,
a very large amount of this one specific sequence.
So, it occurred to me that if there was any possibility
of doing in situ hybridization,
this was the case that would work.
And so I took a radioactive ribosomal RNA
and hybridized it to cells of this sort,
and I was quite pleased
to see in the very first experiments
that we got good in situ hybridization.
Here, this cell is black
because of the silver grains in the photographic emulsion
that had been exposed by the radioactivity
of the hybridizing probe.
So, this is a case where we have hybridized
radioactive RNA - ribosomal RNA -
to the ribosomal DNA in the cell.
So, that led immediately to a publication
with my student Mary Lou Pardue,
who joined me in this work,
and this was the first successful in situ hybridization.
Now, we wanted to try it
on some other tissue or DNA,
but there were very few examples,
because at that time it was not possible
to isolate specific DNA or RNA sequences.
What permitted us to look at another situation
came from studies that had been carried out
on mouse DNA,
and investigators had found that
when mouse DNA was spun
in a cesium chloride (CsCl) gradient,
it separated into two fractions,
a main peak and a satellite peak
- called satellite simply because
it had a slightly different density
and separated from the bulk of the DNA.
We took this satellite DNA,
made it radioactive,
and we hybridized it to mouse chromosomes,
not knowing at all whether it would hybridize,
whether it was even in the chromosomes for that matter,
it might have been a virus or some other infective particle,
but we were very pleased to see that
the mouse satellite DNA hybridized
specifically to one portion of each chromosome,
and you can see on this slide
that each of the chromosomes
has a bit of radioactivity
at the very end of the chromosome.
This region of the chromosome
was known as the heterochromatin,
and had been known cytologically
as a specific region of the chromosome
for a number of years.
This led us immediately to think
that we should be able to try the same technique
and get similar results
looking at the chromosomes of Drosophila,
and the chromosomes of Drosophila
were especially exciting
because it was known from genetics
that the genes were all in
the so-called euchromatic regions of the chromosome,
namely this region from here to here, or over here...
these lighter stained regions...
all of the genes were located in those regions,
and it was known from genetics
that the heterochromatic regions, like here and here,
were devoid of genes.
So, it occurred to us that Drosophila
must have a lot of DNA
similar to the satellite DNA of the mouse.
So, we centrifuged that DNA
in a cesium chloride gradient,
and we found, indeed,
that Drosophila had a lot of satellite DNA.
This species of Drosophila virilis
that we were working on
had three huge satellite peaks
- here, here, and here -
in addition to the main peak of DNA, shown here.
So, we took the satellite DNA,
we made it radioactive,
we hybridized it to chromosomes,
and, sure enough, it hybridized as shown
on these images here,
to the heterochromatic regions.
And this was particular interesting,
as I said,
because it showed that the heterochromatic regions,
that we knew from genetics had no genes,
consisted of this satellite-type DNA,
which our later studies showed
consisted of very simple sequences,
which were incapable of coding for genes,
or were incapable of being genes and coding for proteins.
So, the in situ hybridization technique
at this point was limited
pretty much to sequences
that were present in large amount in cells.
Eventually, it became possible,
by having more and more sensitive probes,
in particular the later techniques were worked out
for making fluorescent probes
to do in situ hybridization...
so, in situ hybridization,
ISH or "ish",
became fluorescent in situ hybridization,
or "fish", FISH,
which is the was it's usually practiced now.
And techniques have gotten
more and more sensitive,
to the point where, nowadays,
it's possible to detect individual,
single DNA and RNA molecules in tissue sections.