In Situ Hybridization

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00:00:07.18 My name is Joe Gall.
00:00:09.18 I'm at the Carnegie Institution in Baltimore
00:00:12.13 in the Department of Embryology,
00:00:14.15 and I'm going to talk today
00:00:16.15 about in situ hybridization.
00:00:19.06 In situ hybridization is the technique
00:00:22.16 by which a labeled, single-stranded
00:00:25.26 RNA or DNA molecule in solution
00:00:29.09 is hybridized to immobilized
00:00:32.12 single-stranded RNA or DNA in a tissue
00:00:35.18 or tissue section.
00:00:37.24 And by having a tag on the molecule in solution,
00:00:43.20 eventually the hybrid is visible
00:00:46.20 in the tissue section
00:00:48.11 as viewed through a microscope.
00:00:50.14 This technique of in situ hybridization
00:00:52.10 was first carried out successfully
00:00:54.07 in my laboratory in 1968.
00:00:57.07 The origin of in situ hybridization,
00:00:59.29 at least as an intellectual phenomenon,
00:01:03.28 derives from experiments
00:01:05.27 that had been carried out much earlier,
00:01:09.06 mainly around 1950,
00:01:11.19 by two investigators named Coons and Kaplan,
00:01:14.24 and what Coons and Kaplan had done
00:01:17.20 was to develop the technique of immunocytochemistry,
00:01:22.05 a technique by which
00:01:25.01 a fluorescent antibody
00:01:28.16 was made to bind to the antigen
00:01:31.21 immobilized in a tissue section,
00:01:34.07 so that one could detect the position of
00:01:37.28 specific protein molecules in cells
00:01:41.16 by their fluorescence.
00:01:43.14 And shown here is a modern image.
00:01:46.13 This is a nuclear body from a salamander oocyte.
00:01:52.01 Here, shows immunostaining
00:01:54.27 with an antibody against the protein Coilin.
00:02:00.01 Here's immunostaining with an antibody
00:02:02.01 against an Sm protein.
00:02:04.13 This is a slide which I made more recently,
00:02:07.21 using the Coons and Kaplan technique.
00:02:11.14 The point, however, is that
00:02:14.15 they had shown that it was possible to detect
00:02:16.28 specific protein molecules in tissues,
00:02:19.12 and it occurred to me that it would be wonderful
00:02:21.15 if one could detect specific nucleic acids in tissues.
00:02:26.16 Actually, I was thinking less about nucleic acids
00:02:29.09 as I was thinking about genes,
00:02:31.19 because I had been studying the giant chromosomes
00:02:33.28 of salamanders, shown here,
00:02:36.14 or the giant chromosomes of Drosophila,
00:02:38.22 shown here,
00:02:40.11 and in these chromosomes
00:02:42.18 one can see bands
00:02:45.10 that were pretty much regarded as genes
00:02:48.18 by people working with them at that time.
00:02:50.24 We now know this is a little bit more complicated than that,
00:02:53.11 but it seemed that we had here a tissue
00:02:58.11 - a chromosome -
00:03:00.19 where specific genes
00:03:02.22 - specific nucleic acids...
00:03:05.12 it should be possible to do something comparable to the
00:03:08.15 immunofluorescent staining technique
00:03:11.12 to detect specific genes in these tissues.
00:03:15.12 So, actually,
00:03:19.17 I kept this in my mind for quite a long time,
00:03:23.20 and it became possible to think about doing this kind of thing
00:03:27.23 in the early 1960s,
00:03:29.29 when investigators discovered that
00:03:33.14 one could take double-stranded DNA,
00:03:36.27 denature it into its single strands,
00:03:40.02 and immobilize those strands
00:03:42.24 on a nitrocellulose filter.
00:03:46.21 This was done by a couple of investigators
00:03:50.03 named Gillespie and Spiegelman,
00:03:52.07 and what they showed was that,
00:03:54.11 having the denatured DNA on a filter,
00:03:57.11 one could then hybridize
00:04:00.02 radioactive RNA to it
00:04:02.18 and actually determine, quantitatively,
00:04:05.17 the amount of a particular gene sequence on the filter.
00:04:09.07 So, I thought,
00:04:11.21 "Well, if they can do it on a filter
00:04:13.13 you ought to be able to do it on a slide".
00:04:15.17 So, I tried denaturing the DNA on a slide,
00:04:21.18 that is, in cells - nuclei -
00:04:25.07 that were squashed out onto a slide,
00:04:27.28 and I found that I could denature the DNA quite adequately,
00:04:31.13 without damaging the tissue,
00:04:33.24 but then when I tried to hybridize
00:04:36.02 a radioactive RNA to it,
00:04:38.15 I got no signal.
00:04:40.16 In this case,
00:04:42.11 I was looking for the radioactivity by autoradiography,
00:04:45.28 that is by covering the tissue
00:04:48.11 with a thin layer of photographic emulsion
00:04:51.16 and looking for silver grains in the emulsion
00:04:54.27 caused by the radioactivity of the probe.
00:04:58.24 As I said, nothing happened,
00:05:01.00 and I more or less gave up on that technique.
00:05:04.29 That was in the early to mid 1960s.
00:05:08.21 Now, a break came, quite accidentally,
00:05:14.06 from studies that I and others
00:05:16.14 had been carrying out
00:05:19.24 on salamander oocytes and frog oocytes,
00:05:22.17 and what we found was that
00:05:27.03 in the giant oocyte nucleus,
00:05:29.19 which is shown here,
00:05:32.06 derived from this large oocyte here
00:05:36.06 - the oocyte is the egg of the frog -
00:05:39.06 inside of this giant nucleus
00:05:42.07 are of course the chromosomes,
00:05:44.08 but in addition we found that
00:05:47.20 there were several hundred to a thousand nucleoli,
00:05:50.25 these round bodies here shown on this particular slide.
00:05:55.08 Now, the interesting thing about these nucleoli
00:06:00.22 that we discovered was that
00:06:03.13 they contained DNA,
00:06:05.23 and it was specifically the genes
00:06:08.07 that code for ribosomal RNA
00:06:10.19 that were in these nucleoli.
00:06:13.05 This was a most unusual situation
00:06:14.23 to have DNA outside of the chromosomes,
00:06:17.06 but it was quite clear from work
00:06:19.09 that had been carried out in my laboratory,
00:06:21.09 as well as in the laboratory of Don Brown
00:06:23.09 and of Oscar Miller,
00:06:25.21 that there was extrachromosomal DNA
00:06:28.18 outside in these nucleoli.
00:06:32.00 And I studied the origin of that DNA
00:06:35.04 and what I found was that
00:06:38.18 it actually was synthesized at one specific time
00:06:41.19 in the early history of the formation of the oocyte,
00:06:47.15 and one could see in these early cells
00:06:52.11 - early nuclei -
00:06:54.11 that there was a cap of DNA here...
00:06:57.18 and you can see the cap over here,
00:07:00.09 you can see the chromosomes here
00:07:02.18 and down here you can see the chromosomes in this nucleus...
00:07:06.10 so, in these two nuclei
00:07:08.09 you have the chromosomes,
00:07:10.03 but then you have this huge cap of
00:07:13.09 extra DNA which in fact
00:07:16.21 was the ribosomal genes
00:07:19.26 that had been somehow...
00:07:22.02 had somehow come out of the chromosome
00:07:23.24 and amplified to give you
00:07:26.14 this one specific sequence,
00:07:28.14 a very large amount of this one specific sequence.
00:07:31.13 So, it occurred to me that if there was any possibility
00:07:33.18 of doing in situ hybridization,
00:07:35.23 this was the case that would work.
00:07:38.05 And so I took a radioactive ribosomal RNA
00:07:42.04 and hybridized it to cells of this sort,
00:07:46.18 and I was quite pleased
00:07:48.29 to see in the very first experiments
00:07:51.22 that we got good in situ hybridization.
00:07:54.02 Here, this cell is black
00:07:56.09 because of the silver grains in the photographic emulsion
00:07:59.19 that had been exposed by the radioactivity
00:08:02.20 of the hybridizing probe.
00:08:04.19 So, this is a case where we have hybridized
00:08:09.22 radioactive RNA - ribosomal RNA -
00:08:12.26 to the ribosomal DNA in the cell.
00:08:18.08 So, that led immediately to a publication
00:08:20.20 with my student Mary Lou Pardue,
00:08:23.05 who joined me in this work,
00:08:27.00 and this was the first successful in situ hybridization.
00:08:30.28 Now, we wanted to try it
00:08:33.02 on some other tissue or DNA,
00:08:35.20 but there were very few examples,
00:08:38.05 because at that time it was not possible
00:08:41.09 to isolate specific DNA or RNA sequences.
00:08:47.07 What permitted us to look at another situation
00:08:51.04 came from studies that had been carried out
00:08:54.15 on mouse DNA,
00:08:57.14 and investigators had found that
00:09:00.09 when mouse DNA was spun
00:09:02.19 in a cesium chloride (CsCl) gradient,
00:09:04.02 it separated into two fractions,
00:09:05.29 a main peak and a satellite peak
00:09:09.08 - called satellite simply because
00:09:11.26 it had a slightly different density
00:09:14.17 and separated from the bulk of the DNA.
00:09:18.08 We took this satellite DNA,
00:09:21.03 made it radioactive,
00:09:23.07 and we hybridized it to mouse chromosomes,
00:09:26.01 not knowing at all whether it would hybridize,
00:09:29.13 whether it was even in the chromosomes for that matter,
00:09:31.28 it might have been a virus or some other infective particle,
00:09:34.28 but we were very pleased to see that
00:09:38.19 the mouse satellite DNA hybridized
00:09:41.04 specifically to one portion of each chromosome,
00:09:45.00 and you can see on this slide
00:09:47.26 that each of the chromosomes
00:09:50.15 has a bit of radioactivity
00:09:53.00 at the very end of the chromosome.
00:09:55.04 This region of the chromosome
00:09:57.05 was known as the heterochromatin,
00:09:59.11 and had been known cytologically
00:10:02.03 as a specific region of the chromosome
00:10:05.06 for a number of years.
00:10:07.17 This led us immediately to think
00:10:10.09 that we should be able to try the same technique
00:10:13.19 and get similar results
00:10:15.29 looking at the chromosomes of Drosophila,
00:10:18.16 and the chromosomes of Drosophila
00:10:20.26 were especially exciting
00:10:22.25 because it was known from genetics
00:10:25.22 that the genes were all in
00:10:31.27 the so-called euchromatic regions of the chromosome,
00:10:35.04 namely this region from here to here, or over here...
00:10:41.14 these lighter stained regions...
00:10:43.15 all of the genes were located in those regions,
00:10:45.13 and it was known from genetics
00:10:47.20 that the heterochromatic regions, like here and here,
00:10:51.28 were devoid of genes.
00:10:54.16 So, it occurred to us that Drosophila
00:10:57.18 must have a lot of DNA
00:11:00.15 similar to the satellite DNA of the mouse.
00:11:03.23 So, we centrifuged that DNA
00:11:07.05 in a cesium chloride gradient,
00:11:09.18 and we found, indeed,
00:11:11.25 that Drosophila had a lot of satellite DNA.
00:11:14.29 This species of Drosophila virilis
00:11:17.06 that we were working on
00:11:19.28 had three huge satellite peaks
00:11:22.06 - here, here, and here -
00:11:25.15 in addition to the main peak of DNA, shown here.
00:11:31.04 So, we took the satellite DNA,
00:11:33.27 we made it radioactive,
00:11:35.27 we hybridized it to chromosomes,
00:11:37.19 and, sure enough, it hybridized as shown
00:11:40.19 on these images here,
00:11:42.01 to the heterochromatic regions.
00:11:44.03 And this was particular interesting,
00:11:46.06 as I said,
00:11:48.03 because it showed that the heterochromatic regions,
00:11:50.29 that we knew from genetics had no genes,
00:11:53.14 consisted of this satellite-type DNA,
00:11:57.22 which our later studies showed
00:12:00.16 consisted of very simple sequences,
00:12:03.07 which were incapable of coding for genes,
00:12:07.28 or were incapable of being genes and coding for proteins.
00:12:11.17 So, the in situ hybridization technique
00:12:13.27 at this point was limited
00:12:16.09 pretty much to sequences
00:12:18.24 that were present in large amount in cells.
00:12:21.18 Eventually, it became possible,
00:12:24.21 by having more and more sensitive probes,
00:12:27.17 in particular the later techniques were worked out
00:12:31.22 for making fluorescent probes
00:12:34.04 to do in situ hybridization...
00:12:36.24 so, in situ hybridization,
00:12:38.17 ISH or "ish",
00:12:40.09 became fluorescent in situ hybridization,
00:12:42.16 or "fish", FISH,
00:12:45.08 which is the was it's usually practiced now.
00:12:48.22 And techniques have gotten
00:12:51.02 more and more sensitive,
00:12:52.27 to the point where, nowadays,
00:12:54.13 it's possible to detect individual,
00:12:56.22 single DNA and RNA molecules in tissue sections.

This Talk

Speaker: Joseph Gall

Audience:

Student
Educators of Adv. Undergrad / Grad
Researcher
Educators

Recorded: January 2014

Talk Overview

In in situ hybridization, RNA or DNA molecules can be visualized in an immobilized cell, tissue, or tissue section. This technique, which was pioneered by Joseph Gall in 1968, works by hybridizing a labeled RNA or DNA molecule that is complementary to the RNA or DNA of interest. In his talk, Gall describes the experiments that inspired him to develop this technique, and the key experiments that led to the first visualization of RNA and DNA inside the cell.

Speaker Bio

Joseph Gall

Joseph Gall is a Professor at the Carnegie Institution for Science and is a leader in the field of cell biology. He has spent much of his career studying the nucleus and its contents, and has made numerous significant contributions to our understanding of chromosome structure and function. Gall, a distinguished microscopist, is one of the… Continue Reading