The Questions of Developmental Biology

In situ hybridization
But where was this gene expressed in the eye? Northern blot analysis (which uses mRNA
extracted from pieces of tissue that have been removed from the embryo) can give only an
approximate location and time for gene expression. A more detailed map of gene expression
patterns can be obtained by using a process called in situ hybridization. Instead of using a DNA
probe to seek mRNA on a filter, the probe is hybridized with the mRNA in the organ itself.
Embryos or organs are fixed to preserve their structure and to prevent the RNA from being
degraded, then sectioned for microscopy and placed on a slide. When the radioactive DNA probe
is added, it binds only where the complementary mRNA is present. After any unbound probe is
washed off, the slide is covered with a transparent photographic emulsion for autoradiography.
By means of computer-mediated bright-field imaging, the reduced silver grains can be shown in a
color that contrasts with the background stain. Thus, we can visualize those cells (or even regions
within cells) that have accumulated a specific type of mRNA. Figure 4.15 shows an in situ
hybridization for Pax6 mRNA in mice. One can see that Pax6 mRNA is found in the region
where the presumptive retina meets the presumptive lens tissue. As development proceeds, it is
seen in the developing retina, lens, and cornea of the eye.
The polymerase chain reaction
The polymerase chain reaction (PCR) is a method of in vitro gene cloning that can
generate enormous quantities of a specific DNA fragment from a small amount of starting
material (Saiki et al. 1985). It can be used for cloning a specific gene or for determining whether
a specific gene is actively transcribing RNA in a particular organ or cell type. The standard
methods of cloning use living microorganisms to amplify recombinant DNA. PCR, however, can
amplify a single region of a DNA molecule several million times in a few hours, and can do it in
a test tube. This technique has been extremely useful in cases in which there is very little nucleic
acid to study. Preimplantation mouse embryos, for instance, have very little mRNA, and we
cannot obtain millions of such embryos to study. If we wanted to know whether the
preimplantation mouse embryo contained the mRNA for a particular protein, it would be very
difficult to find out using standard methods. We would have to lyse thousands of mouse embryos
to get enough mRNA. However, the PCR technique allows us to find this message in a few
embryos by specifically amplifying only that message millions of times (Rappolee et al. 1988).
The use of PCR for finding rare mRNAs is illustrated in Figure 4.17. First, the mRNAs
from a group of cells are purified and converted into complementary DNA (cDNA) using an
enzyme called reverse transcriptase. By using DNA polymerase and S1 nuclease, this
population of single-stranded cDNAs is then made into double-stranded cDNAs. Next a specific
cDNA is targeted for amplification. In preparation for this step, the cDNA double helices are
separated, or denatured, by heating them.