The Questions of Developmental Biology

The idea that the genes of chromosomes were differentially expressed in different cell
types was confirmed using DNA-RNA hybridization (Figure 4.13C). This technique involves
annealing single-stranded pieces of RNA and DNA to allow complementary strands to form
double-stranded hybrids. While some mRNAs from one cell type were also found in other cell
types (as expected for mRNAs encoding enzymes concerned with cell metabolism), many
mRNAs were found to be specific for a particular type of cell and were not expressed in other cell
types, even though the genes encoding them were present (Wetmur and Davidson 1968). Thus,
differential gene expression was shown to be the way a single genome derived from the fertilized
egg could generate the hundreds of different cell types in the body. The question then became,
How does this differential gene expression occur? The answers to that question will be the topic
of the next chapter. To understand the results that will be presented there, however, one must
become familiar with some of the techniques of molecular biology that are being applied to the
study of development. These include techniques to determine the spatial and temporal location of
specific mRNAs, as well as techniques to determine the functions of these messages.
RNA Localization Techniques
Northern blotting
We can determine the temporal and spatial locations where RNAs are expressed by
running an RNA blot (often called a Northern blot). First, one extracts the RNA from embryos at
different stages of development, or from different organs of the same embryo. The investigator
then places the RNA samples side by side at one end of a gel and runs an electrical current
through the gel. The smaller the RNA, the faster it moves through the gel. Thus, different RNAs
are separated by their sizes. This technique is called electrophoresis.* The investigator then
transfers the separated RNAs to a nitrocellulose paper or nylon membrane filter, and incubates
the RNA-containing filter in a solution containing a radioactive single-stranded DNA fragment
from a particular gene (Figure 4.14A). This radioactively labeled DNA probe binds only to
regions of the filter where the complementary RNA is located. If the mRNA for that gene is
present in a sample, the labeled DNA will bind to it and can be detected by autoradiography.
X-ray film is placed above the filter and
incubated in the dark.
The localized radioactivity in the probe
reduces the silver in the X-ray film, and
grains form. The resulting spots, which
appear directly above the places where
the radioactive DNA has bound, appear
black when viewed directly.
Autoradiographs of this type, in which RNAs from several stages
or tissues are compared simultaneously, are called developmental
Northern blots.
Figure 4.14F shows a developmental Northern blot used to
investigate the expression of Pax6 expression in the mammalian embryo.
Pax6 is a protein that is critical for normal eye development; mutations in
this gene give small eyes (in heterozygous mice) or no eyes or nose
(in mice or humans homozygous for the loss-of-function mutation).
The Northern blot shows that this gene is expressed in the embryo in
the brain, eyes, and pancreas, but in no other tissue.