Biotechnology Provides Tools for the Study of Flounder Development
Bruno Soffientino, Doctoral Candidate
Graduate School of Oceanography
The word "biotechnology" is used often
these days, but what does it mean? Biotechnology is a broad term that refers
to the techniques and methods used to study and manipulate organisms at the
cellular and molecular level and to the applications of those methods in research
and industry. Many scientific fields make use of biotechnology: medicine, agriculture,
forensic science, ecology, and oceanography, to mention just a few. The range
of organisms studied is just as wide: from bacteria, flies, sea urchins and
round worms, to mice, frogs, fish, and, of course, humans. In my research, the
development of summer flounder, biotechnology tools help answer important questions
on the regulation of stomach and intestine development in vertebrates.
Vertebrate development comprises all the coordinated
and regulated events of cellular proliferation, differentiation, movement, and
death that take place when an organism transforms from a single-cell fertilized
egg into an organism made up of billions of cells and hundreds of cell types.
After fertilization, the egg begins to divide and cells increase in number;
they differentiate (take on specialized characteristics) and organize into subpopulations
that have specific locations and functions. These subpopulations eventually
give rise to body tissues and organs. The information that specifies the timing,
location, and types of changes that occur is embedded in the genes whose expression
is coordinately (with respect to the other genes participating in the process)
turned on and off or up and down during development. When a gene is expressed,
it usually means that it is translated by the cell's machinery into a protein
with a specific function. It is the appearance and function of the protein products
that result in developmental changes. In other words, genes are the units being
coordinately regulated, but it is their product that carries out the function.
This unfolding of information is often described as a "genetic cascade."
In a cascade, changes are initiated by the activation of "master genes"
that turn other genes on or off, producing a sequence of gene regulation that
ultimately results in changes at the cellular, morphological, and functional
level.
Examples of master genes are provided by the
Hox gene family. These genes specify the position of structures, like limbs
and organs, along the anteroposterior axis of the body in all vertebrate and
arthropod species. Their overexpression or deletion results in morphological
deformities like multiple or missing body sections. The factors that coordinate
and regulate the timing of developmental events depend on the stage of development.
Early on, when the embryo is small, tissues can "communicate" and
coordinate events by releasing chemical signals that diffuse across cell layers.
Later, when the developing animal is much larger, most tissues are no longer
in direct contact with each other, so the process is carried out largely by
the release of hormones that are regulated by the nervous system. Hormones are
chemical signals that travel through the blood to their target tissues, where
they initiate the genetic cascades that result in the appearance of new organs
and structures.
Animal development is a complex continuum of
morphological and functional transformations that are the result of coordinated
regulation of gene expression via diffusible chemical signals early in development,
and via hormones during late development. Summer flounder development is characterized
by a metamorphosis that transforms the larva into a flat juvenile fish (see
Fig. 1). A less visible but important event that occurs during metamorphosis
is the formation of a functional stomach and of pyloric cecae in the anterior
intestine. The stomach, much like our own, contains gastric glands that secrete
hydrochloric acid and the enzyme pepsin for protein digestion. The cecae are
blind sacs, or diverticula, that increase the surface area of the intestine
and slow down the passage of food, thus increasing the absorptive efficiency
of the gut.
The successful completion of metamorphosis and
gastrointestinal development requires the presence of Thyroid Hormone (TH),
but just how the hormone mediates the observed changes is not known. Thyroid
hormone is also involved in gastrointestinal development in mammals, including
humans. I'm studying the summer flounder as a model for gastrointestinal formation
in vertebrates, looking at events that take place during late development that
are, therefore, regulated by hormones (in this case TH). My goal is to understand
the development of the stomach and intestine in terms of the changes in cell
proliferation and differentiation. This will be useful in understanding the
sequence of events that begins with the induction of genes by TH and ends with
a remodeled gastrointestinal system. Biotechnology provides the necessary tools
to carry out this work.
Cell proliferation is the rate at which cells
divide in a tissue. To measure its changes in the stomach and intestine of metamorphosing
flounder, I adapted a technique commonly used in oncology to assess the growth
potential of tumors. The principal is simple: Cells need to replicate their
DNA before dividing, so they take up DNA precursors. The cells are exposed to
a slightly modified DNA precursor, called a DNA base analog, that can be detected
in tissue preparations, and that will be taken up only by the replicating cells,
providing a way to quantify the proliferation rate. The DNA precursor, BrdU,
is a synthetic analog of the DNA base thymidine. To detect the presence of BrdU
in the cells, an anti-BrdU antibody is used.
Antibodies are proteins produced by the immune
systems of all vertebrates in response to the entry of a foreign biological
substance or organism. The most important feature of antibodies is that they
bind strongly and specifically to the substance that caused their production.
The normal function of antibodies is to "tag" the foreign substance
or to precipitate it so that it can be removed from the body. Antibodies against
BrdU that are available commercially are produced in rats and mice by injecting
them with BrdU, collecting the blood, and isolating the specific antibody. Antibodies
against millions of different proteins, biological substances, and pathogens
are produced this way and are routinely used in research and medicine. Antibodies
represent the basic reagents for commercial diagnostic kits like pregnancy tests.
The antibody I use has been further modified
to make it suitable for colorimetric detection of BrdU. It has been attached
to an enzyme, a protein that catalyzes a specific chemical reaction. The enzyme,
horseradish peroxidase, converts the chemical diaminobenzidine to a readily
visible brown precipitate (see Fig. 2). Thin sections of tissues are incubated
with the BrdU antibody solution. The antibody binds on the cell nuclei that
contain BrdU. The preparation is then washed with diaminobenzidine, which is
converted to the brown precipitate over the BrdU-labeled nuclei. Finally, the
sections are stained with a nonspecific blue dye, hematoxylin, that stains all
the cell nuclei. The ratio of brown to blue cells is a direct measure of cell
proliferation. This assay can be used to probe deeper into the mechanisms that
control cell proliferation. By sequentially exposing the fish to two different
DNA base analogs and detecting the proportion of single labeled and double labeled
cells with two different antibodies and color systems, it is possible to estimate
the duration of the cell cycle phases. These parameters, measured in fish of
different developmental stages, provide information on which cell cycle proteins
(and, therefore, genes) are part of the regulatory cascade initiated by TH.
I'm also interested in cell differentiation,
because it represents the endpoint of the genetic cascade. Differentiation is
the visible result of the expression of a particular collection of proteins
that give the cell specific functional characteristics. Consequently, such cell
type-specific proteins can be used as differentiation markers; pepsin, for example,
is a good marker for differentiation of gastric gland cells in the stomach.
The specificity of antibodies can be used to look for differentiation markers.
Because often there are no commercially available antibodies against such proteins,
they have to be made. Pepsin was isolated, identified, and purified, then injected
with immune system stimulants into a rabbit. After a "booster shot"
two months later, the antibody was present in the rabbit's blood in sufficient
quantity to be collected. The enzyme necessary for the color reaction is attached
to the anti-pepsin antibody and the preparation can be used for a specific pepsin
assay.
Biotechnology provides techniques and reagents
that can answer many questions about development, metabolism, reproduction,
and population dynamics in just about any organism. That's what I find so fascinating---what
works with mice, monkeys, or rabbits works just as well with humans, sea urchins,
or fish.