Fate Map and Fate of Germ Layers

Academic Script

In the year 1817, Christian Pander, a doctoral student at the University of Würzburg, Germany, first
recognized the existence of germ layers in chicks (Gallus gallus). A germ layer is a group of cells in
an embryo that interact with each other and contribute to the formation of all organs and organs.
Development of the germ layer begins at the later stages of fertilization, wherein the formation of
zygote takes place as a result of fusion of male and female gametes, i.e. spermatocyte and oocyte,
respectively. The zygote then undergoes subsequent cleavage by mitotic division, wherein one cell
is divided in to two, four, eight, and so on and transforms into a hollow ball of cells called blastula.
The blastula then undergoes an important process called gastrulation, wherein the formation of
germ layers, such as ectoderm, mesoderm and endodermtakes place. Together, the three germ
layers will give rise to all the organs in the body. Based on the presence of number of germ layers
animals are classified into the diploblastic and triploblastic animals. A diploblastic organism has only
two primary germ layers, namely ectoderm and endoderm, whereas a triploblastic organism has an
additional layer, called mesoderm.

1. Formation of zygote by fertilization:

Fertilization is a process in which the fusion of male and female gametes results in the formation of
zygote. In humans, after about the third day of fertilization, the single celled zygote undergoes
multiple cleavages by mitotic cell division to form a solid mass of cells called morula. The morula is
then transformed in to blastocyst, consisting of an outer layer called as trophoblast, and an inner
mass called as embryoblast. The embryoblast initially has two layers, namely
the hypoblast and epiblast. At the end of the second week, a primitive streak appears and the
epiblast in this region moves towards the primitive streak, jumps down into it, and forms a new layer,
called the endoderm, pushing the hypoblast towards the exterior. The epiblast keeps on moving and
forms a second layer called asmesoderm and the top layer is considered as the ectoderm.

2. Gastrulation: the embryological stage of germ layer formation:

During early embryonic development, the overall architecture of the body and the major axes are
established through the process called gastrulation. This, in fact in its original term,referstothe
formation of a gastric cavity, the archenteron.However, today the term gastrulation is often used as
a synonym of endoderm and mesoderm formation. During gastrulation, a hollow cluster of cells
called a blastula reorganizes into two primary germ layers: an inner layer, called endoderm, and an
outer layer, called ectoderm.In all vertebrates, these progenitor cells differentiate into all adult
tissues and organs.
Gastrulation is a dynamic process in which cell migration and an epithelial to mesenchymal
transition (EMT) occurs in an organized way by switching the cell adhesion molecules. The
gastrulation step begins with the extensive cell mixing followed by migration of mesenchymal cells
to the presumptive midline to form the primitive streak. It is the site at which epiblast cells undergo
EMT and initiatethe formation of distinct cell layers that will form the primary germ layers, mesoderm
and endoderm. Epiblast cells that do not enter EMT, undergoes differentiation to become ectoderm.
Several transcriptional programs that influence cell migration during gastrulation and subsequent
cell differentiation into the three germ layers are activated by intercellular signaling mediated by
Wnt, FGF, and Nodal/TGF-beta ligands. In most animals, endoderm formation goes parallel with the
formation of the third germ layer, the mesoderm.

2.2. Development of germ layers, organs and tissues:

2.2.1. The endoderm:
In general, the blastocytes grows in size by obtaining nutrition from the uterus and a few cells
separate from the inner mass to form endoderm in blastocoels. The cells migrating inward along the
archenteron form the inner layer of the gastrula, which develops into the endoderm.The endoderm
then differentiates into the primitive guts; a part of it gives rise to digestive tract and the other portion
forms yolk.After the formation of endoderm the remaining cell of inner mass forms embryonic discs,
which has three parts, namely cephalic margin, embryonic disc and caudal margin.

In the beginning, the endoderm consists of flattened cellsthatsubsequently become columnar. It

forms the epithelial lining of the whole of the digestive tract except part of the mouth and pharynx
and the terminal part of the rectum. It also forms the lining cells of all the glands which open into the
alimentary canal. Together, the endoderm forms the epithelial parts of trachea, the lungs, the
pharynx, the thyroid, the parathyroid,the liver, the pancreas, the stomach, the
colon,theintestines,and the urinary bladder.

2.2.2. The mesoderm

The formation of mesoderm in the embryos is the characteristic feature of triploblastic animals.
During gastrulation some of the cells migrating inward contribute to the mesoderm, an additional
layer between the endoderm and theectoderm. The formation of a mesoderm leads to the
development of the coelom. The coelom that fills with the fluid acts as a cushion and protect the
organs that formed inside the coelom from shocks. Thus, the organs in the coelom have the
freedom to move, grow on its own pace, and develop independently of the body wall.
The mesoderm has 4 different components such as intermediate, paraxial, lateral plate, and chorda-
mesoderm. The mesodermal cells begin the process of differentiation through interactions with the
ectodermal and endodermal cells using different cell signaling cascade mechanisms. The
intermediate mesoderm gives rise to kidneys,and gonads; the paraxial mesoderm forms into
cartilage, skeletal muscle, and dermis; the lateral plate mesoderm is responsible fordevelopment of
the circulatory system, the wall of the gut, and wall of the human body; and finally the
chordamesoderm forms the notochord. In brief, the mesoderm forms muscle, bone, cartilage,
connective tissue, adipose tissue, circulatory system, lymphatic system, dermis, genitourinary
system, serous membranes, and notochord.

2.2.3. Ectoderm
The embryo’s epiblast gives rise to the formation of the ectoderm, which forms the outer layer of the
embryo. The ectoderm develops into the surface ectoderm, neural crest, and the neural tube. The
surface ectoderm develops into epidermis, hair, nails, lens of the eye, sebaceous
glands, cornea, tooth enamel, the epithelium of the mouth and nose.The neural crest, which derives
from the ectoderm,is often considered asa fourth germ layer owing to its great importance in the
development of peripheral nervous system, adrenal medulla, melanocytes, facial cartilage
and dentine of teeth. The neural tube of the ectoderm develops into brain, spinal cord, posterior
pituitary gland, motor neurons, and retina.

3. Fate mapping
The development of an organism from a single cell, crossing through different stages of embryo,
morula and blastula to a recognizable physical formthat slowly shapes into a small animal is an
orchestrated event of different mechanisms that occurs at a time-bound harmonized way. It is
indeed a miracle to not only a common man but also to the developmental biologist, who astonished
by the finely tuned developmental process termed morphogenesis. It is simple to ask as “how this
process is finely tuned and what controls the regulation? A classic way to approach such a question
is to generate a fate map. This area of developmental biology is the recent focus and
developmental biologists have carefully characterized the stages of morphogenesis and have
identified key transitional periods: blastula, gastrula, and neurula stages through varied imaging
methods, collectively called fate mapping.

A fate map is a graphic representation of the cells within the embryos that are developing in
to specific tissues or organs at a given specific point during developmental stages. For example,
studying the future position of a dividing cell from its origin from a mass of cell i.e. cell lineage, or
studying the developmental differentiation of a particular cell forward from its position in one of the
three germ layers. Labeling individual cells within their germ layers will allow for a pictorial
interpretation of gastrulation. This chart or graphical representation that explains the fate of each
part of an early embryo is referred to as a fate map.
Constructing a fate map is animportant part of understanding the developmental pathway of an
organism. Determining the architectural assembly of an organism could possibly lead to establish
the function of each specific region. Furthermore, understanding the lineage and migration of
progenitor cells, help in discovering gene regulatory networks and signaling pathways. The
possibility of new developmental discoveries comes with the creation of each new fate map.

3.1. Construction of a Fate Map

A fate map is an experimental tool to explore the reorganization of cells from one stage of
development to a later stage. Briefly, the step involves labeling of a cell at a recorded location and
observing the same cell or its progeny that are relocated at a later stage. If cell rearrangements are
conventional from embryo to embryo, then a map can be constructed by repeating this procedure
several times from different recorded positions. This approach has been widely used, employing a
variety of ways to label the cells and varying degrees of complexity in classifying final cell fate,
ranging from scoring their location by tissues or organ or analyses of gene expression patterns. In
recent times various techniques have been devised for the construction of fate map. Of these,
tracing the course with natural colours and artificial markings are most important.

3.1.1. Natural markings:

Fate mapping can be performed utilizing the natural markings or pigments that are present in an
organism. For example, the cytoplasm of certain eggs ofascidians has natural pigments. Thus, in
the fertilized egg four coloured centres can be seen as an upper hemisphere of tight protoplasm, a
yellow crescent postero-ventrally, a grey crescent antero-dorsally and a vegetal area of dark grey
yolky substance. The fate of these areas can be followed very easily and has has been revealed
that the upperclear cytoplasm contains the material for epidermal ectoderm. The grey crescent area
differentiates into the prospective neuro-ectoderm and notochord. The yellow crescent becomes the
prospective mesoderm and the dark grey yolky area forms the prospective endoderm.

3.1.2. Artificial Markings:

There are three methods to mark or label only the blastomeres by which their fate can be traced.

3.1.2.1. Vital staining:

This vital staining method was first introduced by Vogt in the year 1928. Vital stains, such as
methylene blue, neutral red, bismark brown, janus green and nile blue sulphate are some of the
common vital stains, which are retained by living cells for considerable period of time. These dyes
are in no way found to interfere with normal life activities. Thus, the persistence of the stains in the
cells makes it possible to determine the ultimate location of the marked areas in the late embryo.
The vital staining technique involves soaking a piece of agar with a vital dye and placing it on a
surface of the embryo in the required location. Upon exposed to tissue, the stain immediately
diffuses into the blastomeres and the cells are stained. The interesting feature is that the
descendents of the marked cells also become stained. Therefore, in the differentiated embryo, the
parts stained indicate that the fates of the original stained blastomeres. For example, if the stained
cells are seen in the gut of the differentiated embryo, the fate of the marked blastomeres is said to
be the development of gut. Similarly, several such stain marks can be made on the surface of the
same embryo using different colours and the various organ forming areas can be marked in this
way.

3.1.2.2. Carbon particle marking:

This technique was introduced by Spratt to demonstrate the process involved in primitive streak
formation in chick. This method consists of applying tiny particles of carbon over the surface of
blastomeres, which stick to the cell surface and enable the investigator to monitor the movements of
the cells, and to determine the fate of these blastomeres.

3.1.2.3. Radioactive isotope labelling:

Radioactive isotopes have long been used in the tracer experiments utilizing the property of
emission of alpha, beta and gamma rays. Briefly, the early blastomeres are labelled with any of the
radioactive isotopes such asC14 and P32and course of these radioactive isotopes can be carefully
monitored to determine the fate of blastomeres.

Summary
To summarise, at the end of this lecture we understood that the germ layers has main role in the
formation of tissues and organs. They form the main body parts in the animals. Without germ layers
it may be difficult for the formation of body organs. With the help of the fate map technique we can
understand the future change in germ layers of tissues and organs in animals. Thank you.