Chapter CD

Molecular Biology

Some Elementary Facts about Control of Development

1. It has been observed that certain regions of the embryo have the ability to influence the
differentiation of neighbouring regions. One of the most interesting instances of this
phenomenon is the influence exerted by the optic vesicle on the overlying skin to form
the lens vesicle. It has been shown that the lens vesicle fails to form if the optic vesicle is
removed. Conversely, if the optic vesicle is transplanted elsewhere (e.g. under the skin of
the abdomen) the overlying skin there begins to form a lens vesicle. These experiments
show that the optic vesicle induces the formation of the lens vesicle.
The influence exerted by such an area (i.e. optic vesicle) is called induction whereas the
area exerting the influence is called an organiser.
2. The first organizer that is recognisable in the embryo is the dorsal lip of the blastopore,
which is, therefore, called the primary organiser. Removal of this region results in total
failure of embryonic development. However, on the other hand if the dorsal lip of the
blastopore is grafted on to an ectopic site of another embryo, it induces the development
of an entire embryo. This indicates that signals for the development of the embryo have
originated from the dorsal lip of the blastopore and have influenced the differentiation of
surrounding tissues. Organisers that appear later in development have correspondingly
lesser effects.
3. It is now known that the organisers exert their influence by elaborating chemical
substances, which are probably complex proteins, including enzymes.
4. The chemical substances elaborated by the organiser may be:
a. inductors which stimulate the tissue to differentiate in a particular manner; or
b. inhibitors which have a restraining influence on differentiation.
The observations recorded above were empirical. With the advent of molecular biology
we know that the production of organisers, inductors and inhibitors are controlled by genes.
In other words we can say that development is controlled by genes. A study of the controlling
mechanisms can be termed Genetic control of development. It can also be described as
Molecular control of development.
Genes exert their influence on cellular functions by synthesis of proteins. The proteins
synthesised differ from cell to cell and within the same cell at different times. This provides

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the basic mechanism for control of any process, including embryonic development. Recent
researches have provided us with a vast amount of information about individual genes, and
about the various factors produced by them to control developmental processes step by step.
As you already know all information in cells is stored in molecules of DNA. To understand
genetic processes we have to first know some facts about DNA structure.

DNA STRUCTURE
Some elementary facts about DNA structure have been described in Chapter 1. Here we will
consider further details.
1. We have seen that each strand of the DNA fibre consists of a chain of nucleotides. The
structure of a nucleotide is shown in Fig. CD-3.1.
2. Each polynucleotide chain has marked ends. In Fig. CD-3.2 observe that, at the upper
end of the chain, the 5th carbon atom of the sugar molecule is not linked to any other
nucleotide. This end is called the 5 or 5P terminus. The other end of the chain ends in a
sugar molecule whose 3rd carbon atom is not linked to any other nucleotide and bears an
3-OH group. This end of the polynucleotide chain is called the 3 end or 3 OH terminus.
3. The DNA molecule is made up of two such polynucleotide chains. These chains lie side
by side but run in opposite directions (antiparallel). One chain runs in 53 direction, the
other in 35direction (Fig. CD-3.3).
4. The two chains are held together by hydrogen bonds between the nitrogenous bases.
5. The pairing between nitrogenous bases is fixed i.e., adenine (A) always pairs with thymine
(T), and cytosine (C) with guanine (G). The specific pairing of bases is due to the fact that
their molecules are complementary and perfect hydrogen bonds are formed easily. A and
T share two hydrogen atoms while C and G are joined by three hydrogen bonds.
6. As there is specific base pairing the two strands of DNA are complementary to each other.

If the sequence of bases on one chain is ATGCA the corresponding region on other chain
will have the sequence TACGT.

Fig. CD-3.1: (A) Schematic diagram of a molecule of nucleotide (P = phosphoric acid. S = sugar.
B = nitrogenous base). (B) Chemical structure of a molecule of nucleotide.

Chapter CD 3 Molecular Biology

Fig. CD-3.2: A polynucleotide chain. (A) Schematic diagram.

(B) Chemical structure of a nucleotide chain.

7. Two complementary chains (polynucleotide chains) of DNA are twisted around each
other to form what is called a double helix.
8. Every protein is made up of polypeptide chains. These chains are made up of a series of
amino acids. The nature of the protein depends upon the amino acids present, and the
sequence in which they are arranged. A sequence of three bases on the DNA strand, codes
for one amino acid. Under the influence of DNA these amino acids are linked together in
a particular sequence to form proteins. Thus the order in which these bases are arranged
along the length of a strand of DNA determines the nature of the protein that can be
synthesized.

Molecular Structure of A Gene

Chemically, a gene is composed of DNA. In simple language, a structural gene can be defined
as a segment of DNA which contains the information (code) for the synthesis of one complete
polypeptide chain. Thus a gene is nothing but a set of instructions for making proteins.

As each polypeptide chain is made of specific amino acids arranged in a specific sequence,
it was expected that in a structural gene DNA sequences coding for these amino acids must
be contiguous. However, it is now known that there are many non-coding sequences (called

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Fig. CD-3.3: Antiparallel polynucleotide chains showing sugar-phosphate backbone and nitrogenous
base pairing. The nitrogenous base Cytosine (C) always pairs with Guanine (G) while Adenine (A) pairs with
Thymine (T). (A) Schematic diagram. (B) Structural diagram.

introns), interposed between the coding sequences (or exons). The number of introns in
various genes is variable and sometimes it may so happen that introns are much larger than
exons (coding sequences). Though introns are transcribed (as described below) they are
not included in mature RNA. A structural gene not only contains the sequence of exons and
introns (explained above) but also possesses flanking regions at ends. These flanking regions
are important for regulation of gene expression (Fig. CD-3.4). At the 5 end the flanking region
is made up of DNA sequences, which controls transcription. This is called the promotor
region. It contains a TATA box that is essential for transcription. Following the promotor
region there is a code for initiation of transcription. This is followed by the code for initiation of
translation (ATG). At the 3 end the flanking region consists of a translation termination codon
(TAA), which is followed by a poly (A) cap codon. For initiation of transcription it is necessary
that the promotor region (TATA Box) should bind to RNA polymerase. However, in order to
bind to this site the polymerase requires additional proteins called transcription factors.
Transcription factors acting in combination with other proteins activate DNA transcription

Chapter CD 3 Molecular Biology

Fig. CD-3.4: A structural gene.

(gene expression). DNA transcription starts at the 5 end and ends at the 3 end of a gene. The
flanking region of the 3 end helps in the stabilization of newly formed mRNA and allows it to
go out of the nucleus.

SYNTHESIS OF PROTEIN
Some facts about protein synthesis have been given in Chapter 1. Further details are given
here.

Two processes are involved in the synthesis of protein. These are (1) Transcription and (2)
Translation.

Transcription
In the process of trancription genetic information stored in the DNA of a gene is transmitted to
messenger RNA (mRNA) (Fig. CD-3.5). This is the first step in the formation of protein.

The steps in the process of transcription are as follows
1. The two strands of the DNA double helix separate from each other. This happens because
of the breakage of hydrogen bonds between nitrogenous base pairs. This is achieved by the
activation of transcription factors and the release of RNA polymerase in the promoter
region of the gene.
2. Only one strand of the DNA double helix is used for the synthesis of an mRNA molecule.
3. Transcription begins at the 5 end and ends at the 3 end of the gene.
4. Each base in the newly synthesized mRNA molecule is complementary to a corresponding
base in the DNA of the gene. Thus information of a particular gene (DNA strand) is
transformed to mRNA unchanged.
5. In a strand of mRNA all the sequences present in a structural gene, i.e., both extrons and
introns, are transcribed.

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Fig. CD-3.5: (A) Transcription of mRNA from DNA. (B). 5 capping and 3 end polyadenylation.
(C), and splicing of mRNA to get mature RNA.

6. The non-coding sequences (introns) intervening between exons are excised (Fig. CD-3.5).
The exons are then joined together to form mature RNA. It is obvious that mature RNA
does not have any introns and therefore becomes shorter. This process of removal of
introns (by cutting them off and joining the ends of exons) is known as splicing.
7. A molecule of methylguanine gets attached to the 5 end. It is called the methylguanine
cap. This 5 cap protects mRNA from degradation and facilitates transport of mRNA to
cytoplasm. Similarly, the 3 end of mRNA bears a poly (A) tail, which also protects mRNA
from degradation and facilitates the transport of mRNA to cytoplasm.
8. The mRNA then migrates from nucleus to cytoplasm where it attaches to ribosomes for
synthesis of protein (translation).

Translation
1. Messenger RNA passes from the nucleus to the cytoplasm. Here it becomes attached to a
ribosome.
2. The cytoplasm also contains another form of RNA called transfer RNA. On one side transfer
RNA becomes attached to an amino acid. On the other side it bears a code of three bases
(anticodon) that are complementary to the bases coding for its amino acid on messenger

Chapter CD 3 Molecular Biology

RNA. Under the influence of the ribosome several units of transfer RNA, along with their
amino acids, become arranged alongside the strand of messenger RNA in the sequence
determined by the code on messenger RNA. This process is called translation.
3. The amino acids now become linked to each other to form a polypeptide chain.

From the above it will be clear that the amino acids are linked up exactly in the order in
which their codes are arranged on messenger RNA, which in turn is based on the code on
the DNA molecule.
4. Chains of amino acids formed in this way constitute polypeptide chains. Proteins are
formed by union of polypeptide chains.
5. The flow of information from DNA to RNA and finally to protein has been described as the
central dogma of molecular biology.

Some Further Details about Genes and Protein Synthesis

1. At one time it was taught that one gene was responsible for the synthesis of only one
protein. However, it is now known that although there are only about 35000 genes in the
human genome, the number of proteins is more than one hundred thousand. It is obvious
that one gene must have the ability to synthesize several proteins.The mechanism by
which a single gene can control the synthesis of many proteins is explained below. The
process is called alternative splicing.

We have seen that a strand of newly formed RNA contains introns and exons. The introns
have to be removed by splicing for the strand to become mature. The nature of protein
synthesized can be influenced by the exact introns removed because the sequence of
amino acids is affected by removal of introns.

In the process of alternative splicing the exons are spliced in different patterns (Fig. CD3.6). As a result, polypeptide chains with differing sequences of amino acids present are
produced. This leads to the synthesis of different proteins.

The function of the protein, synthesized under the influence of mRNA, can also be
modified by its phosphorylation, or by its combination with other proteins. This explains
why the number of proteins exceeds by almost three times the number of genes present in
the human genome.
2. Cells of one type differ from those of other types because they synthesize different proteins,
including enzymes. However, we have also seen that each somatic cell of the body has
exactly the same complement of genetic material as the fertilized ovum (in the form of
DNA). How is it then that different cell types come to produce different types of protein i.e.,
cells in the skin produce keratin, those of the endocrine pancreas synthesize insulin, and
red blood cells produce hemoglobin ?
3. The answer to this question lies in the concept that in any given cell only a few of its
genes are active, and the others are resting. A gene that is active is said to be expressed.
Differentiation of cells takes place because of the expression of a small number of
developmental regulatory genes (master genes) acting at specific times of development.
4. In addition to the protein coding sequences (of bases) DNA also bears other regions that
have a controlling function. These regions provide signals for initiation and termination

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Fig. CD-3.6: Diagram showing the process of alternative splicing. (A) Transcription of a structural gene
may form mature mRNA in which all exons are present; and (B and C) where one exon is excluded in
the process of splicing. In this way a single gene can form three different kinds of protein.

of the process of transcription, or for the control of the process in other ways. The DNA
sequence that provides the signal for initiation of transcription is called the promoter.
Binding of RNA polymerase to the promoter causes the DNA fibre to uncoil, and makes it
possible for RNA polymerase to reach the fibre. The process transcription begins in this way.
However, to bind to the promoter region the RNA polymerase also needs a transcription
factor. Transcription factors (gene regulatory proteins) are present in the nucleus. They
determine the region of the DNA to be transcribed. Transcription ends when a signal for
termination of transcription is encountered.

MOLECULAR CONTROL OF GROWTH AND

DIFFERENTIATION DURING DEVELOPMENT
At present it is well known that several genes and gene families play important roles in the
development of the embryo. Most of these genes produce transcription factors (described
above) that control RNA transcription. Transcription factors play an important role in gene
expression as they can switch genes on and off by activating or repressing them. It is believed
that many transcription factors control other genes, which regulate fundamental embryological
processes like induction, segmentation, migration, differentiation and programmed cell
death (apoptosis). These fundamental embryological processes are mediated by growth and
differentiation factors, growth factor receptors and various cytoplasmic proteins.

Several components are required for the expression of a given gene. These are:
1. Growth factors, which act as cell signaling molecules for induction of cellular
differentiation.

Chapter CD 3 Molecular Biology

2. Receptors, which are present in the cell membrane. Their function is to recognise and
respond to growth factors.
3. Activation of signal tranducing proteins within the cell cytoplasm.
4. Activation of transcription factor, which binds to DNA in the nucleus and finally leads to
transcription. In other words the gene is now expressed.
Thus two different categories of molecules play an important role in embryonic
development. These are signaling molecules and transcription factors.

The signaling molecules (like growth factors) are present outside the cell and exert their
effects on neighbouring cells, or on cells located at a distance. These signaling molecules act
by binding to the receptors present on the plasma membrane of the cell and ultimately activate
the transcription factors.

The transcription factors are gene regulatory proteins, which are present in the nucleus.
Transcription factors are responsible for gene expression and are therefore important
molecules for control of embryonic development.

Growth and Differentiation Factors

The term growth factor refers to a naturally occurring protein capable of stimulating cellular
proliferation and cellular differentiation. Growth factors are important for regulating a variety
of cellular processes.

There are different factors for different cells. The epidermal growth factor (EGF) stimulates
epidermal cells. The fibroblast growth factor (FGF) stimulates fibroblasts. The platelet derived
growth factor (PDGF) stimulates the proliferation of connective tissues.
Growth factors act typically as signaling molecules between cells in embryos. Some
examples are cytokines and hormones that bind to specific receptors on the surfaces of their
target cells.

As described above, cell to cell signaling is necessary for induction of cellular differentiation.
In this process one group of cells sends signals to another group of cells causing them to
change their morphology and function. The first group of cells, which sends signals, is called
the inducer. The second group of cells, which responds to the signal, is called the responder.
This kind of interaction between tissues is a common occurrence during embryonic
development.

Some examples are:
1. interaction between epithelium and underlying mesenchymal tissue.
2. interactions between two different types of epithelial tissues.

This kind of interaction leads to differentiation of new tissues (or organs). For example,
interaction between the endoderm of the ureteric bud and the mesenchyme of the metanephric
blastima leads to differentiation of nephrons in the kidney. Similarly, induction of the lens by
the epithelium of the optic cup is an example of interaction between two epithelial tissues.

Signals, in the form of growth and differentiation factors, are transmitted from one cell to
another by endocrine, paracrine or juxtacrine interactions.
1. Endocrine signals: These signals are hormones, which travel through the blood to reach a
distant place in the body.

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Fig. CD-3.7: Growth and differentiation factors.

2. Paracrine signals: These signals target cells, which are present in the neighbourhood of
the emitting cell.
3. Juxtacrine signals: In this kind of signaling it is necessary that adjacent cells should be
in cell to cell physical contact. Well known examples of these are signals passing through
gap junctions and notch signaling (described later in this chapter).
4. Sometimes signals may act on the same cell that secreted them. This kind of interaction in
called autocrine.

HEDGEHOG PROTEINS
Embryological development is most influenced by Hedgehog proteins, which act as signaling
molecules. In developing embryos, sonic hedgehog acts as a signaling molecule at many
places. Some of these are:
1. the notochord,
2. the neuro-ectoderm,

Chapter CD 3 Molecular Biology

3. the primitive node,

4. the zone of polarising activity in a limb,
5. the genital tubercle,
6. the retina,
7. hair buds, and
8. lung buds.
This molecule is very active throughout the process of embryogenesis. Mutation of the
sonic hedgehog gene causes incomplete cleavage of the developing brain into right and left
cerebral hemisphere and cyclopia (holoprosencephaly).

Growth Factor Receptors

Molecules that carry a signal to a receptor are called ligands. A ligand may be a hormone, a
cytokine or a growth factor. The main function of a receptor is to recognise and respond to
specific ligands such as growth factors and hormones.
The transmembrane receptors are protein in nature. They reside within the plasma
membrane of a cell. They have an extracellular domain (that binds the ligand), a transmembrane domain and a cytoplasmic domain. They bind to the specific signaling molecules
on the outer side of the membrane and initiate tyrosine kinase activity on the inner side of the
membrane. This is followed by the activation of cytoplasmic protein kinases.

Notch Receptors
The notch receptor is another kind of surface receptor. These receptors respond to juxtacrine
signaling and play an important role in embryonic development. In juxtacrine signaling a
protein on one cell surface interacts with a receptor on an adjacent cell surface. The notchsignaling pathway is an important mechanism of neuronal differentiation, blood vessel
specification, and somite segmentation. In the mechanism of neuronal differentiation, in a
population of developmentally equivalent cells only a few cells develop into neurons while
many adjoining cells develop into glial cells. The maturing neuronal cells are dominant. They
inhibit the maturation of neighbouring cells into neurons, and make them develop into glial
cells. This phenomenon is known as lateral inhibition.

Transcription Factors
Transcription factors regulate gene expression by acting on promoter or enhancer regions
of specific genes. They bind to promoter region of the gene (along with RNA polymerase) to
initiate the process of transcription.

HOX Genes
In humans, HOX genes encode special kinds of transcription factors that are involved in the
regulation of segmentation, of the patterning of the hind brain, and of the formation of the axis
of the embryo (including limb bud axis and genital axis).

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The HOX genes are responsible for cranial to caudal patterning of the derivatives of germ
layers (ectoderm, mesoderm and endoderm).

HOX genes regulate the differentiation of somites, vertebrae and hindbrain segmentation.

Some other factors and genes that affect embryonic growth are as follows.
1. Vitamin A (retinoic acid) has been identified as an important regulatory substance in
embryonic development. It acts as a transcription factor for specific genes that are involved
in embryonic patterning.
2. The PAX genes (or paired box genes) influence the development of sense organs (eye and
ear) and of the nervous system.
3. The POU genes play a vital role in cleavage of early embryonic cells.
4. The Lim genes regulate muscle differentiation.
5. The Dlx genes are involved in morphogenesis of the jaw and of the internal ear.