Some Elementary Facts about Control of Development

1. It has been observed that certain regions of the embryo have the ability to infl uence the diff erentiation of neighbouring regions. One of the most interesting instances of this phenomenon is the infl uence 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. Th ese experiments show that the optic vesicle induces the formation of the lens vesicle.Th e infl uence exerted by such an area (i.e. optic vesicle) is called induction whereas the area exerting the infl uence is called an organiser.

2. Th e fi rst 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. Th is indicates that signals for the development of the embryo have originated from the dorsal lip of the blastopore and have infl uenced the diff erentiation of surrounding tissues. Organisers that appear later in development have correspondingly lesser eff ects.

3. It is now known that the organisers exert their infl uence by elaborating chemical substances, which are probably complex proteins, including enzymes.

4. Th e chemical substances elaborated by the organiser may be: a. inductors which stimulate the tissue to diff erentiate in a particular manner; or b. inhibitors which have a restraining infl uence on diff erentiation. Th e 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 infl uence on cellular functions by synthesis of proteins. Th e proteins synthesised diff er from cell to cell and within the same cell at diff erent times. Th is provides

Molecular Biology

Chapter CD 3

Human Embryology

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 5’P 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 5’–3’ direction, the other in 3’–5’direction (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

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

Fig. CD-3.2: A polynucleotide chain. (A) Schematic diagram. (B) Chemical structure of a nucleotide chain.

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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

(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.

Fig. CD-3.4: A structural gene.

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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

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.

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. CD-3.6). 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

Human Embryology

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.

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.

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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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,

Fig. CD-3.7: Growth and differentiation factors.

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 trans-membrane 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 notch-signaling 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).

Human Embryology

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.