Molecular Control of Development

Some elementary facts about control of development

1. It has been observed that certain regions of the embryo have the ability to inuence the differentiation of neighbouring regions. One of the most interesting instances of this phenomenon is the inuence 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 inuence exerted by such an area (i.e. optic vesicle) is called induction whereas the area exerting the inuence is called an organiser. 2. The 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. This indicateds that signals for the development of the embryo have originated from the dorsal lip and have inuenced the differentiation of surrounding tissues.

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Organisers that appear later in development have correspondingly lesser effects. 3. It is now known that the organisers exert their inuence 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 inuence 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 described as Molecular control of development. Genes exert their inuence 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 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 alredy know all information in cells is stored in molecules of DNA. To

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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.

understand genetic processes we have to rst know some facts about DNA structure.

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

DNA STRUCTURE
Some elementary facts about the structure have been given in Chapter 1. Here we will consider further details. 1. We have seen that each strand of the DNA bre 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 which 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 xed i.e., adenine (A) always pairs with thymine (T), and cytosine (C) with guanine (G). The specic pairing 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 specic base pairing the two strands of DNA are complementary to each

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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.

other. If the sequence of bases on one chain is ATGCA then the corresponding region on other chain will have the sequence TACGT. 7. Two complementary chains (polynucleotide chains) of DNA are twisted around each other to form what is called double helix. 8. Every protein is made up of polypeptide chains, which in-turn 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 inuence 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 dened as a segment of DNA which contains the information (code) for the synthesis of one complete polypeptide chain (or an enzyme). Thus a gene is nothing but a set of instructions for making proteins.

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As each polypeptide chain is made of sequential arrangement of specic amino acids, it was expected that there must be a contiguous sequence of DNA coding for these amino acids in a structural gene. However, it is now known that there are many non-coding sequences, which are called 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) but are not included in mature RNA. A structural gene not only contains the sequence of exons and introns (explained above) but also possesses anking regions at ends. These anking regions are important for regulation of gene expression (Fig. CD-3.4). At the 5 end the anking region is made up of DNA sequences, which controls transcription. This region 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, which is followed by the code for initiation of translation (ATG). At the 3 end the anking 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 (gene expression). DNA transcription starts at the 5 end and ends at the 3 end of a gene. The anking region of the3 end helps in the stabilization of newly formed mRNA and allows it to go out of the nucleus.

SYNTHESIS OF PROTEIN
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 rst 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 are separated from each other. This happens because of the breakage of hydrogen bonds between nitrogenous base pairs, This is achieved by the activation of transcription

Fig. CD-3.4. Structure of a structural gene.

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Transcription initiation 5 3 Transcription starts TATA Exon Intron

Transcription termination 3 5 Transcription stops

B Primary mRNA 3 5 cap Splicing C Mature mRNA Poly (A)

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

2.

3. 4.

5.

6.

factors and release of RNA polymerase in the promoter region of the gene. Only one strand of the DNA double helix is used for the synthesis of an mRNA molecule. Transcription begins at the 5 end and ends at the 3 end of the gene. Each base in 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. In a strand of mRNA all the sequences present in a structural gene, i.e., both extrons and introns, are transcribed. 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 is therefore shorter. This process of removal of introns (by cutting them off and joining the ends of extrons) 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. As stated above, in the cytoplasm messenger RNA 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 RNA. Under the inuence of the ribosome several units

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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 ow of information from DNA to RNA and nally to protein has been described as the central dogma of molecular biology.

Some Further Details About Genes And Protein Synthesis

(a) At one time it was taught that one gene produced only one protein or enzyme. However, it is now known that although there are only about 35000 genes in the human genome, proteins number more than 100000. The mechanism by which a single gene can give rise to many proteins is explained below.

As stated earlier, a single gene can synthesise more then one protein. This is achieved by the process called alternative splicing. In this process the exons are spliced in different patterns (Fig. CD-3.6). The absence of one or two exons in mRNA changes the sequence of amino acids present in the resulting polypeptide chain. In this way different proteins are formed. Similarly, the function of the protein, made from the mRNA, can also be modied 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. (b) Another important fact about gene regulation of development is that cells of one type differ from those of other types because they synthesize different proteins, including enzymes. However, we have also seen that each cell of the body (except a germ cell) has exactly the same complement of genetic material (in the form of DNA) as the fertilized ovum. How is it then that different cell types come to produce different types of protein i.e., skin cells produce keratin, those of endocrine pancreas synthesize insulin and red blood cells produce hemoglobin etc.?

Ribosome mRNA 5 AUG UUC GCC UAC tRNA AUG UUC GCC AGG CCG GUG UCC UAG 3 AGG

Met Amino acid

Met Ser

Gly

Ph

Leu Ser
Polypeptide chain

1.Initiation

2.Elongation

3.Termination

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

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Fig. CD-3.7. Diagram showing the process of alternative splicing. Transcription of a structural gene may form a mature mRNA in which all exons are present (A); where one exon is exclued in the process of splicing (B and C). Thus a single gene can form three different kinds of protein.

(c) The answer to this question lies in the concept that in any given cell only a few of its genes are active and others 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 specic times of development. (d) 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 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 bre to uncoil, and thus makes it possible for RNA polymerase to reach the bre and to begin the process of transcription. However, to bind to the promoter region the RNA polymerase also needs transcription factor. The transcription factors (gene regulatory proteins) are present in the nucleus. They

determine the region of the DNA to be transcribed. Transcription continues up to the region of the DNA bre that bears a code that gives a signal for termination of transcription.

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), which 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 many other genes, which regulate fundamental embryological processes like induction, segmentation, migration, differentiation and apoptosis (programmed cell death). The above fundamental embryological processes are mediated by growth and differentiation factors, growth factor receptors and various cytoplasmic proteins.

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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. 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 nally leads to transcription. In other words the gene is now expressed. Thus two different categories of molecules play an important role in embryonic development i.e., signaling molecules and transcription factors. The signaling molecules, like growth factors, are present outside the cell and exert their effects on the neighboring cells or 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.

specic 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 to change their morphology and function. The rst group of cells which sends signals is called the inducer and the second group which responds to the signal is called responder. During embryonic development this kind of the interaction between tissues is a common occurrence. For example interaction between epithelium and underlying mesenchymal tissue, and interactions between two different types of epithelial tissues are most common types of interactions. This kind of interaction leads to differentiation of new tissues (or organs). For example, interaction between endoderm of ureteric bud and the mesenchyme of metanephric blastima leads to differentiation of nephrons in the kidney. Similarly, induction of 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 (Fig. CD-3.8). Endocrine signals: These signals are hormones, which travel through the blood to reach a distant place in the body. Paracrine signals: These signals target cells, which are present in the neighbourhood of the emitting cell. 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). Sometimes signals may act on the same cell that secreted them. This kind of interaction in called as autocrine. Some common growth and differentiation factors are given in Table CD-3.1.

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. These factors are different for different cells e.g., the epidermal growth factor (EGF) stimulates epidermal cells; the broblast growth factor (FGF) stimulates broblasts; the platelet derived growth factor (PDGF) stimulates the proliferation of connective tissues. Growth factors typically act as signaling molecules between cells in embryos. Examples are cytokines and hormones that bind to

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Endocrine 9 land Hormone Blood verssel Receptoss Target cells situated at a distance (A) Enderine signals reach to target cell through blood at a distance

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 specic 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 specic 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.

Receptor

Paracrine cells

Target cells

(B) Paracrine cells are present closer to target cells

Gap junction

Notch Receptors
Another kind of surface receptor is called the notch 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 specication, 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 neighbouring cell 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.

Juxtacrine cells (C) Juxtacrine signals pass in adjecent cells which are in physical contacts with each other

Fig. CD-3.8. Different types of cell to cell signals. A - Endocrine. B - Paracrine. C - Juxtacrine.

HEDGEHOG PROTEINS
Embryological development is most inuenced by Hedgehog proteins, which act as signaling molecules. Sonic hedgehog acts as a signaling molecule in developing embryos at places like the notochord, the neuro-ectoderm, the primitive node, the zone of polarising activity in a limb, the genital tubercle, the retina, hair buds, the lung buds and others. This molecule is very active throughout the process of embryogenesis. Mutation of the sonic hedgehog gene causes incomplete cleavage of the developing brain into

Transcription factors
Transcription factors regulate gene expression by acting on promoter or enhancer regions

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Table CD-3.1. Growth and differentiation factors.

Growth Factor Families 1. Epidermal Growth Factor(EGF) 2. Transforming Growth Factors (TGFs)TGFB1 to TGFB5 Bone Morphogenetic Factors (BMP 1 to 9) Mullerian Inhibiting Factor (MIF) Nodal Lefty 3. Hedgehog Proteins Sonic Hedgehog (Shh), Desert, Indian 4. WNT Protein 5. Fibroblast Growth Factors (FGFs) Functions Growth and proliferation of cells of ectodermal and mesodermal origin. Forms the extracellular matrix, induces epithelial branching, myoblast proliferation Bone formation, cell division, cell migration and apoptosis Regression of paramesonephric duct. Formation of primitive streak, right-left axial xation, formation of mesoderm. Determination of body asymmetry. Shh controls neural tube formation, somite differentiation, gut formation, limb development, growth of genital tubercle. Midbrain development, somite and urogenital differentiation, limb patterning. Mesoderm differentiation, angiogenesis, axon growth, limb development, development of various parts of brain, early liver induction, mesenchymal proliferation in jaw, induction of prostate gland, outgrowth of genital tubercle. IGF-1 acts as a factor for bone growth, IGF-2 is a fetal growth factor.

6. Insulin-like Growth Factors (IGFs)

of specic genes (i.e., they bind to promoter region of the gene along with RNA polymerase to initiate the process of transcription).

It acts as transcription factor for specic genes that are involved in embryonic patterning.

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

PAX Genes (paired box genes)

These genes shape the development of sense organs (eye and ear) and the nervous system..

POU Genes
POU genes play a vital role in cleavage of the early embryonic cells.

Lim Proteins
Lim genes regulate muscle differentiation.

Retinoic acid
Vitamin A has been identied as an important regulatory substance in embryonic development.

Dlx Genes
These genes are involved im morphogenesis of the jaw and of the internal ear.

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