Molecular biology has opened the doors to new ways to study embryology and to enhance our understanding of normal and abnormal development. Sequencing the human genome, together with creating techniques to investigate gene regulation at many levels of complexity, has taken embryology to the next level. Thus, from the anatomical to the biochemical to the molecular level, the story of embryology has progressed, and each chapter has enhanced our knowledge.
There are approximately 23,000 genes in the human genome, which represents only one fifth of the number predicted prior to completion of the Human Genome Project. Because of various levels of regulation, however, the number of proteins derived from these genes is closer to the original predicted number of genes. What has been disproved is the one-gene–one- protein hypothesis. Thus, through a variety of mechanisms, a single gene may give rise to many proteins.
Gene expression can be regulated at several levels: (1) different genes may be transcribed, (2) nuclear deoxyribonucleic acid (DNA) transcribed from a gene may be selectively processed to regulate which RNAs reach the cytoplasm to become messenger RNAs (mRNAs), (3) mRNAs may be selectively translated, and (4) proteins made from the mRNAs may be differentially modified.
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Gene Transcription
Genes are contained in a complex of DNA and proteins (mostly histones) called chromatin, and its basic unit of structure is the nucleosome. Each nucleosome is composed of an octamer of histone proteins and approximately 140 base pairs of DNA. Nucleosomes themselves are joined into clusters by binding of DNA existing between nucleosomes (linker DNA) with other histone proteins. Nucleosomes keep the DNA tightly coiled, such that it cannot be transcribed. In this inactive state, chromatin appears as beads of nucleosomes on a string of DNA and is referred to as heterochromatin. For transcription to occur, this DNA must be uncoiled from the beads. In this uncoiled state, chromatin is referred to as euchromatin.
Genes reside within the DNA strand and contain regions called exons, which can be translated into proteins, and introns, which are interspersed between exons and which are not transcribed into proteins. In addition to exons and introns, a typical gene includes the following: a promoter region that binds RNA polymerase for the initiation of transcription; a transcription initiation site; a translation initiation site to designate the first amino acid in the protein; a translation termination codon; and a 3' untranslated region that includes a sequence (the poly A addition site) that assists with stabilizing the mRNA, allows it to exit the nucleus, and permits it to be translated into protein. By convention, the 5' and the 3' regions of a gene are specified in relation to the RNA transcribed from the gene. Thus, DNA is transcribed from the 5' to the 3' end, and the promoter region is upstream from the transcription initiation site. The promoter region, where the RNA polymerase binds, usually contains the sequence TATA, and this site is called the TATA box.
In order to bind to this site, however, the polymerase requires additional proteins called transcription factors. Transcription factors also have a specific DNA-binding domain plus a transactivating domain that activates or inhibits transcription of the gene whose promoter or enhancer it has bound. In combination with other proteins, transcription factors activate gene expression by causing the DNA nucleosome complex to unwind, by releasing the polymerase so that it can transcribe the DNA template, and by pre- venting new nucleosomes from forming.
Enhancers are regulatory elements of DNA that activate utilization of promoters to control their efficiency and the rate of transcription from the promoter. Enhancers can reside anywhere along the DNA strand and do not have to reside close to a promoter. Like promoters, enhancers bind transcription factors (through the transcription factor’s transactivating domain) and are used to regulate the timing of a gene’s expression and its cell-specific location. For example, separate enhancers in a gene can be used to direct the same gene to be expressed in different tissues. The PAX6 transcription factor, which participates in pancreas, eye, and neural tube development, contains three separate enhancers, each of which regulates the gene’s expression in the appropriate tissue. Enhancers act by altering chromatin to expose the promoter or by facilitating binding of the RNA polymerase. Sometimes, enhancers can inhibit transcription and are called silencers. This phenomenon allows a transcription factor to activate one gene while silencing another by binding to different enhancers. Thus, transcription factors themselves have a DNA-binding domain specific to a region of DNA plus a transactivating domain that binds to a promoter or an enhancer and activates or inhibits the gene regulated by these elements.
DNA Methylation Represses Transcription
Methylation of cytosine bases in the promoter regions of genes represses transcription of those genes. Thus, some genes are silenced by this mechanism. For example, one of the X chromosomes in each cell of a female is inactivated (X chromosome inactivation) by this methylation mechanism. Similarly, genes in different types of cells are repressed by methylation, such that muscle cells make muscle proteins (their promoter DNA is mostly unmethylated), but not blood proteins (their DNA is highly methylated). In this manner, each cell can maintain its characteristic differentiated state. DNA methylation is also responsible for genomic imprinting in which only a gene inherited from the father or the mother is expressed, while the other gene is silenced. Approximately 40 to 60 human genes are imprinted and their methylation pat- terns are established during spermatogenesis and oogenesis. Methylation silences DNA by inhibiting binding of transcription factors or by altering histone binding resulting in stabilization of nucleosomes and tightly coiled DNA that cannot be transcribed.
Other Regulators Of Gene Expression
The initial transcript of a gene is called nuclear RNA (nRNA) or sometimes premessenger RNA. nRNA is longer than mRNA because it contains introns that are removed (spliced out) as the nRNA moves from the nucleus to the cytoplasm. In fact, this splicing process provides a means for cells to produce different proteins from a single gene. For example, by removing different introns, exons are “spliced” in different patterns, a process called alternative splicing. The process is carried out by spliceosomes, which are complexes of small nuclear RNAs (snRNAs) and proteins that recognize specific splice sites at the 5' or the 3' ends of the nRNA. Proteins derived from the same gene are called splicing isoforms (also called splice variants or alternative splice forms), and these afford the opportunity for different cells to use the same gene to make proteins specific for that cell type. For example, isoforms of the WT1 gene have different functions in gonadal versus kidney development.
Even after a protein is made (translated), there may be post-translational modifications that affect its function. For example, some proteins have to be cleaved to become active, or they might have to be phosphorylated. Others need to combine with other proteins or be released from sequestered sites or be targeted to specific cell regions. Thus, there are many regulatory levels for synthesizing and activating proteins, such that although only 23,000 genes exist, the potential number of proteins that can be synthesized is probably closer to five times the number of genes.
Induction And Organ Formation
Organs are formed by interactions between cells and tissues. Most often, one group of cells or tissues causes another set of cells or tissues to change their fate, a process called induction. In each such interaction, one cell type or tissue is the inducer that produces a signal, and one is the responder to that signal. The capacity to respond to such a signal is called competence, and competence requires activation of the responding tissue by a competence factor. Many inductive interactions occur between epithelial and mesenchymal cells and are called epithelial–mesenchymal interactions. Epithelial cells are joined together in tubes or sheets, whereas mesenchymal cells are fibroblastic in appearance and dispersed in extracellular matrices. Examples of epithelial–mesenchymal interactions include the following: gut endoderm and surrounding mesenchyme to produce gut-derived organs, including the liver and pancreas; limb mesenchyme with overlying ectoderm (epithelium) to produce limb outgrowth and differentiation; and endoderm of the ureteric bud and mesenchyme from the metanephric blastema to produce nephrons in the kidney. Inductive interactions can also occur between two epithelial tissues, such as induction of the lens by epithelium of the optic cup. Although an initial signal by the inducer to the responder initiates the inductive event, crosstalk between the two tissues or cell types is essential for differentiation to continue.
Cell Signaling
Cell-to-cell signaling is essential for induction, for conference of competency to respond, and for crosstalk between inducing and responding cells. These lines of communication are established by paracrine interactions, whereby proteins synthesized by one cell diffuse over short distances to interact with other cells, or by juxtacrine interactions, which do not involve diffusable proteins. The diffusable proteins responsible for paracrine signaling are called paracrine factors or growth and differentiation factors (GDFs).
Signal Transduction Pathways
Paracrine Signaling
Paracrine factors act by signal transduction pathways either by activating a pathway directly or by blocking the activity of an inhibitor of a pathway (inhibiting an inhibitor, as is the case with hedgehog signaling). Signal transduction pathways include a signaling molecule (the ligand) and a receptor. The receptor spans the cell membrane and has an extracellular domain (the ligand-binding region), a transmembrane domain, and a cytoplasmic domain. When a ligand binds its receptor, it induces a conformational change in the receptor that activates its cytoplasmic domain. Usually, the result of this activation is to confer enzymatic activity to the receptor, and most often this activity is a kinase that can phosphorylate other proteins using ATP as a substrate. In turn, phosphorylation activates these proteins to phosphorylate additional proteins, and thus a cascade of protein interactions is established that ultimately activates a transcription factor. This transcription factor then activates or inhibits gene expression. The pathways are numerous and complex and in some cases are characterized by one protein inhibiting another that in turn activates another protein (much like the situation with hedgehog signaling).
Juxtacrine Signaling
Juxtacrine signaling is mediated through signal transduction pathways as well but does not involve diffusable factors. Instead, there are three ways juxtacrine signaling occurs: (1) A protein on one cell surface interacts with a receptor on an adjacent cell in a process analogous to paracrine signaling. The Notch pathway represents an example of this type of signaling. The Notch receptor protein extends across the cell membrane and binds to cells that have Delta, Serrate, or Jagged proteins in their cell membranes. Binding of one of these proteins to Notch causes a conformational change in the Notch protein such that part of it on the cytoplasmic side of the membrane is cleaved. The cleaved portion then binds to a transcription factor to activate gene expression. Notch signaling is especially important in neuronal differentiation, blood vessel specification, and somite segmentation. (2) Ligands in the extracellular matrix secreted by one cell interact with their receptors on neighboring cells. The extracellular matrix is the milieu in which cells reside. This milieu consists of large molecules secreted by cells including collagen, proteoglycans (chondroitin sulfates, hyaluronic acid, etc.), and glycoproteins, such as fibronectin and laminin.
These molecules provide a substrate for cells on which they can anchor or migrate. For example, laminin and type IV collagen are components of the basal lamina for epithelial cell attachment, and fibronectin molecules form scaffolds for cell migration. Receptors that link extracellular molecules such as fibronectin and laminin to cells are called integrins. These receptors “integrate” matrix molecules with a cell’s cytoskeletal machinery (e.g., actin microfilaments) thereby creating the ability to migrate along matrix scaffolding by using contractile proteins, such as actin. Also, integrins can induce gene expression and regulate differentiation as in the case of chondrocytes that must be linked to the matrix to form cartilage. (3) There is direct transmission of signals from one cell to another by gap junctions. These junctions occur as channels between cells through which small molecules and ions can pass. Such communication is important in tightly connected cells like epithelia of the gut and neural tube because they allow these cells to act in concert. The junctions themselves are made of connexin proteins that form a channel, and these channels are “connected” between adjacent cells.
It is important to note that there is a great amount of redundancy built into the process of signal transduction. For example, paracrine signaling molecules often have many family members such that other genes in the family may compensate for the loss of one of their counterparts. Thus, the loss of function of a signaling protein through a gene mutation does not necessarily result in abnormal development or death. In addition, there is crosstalk between pathways, such that they are intimately interconnected. These connections provide numerous additional sites to regulate signaling.
Paracrine Signaling Factors
There are a large number of paracrine signaling factors acting as ligands, which are also called GDFs. Most are grouped into four families, and members of these same families are used repeatedly to regulate development and differentiation of organ systems. Furthermore, the same GDFs regulate organ development throughout the animal kingdom from Drosophila to humans. The four groups of GDFs include the fibroblast growth factor (FGF), WNT, hedgehog, and transforming growth factor-b (TGF-b) families. Each family of GDFs interacts with its own family of receptors, and these receptors are as important as the signal molecules themselves in determining the outcome of a signal.
Fibroblast Growth Factors
Originally named because they stimulate the growth of fibroblasts in culture, there are now approximately two dozen FGF genes that have been identified, and they can produce hundreds of protein isoforms by altering their RNA splicing or their initiation codons. FGF proteins produced by these genes activate a collection of tyrosine receptor kinases called fibroblast growth factor receptors (FGFRs). In turn, these receptors activate various signaling pathways. FGFs are particularly important for angiogenesis, axon growth, and mesoderm differentiation. Although there is redundancy in the family, such that FGFs can sometimes substitute for one another, individual FGFs may be responsible for specific developmental events. For example, FGF8 is important for development of the limbs and parts of the brain.
Hedgehog Proteins
The hedgehog gene was named because it coded for a pattern of bristles on the leg of Drosophila that resembled the shape of a hedgehog. In mammals, there are three hedgehog genes, Desert, Indian, and sonic hedgehog. Sonic hedgehog is involved in a number of developmental events including limb patterning, neural tube induction and patterning, somite differentiation, gut regionalization, and others. The receptor for the hedgehog family is Patched, which binds to a protein called Smoothened. The Smoothened protein transduces the hedgehog signal, but it is inhibited by Patched until the hedgehog protein binds to this receptor. Thus, the role of the paracrine factor hedgehog in this example is to bind to its receptor to remove the inhibition of a transducer that would normally be active, not to activate the transducer directly.
WNT Proteins
There are at least 15 different WNT genes that are related to the segment polarity gene, wingless in Drosophilia. Their receptors are members of the frizzled family of proteins. WNT proteins are involved in regulating limb patterning, midbrain development, and some aspects of somite and urogenital differentiation among other actions.
The TGF-b Superfamily
The TGF-b superfamily has more than 30 members and includes the TGF-bs, the bone morphogenetic proteins, the activin family, the Müllerian inhibiting factor (MIF, anti-Müllerian hormone), and others. The first member of the family, TGF-b1, was isolated from virally transformed cells. TGF-b members are important for extracellular matrix formation and epithelial branching that occurs in lung, kidney, and salivary gland development. The BMP family induces bone formation and is involved in regulating cell division, cell death (apoptosis), and cell migration among other functions.
Other Paracrine Signaling Molecules
Another group of paracrine signaling molecules important during development are neurotransmitters, including serotonin and norepinephrine, that act as ligands and bind to receptors just as proteins do. These molecules are not just transmitters for neurons, but also provide important signals for embryological development. For example, serotonin (5HT) acts as a ligand for a large number of receptors, most of which are G protein–coupled receptors. Acting through these receptors, 5HT regulates a variety of cellular functions, including cell proliferation and migration, and is important for establishing laterality, gastrulation, heart development, and other processes during early stages of differentiation. Norepinephrine also acts through receptors and appears to play a role in apoptosis (programmed cell death) in the interdigital spaces and in other cell types.
Summary
During the past century, embryology has progressed from an observational science to one involving sophisticated technological and molecular advances. Together, observations and modern techniques provide a clearer understanding of the origins of normal and abnormal development and, in turn, suggest ways to prevent and treat birth defects. In this regard, knowledge of gene function has created entire new approaches to the subject.
There are approximately 23,000 genes in the human genome, but these genes code for approximately 100,000 proteins. Genes are contained in a complex of DNA and proteins called chromatin, and its basic unit of structure is the nucleosome. Chromatin appears tightly coiled as beads of nucleosomes on a string and is called heterochromatin. For transcription to occur, DNA must be uncoiled from the beads as euchromatin. Genes reside within strands of DNA and contain regions that can be translated into proteins, called exons, and untranslatable regions, called introns. A typical gene also contains a promoter region that binds RNA polymerase for the initiation of transcription; a transcription initiation site, to designate the first amino acid in the protein; a translation termination codon; and a 3' untranslated region that includes a sequence (the poly A addition site) that assists with stabilization of the mRNA. The RNA polymerase binds to the promoter region that usually contains the sequence TATA, the TATA box. Binding requires additional proteins called transcription factors. Methylation of cytosine bases in the promoter region silences genes and prevents transcription. This process is responsible for X chromosome inactivation whereby the expression of genes on one of the X chromosomes in females is silenced and also for genomic imprinting in which either a paternal or a maternal gene’s expression is repressed.
Different proteins can be produced from a single gene by the process of alternative splicing that removes different introns using spliceosomes. Proteins derived in this manner are called splicing isoforms or splice variants. Also, proteins may be altered by post-translational modifications, such as phosphorylation or cleavage.
Induction is the process whereby one group of cells or tissues (the inducer) causes another group (the responder) to change their fate. The capacity to respond is called competence and must be conferred by a competence factor. Many inductive phenomena involve epithelial–mesenchymal interactions.
Signal transduction pathways include a signaling molecule (the ligand) and a receptor. The receptor usually spans the cell membrane and is activated by binding with its specific ligand. Activation usually involves the capacity to phosphorylate other proteins, most often as a kinase. This activation establishes a cascade of enzyme activity among proteins that ultimately activates a transcription factor for initiation of gene expression.
Cell-to-cell signaling may be paracrine, involving diffusable factors, or juxtacrine, involving a variety of nondiffusable factors. Proteins responsible for paracrine signaling are called paracrine factors or growth and differentiation factors (GDFs). There are four major families of GDFs: FGFs, WNTs, hedgehogs, and TGF-bs. In addition to proteins, neurotransmitters, such as serotonin (5HT) and norepinephrine, also act through paracrine signaling, serving as ligands and binding to receptors to produce specific cellular responses. Juxtacrine factors may include products of the extracellular matrix, ligands bound to a cell’s surface, and direct cell-to-cell communications.