lock and key hypothesis enzymes

Lock-and-key model

strong>Lock-and-key model n., [lɑk ænd ki ˈmɑdl̩] Definition: a model for enzyme-substrate interaction

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Lock-and-key model Definition

Lock-and-key model is a model for enzyme-substrate interaction suggesting that the enzyme and the substrate possess specific complementary geometric shapes that fit exactly into one another. In this model, enzymes are depicted as highly specific. They must bind to specific substrates before they catalyze chemical reactions . The term is a pivotal concept in enzymology to elucidate the intricate interaction between enzymes and substrates at the molecular level. In the lock-and-key model, the enzyme-substrate interaction suggests that the enzyme and the substrate possess specific complementary geometric shapes that fit exactly into one another. Like a key  into a  lock , only the correct size and shape of the substrate ( the key ) would fit into the  active site  ( the keyhole ) of the enzyme ( the lock ).

Compare: Induced fit model   See also: enzyme , active site , substrate

Lock-and-key vs. Induced Fit Model

At present, two models attempt to explain enzyme-substrate specificity; one of which is the lock-and-key model , and the other is the Induced fit model . The lock and key model theory was first postulated by  Emil Fischer   in 1894. The lock-and-key enzyme action proposes the high specificity of enzymes. However, it does not explain the stabilization of the transition state that the enzymes achieve. The induced fit model (proposed by Daniel Koshland in 1958) suggests that the active site continues to change until the substrate is completely bound to the active site of the enzyme, at which point the final shape and charge are determined. Unlike the lock-and-key model, the induced fit model shows that enzymes are rather flexible structures. Nevertheless, Fischer’s Lock and Key theory laid an important foundation for subsequent research, such as during the refinement of the enzyme-substrate complex mechanism, as ascribed in the induced fit model. The lock-and-key hypothesis has opened ideas where enzyme action is not merely catalytic but incorporates a rather complex process in how they interact with the correct substrates with precision.

Key Components

Components of the lock and key model:

• Enzyme : the enzyme structure is a three-dimensional protein configuration, with an active site from where the substrate binds.
• Substrate : often an organic molecule, a substrate possesses a structural feature that complements the geometry of the enzyme’s active site.

In the lock and key model, both the enzymes and the substrates facilitate the formation of a complex that lowers the activation energy needed for a chemical transformation to occur. Such reduction in the activation energy allows the chemical reaction to proceed at a relatively faster rate, making enzymes crucial in various biological and molecular processes.

Lock-and-key Model Examples

Some of the common examples that are often discussed in the context of the Lock and Key Model are as follows:

• Enzyme lactate dehydrogenase with a specific active site for its substrates, pyruvate and lactate. The complex facilitates the interconversion of pyruvate and lactate during anaerobic respiration
• Enzyme carbonic anhydrase with a specific active site for the substrates carbon dioxide and water. The complex facilitates the hydration of carbon dioxide, forming bicarbonate
• Enzyme lysozyme binding with a bacterial cell wall peptidoglycan, which is a vital immune function

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• Aryal, S. and Karki, P. (2023).  “Lock and Key Model- Mode of Action of Enzymes”. Microbenotes.com. https://microbenotes.com/lock-and-key-model-mode-of-action-of-enzymes/
• Farhana, A., & Lappin, S. L. (2023, May).  Biochemistry, Lactate Dehydrogenase . Nih.gov; StatPearls Publishing. https://www.ncbi.nlm.nih.gov/books/NBK557536/

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Last updated on January 11th, 2024

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Microbe Notes

Lock and Key Model- Mode of Action of Enzymes

Enzymes are biological catalysts. These are commonly proteins but also include RNA (ribozymes) molecules that catalyze chemical reactions by lowering the activation energy of a reaction. These are known to speed up the rate of a reaction millions of times faster than the reaction without enzymes. Nearly all biological reactions require enzymes to transform substrate into products. The substrate is the reactant molecule upon which enzymes act during a chemical reaction, and products are the substances formed as a result of a chemical reaction. A single reactant molecule can decompose to give multiple products. Similarly, two reactants can enter into a reaction to yield products. These are reusable even after the completion of the reaction. Chemical properties such as charge and pH are vital in enzymatic reactions.

Binding between enzymes and reactant molecules takes place in such a way that chemical bond-breaking and bond-forming processes occur more readily. Meanwhile, no change in ∆the G value of a reaction takes place, thereby not altering the energy-releasing or energy-absorbing process of the reaction. However, it lowers the energy of the transition state, the topmost unstable state where the activated complex is formed from reactants that later give products.

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Interesting Science Videos

Enzyme’s Active site and Substrate Specificity

Enzymes are relatively larger than the substrates, whose only a small fraction is involved in catalysis by reducing chemical activation energy, also known as the catalytic site, and the other portion for binding with the substrate and orienting them also known as the binding site. The catalytic site and binding site altogether form the active site of an enzyme. Usually, there are two active sites in an enzyme.

•  The active site of enzymes is a cleft portion, composed of a small number of a unique combination of amino acid residues, usually three to four in number, which make up only ~10-20% of the volume of an enzyme. 
• The remaining amino acids are used to maintain tertiary structure by proper scaffold folding through non-covalent interactions.
• Non-covalent interaction between enzyme and substrate in correct orientation favors their reaction. These interactions include hydrogen bonds , hydrophobic bonds, ionic interactions, and Van der Waal’s interactions.
• However, transient covalent bonds between enzymes and substrates are also formed during the time of reaction.
• Side chains of amino acids play an important role in highly specific three-dimensional conformation at the level of the active site. These are large or small, hydrophilic or hydrophobic, acidic or basic.
• The specific shape, size, and chemical behavior of enzymes are determined by the nature of amino acids and their 3D space in the active site.

Specificity is a distinctive feature of enzymes where they have a unique ability to choose an exact substrate from a group of similar chemical molecules. Their specificity towards their substrate varies to a different extent. These are of different types, namely: Bond specificity, Group specificity. Substrate specificity, Stereospecificity, Geometrical specificity, and Co-factor specificity.

Substrate specificity is also k/a absolute specificity for the enzyme’s specificity towards one substrate and one reaction. For e.g., Lactase acts on the B-1-4 glycosidic linkage of lactose to yield galactose and glucose. The restrictive nature of enzymes towards the choice of substrate can be attributed to the enzymatic activity of two oxidoreductase enzymes. Alcohol dehydrogenase uses its substrate alcohol while lactic acid dehydrogenase act on lactic acid. Although these two enzymes function with the mechanism of oxidation and reduction reaction, their substrates can’t be used interchangeably. This is because the different structure of each substrate prevents their fitting into the active site of the alternative enzyme.

In most cases, cofactors, the non-protein molecules, are required to ensure an efficient enzyme-facilitated chemical reaction. These function to bind with enzymes via either ionic interaction or covalent interactions. Metal ions (such as minerals) and co-enzymes (vitamin derivatives) are cofactors.

Lock and Key Model

A German scientist, Emil Fischer postulated the lock and key model in 1894 to explain the enzyme’s mode of action. Fischer’s theory hypothesized that enzymes exhibit a high degree of specificity towards the substrate. This model assumes that the active site of the enzyme and the substrate fit perfectly into one another such that each possesses specific predetermined complementary geometric shapes and sizes. Thus, the shape of the enzyme and substrate do not influence each other. This specificity is analog to the lock and key model, where the lock is the enzyme, and the key is the substrate. In certain circumstances, if a second substrate similar in shape and size to the primary substrate is made to bind to the enzyme, this second substrate also fits in the active site too.

How does Lock and Key Model work?

• Binding of the substrate(s) to the enzyme at their active site takes place, thereby forming an enzyme-substrate complex.
• Enzymes catalyze the chemical reaction to take place, which can either be a synthesis reaction (favors bond formation) or a decomposition reaction (favors bond breakage).
• As a result, the formation of one or more products takes place, and the enzymes are released for their reuse in the next reaction.

Limitations of Lock and Key Model

• It doesn’t explain how the enzyme-substrate complex is stabilized in the transition state.
• This model supposes the enzyme is a rigid structure whose shape does not change upon binding with a suitable substrate. However, this is not supported by recent research, which states that there is a change in conformation of the active site of the enzyme upon binding of substrate.
• It does not describe the condition for binding multiple substrates to the enzyme.

Later, it was found that enzyme specificity toward one substrate is not always true. Although there are enzymes that specifically bind with only one substrate, there are also enzymes that exhibit broad specificity towards different but similarly structured substrates, such as lipase that can bind to different types of lipids. Similarly, proteases such as trypsin and chymotrypsin can degrade multiple types of proteins. Thus, the lock and key model is flawed, and the induced fit model was introduced to give a more refined view of the mode of enzymatic action.

• Blanco, A., & Blanco, G. (2017). Medical Biochemistry. Academic Press. https://www.khanacademy.org/science/ap-biology/cellular-energetics/enzyme-structure-and-catalysis/a/enzymes-and-the-active-site
• https://www.biologyonline.com/dictionary/substrate-specificity
• https://www.britannica.com/science/protein/The-mechanism-of-enzymatic-action
• https://www.biologyonline.com/dictionary/lock-and-key-model
• https://study.com/learn/lesson/lock-key-model-vs-induced-fit-model.html
• https://chem.libretexts.org/Bookshelves/Organic_Chemistry/Organic_Chemistry_(McMurry)/26%3A_Biomolecules-_Amino_Acids_Peptides_and_Proteins/26.11%3A_Enzymes_and_Coenzymes
• https://en.wikibooks.org/wiki/Structural_Biochemistry/Protein_function/Lock_and_Key
• https://ib.bioninja.com.au/higher-level/topic-8-metabolism-cell/untitled-6/models-of-action.html

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Chapter 13: Chemical Kinetics

Back to chapter, previous video 13.11: catalysis.

Enzymes are biological catalysts that accelerate the rate of biochemical reactions. Most enzymes are proteins, made up of amino acids; while some are RNA molecules known as ribozymes. 

Enzymes act by lowering the activation energy of a reaction, thereby increasing the reaction rate. They can catalyze both synthesis and breakdown of chemical bonds, but do not affect the direction or equilibrium of the reaction. 

Each enzyme binds to a specific reactant, called the substrate, and catalyzes a particular reaction. The substrate binds to a distinct catalytic region of the enzyme called the active site through interactions such as intermolecular forces and transient covalent bonds, resulting in an enzyme-substrate complex.

This binding is highly specific due to the conformational complementarity required between the enzyme and its substrate. Thus, a particular enzyme can only catalyze specific reactions based on its conformation.  

A simple way of understanding complex formation is the lock-and-key model, which hypothesizes that the substrate fits into the enzyme's active site, analogous to a 'key' fitting into its corresponding 'lock'. 

However, another model, the induced-fit model, takes into account the dynamic nature of the complex. This model states that when a substrate binds, it induces small conformational changes, resulting in a tighter fit that favors the reaction. 

The activation energy for a reaction can be lowered by various methods. 

Common mechanisms include inducing conformational changes in the substrate that allow a bond to be broken more easily or bringing the reactive groups of two substrates in close proximity, thereby promoting bond formation.Enzyme activity can be temporarily or permanently suppressed by natural or synthetic molecules called inhibitors.

For example, a competitive inhibitor competes with the substrate to bind to the enzyme's active site, thereby preventing substrate binding.

On the other hand, a noncompetitive inhibitor binds to another location on the enzyme, which causes a conformational change at the active site, reducing the enzyme’s catalytic activity.

Inside living organisms, enzymes act as catalysts for many biochemical reactions involved in cellular metabolism. The role of enzymes is to reduce the activation energies of biochemical reactions by forming complexes with its substrates. The lowering of activation energies favor an increase in the rates of biochemical reactions.

Enzyme deficiencies can often translate into life-threatening diseases. For example, a genetic abnormality resulting in the deficiency of the enzyme G6PD (glucose-6-phosphate dehydrogenase) adversely affects the metabolic pathway supplying NADPH to cells.

A disruption in this metabolic pathway can reduce glutathione in red blood cells causing damage to other enzymes and proteins like hemoglobin. The excessive metabolization of hemoglobin builds up the bilirubin level, which leads to jaundice, a condition that can become severe. Hence, people who suffer from G6PD deficiency must avoid certain foods and medicines containing chemicals that could trigger damage to their glutathione-deficient red blood cells.

Enzyme Function and Structure

Enzymes are grouped into different classes based on the specific function they perform. For example, oxidoreductases are involved in redox reactions, while transferases catalyze the transfer of functional groups. Bond formation with ATP hydrolysis requires ligases, whereas hydrolysis reactions and double bonds formation are catalyzed by hydrolases and lyases, respectively. Isomerase enzymes usually catalyze isomerization reactions.

Enzymes generally possess active sites. These are specific regions on the molecule with a conformation that favors the enzyme to bind to a specific substrate (a reactant molecule) to form an enzyme-substrate complex or the reaction intermediate.

Two models—the lock-and-key model and the induced-fit model—attempt to explain the working of an active site (Figure 1). The most simplistic lock-and-key hypothesis suggests that the active site and the molecular shape of the substrate are complementary—fitting together like a key in a lock (Figure 1a). On the other hand, the induced-fit hypothesis suggests that the enzyme molecule is flexible and changes shape to accommodate a bond with the substrate (Figure 1b).

However, both the lock-and-key model and the induced-fit model account for the fact that enzymes can only bind with specific substrates and catalyzes only a particular reaction.

Figure 1 (a) According to the lock-and-key model, the shape of an enzyme’s active site is a perfect fit for the substrate. (b) According to the induced fit model, the active site is somewhat flexible, and can change shape in order to bond with the substrate.

Enzyme Inhibitors

The activity of enzymes can also be interrupted by the process of enzyme inhibition. There are several common types of enzyme inhibition.

During competitive inhibition, a molecule (natural or synthetic) other than the substrate directly binds to the enzyme's active site. The structural and chemical similarity of the inhibitor to the substrate facilitates its binding to the active site. Such competitive inhibitors, thus, compete with substrates, preventing them from binding to the enzyme. Most often, increasing substrate concentration can suppress the effects of competitive inhibition.

In non-competitive inhibition, a molecule (natural or synthetic) binds to an allosteric (other) region of the enzyme, different from its active site. The inhibitor-binding causes a conformational change to the enzyme's active site, resulting in a decrease in the enzyme's ability to catalyze the reaction. Unlike competitive inhibition, an increase in substrate concentration does not mitigate the inhibitory effects of non-competitive inhibition.

Part of this text is adapted from Openstax, Chemistry 2e, Section 12.7: Catalysis.

Structural Biochemistry/Protein function/Lock and Key

In the Lock and Key Model, first presented by Emil Fisher, the lock represents an enzyme and the key represents a substrate. It is assumed that both the enzyme and substrate have fixed conformations that lead to an easy fit. Because the enzyme and the substrate are at a close distance with weak attraction, the substrate must need a matching shape and fit to join together. At the active sites, the enzyme has a specific geometric shape and orientation that a complementary substrate fits into perfectly. The theory behind the Lock and Key model involves the complementarity between the shapes of the enzyme and the substrate. Their complementary shapes make them fit perfectly into each other like a lock and a key. According to this theory, the enzyme and substrate shape do not influence each other because they are already in a predetermined perfectly complementary shape. As a result, the substrate will be stabilized. This theory was replaced by the induced fit model which takes into account the flexibility of enzymes and the influence the substrate has on the shape of the enzyme in order to form a good fit.

The active site is the binding site for catalytic and inhibition reaction of the enzyme and the substrate; structure of active site and its chemical characteristic are of specificity for binding of substrate and enzyme. Three models of enzyme-substrate binding are the lock-and-key model, the induced fit model, and the transition-state model. The lock-and-key model assumes that active site of enzyme is good fit for substrate that does not require change of structure of enzyme after enzyme binds substrate.

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The Lock and Key Theory: Understanding Enzyme Specificity and Catalysis

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The Lock and Key Theory, introduced by Emil Fischer, is a fundamental concept in biochemistry that explains enzyme specificity. It compares the enzyme's active site to a lock and the substrate to a key, illustrating how only the correct substrate can initiate a reaction. This theory is pivotal in understanding biochemical pathways, organic chemistry, and pharmaceuticals, influencing drug design by targeting enzyme active sites to treat diseases.

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Introduction to the Lock and Key Theory

Definition of the lock and key theory.

The Lock and Key Theory explains the specificity of enzyme action by comparing the enzyme's active site to a lock and the substrate to a key

Elements of the Lock and Key Theory

Enzyme and its active site

Enzymes are specialized proteins with a three-dimensional pocket, called the active site, designed to bind specific substrates

Substrates are molecules that bind to the enzyme's active site, triggering a specific reaction

Enzyme-substrate complex and product

The binding of the substrate to the active site forms an enzyme-substrate complex, which facilitates the conversion of the substrate into the product

Reaction sequence in the Lock and Key Theory

The Lock and Key Theory can be summarized by the reaction sequence \(E + S \rightarrow ES \rightarrow E + P\), where \(E\) represents the enzyme, \(S\) the substrate, \(ES\) the enzyme-substrate complex, and \(P\) the product

Applications of the Lock and Key Theory

Importance in biochemistry and pharmaceutical industry.

The Lock and Key Theory is a fundamental concept in biochemistry and is crucial in drug design for developing specific and effective pharmaceutical agents

Comparison with the Induced Fit Theory

The Induced Fit Theory complements the Lock and Key model by suggesting that the active site is dynamic and can adapt to accommodate slight variations in substrate structure

Educational resources for learning the Lock and Key Theory

Detailed diagrams and glossaries are valuable tools for understanding the step-by-step process of enzyme action and the key terms associated with the Lock and Key Theory

Significance of the Lock and Key Theory

Influence on our understanding of enzymatic function.

The Lock and Key Theory has greatly contributed to our understanding of enzymatic function and the complex chemical processes that sustain life

Practical applications in drug design

The Lock and Key Theory is essential in the development of enzyme inhibitors for treating diseases by disrupting pathological processes

Role in biochemistry education and research

The Lock and Key Theory remains a fundamental concept in biochemistry education and research, providing a starting point for exploring the complexities of enzyme behavior

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Enzymes, which are specialized ______, work as catalysts and their action is explained by the ______ and Key Theory.

proteins Lock

Describe the active site of an enzyme.

Three-dimensional pocket on enzyme surface; binds substrate with high specificity; involves non-covalent interactions like hydrogen bonds, ionic interactions, van der Waals forces.

Explain the enzyme-substrate complex.

Transient complex formed when substrate binds to enzyme's active site; facilitates substrate's conversion into product.

Summarize the reaction sequence in the Lock and Key Theory.

E + S -> ES -> E + P; E is enzyme, S is substrate, ES is enzyme-substrate complex, P is product; enzyme remains unchanged post-reaction.

The ______ and Key Theory is crucial for understanding enzyme catalysis in organic chemistry and drug design in the pharmaceutical industry.

Originator of Induced Fit Theory

Daniel Koshland proposed the Induced Fit Theory in 1958.

Characteristic of enzyme's active site in Induced Fit Theory

Active site is dynamic, molds around substrate upon binding.

Role of substrate structure variability in enzyme specificity

Enzyme adaptability allows accommodation of substrates with slight structural variations.

Educational materials like ______ and glossaries are crucial for grasping the ______ Theory.

detailed diagrams Lock and Key

Lock and Key Theory - Basic Concept

Theory where enzymes and substrates fit together precisely like a lock and key, explaining enzyme specificity.

Enzyme Specificity - Importance

Critical for enzymes to catalyze only the correct reactions, ensuring proper metabolic function.

Drug Design - Lock and Key Relevance

Lock and Key Theory guides creation of enzyme inhibitors that mimic substrates, blocking unwanted reactions in disease.

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The Fundamentals of Enzyme Specificity: Lock and Key Theory

Exploring the Components and Dynamics of the Lock and Key Model

The role of the lock and key theory in organic chemistry and drug discovery, lock and key versus induced fit: diverse models of enzyme specificity, educational resources for grasping the lock and key theory, the enduring influence of the lock and key theory on biochemical science.

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Question Video: Describing the Lock and Key Theory of Enzyme Action Biology • First Year of Secondary School

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Which of the following best describes the lock and key theory of enzyme action? [A] The substrate is the “lock” into which the enzyme, or the “key,” fits. [B] The enzyme and substrate have identical shapes, like a “lock and key.” [C] Once the enzyme and substrate have joined, they are locked together and cannot be separated. [D] The enzyme is the “lock” into which the substrate, or the “key,” fits.

Video Transcript

Which of the following best describes the lock-and-key theory of enzyme action? Option (A) the substrate is the lock into which the enzyme, or the key, fits. Option (B) the enzyme and substrate have identical shapes, like a lock and key. Option (C) once the enzyme and substrate have joined, they are locked together and cannot be separated. Option (D) the enzyme is the lock into which the substrate, or the key, fits.

To answer this question, we need to define the key terms enzyme and substrate and understand the lock-and-key theory of enzyme action. An enzyme is a biological catalyst. It speeds up reactions. Enzymes are proteins that are usually globular in shape. And every enzyme has a specific region called an active site. The active site is unique to each enzyme and specific for a certain substrate.

Now, recall that the substrate refers to the specific molecule that an enzyme acts on. The substrate must be complementary to its particular enzyme’s active site as this is where it binds. Upon binding of the substrate to the enzyme’s active site, it is referred to as the enzyme–substrate complex.

The last part of our question that we need to consider is the lock-and-key theory. The lock-and-key theory of enzyme action proposes that the enzyme’s active site and the shape of the substrate molecule are complementary to one another. This allows the substrate to fit into the enzyme, like how a key would fit into a lock. If the substrate doesn’t fit, then the enzyme will not act on it. Only the correct substrate will bind with the active site. When they fit together correctly, the reaction proceeds and converts the substrate into its products. Recall that enzymes are not consumed during this process.

Now that we’ve reviewed some terminology, let’s take a look at our answers. Because lock-and-key theory of enzyme action refers to a substrate fitting into an enzyme’s active site, we need to find an answer that corresponds to this description. Answer (D) states that the enzyme is the lock into which the substrate, or key, fits. This answer is consistent with the lock-and-key theory, so option (D) is the correct choice.

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key-lock hypothesis

Learn about this topic in these articles:, chromatography.

Very specific intermolecular interactions, “lock and key,” are known in biochemistry. Examples include enzyme-protein, antigen-antibody, and hormone-receptor binding. A structural feature of an enzyme will attach to a specific structural feature of a protein. Affinity chromatography exploits this feature by binding a

…and enzyme, called the “key–lock” hypothesis, was proposed by German chemist Emil Fischer in 1899 and explains one of the most important features of enzymes, their specificity. In most of the enzymes studied thus far, a cleft, or indentation, into which the substrate fits is found at the active…

The key–lock hypothesis ( see above The nature of enzyme-catalyzed reactions) does not fully account for enzymatic action; i.e., certain properties of enzymes cannot be accounted for by the simple relationship between enzyme and substrate proposed by the key–lock hypothesis. A theory called the induced-fit theory retains…

Enzyme Action ( OCR A Level Biology )

Revision note.

Biology & Environmental Systems and Societies

Mechanism of Enzyme Action

• Enzymes have an active site where specific substrates bind forming an enzyme-substrate complex
• The active site of an enzyme has a specific shape to fit a specific substrate
• Extremes of heat or pH can change the shape of the active site, preventing substrate binding – this is called denaturation (the enzyme is said to be denatured )
• Substrates collide with the enzymes active site and this must happen at the correct orientation and speed in order for a reaction to occur

The active site of an enzyme has a specific shape to fit a specific substrate (when the substrate binds an enzyme-substrate complex is formed)

Enzyme specificity

• The specificity of an enzyme is a result of the complementary nature between the shape of the active site on the enzyme and its substrate (s)
• Proteins are formed from chains of amino acids held together by peptide bonds
• The order of amino acids determines the shape of an enzyme
• If the order is altered, the resulting three-dimensional shape changes

An example of enzyme specificity – the enzyme catalase can bind to its substrate hydrogen peroxide as they are complementary in shape, whereas DNA polymerase is not

The enzyme-substrate complex

• An enzyme-substrate complex forms when an enzyme and its substrate join together
• The enzyme-substrate complex is only formed temporarily before the enzyme catalyses the reaction and the product(s) are released

The temporary formation of an enzyme-substrate complex

The lock-and-key hypothesis

• Enzymes are globular proteins
• This means their shape (as well as the shape of the active site of an enzyme) is determined by the complex tertiary structure of the protein that makes up the enzyme and is therefore highly specific
• He suggested that both enzymes and substrates were rigid structures that locked into each other very precisely , much like a key going into a lock
• This is known as the ‘ lock-and-key hypothesis ’

The induced-fit hypothesis

• The lock-and-key model was later modified and adapted to our current understanding of enzyme activity, permitted by advances in techniques in the molecular sciences
• The modified model of enzyme activity (first proposed in 1959) is known as the ‘ induced-fit hypothesis ’
• The enzyme and its active site (and sometimes the substrate) can change shape slightly as the substrate molecule enters the enzyme
• These changes in shape are known as conformational changes
• The conformational changes ensure an ideal binding arrangement between the enzyme and substrate is achieved
• This maximises the ability of the enzyme to catalyse the reaction

Enzymes and the lowering of activation energy

• All chemical reactions are associated with energy changes
• For a reaction to proceed there must be enough activation energy
• Enzymes speed up chemical reactions because they reduce the stability of bonds in the reactants
• The destabilisation of bonds in the substrate makes it more reactive
• Rather than lowering the overall energy change of the reaction, enzymes work by providing an alternative energy pathway with a lower activation energy
• Enzymes avoid the need for these extreme conditions (that would otherwise kill cells )

The activation energy of a chemical reaction is lowered by the presence of a catalyst (i.e. an enzyme)

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Author: Alistair

Alistair graduated from Oxford University with a degree in Biological Sciences. He has taught GCSE/IGCSE Biology, as well as Biology and Environmental Systems & Societies for the International Baccalaureate Diploma Programme. While teaching in Oxford, Alistair completed his MA Education as Head of Department for Environmental Systems & Societies. Alistair has continued to pursue his interests in ecology and environmental science, recently gaining an MSc in Wildlife Biology & Conservation with Edinburgh Napier University.

The Hydrogen Bond: A Hundred Years and Counting

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• Published: 18 November 2019
• Volume 100 , pages 61–76, ( 2020 )

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Since its original inception, a great deal has been learned about the nature, properties, and applications of the H-bond. This review summarizes some of the unexpected paths that inquiry into this phenomenon has taken researchers. The transfer of the bridging proton from one molecule to another can occur not only in the ground electronic state, but also in various excited states. Study of the latter process has developed insights into the relationships between the nature of the state, the strength of the H-bond, and the height of the transfer barrier. The enormous broadening of the range of atoms that can act as both proton donor and acceptor has led to the concept of the CH···O HB, whose properties are of immense importance in biomolecular structure and function. The idea that the central bridging proton can be replaced by any of various electronegative atoms has fostered the rapidly growing exploration of related noncovalent bonds that include halogen, chalcogen, pnicogen, and tetrel bonds.

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Forty years of progress in the study of the hydrogen bond

Hydrogen Bonding Motifs: New Progresses

What Does Hydrogen Bonding Say About the Nature of the Chemical Bond?

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Scheiner, S. The Hydrogen Bond: A Hundred Years and Counting. J Indian Inst Sci 100 , 61–76 (2020). https://doi.org/10.1007/s41745-019-00142-8

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From molecules to mastication: the development and evolution of teeth

Andrew h. jheon.

1 Department of Orofacial Sciences and Program in Craniofacial and Mesenchymal Biology, University of California San Francisco, San Francisco, USA

Kerstin Seidel

Brian biehs, ophir d. klein.

2 Department of Pediatrics and Institute for Human Genetics, University of California San Francisco, San Francisco, USA

Teeth are unique to vertebrates and have played a central role in their evolution. The molecular pathways and morphogenetic processes involved in tooth development have been the focus of intense investigation over the past few decades, and the tooth is an important model system for many areas of research. Developmental biologists have exploited the clear distinction between the epithelium and the underlying mesenchyme during tooth development to elucidate reciprocal epithelial/mesenchymal interactions during organogenesis. The preservation of teeth in the fossil record makes these small organs essential for the work of paleontologists, anthropologists, and evolutionary biologists. In addition, with the recent identification and characterization of dental stem cells, teeth have become of interest to the field of regenerative medicine. Here, we review the major research areas and studies in the development and evolution of teeth, including morphogenesis, genetics and signaling, evolution of tooth development, and dental stem cells. Brief discussions of microRNAs and human disease as they apply to teeth are also included.

I. MORPHOGENESIS AND DEVELOPMENT

The formation of a head with complex jaws and networked sensory organs was a central innovation in the evolution of vertebrates, allowing the shift to an active predatory lifestyle. 1 The earliest vertebrates were jawless fish (agnathans); the jaw-bearing gnathostomes arose later and have been more successful evolutionarily. An important event in head evolution was the emergence of specialized dentition. To function properly in grasping and crushing food, teeth must be of adequate hardness, proper shape, and anchored to underlying bone ( Fig. 1 ). Tooth number, shape, and size vary significantly among species, because of natural selection in response to the environmental pressures provided by various types of food.

The crown (part of the tooth covered by enamel) and the root are shown. The tooth and its supporting structure, the periodontium, contain all four mineralized tissues in the body, bone (B), cementum (Ce), dentin (De), and enamel (En). The tooth is attached to the underlying bone via periodontal ligaments (pl) in humans. The pulp chamber (p) houses the blood vessels and nerves (not shown) as well as the putative odontoblast stem cells. The gingiva (G) is the oral mucosa that overlies alveolar bone (B).

Teeth, or tooth-like structures called odontodes or denticles, are present in all vertebrate groups, although they have been lost in some lineages. Most fish and reptiles, and many amphibians, possess dentitions that contain a large number of teeth (polyodont) of similar shape (homodont) that undergo continuous replacement (polyphyodont 2 ). These teeth are comprised of dentin and enamel or an enamel-like structure, are rootless, and are attached directly to bone by ankylosis or fibrous tissue. In contrast, mammalian teeth are rooted and are connected to the jaws through interactions between the periodontal ligaments and alveolar sockets ( Fig. 1 ).

Egg-laying monotremes, the most basal living mammals, possess a rudimentary unpaired egg tooth, similar to reptiles and birds, for use during hatching. 3 Adult monotremes have horny plates as opposed to teeth. Therian mammals, which include all living mammals except monotremes, typically are heterodonts, meaning that the teeth have different shapes. Four types of teeth are present in mammals: incisors, canines, premolars and molars. In contrast to non-mammalian vertebrates, only two generations of teeth (diphyodonty) are present in mammals. The ancestral dental pattern for eutherian placental mammals in each quadrant is three incisors, one canine, four premolars, and three molars, and the premolars and molars are typically multicuspid. 4 In most extant mammals, tooth number is reduced relative to this ancestral pattern (e.g., Fig. 2 ).

The maxillary (A, C) and mandibular (B, D) dental arches show the reduced dentitions in adult human (A, B) and mouse (C, D). Both species are derived from a common mammalian ancestor that is thought to have had 6 incisors, 2 canines, 8 premolars, and 6 molars in each dental arch. The third molar or wisdom tooth (M3) is absent in the human specimen. I, incisor; I1, central incisor; I2, lateral incisor; C, canine; PM1, first premolar; PM2, second premolar; M1, first molar, M2, second molar; M3, third molar; D, diastema. Images are courtesy of Dr. Kyle Burke Jones (UCSF).

The dentition is highly specialized in mice, which are the most commonly used model to study tooth development. In each quadrant, a single incisor is separated from three molars by a toothless region called the diastema ( Fig. 2C, D ). Rodent incisors are unusual because they grow continuously throughout the life of the animal, a property attributed to the presence of populations of adult stem cells. Such stem cell-fueled continuous growth of rodent teeth is discussed in more detail below. Also, rodents possess no replacement teeth, unlike humans, who have two sets of teeth.

A. STAGES OF TOOTH DEVELOPMENT

The tissues required for tooth development originate from two principal sources. The epithelium is derived from oral ectoderm and potentially pharyngeal endoderm, 5 , 6 whereas the mesenchyme is derived from cranial neural crest cells. Neural crest cells arise from the margins of the neuro-epithelium and migrate laterally and ventrally to fill the facial prominences with mesenchyme. 7 The neural crest-derived mesenchyme (hereafter referred to as mesenchyme) eventually forms the facial and jaw skeletons, as well as most of the soft and hard tissues in teeth, including dentin, dental pulp, alveolar bone, and periodontal ligament; 8 these tooth-specific tissues are discussed in greater detail below.

In mice, initiation of tooth development occurs between embryonic day (E) 8.5–10, when the sites of tooth formation are first apparent based on the expression of several genes. The first morphological sign of odontogenesis is a thickening of the oral epithelium at E11 in the mouse (week 7 of gestation in humans) ( Fig. 3 ). During the subsequent bud stage at E12.5-E13.5, cells of the thickened oral epithelium proliferate and form a dental lamina that invaginates into the mesenchyme. Mesenchymal cells condense underneath the forming epithelial bud to generate the dental papilla. During the cap stage at E14, the epithelial bud extends further and begins to surround the dental papilla. Cells of the dental mesenchyme located adjacent to the dental papilla or outside the epithelial organ form the dental follicle or sac. Also, the primary enamel knot, a transient signaling center that regulates tooth shape, is present in the dental epithelium. During the bell stage beginning at E16, the tooth germ increases further in size, and the final shape of the tooth crown becomes increasingly apparent. In the forming molars, secondary enamel knots, which are the signaling centers in areas of epithelial folding whose initiation is controlled by the primary enamel knot, determine the sites of tooth cusp formation. 9 Finally, tooth-specific cell types, such as ameloblasts and odontoblasts, begin to differentiate.

The various stages of mouse molar (A) and incisor (B) development, and the adult mouse mandible (C) are depicted in sagittal views. The oral epithelium thickens at the placode stage and invaginates into the neural crest-derived mesenchyme. Mesenchymal condensation occurs at the bud stage and the enamel knot, a central signaling area, first appears at the cap stage. The extracellular matrices of dentin and enamel are secreted with the differentiation of ameloblasts and odontoblasts during the bell stage. The matrix will eventually mineralize forming the tooth crown and is followed by tooth eruption. Similar developmental events occur in incisors and molars with notable differences being the presence of a vestibular lamina (VL), as well as the labial and lingual cervical loops (laCL and liCL, respectively), during incisor development, and the presence of secondary enamel knots, the future site of cusps, during molar development.

The enamel-producing ameloblasts are generated from epithelial cells adjacent to the dental papilla called the inner enamel epithelium (IEE), and these cells secrete enamel matrix that eventually mineralizes. Dentin-producing odontoblasts differentiate from the outermost layer of the dental papilla and gradually migrate to the center of the dental papilla as they secrete dentin matrix. Root formation coincides with tooth eruption after formation of the crown, which is the part of the tooth covered by enamel. 10 Cementum, periodontal ligament, and alveolar bone are all derived from the dental follicle, which has a mesenchymal origin, and are involved in anchoring the teeth to the jaws. 11 , 12 Rodent incisors do not have a typical crown or root but rather possess a crown-like labial (near the lip) surface covered by enamel and a root-like lingual (near the tongue) surface where enamel is absent. 13 The first teeth to erupt in mice, the mandibular incisors, become visible at around postnatal day (P) 9, followed shortly by the maxillary incisors. Eruption of molar teeth begins at P15 with the first mandibular molars.

B. EPITHELIAL-MESENCHYMAL INTERACTIONS: THE TOOTH AS A MODEL FOR DEVELOPMENTAL BIOLOGISTS

Reciprocal interactions between the epithelium and the underlying mesenchyme regulate tooth morphogenesis, and studies of these interactions have made the tooth an important model for developmental biologists. The ability to form teeth, also called the odontogenic potential, was shown by classical tissue recombination experiments to reside in the epithelium at the placode stage. In these studies, oral epithelium of murine embryos between E9 and E11.5 induced tooth formation in non-dental mesenchyme. 14 , 15 After E11.5, the dental mesenchyme was able to induce non-dental epithelia to participate in odontogenesis, 16 whereas the ability by the epithelium to induce tooth formation appeared to be lost at this stage. Thus, these early experiments suggested that, during early tooth development, the odontogenic potential shifts from the epithelium to the mesenchyme. During the cap stage and beyond, the dental mesenchyme regulates tooth shape formation and can induce formation of ameloblasts and enamel matrix secretion in non-dental epithelium. 16 , 17

C. ADULT TEETH AND TOOTH-SPECIFIC MINERALIZED TISSUES

In terms of hard tissues, the tooth is comprised of enamel, dentin and cementum, and it is anchored to the alveolar bone by periodontal ligaments; thus, the tooth and supporting structure, called the periodontium, contain all of the four types of mineralized tissues found in the vertebrate body ( Fig. 1 ). The two main parts of an adult tooth are the crown and the root. The crown is covered by enamel, which is a highly mineralized, acellular substance secreted by cells derived from dental epithelium. Enamel is the hardest structure found in the body, and it consists primarily of hydroxyapatite, a crystalline calcium phosphate, which is also in the major component of dentin, cementum, and bone. The organization of mineral in enamel is unique, as this material is formed of rods of hydroxyapatite crystals running from the dentin-enamel junction to the surface of the tooth. Underlying dentin supports both the enamel layer of the tooth crown and the cementum layer of the tooth root. Dentin is less mineralized and less brittle than enamel and is necessary for the support of enamel in the crown and cementum in the roots. The individual collagen fibrils of the periodontal ligaments originate from the cementum and cementum-dentin junction and attach directly to the alveolar bone of the jaws ( Fig. 1 ). 18 The dental pulp is a mass of vascularized connective tissue enclosed by dentin in the central part of the tooth. The apical foramen, an opening in the area of the root apex, allows the supply of the dental pulp with blood vessels and nerves.

II. GENETICS AND SIGNALING

A number of signaling pathways work in concert to orchestrate tooth development, and this section summarizes some of the major pathways. Signaling cascades involved in development, response to intercellular signals and environment, cell cycle control, and pathogenesis require transcription factors that interact with DNA to regulate gene expression; some of the transcription factors involved in tooth development are summarized. Newly discovered functions for microRNAs are also briefly discussed. Expression data of the molecular factors discussed here at various stages of tooth development can be found at www.bite-it.helsinki.fi .

Fgf8 and Fgf9 are amongst the earliest genes to be expressed in the oral epithelium. The conditional inactivation of Fgf8 in ectoderm caused defects in structures derived from the first pharyngeal arch including teeth, jaws, lateral skull wall, and middle ear, as well as part of the tongue and other soft tissues. 19 Although molars and the proximal mandible were absent, the distal-most structures such as lower incisors were present. These results suggested that a large proximal derivative of the first pharyngeal arch primordium is specified by FGF8, but a small distal region depends on other signaling molecules for its outgrowth and patterning. 19 In mice over-expressing a dominant negative form of Fgfr2b , tooth development did not progress beyond the bud stage. 20 Fgf4 and Fgf9 , which are expressed in the enamel knot, are thought to stimulate proliferation in adjacent epithelial and mesenchymal tissues. 9 , 21 Deletion of Fgf3 and Fgf10 in mice results in smaller teeth with aberrant cusp morphology 22 , but Fgf3 and Fgf10 do not appear to be required individually for ameloblast differentiation. 22 , 23 The inactivation of sprouty genes, which are inhibitors of FGF signaling, results in the formation of supernumerary teeth 24 and the generation of ectopic enamel on the lingual surface of the incisor. 25 FGF signaling is also important in zebrafish tooth morphogenesis, and decreases in FGF signaling have been proposed to lead to the loss of oral teeth. 26 , 27

BMPs function at multiple stages during odontogenesis. BMP4, in particular, is an important mediator of signaling between epithelial and mesenchymal tissues. 28 During initiation of tooth formation, BMP signaling in the oral epithelium antagonizes FGF signaling, which is thought to determine the sites of tooth formation. 29 – 31 Mesenchymal BMP4 regulates Shh expression 30 and is critical for the transition from tooth bud to cap stage and for induction of the enamel knot in the epithelium. 32 , 33 The inactivation of activin or Bmpr1a in either epithelium or mesenchyme results in the arrest of tooth development after the bud stage. 34 – 36 During the cap stage, BMP4 induces the cyclin dependent kinase inhibitor, p21 , and the expression of Bmp4 and p21 is associated with differentiation as well as apoptosis of the primary enamel knot. 33 Therefore, BMP signaling regulates patterning of the cusps and ultimately, the shape of the tooth crown. Fst1 , which acts as an antagonist of BMP and INHBA (formerly known as activin) signaling, and Sostdc1 , an inhibitor of both BMP and WNT signaling, are important regulators of enamel knot formation. 37 , 38 BMP signaling is also known to function during root formation 39 and during differentiation of odontoblasts and ameloblasts. 38 , 40 , 41

During initiation of tooth formation, Shh is expressed in specific regions of the epithelium of the molar and incisal placodes. 42 At this early stage, the mitogenic activity of SHH is thought to stimulate proliferation in the placode epithelium, which enables invagination into the underlying mesenchyme and formation of the epithelial bud. 43 , 44 Analysis of expression of the receptor Ptch1 and the transcription factor Gli1 , which are both downstream targets of SHH signaling, showed that SHH signals to the mesenchyme as well as the epithelium. 44 , 45 Expression of Shh is retained at the tip of the epithelial bud, becomes down-regulated towards the end of the bud stage, is re-induced in the enamel knot, and remains expressed in the epithelium throughout ameloblast differentiation. The regulation of these interesting expression patterns is not well understood and remains the subject of much interest.

SHH from the enamel knot regulates crown formation by stimulating proliferation in epithelial and mesenchymal tissues adjacent to the signaling center. Conditional inactivation of Shh or the signal transducer smoothened from the epithelium, or inhibition of signaling using an antibody against SHH, demonstrated that SHH signaling regulates tooth separation, size and morphology as well as cytological organization of matrix secreting cells. 46 – 48 Primary cilia exert a negative regulatory effect on SHH activity and function to repress tooth formation. 49 In zebrafish, SHH signaling is required continuously throughout tooth development from initiation to morphogenesis. 50 Despite gene duplication and differences in the location of where teeth form between mice and zebrafish, the role of SHH signaling in tooth development appears to be conserved between these two species. 50

The modulation of WNT signaling leads to variations in tooth number. Supernumerary teeth arise with the up-regulation of WNT signaling. Multiple ectopic teeth in the molar region were observed after constitutive activation of the transcriptional effector CTNNB1 (beta catenin), 51 , 52 and mis-expression in epithelial tissues of Lef1 , the binding partner of CTNNB1, led to multiple teeth in the incisor region. 53 Overexpression of the zinc finger protein-encoding gene, Sp6 , led to an increase in WNT signaling and mice with up to 50 teeth. 54 Inactivation of WNT antagonists such as Apc 55 , 56 and Sostdc1 37 , 57 , 58 also led to increases in the number of teeth. Several of these studies demonstrated that the dental epithelium undergoes multiple invaginations leading to the formation of extra enamel knots and ultimately, supernumerary teeth. 51 , 54 , 56 , 59 Conversely, there is evidence in humans that decreases in WNT signaling lead to tooth loss. 60 , 61

The mechanism by which WNT signaling regulates tooth number is still unclear. Surprisingly, Msx1 , which is required for normal tooth development, was dispensable for WNT-mediated supernumerary tooth formation, whereas Fgf8 was identified as a direct target of WNT signaling. 56 WNT signaling regulates Shh 48 , 62 and Bmp4 expression, 63 and it affects multiple stages of tooth development such as bud to cap transition, formation of the enamel knot, molar tooth size, and dentinogenesis. 41 , 52 , 54 , 62 , 64 Conditional inactivation in the dental mesenchyme of Smad4 , which encodes a BMP signal transducer, led to an up-regulation of CTNNB1 and down-regulation of WNT antagonist genes such as Dkk1 and Sfrp1 . 41 In these mice, enamel appeared normal whereas dentin formation was compromised, an observation that challenges the traditional notion that ameloblast differentiation is dependent upon odontoblast differentiation. 65

Components of the Notch signaling pathway, which include four transmembrane Notch receptors ( Notch1 - 4 ) and 5 transmembrane ligands ( Jag1 , Jag2 , Dll1 , Dll3 , and Dll4 ), are expressed during tooth development and affect several aspects of tooth formation. Notch signaling was demonstrated to regulate tooth morphogenesis and ameloblast differentiation. 66 Specifically, inactivation of the Notch-interacting domain of JAG2 in mice caused abnormal molar shapes, additional cusps, and inhibition of ameloblast differentiation and enamel matrix deposition. 66

The stratum intermedium (SI), a layer of cells that is subjacent to the ameloblast layer during enamel formation and whose function is still unclear, expresses Notch1 and its downstream target, Hes1 , whereas the IEE and ameloblasts express Jag1 . 67 In HAT-7 cells, a dental epithelial cell line, treatment with exogenous JAG1 led to the differentiation of SI cells, and this effect was neutralized with an anti-JAG1 antibody, pointing to the importance of Notch signaling in the SI. 67

Ectodysplasin-A ( Eda ) is a member of the tumor necrosis factor (TNF) superfamily of signaling molecules. The EDA-A1 isoform, its receptor EDAR, and the adapter protein EDARADD act in a linear fashion and activate canonical NFKB signaling as well as other pathways. 68 The EDA-A2 isoform signals through another receptor, EDA2R (formerly known as XEDAR), rather than EDAR, and activates similar pathways but appears to play a less important role in development. 69 Mice with mutations in Eda , Edar or Edaradd (initially found as the spontaneous mutants tabby , downless , and crinkled , respectively) all have a decrease in the number of teeth with abnormal cusp morphology. 68

During early odontogenesis, EDA signaling is crucial for determining the size of the tooth field and the number of teeth generated. Specifically, mutations in Eda or Edar resulted in formation of smaller teeth and frequently the absence of third molars. 70 In contrast, increased levels of Eda expression, or expression of a constitutively active form of Edar , led to the formation of a supernumerary tooth in the diastema region. 71 , 72 In zebrafish, mutations in eda and edar led to defects in ectodermal structures such as scales and glands and partial or complete loss of pharyngeal teeth. 73

EDA signaling also affects tooth shape. Mutations in any of the three pathway components in mice result in molars with reduced cusp number and rounded cusps. Eda is expressed in oral and dental epithelium throughout tooth formation, whereas Edar and Edaradd are expressed in the enamel knot. The enamel knots in tabby or crinkled mutants were smaller, 74 , 75 whereas loss of Edar in downless mice led to the formation of an elongated-rope like enamel knot. 76 Interestingly, overexpression of Edar but not of Eda resulted in formation of extra cusps. 71 , 72 It is clear that the loss of function of Eda versus Edar has distinct effects on tooth size and morphology. This may be due to activation of the EDA2R pathway, which is influenced by Eda but not Edar expression, to interaction between EDAR and a yet unidentified protein, and/or to a ligand-independent activity for EDAR. 77

G. Transcription Factors

The initial patterning as well as the coordinated interplay of signals at each step of tooth development is greatly dependent on the actions of transcription factors. Here, some of the general concepts and recent advances in our understanding of the roles of transcription factors in tooth development are discussed.

At E8.5, prior to any morphological signs of tooth development, Pitx2 is expressed in the stomatodeal epithelium, the precursor to oral and dental epithelium, and it is considered to be the earliest transcription factor expressed during tooth development. 30 , 78 Pax9 expression has been shown to specify the mesenchymal regions at the prospective sites of all teeth at E10. 29 The direct regulation by PITX2 of Dlx2 , a gene that is expressed at E9.5, is attenuated by a physical interaction between DLX2 and PITX2. 79 At later stages, DLX2 and FOXJ1, a transcription factor expressed in the oral epithelium that plays a fundamental role in embryonic development, activate transcription of amelogenin, a tooth-specific protein required in enamel formation and mineralization. 80

During initiation of tooth development, epithelial FGF8 and BMP4 induce the expression of numerous transcription factor genes including Barx1 , Dlx1 , Dlx2 , Msx1 , Msx2 , Pax9 , Pitx1 , and Pitx2 . 29 – 31 The expression in prospective mesenchyme of many non-HOX homeobox-containing genes, such as Barx1 , Dlx1 , Dlx2 , Dlx3 , Dlx4 , Dlx5 , Dlx6 , Lhx6 , Lhx7 , Msx1 , and Msx2 , 81 – 83 led to the proposal of the odontogenic homeobox code model, which postulates that expression of specific combinations of homeobox gene directs the formation of specific tooth types. 84

Msx1 and Pax9 act in the dental mesenchyme to maintain expression of Bmp4 , which is crucial for establishing the enamel knot. 32 , 33 Absence of either of these transcription factors led to an arrest in tooth development at the bud stage, similar to that reported in Lef1 -null embryos. 64 Recently, it was shown that during early tooth formation, mesenchymal condensation (i.e. compression of mesenchyme) alone could regulate expression of Msx1 and Pax9 , as well as Bmp4 . 85

H. microRNAs

There is an emerging role for microRNAs (miRNAs) in the development and evolution of teeth. Small RNAs, and miRNAs in particular, have important effects on development and disease through modulation of specific signaling pathways. 86 miRNAs are endogenously expressed, short (~21 nucleotides), non-coding RNA molecules that affect protein synthesis by posttranscriptional mechanisms. 87 The involvement of miRNAs in various ectodermal derivatives has been demonstrated in skin, 88 , 89 hair, 90 and teeth. 91 – 94 Pitx2 -Cre; Dicer deleted mice showed a multiplication of enamel-free incisors, demonstrating the importance of miRNAs in ameloblast differentiation as well as their role in the regulation of ameloblast stem cells; 91 Dicer is an RNAse III enzyme required for conversion of pre-miRNAs to mature miRNAs. 95 , 96 Krt14 -Cre; Dicer deleted mice showed milder changes in tooth shape, epithelial homeostasis, and enamel formation. 93 The differences in phenotype between the two mutant mice are likely due to the early expression of Pitx2 in stomatodeal epithelium compared to Krt14 expression. The expression of miRNAs in distinct regions of the mouse incisor and pulp was profiled using microarray experiments, laying the groundwork for future investigations. 93 , 94 These initial studies indicate that there is much work ahead in understanding the roles of miRNAs during tooth development.

III. EVOLUTION OF TOOTH DEVELOPMENT

A fundamental question in evolutionary developmental biology is how genetic changes contribute to morphologic variations that are subjected to natural selection. Teeth or tooth-like structures such as odontodes and denticles are invaluable in the study of evolutionary developmental biology for several reasons. First, teeth are ancient structures that are found in multiple locations in the vertebrate body such as the posterior pharynx of extinct jawless fish and extant fish, the dermal surface of sharks and rays, the oral cavity of rodents and humans, and lining the oro-pharyngeal cavity of fish in association with gill arches. 97 , 98 Second, there is great variation in the shape, size, number, and rows of teeth, and these variations are relatively easy to characterize. Third, teeth are readily fossilized vertebrate structures with excellent preservation of morphology due to the hardness of enamel, and thus they provide a large number of specimens for comparative genomic, anatomic, and phylogenetic studies. This section provides an overview of the main ideas and current research in the evolution of tooth development and highlights some of the multi-disciplinary approaches that can be utilized to answer important questions in the evolution and development of teeth.

A. THE ORIGIN OF TEETH IN VERTEBRATES

Teeth are an ancient and key vertebrate innovation, and their origin is a hotly debated question. The first occurrence of tooth-like structures is believed to be in the posterior pharynx of jawless fishes more than 500 million years ago. 98 , 99 With the evolution of jawed vertebrates, teeth developed on oral jaws and helped to establish the dominance of gnathostomes on land and in water.

It is still unclear whether oral teeth evolved with jaws for predation and mastication or first appeared as external dental armor as protection from predation. At least two opposing theories have been put forth regarding the evolution of oral teeth. The ‘outside-in’ theory posits that teeth evolved from ectoderm-derived, skin denticles that folded and integrated into the mouth. 100 The ‘inside-out’ theory suggests that teeth originated from endoderm, with the formation of pharyngeal teeth in jawless vertebrates and moved anteriorly to the oral cavity with the evolution of jaws. 101 However, recent studies suggest that neither theory may be entirely correct. 102

Fate-mapping approaches using transgenic axolotls showed that teeth formed normally regardless of whether the oral epithelium was derived from ectoderm or endoderm. 6 Experiments utilizing chicken embryos, which have lost the ability to form teeth, 103 have demonstrated the dominant role of mesenchyme in the initiation of tooth development. Specifically, transplantation of mouse neural crest cells into developing chicken embryos showed the formation of tooth germ-like structures. 104

Some extant fish, such as certain cichlids, possess both oral and pharyngeal teeth ( Fig. 4 ). Pharyngeal teeth develop on discrete pharyngeal jaws in hox-positive, endoderm-derived sites, whereas oral teeth develop in hox-negative, ectoderm-derived regions. 99 Pharyngeal teeth of jawless vertebrates appear to utilize an ancient gene network that predates the origin of oral jaws, oral teeth, and ectodermal appendages. 99 During mouse development, expression of various genes such as claudin6, Foxa2 , alpha-fetoprotein, Esrp1 (formerly known as Rbm35a ), and Sox2 is observed in the presumptive molar region but not in the incisor region. 5 In Chuk- (formerly known as Ikka ) null mice, there was abnormal epithelial evagination in incisors but not in molars. 105 These and other studies suggest differences in the epithelium from which incisor and molar teeth develop. However, despite distinct developmental environments, which suggest different molecular mechanisms that result in heterodont dentition, both oral and pharyngeal teeth also show striking similarities in their gene regulatory networks. 99

Point A indicates the origin of pharyngeal teeth in extinct ( † ) jawless fish. Oral teeth and jaws are thought to have arisen at point B. The pharyngeal teeth were lost in common ancestors to tetrapods at point C. In some extant teleosts such as cichlids, both oral and pharyngeal teeth are present and pharyngeal jaws are thought to have arisen at point D. Adapted from Fraser et al. 99

Taken together, the studies using axolotl, cichlids, chicken, and mice demonstrate that teeth can form despite different epithelial origins and demonstrate the important role of mesenchyme in the initiation of tooth development, challenging the primacy of oral ectoderm in this role. 14 These studies also demonstrate the conservation of gene regulatory networks across lineages with origins in different germ layers and the role of deep homology 106 in the evolution and development of teeth. Thus, teeth appear to have evolved both ‘inside and out’, wherever and whenever the odontogenic-specific gene network of the mesenchyme was present. 97

B. EVOLUTION OF TOOTH SHAPE, SIZE, NUMBER, AND ROWS

Both humans and rodents evolved from a common mammalian ancestor that is thought to have had a full complement of teeth comprising three incisors, one canine, four premolars, and three molars in each dental quadrant that replaced its teeth a single time. 4 During mammalian evolution, teeth were lost along the lineages that gave rise to both rodents and humans ( Fig. 2 ). Humans have all four of the major classes of teeth but have lost members of several of these classes; for example, we only have two incisors and two premolars ( Fig. 2A, B ). Rodent ancestors underwent a further reduction in dental formula, such that mice have only one incisor and three molars per quadrant, and no replacement teeth. Rodent teeth are considered to be deciduous teeth that do not undergo replacement, 107 – 109 but the potential for replacement teeth in mice appears to have been retained. 109 – 111

In addition to modifications in the number of teeth, the morphology of mammalian teeth is enormously diverse. These modifications involve variations in cusp shape and crest organization, and in the case of a number of species, the evolution of stem cell-fueled continuous growth, as discussed below. Comparative studies of tooth morphology have been greatly advanced by improvements in three-dimensional (3D) imaging techniques such as high-resolution micro-computed tomography. Some recent studies regarding the regulation of tooth shape, size, number, and rows are discussed below.

The various tooth shapes observed in heterodont animals are believed to have evolved from ancestral conical teeth, perhaps similar to canines, through the addition of cones and grooves. 112 Relatively little is known regarding the molecular mechanisms underlying such changes, and therefore they are the subject of much current interest. Decreasing BMP signaling in the incisor region can lead an incisor to acquire a molar-like phenotype. 113 However, it has recently been proposed that the molar-like phenotype was a result of the splitting of the incisor placode rather than a change in tooth identity. 114 Lrp4 -null mice displayed enamel grooves on the labial surface of incisors that exhibited similar molecular characteristics as molar cusps, suggesting that WNT signaling may be involved in cusp development. 112

Two recent studies have provided important information about the developmental regulation of the relative size and number of molars. By using mouse molar cultures, it was proposed that a combination of activators and inhibitors governs the relative relationship between size and number of teeth. 115 Detailed studies of tooth shape indicated that the complexity of the cusps directly reflects the animal’s diet across many mammalian species. 115 , 116 These studies pointed to higher order, generalizable principles that govern tooth shape and size.

Several studies have shown that alterations in signaling pathways can lead to variation in tooth number, and such studies point to mechanisms that may have determined tooth number during mammalian evolution. An example of dramatic tooth loss was highlighted in the cypriniform fish, a group including zebrafish. Zebrafish possess pharyngeal teeth, and fossil evidence suggests that zebrafish lost their oral teeth 50 million years ago ( Fig. 4 ). 117 This was associated with the loss of dlx2a and dlx2b expression in the oral epithelium. Because DLX genes are required for tooth development in mice, 118 changes in trans-acting regulators of DLX genes that may be downstream of FGF signaling have been proposed as candidates responsible for the loss of cypriniform oral teeth. 27 Interestingly, a region in the upstream regulatory element of dlx2b was retained that drives specific expression in the oral epithelium, and the retention of this cis-regulatory element is posited to be due to its requirement in other tissues, as the DLX genes have pleiotropic effects. 119 These studies suggest that teeth lost from specific regions may be relatively easy to reacquire during evolution 119 and they are exciting because they challenge Dollo’s Law of the Irreversibility of Evolution, which states that an organism can never exactly return to a previous evolutionary state 120 because a lost structure cannot reappear in evolution.

A number of mouse mutants with changes in tooth number or pattern have provided tantalizing hints about the evolution of dentition. Supernumerary teeth present in the diastema region of mice ( Fig. 2C, D ), a species with reduced dentition, may represent the revival of teeth present in ancestral species. The following experiments in mice led to supernumerary teeth in the diastema region of the mandible: epithelial overexpression of Eda under the control of the Krt14 promoter; 71 inactivation of the receptor tyrosine kinase antagonists, Spry2 and Spry4 ; 24 production of a hypomorphic allele of the gene encoding Polaris, a protein involved in SHH signaling; 121 a null mutation of the SHH antagonist, Gas1 ; 49 and inactivation of the BMP/WNT pathway inhibitor gene, Sostdc1 . 37 , 57 , 58 , 122 Diastema teeth in Krt14 -Eda mutants and in Spry2 - , Spry4-, polaris-, and Gas1 -null mice have a size and shape characteristic of premolars, a tooth type that was lost in mice around 50–100 million years ago. Interestingly, diastema teeth in Sostdc1 -null mice showed a molar-like phenotype, as well as enlarged enamel knots and altered cusp patterns. 37 , 57 , 58 , 122 In the diastema region, it was previously observed that the tooth primordium was present but failed to further develop because it does not maintain Shh expression. 123 – 126 These studies demonstrate how loss of teeth from specific regions may be relatively easy to reacquire during evolution.

Mice carrying mutations in Sostdc1 , 58 , 127 Lrp4 , 122 or inheriting the Di (duplicate incisor) trait 128 have supernumerary upper or lower incisors that are located lingual to the normal incisor. Decreases in sprouty gene dosages also led to increasing numbers of incisors. 129 The detailed study of Sostdc1 mutants indicated that the supernumerary incisors corresponded to replacement teeth. 127 Splitting of the incisor placode has been observed in Sostdc1 / Fst1 double-null mice, which have bifid incisors. 114 The potential mechanisms by which supernumerary incisors arise in mice include: failure of integration of the ancestral dental primordia; 109 development of replacement teeth; 127 splitting of a large placode into smaller elements; 114 or development of supernumerary tooth germs. 130 These examples of supernumerary teeth may also reflect ancestral rodent dentition, in which a larger number of incisors was found, and highlight potential mechanisms by which humans and mice have evolved their reduced dentition.

Mammals possess a single row of teeth in the upper and lower jaws, unlike the multiple rows observed in some non-mammalian species such as fish and snakes. Teeth are replaced only once in most mammals, whereas in many non-mammalian species, teeth are continuously replaced. Additionally, in rodents, there are no replacement teeth, but there is continuous, stem cell-fueled growth of incisors, as well as of molars in species such as voles. 131 Interestingly, supernumerary teeth developed lingual to the first molars in mice with inactivation of Osr2 (odd-skipped related-2), a gene homologous to the Drosophila transcriptional repressor odd-skipped . 110 Osr2 limits the odontogenic field by suppressing the BMP4-MSX1 signaling cascade. 110 The development of supernumerary teeth in Osr2 mutants may represent a second row of teeth similar to the multiple rows observed in some fish, and it may represent a reawakening of replacement teeth in mice.

C. COMPARATIVE TOOTH MORPHOLOGY AND MAMMALIAN EVOLUTION

Due to the highly mineralized nature of enamel, there is excellent preservation of detailed dental features in teeth from extant and extinct species. Using this vast repository of specimens, detailed 3D images can be constructed to compare subtle differences in tooth morphology. This information can be applied in interesting ways to further our understanding on the evolution of tooth development.

Comparative morphologic studies of mutant mice and various extinct and extant species have shed light on the role of specific genes in the evolution and development of tooth morphology. One such study showed that varying dosages of the Fgf3 gene caused morphological changes in teeth of mutant mice and in human patients ( Fig. 5 ). 132 Using comparisons between mice and humans carrying Fgf3 mutations with primitive rodent and primate fossils, it was observed that decreases in Fgf3 dosage led to tooth phenotypes that resembled the progressive reappearance of ancestral morphologies ( Fig. 5 ). 132 Progressive decreases in sprouty dosage caused increasing numbers of incisors, mimicking the dentition of rodent ancestors. 129 Multidisciplinary approaches that integrate development and evolution can thus help to correlate subtle dental modifications with genetic mutations in a variety of mammalian lineages.

As Fgf3 dosage is decreased in mice, the mesio-lingual (ML) cusp of the upper first molar is transformed into the ML crest ( Fgf3 +/− ) and is eventually lost ( Fgf3 −/− ), whereas the mesio-distal (MD) crest appears ( Fgf3 −/− ). Comparisons of mutant and wild-type mice with fossil rodents such as Democricetodon , Myocricetodon , and Potswarmus show several features: during the transition from ancestral to derived morphologies, there is a loss of the MD crest, an emergence of the DL cusp, and an emergence of the ML crest that is transformed into the ML cusp in mice. The arrow indicating the relative levels of FGF signaling applies only to the allelic series of Fgf3 mutant mice, as the expression levels of Fgf3 in muroid ancestors is unknown. The following abbreviations are used for orientation: M, mesial; D, distal; V, vestibular; L, lingual. Figure adapted from Charles et al. 132

A large amount of information can be extracted from the analysis of fossilized teeth. For example, a record of growth can be attained from the enamel and dentin that allows the reconstruction of the developmental history and the timing of crown and root formation. Measurements of daily enamel cross-striations can be used to infer information about the timing and rate of enamel/crown formation; accentuated neonatal lines in the enamel of deciduous and permanent molars may denote the time of birth; incremental markings in the dentin indicate the timing of root completion; and the quality of the enamel-dentin junction, where the crown meets the root, provides a window to tooth development and the actions of the enamel knot. 133 Using such techniques, tooth development in Neanderthals was shown to closely resemble that of human populations, underscoring the similarities between humans and Neanderthals. 133

In another study utilizing fossilized teeth to understand the evolution of species, the worn cusp apices of teeth (mesowear) from North American horses for the past 55.5 million years was analyzed. Hypsodonty (high-crowned teeth) was correlated with mesowear, thereby strengthening the argument that the evolution of hypsodonty relative to brachydonty (short-crowned teeth) was adapted for abrasive diets associated with the spread of grasslands in North America. 134 In brachydont species such as humans, the tooth crown is entirely above the level of the jaw bone upon initial eruption, whereas in hypsodont species, some of the tooth crown is retained below the level of the jaw bone. 135 In rodents, hypsodonty is posited to be an intermediate stage on the evolutionary path towards hypselodonty (ever-growing teeth). 135

Thus, by utilizing model and non-model organisms to analyze genetic and signaling pathways, along with detailed 3D reconstructions of teeth in extant and extinct species and in combination with ecological data, some of the exciting multi-disciplinary studies discussed above are at the forefront of research in the evolution of tooth development.

IV. DENTAL STEM CELLS

The regenerative ability of many adult tissues is dependent on tissue-specific stem cell populations that maintain stable numbers by self-renewal and that possess the capacity to differentiate into distinct cell lineages. Regeneration and renewal in adult mammals has been studied in several organs, including the blood, gut, brain, skin, and hair. Here, we describe the advent of the continuously growing mouse incisor as an adult stem cell model system. Although the study of incisor stem cells is a relatively new field, advances have recently been made in the identification of these cells, the understanding of their function, and the characterization of molecular mechanisms that regulate their behavior.

In mice, both molars and incisors go through similar developmental stages at early stages of odontogenesis, but incisors continue to grow throughout postnatal life, whereas molars cease growth in the perinatal period. The ability of the incisor to grow continuously is dependent on the presence of epithelial and mesenchymal stem cells that have the capacity to self-renew and differentiate into all of the cell types of the adult tooth, including ameloblasts, odontoblasts, and the SI. Importantly, in the wild-type rodent incisor, the labial CL, but not the lingual CL, contains stem cells that give rise to ameloblasts and the SI ( Fig. 6 ). Labeling experiments demonstrated that cells in the dental epithelium move in a proximal to distal direction. 136 In the labial CL, the stem cell progeny contribute to a population of transit amplifying (T-A) cells ( Fig. 6B ). T-A cells undergo several rounds of cell division before they move distally and differentiate into ameloblasts. Early labeling experiments examining the rate of ameloblast and odontoblast migration in mice and rats gave the first clues that turnover of these specialized cells was rapid, underlining the need for progenitor pools to resupply differentiated cell populations. 136 , 137 Mouse incisor epithelia appear to function as a “conveyor belt”, moving cells from a proximal, undifferentiated source to regularly repopulate the tooth with specialized cell types. Initial studies using explant cultures of the CL region from 2 day-old mice showed that new epithelial structures could be generated in culture, indicating that the labial CL housed the dental stem cells that give rise to ameloblasts and the SI. 138

The lower incisor is shown in sagittal view (A–D). (A) The diagram indicates the two stem cell compartments in the lingual (liCL) and labial (laCL) cervical loops. Also shown are the inner enamel epithelium (IEE), from which the transit-amplifying (T-A) cells and ameloblasts (Am) arise, the outer enamel epithelium (OEE) that house the enamel stem cells in the laCL, stellate reticulum (SR), stratum intermedium (SI), odontoblasts (Od), dentin (De), enamel (En), and blood vessels (BV). (B) Adult mice were injected with BrdU for 1.5 h. BrdU-positive cells indicate rapidly proliferating cells in the T-A region. (C–D) Images from incisors of Krt5 -tTA; H2B-GFP mice. In the absence of doxycycline (no Dox; C), GFP is present in all the cells expressing Krt5 , which includes the OEE, IEE, SR, SI, and Am. In the presence of doxycycline (+ Dox; D) for 8 weeks, H2B-GFP expression was turned off, leading to the retention of GFP in the slowly proliferating label-retaining cells (LRCs) of the OEE. The LRCs are putative dental epithelial stem cells.

Identification of organ-specific adult stem cell populations can be challenging, because stem cells often reside in heterogeneous niches intermingled with support cells. A useful character of stem cells that has aided in their identification in vivo is the relatively slow cell-division kinetics of many stem cells relative to surrounding tissue. 139 Slow-cycling cell populations have largely been identified through label retention experiments, traditionally utilizing BrdU incorporation, because cells that divide slowly do not dilute the BrdU label as quickly as their rapidly dividing neighbors. Using this technique, BrdU label-retaining cells (LRCs) were identified in the labial CL of cultured perinatal incisors and in adult incisors in situ . 138 , 119 Another approach to label retention is the use of transgenic mice harboring a tetracycline-sensitive, histone H2B-GFP cassette under the control of a tissue specific trans-activator ( Fig. 6C, D ). 140 Expression of H2B-GFP is initially activated in all cells of the tissue of interest followed by a “chase” period when the transgene is repressed by exposure of the animal to doxycycline, such that rapidly dividing cells dilute the label. This technique was used to identify LRCs in the outer enamel epithelium (OEE) of the adult labial CL. 141 The LRCs of the dental epithelium expressed Gli1 , a target of SHH signaling, and lineage tracing experiments demonstrated that the Gli1 -expressing cells were indeed stem cells. 141 More recently, identification of LRCs in non-mammalian vertebrates has been pursued. 142 , 143

Understanding the regulation of adult stem cell populations is key to future utilization of such cells for clinical therapies. How stem cells are maintained at the appropriate number, what signals regulate their differentiation, and how they are established within the context of the developing organism are important questions in stem cell research. Many signaling molecules and pathways are implicated in development and homeostasis of the incisor, including WNTs, BMPS and FGFs. One theme that has emerged from several recent studies is the convergence of distinct FGF signaling pathways that maintain the size and shape of the CL through regulation of cell division and death. Expression analyses first indicated that components of the FGF pathways may play major roles in the mouse incisor. 22 , 138 Specifically, Fgf3 and Fgf10 are expressed in the mesenchyme immediately adjacent to and surrounding the labial CL, whereas Fgfr1b and Fgfr2b are enriched in the epithelium. Incisors of Fgf3 null mice at P0 are indistinguishable from those in wild-type mice, yet Fgf3 −/− ;Fgf10 +/− compound mutants reveal a severely hypoplastic labial CL, indicating that precise levels of FGF signaling are responsible for regulating the size and shape of the stem cell niche. 22 Consistent with this result, attenuation of signaling via FGFR2B in the embryo using a tetracycline-inducible dominant negative system gave similar results in E18.5 embryos. 144

It appears that several additional signaling pathways either directly or indirectly converge on FGF signaling via regulation of Fgf3 to control proliferation in developing incisors. Mis-expression of Fst throughout the dental epithelium under the control of the Krt14 promoter resulted in complete down-regulation of mesenchymal Fgf3 expression with reduction in epithelial proliferation and severely hypoplastic labial CLs. 22 Conversely, loss of Fst in the epithelium of incisors led to up-regulated Fgf3 expression in mesenchyme adjacent to the lingual CL, causing increased proliferation and expansion of the lingual CL. 22 Deletion of Tgfbr1 (formerly known as Alk5 ) in the mesenchyme led to down-regulation of Fgf3 , Fgf9 , Fgf10 , and a reduced labial CL size, likely due to proliferation defects. 145 . Notably, fewer LRCs survived in this mutant, and the defect could be reversed by the addition of exogenous FGF10. Thus, several lines of evidence indicate that FGF signaling is involved in the maintenance of incisor stem cell number.

The sprouty genes encode negative feedback regulators of FGF signaling that are expressed in both the lingual and labial epithelia as well as in the mesenchyme adjacent to the labial CL. Loss of sprouty function in the incisor resulted in up-regulation of FGF gene expression in the lingual epithelium and mesenchyme and the presence of ameloblasts in the lingual epithelium. 25 This study highlighted the importance of balanced FGF signaling in the incisor to maintain asymmetric production of ameloblasts on the labial side, while preventing ameloblast formation and activity on the lingual side.

In addition to FGF signaling, incisor stem cells require the activity of the Notch pathway to ensure development and survival. Three Notch receptors are expressed in the CL regions in the developing incisor. Notch1 and Notch2 are expressed in the epithelium and mesenchyme, whereas Notch3 is restricted to the mesenchyme. 138 The Notch ligand encoded by Jag2 is strongly expressed in the epithelium, and Jag2 -null mice show incisors with defects in cellular morphology. 66 Inhibition of Notch signaling with DAPT led to a reduction in the size of the labial CL in explant experiments. 146

Comparative analysis of two different rodent species provides a potential mechanism for the evolution of ever-growing teeth. The sibling vole ( Microtus rossiaemeridionalis ), in contrast to mice, possesses ever-growing incisors and molars. 131 During development, CLs in mouse molars undergo a transition to the root fate and cease producing enamel. Vole molars do not undergo such a transition, and enamel is continuously generated for the life of the animal. 131 Interestingly, mouse and vole molars are practically identical in morphology and distribution of developmental markers until E17, when the molars are in the late bell stage. Given the roles of FGF and Notch signaling in maintenance of the stem cell niche in the mouse incisor, these pathways were compared at stages during which mouse molars initiate root formation. 131 The development of roots in the mouse coincides with loss of epithelial Notch and mesenchymal FGF signaling, whereas vole molars continue to express key signaling components and partially bypass the root fate. This idea is substantiated by evidence showing that the maintenance of Notch signaling in the cervical loops of mouse molars grown in vitro resulted in continuous crown development in lieu of root formation. 147 Although correlative, these studies suggest that vole molars have evolved mechanisms that maintain the necessary morphology for continuous growth fueled by molar cervical loops, which may be analogous to the stem cell niche of the mouse incisor.

CONCLUDING REMARKS

Important advances have been made in our understanding of the development and evolution of teeth. The tooth provides a valuable model for the elucidation of major biological questions, and the identification and initial characterization of dental stem cells have been exciting recent developments. Many tooth-specific defects in model and non-model organisms mirror conditions present in humans and provide a means to study their genetics, development, and pathology. For a comprehensive review on the diseases of the tooth, please refer to the review by XX et al. However, much work remains to be done, and the utilization of cellular, molecular, and genetic approaches, as well as anthropological and paleontological techniques, will enable continued progress. The rapid increases in our understanding of dental development in extant and extinct vertebrate species using techniques including 3D imaging, genetic manipulations, omics analyses, and genome-wide association studies make this an exciting time to study the development and evolution of teeth.

Acknowledgments

We apologize to those colleagues whose work we were unable to cite due to space constraints. We would like to thank our colleagues in the UCSF Craniofacial and Mesenchymal Biology Program and Jukka Jernvall, Irma Thesleff, Renata Peterkova, Herve Lesot, Vagan Mushegyan, Cyril Charles, Laurent Viriot, Ann Huysseune, and Gail Martin for helpful discussions. The authors are funded in part by the National Institutes of Health (DP2-OD007191 and R01-DE021420 to O.D.K., and K99-DE022059 to A.H.J.).

Abbreviations used

SUPPORTING INFORMATION

Gene and protein names in the mouse and human genome databases are available from the NCBI website, http://www.ensembl.org/index.html .