Fundamental Molecular Processes in Development

From a functional standpoint, many of the important molecules that guide embryonic development can be grouped into relatively few categories. Some of them remain in the cells that produced them and act as transcription factors (Fig. 4.2). Transcription factors are proteins possessing domains that bind to the DNA of promoter or enhancer regions of specific genes. They also possess a domain that interacts with RNA polymerase II or other transcription factors and consequently regulates the amount of messenger RNA (mRNA) produced by the gene.

Other molecules act as intercellular signaling molecules. Such molecules leave the cells that produce them and exert their effects on others, which may be neighboring cells or cells located at greater distances from the cells that produce the signaling molecules. Many signaling molecules are members of large families of related proteins, called growth factors. To exert their effect, signaling molecules typically bind as ligands to receptor molecules that are often transmembrane proteins protruding through the plasma membrane of the cells that they affect. When these receptor molecules form complexes with signaling molecules, they set off a cascade of events in a signal transduction pathway that transmits the molecular signal to the nucleus of the responding cell. This signal influences the nature of the gene products produced by that cell and often the cell’s future course of development.

Transcription Factors

Many families of molecules act as transcription factors. Some transcription factors are general ones that are found in virtually all cells of an organism. Other transcription factors are specific for certain types of cells and stages of development. Specific transcription factors are often very important in initiating patterns of gene expression that result in major developmental changes. They typically do so by acting on promoters or enhancers to activate or repress the transcription of specific genes. Based on their structure and how they interact with DNA, transcription factors can be subdivided into several main groups, the most important of which are introduced here.

Homeobox-Containing Genes and Homeodomain Proteins

One of the most important types of transcription factors is represented by the homeodomain proteins. These proteins contain a highly conserved homeodomain of 60 amino acids; a homeodomain is a type of helix-loop-helix region (Fig. 4.3). The 180 nucleotides in the gene that encode the homeodomain are collectively called a homeobox. Homeobox regions were first discovered in the homeotic genes of the antennapedia and bithorax complex in Drosophila (see Fig. 4.1), hence their name. This designation sometimes confuses students because, since their initial description, homeoboxes have been found in several more distantly related genes outside the homeotic gene cluster. Many other gene families contain not only a homeobox but also other conserved sequences (Fig. 4.4).

HOX Genes

The Drosophila antennapedia-bithorax complex consists of 8 homeobox-containing genes located in 2 clusters on one chromosome. Mice and humans possess at least 39 homologous homeobox genes (called Hox genes in vertebrates [HOX in humans]), which are found in 4 clusters on 4 different chromosomes (Fig. 4.5). The Hox genes on the 4 mammalian chromosomes are arranged in 13 paralogous groups.

Vertebrate Hox genes play a prominent role in the craniocaudal segmentation of the body, and their spatiotemporal expression proceeds according to some remarkably regular rules. The genes are activated and expressed according to a strict sequence in the 3′ to 5′ direction, corresponding to their positions on the chromosomes. Consequently, in Drosophila and mammals, 3′ genes are expressed earlier and more anteriorly than are 5′ genes (Fig. 4.6). Mutations of Hox genes result in morphological transformations of the segmental structures in which a specific gene is normally expressed. Generally, loss-of-function mutations result in posterior-to-anterior transformations (e.g., cells of a given segment form the structural equivalent of the next most anterior segment), and gain-of-function mutations result in anterior-to-posterior structural transformations. Figure 4.7 illustrates an experiment in which injection of an antibody to a homeodomain protein into an early frog embryo resulted in the transformation of the anterior spinal cord into an expanded hindbrain.

Although Hox genes were originally described to operate along the main body axis, sequential arrays of expression are found in developing organs or regions as diverse as the gut, the limbs, and the internal and external genitalia. The expression of isolated Hox genes also occurs in locations such as hair follicles, blood cells, and developing sperm cells. The principal function of the Hox genes is involved in setting up structures along the main body axis, but ordered groups of Hox genes are later reused in guiding the formation of several specific nonaxial structures. In mammals, individual members of a paralogous group often have similar functions, so that if one Hox gene is inactivated, the others of that paralogous group may compensate for it. If all members of a paralogous group are inactivated, profound morphological disturbances often result (see p. 171).

The regulation of Hox gene expression is complex. A major regulator along parts of the anteroposterior axis of the developing central nervous system is retinoic acid, but this effect is mediated by other genes. At a different level, Hox expression is influenced by modifications of chromatin and the three-dimensional organization of the chromosomes. Even after the transcription has occurred, microRNAs (miRNAs) may cleave Hox mRNAs and inactivate them.

Pax Genes

The Pax gene family, consisting of 9 known members, is an important group of genes that are involved in many aspects of mammalian development (Fig. 4.8). The Pax genes are homologous to the Drosophila pair-rule segmentation genes (see Fig. 4.1). All Pax proteins contain a paired domain of 128 amino acids that binds to DNA. Various members of this group also contain entire or partial homeobox domains and a conserved octapeptide sequence. Pax genes play a variety of important roles in the sense organs and developing nervous system, and outside the nervous system they are involved in cellular differentiative processes when epithelial-mesenchymal transitions occur.

Other Homeobox-Containing Gene Families

The POU gene family is named for the acronym of the first genes identified: Pit1, a gene uniquely expressed in the pituitary; Oct1 and Oct2; and Unc86, a gene expressed in a nematode. Genes of the POU family contain, in addition to a homeobox, a region encoding 75 amino acids, which also bind to DNA through a helix-loop-helix structure. As described in Chapter 3 (see p. 42), Oct-4 plays an important role during early cleavage.

The Lim proteins constitute a large family of homeodomain proteins, some of which bind to the DNA in the nucleus and others of which are localized in the cytoplasm. Lim proteins are involved at some stage in the formation of virtually all parts of the body. The absence of certain Lim proteins results in the development of headless mammalian embryos (see p. 83).

The Dlx gene family, similar to the Hox gene family, is a group of genes that have been phylogenetically conserved. The six members of this group in mammals are related to the single distalless gene in Drosophila, and they play important roles in patterning, especially of outgrowing structures, in early embryos. The mammalian Dlx genes operate in pairs, which are closely associated with Hox genes. Dlx5 and Dlx6 are located 5′ to Hoxa13; Dlx3 to Dlx7 are 5′ to Hoxb13; and Dlx1 and Dlx2 are 5′ to Hoxd13. In addition to being involved in appendage development, Dlx gene products are involved in morphogenesis of the jaws and inner ear and in early development of the placenta.

The Msx genes (homologous to the muscle-segment homeobox [msh] gene in Drosophila) constitute a small, highly conserved family of homeobox-containing genes, with only two representatives in humans. Nevertheless, the Msx proteins play important roles in embryonic development, especially in epitheliomesenchymal interactions in the limbs and face. Msx proteins are general inhibitors of cell differentiation in prenatal development, and in postnatal life they maintain the proliferative capacity of tissues.

T-Box Gene Family

The T-box (Tbx) genes take their names from the brachyury (T) locus, which was recognized as early as 1927 to cause short tails in heterozygotic mice. In 1990, the gene was cloned and found to contain a conserved region (the T-box), coding 180 to 200 amino acids, which binds to a specific nucleotide sequence in the DNA. Initially thought to be a single gene, an entire family of T-box genes with more than 100 members (18 genes in the human genome) has been described. Genes of this family play important roles in development, such as inducing the mesodermal germ layer and in coordinating outgrowth of either the arm or the leg.

Helix-Loop-Helix Transcription Factors

Basic Helix-Loop-Helix Proteins

The transcription factors of the basic helix-loop-helix type are proteins that contain a short stretch of amino acids in which two α-helices are separated by an amino acid loop. This region, with an adjacent basic region, allows the regulatory protein to bind to specific DNA sequences. The basic regions of these proteins bind to DNA, and the helix-loop-helix domain is involved in homodimerization or heterodimerization. This configuration is common in numerous transcription factors that regulate myogenesis (see Fig. 9.33).

Forkhead Gene Family

Another large family of transcription factors (>100 members, with 30 in mice) constitutes the forkhead (Fox) genes. As a variant of the helix-loop-helix theme, a common element among the forkhead proteins is the forkhead DNA-binding region, which is constituted as a winged helix structure. The Fox genes are expressed in many developing organs throughout the body. They tend to have microscopically distinct domains within a developing organ and can work together to direct the morphogenesis of a structure.

Zinc Finger Transcription Factors

The zinc finger family of transcription factors consists of proteins with regularly placed cystidine and histidine units that are bound by zinc ions to cause the polypeptide chain to pucker into fingerlike structures (Fig. 4.9). These “fingers” can be inserted into specific regions in the DNA helix.

Sox Genes

The Sox genes comprise a large family (>20 members) that have in common an HMG (high-mobility group) domain on the protein. This domain is unusual for a transcription factor in that, with a partner protein, it binds to 7 nucleotides on the minor instead of the major groove on the DNA helix and causes a pronounced conformational change in the DNA. Sox proteins were first recognized in 1990, when the SRY gene was shown to be the male-determining factor in sex differentiation (see p. 389), and the name of this group, Sox, was derived from Sry HMG box. One characteristic of Sox proteins is that they work in concert with other transcription factors to influence expression of their target genes (Fig. 4.10). As may be expected from their large number, Sox proteins are expressed by most structures at some stage in their development.

WT1

WT1 (Wilms tumor suppressor gene) is an isolated gene that in prenatal life plays a prominent role in formation of both the kidneys and gonads. It is crucial for the development of the early forms of the kidney and for the formation of the definitive adult kidney. In addition, WT1 is necessary for formation of the gonads. Its name derives from Wilms tumor, a prominent type of kidney tumor in young children.

Signaling Molecules

Much of embryonic development proceeds on the basis of chemical signals sent from one group of cells and received and acted on by another. A significant realization is that the same signaling molecule can be used at many different times and places as the embryo takes shape. Locally controlled factors, such as the concentration or duration of exposure to a signaling molecule, are often important determinants of the fate of a group of responding cells. This situation reduces greatly the number of signaling molecules that need to be employed. Most signaling molecules are members of several, mostly large, families. The specific sequence of signaling molecule (ligand) → receptor → signal transduction pathway is often called a signaling pathway. This section outlines the major families of signaling molecules that guide embryonic development.

Transforming Growth Factor-β Family

The transforming growth factor- β (TGF-β) superfamily consists of numerous molecules that play a wide variety of roles during embryogenesis and postnatal life. The TGF family was named because its first-discovered member (TGF-β₁) was isolated from virally transformed cells. Only later was it realized that many signaling molecules with greatly different functions during embryonic and postnatal life bear structural similarity to this molecule. Table 4.1 summarizes some of these molecules and their functions.

Table 4.1

Members of the Transforming Growth Factor-β Superfamily Mentioned in This Text

Member	Representative Functions	Chapters
TGF-β1 to TGF-β5	Mesodermal induction	5
	Myoblast proliferation	9
	Invasion of cardiac jelly by atrioventricular endothelial cells	17
Activin	Granulosa cell proliferation	1
	Mesodermal induction	5
Inhibin	Inhibition of gonadotropin secretion by hypophysis	1
Müllerian inhibiting substance	Regression of paramesonephric ducts	16
Decapentaplegic	Signaling in limb development	10
Vg1	Mesodermal and primitive streak induction	5
BMP-1 to BMP-15	Induction of neural plate, induction of skeletal differentiation, and other inductions	5, 9, 10
Nodal	Formation of mesoderm and primitive streak, left-right axial fixation	5
Glial cell line–derived neurotrophic factor	Induction of outgrowth of ureteric bud, neural colonization of gut	16, 12
Lefty	Determination of body asymmetry	5

BMP, bone morphogenetic protein; TGF-β, transforming growth factor-β.

The formation, structure, and modifications of TGF-β₁ are representative of many types of signaling molecules and are used as an example (Fig. 4.11). Similar to many members of this family, TGF-β₁ is a disulfide-linked dimer, which is synthesized as a pair of inactive 390-amino acid precursors. The glycosylated precursor consists of a small N-terminal signal sequence, a much larger proregion, and a 112-amino acid C-terminal bioactive domain. The proregion is enzymatically cleaved off the bioactive domain at a site of 4 basic amino acids adjoining the bioactive domain. After secretion from the cell, the proregion of the molecule remains associated with the bioactive region, thus causing the molecule to remain in a latent form. Only after dissociation of the proregion from the bioactive region does the bioactive dimer acquire its biological activity.

Among the most important subfamilies of the TGF-β family are the bone morphogenetic proteins (BMPs). Although BMP was originally discovered to be the active agent in the induction of bone during fracture healing, the 15 members of this group play important roles in the development of most structures in the embryo. BMPs often exert their effects by inhibiting other processes in the embryo. To make things even more complicated, certain very important interactions in embryonic development (e.g., induction of the central nervous system; see p. 84) occur because of the inhibition of BMP by some other molecule. The net result is an effect caused by the inhibition of an inhibitor. Molecules that inhibit or antagonize the action of BMPs are listed in Table 4.2. These molecules bind to secreted BMP dimers and interfere with their binding to specific receptors.

Table 4.2

Major Molecular Antagonists of Growth Factors

BMPs	Wnt
Noggin Chordin Chordinlike Twisted gastrulation Follistatin FSRP (follistatin-related protein) DAN/cerberus Gremlin Ectodin Coco

Axin-1, Axin-2

Dickkopf

Cerberus

Wif (Wnt inhibitory factor-1)

Sfrp (Secreted frizzled-related protein)

Wise (Wnt modulator in surface ectoderm)

SHH FGF (FGFR) Cyclopamine (in plants) Sprouty   Nodal   Lefty-1
Cerberus-like

Fibroblast Growth Factor Family

Fibroblast growth factor (FGF) was initially described in 1974 as a substance that stimulates the growth of fibroblasts in culture. Since then, the originally described FGF has expanded into a family of 22 members, each of which has distinctive functions. Many members of the FGF family play important roles in a variety of phases of embryonic development and in fulfilling functions, such as the stimulation of capillary growth, in the postnatal body. Some of the functions of the FGFs in embryonic development are listed in Table 4.3. Secreted FGFs are closely associated with the extracellular matrix and must bind to heparan sulfate to activate their receptors.

Similar to other signaling molecules, FGF activity is regulated in many ways. In contrast to the BMPs, which are regulated by several molecules that bind to them in the extracellular space, FGFs are mainly regulated farther downstream. Means of FGF regulation include the following: (1) modifications of their interaction with heparan proteoglycans in the receptor complex; (2) regulation at the membrane of the responding cell through the actions of transmembrane proteins; and (3) intracellular regulation by molecules, such as sprouty, which complex with parts of the signal transduction machinery of the responding cell. A main theme in the role of signaling molecules in embryonic development is variation, both in the variety of forms of signal molecules in the same family and in the means by which their activity is regulated. Most of the details of these are beyond the scope of this book, but for the beginning student it is important to recognize that they exist.

Hedgehog Family

The hedgehog signaling molecules burst on the vertebrate embryological scene in 1994 and are among the most important signaling molecules known (Table 4.4). Related to the segment-polarity molecule, hedgehog, in Drosophila, the three mammalian hedgehogs have been given the whimsical names of desert, Indian, and sonic hedgehog. The name hedgehog arose because mutant larvae in Drosophila contain thick bands of spikey outgrowths on their bodies.

Sonic hedgehog (shh) is a protein with a highly conserved N-terminal region and a more divergent C-terminal region. After its synthesis and release of the propeptide from the rough endoplasmic reticulum, the signal peptide is cleaved off, and glycosylation occurs on the remaining peptide (Fig. 4.12). Still within the cell, the shh peptide undergoes autocleavage through the catalytic activity of its C-terminal portion. During cleavage, the N-terminal segment becomes covalently bonded with cholesterol. The 19-kD N-terminal peptide is secreted from the cell, but it remains bound to the surface of the cell that produced it. All the signaling activity of shh resides in the N-terminal segment. Through the activity of another gene product (disp [dispatched] in Drosophila), the N-terminal segment of shh, still bound with cholesterol, is released from the cell. The C-terminal peptide plays no role in signaling.

At the surface of a target cell, shh, still complexed with cholesterol, binds to a receptor, Patched (Ptc), closely associated with another transmembrane protein, smoothened (smo). Ptc normally inhibits the signaling activity of smo, but shh inhibits the inhibitory activity of Ptc, thus allowing smo to give off an intracellular signal. Through the mediation of several other molecules, which are normally bound to microtubules, smo ultimately activates the 5-zinc finger transcription factor, Gli, which moves to the nucleus, binds to specific sites on the DNA of that cell, and thereby affects gene expression of the target cell.

Wnt Family

The Wnt family of signaling molecules is complex, with 18 members represented in the mouse. Related to the segment-polarity gene Wingless in Drosophila, Wnts play dramatically different roles in different classes of vertebrates. In amphibians, Wnts are essential for dorsalization in the very early embryo, whereas their role in preimplantation mouse development seems to be minimal. In mammals, Wnts play many important roles during the period of gastrulation. As many organ primordia begin to take shape, active Wnt pathways stimulate the cellular proliferation that is required to bring these structures to their normal proportions. Later in development, Wnts are involved in a variety of processes relating to cellular differentiation and polarity.

Wnts have been described as being “stickier” than other signaling molecules, and they often interact with components of the extracellular matrix. Their signaling pathway is complex and is still not completely understood (see Fig. 4.16). Similar to most other signaling molecules, the activity of Wnts can be regulated by other inhibitory molecules (see Table 4.2). Some inhibitory molecules, such as Wnt-inhibitory factor-1 (WIF-1) and cerberus, directly bind to the Wnt molecule. Others, such as dickkopf, effect inhibition by binding to the receptor complex.

Other Actions of Signaling Molecules

An important and more recent realization in molecular embryology is how often signaling molecules act by inhibiting the actions of other signaling molecules. For example, the signaling molecules chordin, noggin, and gremlin all inhibit the activity of BMP, which itself often acts as an inhibitor (see Table 4.2).

Evidence from several developing organ systems indicates that some signaling molecules (e.g., shh and members of the FGF family) are positive regulators of growth, whereas others (e.g., some members of the BMP family) serve as negative regulators of growth. Normal development of a variety of organs requires a balance between the activities of these positive and negative regulators. Such interactions are described later in the text for developing organ systems as diverse as limbs, hair (or feathers), teeth, and the branching of ducts in the lungs, kidneys, and prostate gland.

Receptor Molecules

For intercellular signaling molecules to exert an effect on target cells, they must normally interact with receptors in these cells. Most receptors are located on the cell surface, but some, especially those for lipid-soluble molecules, such as steroids, retinoids, and thyroid hormone, are intracellular.

Cell surface receptors are typically transmembrane proteins with extracellular, transmembrane, and cytoplasmic domains (see Fig. 4.2). The extracellular domain contains a binding site for the ligand, which is typically a hormone, cytokine, or growth factor. When the ligand binds to a receptor, it effects a conformational change in the cytoplasmic domain of the receptor molecule. Cell surface receptors are of two main types: (1) receptors with intrinsic protein kinase activity and (2) receptors that use a second messenger system to activate cytoplasmic protein kinases. An example of the first type is the family of receptors for FGFs, in which the cytoplasmic domain possesses tyrosine kinase activity. Receptors for growth factors of the TGF-β superfamily are also of this type, but in them the cytoplasmic domain contains serine/threonine kinase activity. In cell surface receptors of the second type, the protein kinase activity is separate from the receptor molecule itself. This type of receptor is also activated by binding with a ligand (e.g., neurotransmitter, peptide hormone, growth factor), but a series of intermediate steps is required to activate cytoplasmic protein kinases. A surface receptor, Notch, is introduced in greater detail in Box 4.2 as a specific example of a receptor that plays many important roles in embryonic development.

Box 4.2   Lateral Inhibition and the Notch Receptor

The normal development of many tissues begins with a population of developmentally equivalent cells. At some point, one of these cells begins to differentiate into a dominant mature cell type, such as a neuron, and, in doing so, it transmits to its neighboring cells a signal that prevents them from differentiating into that same cell type. As a consequence, these neighboring cells are forced to differentiate into a secondary cell type, such as a glial cell in the central nervous system (Fig. 4.13). This type of signaling of a dominant cell to its subservient neighbors is called lateral inhibition.

The common mechanism of lateral inhibition is the Notch signaling pathway, which is so basic that it has been preserved largely unchanged throughout the animal kingdom. Notch is a 300-kD cell surface receptor with a large extracellular domain and a smaller intracellular domain. The Notch receptor becomes activated when it combines with ligands (Delta or Jagged in vertebrates) that extend from the surface of the dominant cell. This sets off a pathway that inhibits the neighboring cell from differentiating into the dominant phenotype.

An abbreviated version of this pathway is as follows (Fig. 4.14): The complexing of Notch with its ligand (e.g., Delta) stimulates an intracellular protease reaction that cleaves off the intracellular domain of the Notch molecule. The liberated intracellular domain of Notch becomes translocated to the nucleus, but on its way it may become associated with regulatory proteins, such as Deltex. Within the nucleus, the intracellular domain of Notch combines with several helix-loop-helix transcription factors, and this complex binds to the DNA of a gene called enhancer of split. The product of this gene is another transcription factor that regulates other genes. It represses certain genes of the Achaete-Scute complex, whose function is to promote neuronal development. By this complex pathway, the subservient cells, in the nervous system for example, are denied the opportunity to differentiate into neurons and instead follow a secondary pathway, which leads to their becoming glial cells.

As complex as it seems, this description is a greatly abbreviated version of this inhibitory pathway and its controlling elements. When more is learned about all the elements involved in this pathway, it will likely look like a component of an immense network of regulatory pathways that interact in very complex ways to integrate internal and external environmental influences that determine the ultimate developmental fate of a cell.

Signal Transduction

Signal transduction is the process by which the signal provided by the first messenger (i.e., the growth factor or other signaling molecule) is translated into an intracellular response. Signal transduction is very complex. It begins with a response to binding of the signaling molecule to its receptor and the resulting change in the conformation of the receptor. This process sets off a chain reaction of activation or inhibition of a string of cytoplasmic molecules whose function is to carry the signal to the nucleus, where it ultimately influences gene expression. It is common to speak about signal transduction pathways as though they are straight lines, but in reality signal transduction should be viewed as a massive network subject to a wide variety of modulating influences. Despite this complexity, signal transduction can be viewed as linear pathways for purposes of introduction. Several major pathways of relevance to signaling molecules treated in this text are summarized here.

Members of the FGF family connect with the receptor tyrosine kinase (TRK) pathway (Fig. 4.15A). After FGF has bound to the receptor, a G protein near the receptor becomes activated and sets off a long string of intracytoplasmic reactions, starting with RAS and ending with the entry of ERK into the nucleus, and its interaction with transcription factors. Members of the TGF-β family first bind to a type II serine/threonine kinase receptor, which complexes with a type I receptor (Fig. 4.15B). This process activates a pathway dominated by Smad proteins. Two different Smads (R-Smad and Co-Smad) dimerize and enter the nucleus. The Smad dimer binds with a cofactor and is then capable of binding with some regulatory element on the DNA.

The hedgehog pathway was already introduced in Figure 4.12. The complex Wnt pathway first involves binding of the Wnt molecule to its transmembrane receptor, Frizzled. In a manner not yet completely understood, Frizzled interacts with the cytoplasmic protein Disheveled, which ties up a complex of numerous molecules (destruction complex), which in the absence of Wnt cause the degradation of an important cytoplasmic protein, β-catenin (Fig. 4.16). If β-catenin is not destroyed, it enters the nucleus, where it acts as a powerful adjunct to transcription factors that determine patterns of gene expression.

The more recently discovered Hippo pathway, highly conserved in phylogeny, is proving to be very important in regulating organ growth throughout the animal kingdom. Loss of Hippo function results in unrestrained growth of structures ranging from the cuticle of Drosophila to the liver of mammals. In mammals, Hippo restricts cellular proliferation and promotes the removal of excess cells through apoptosis. It is involved in maintaining the balance between stem cells and differentiated cells both prenatally and postnatally.

These and other less prominent signal transduction pathways are the intracellular effectors of the many signaling events that are necessary for the unfolding of the numerous coordinated programs that guide the orderly progression of embryonic development. Specific examples involving these signaling pathways are frequently mentioned in subsequent chapters.

Small RNAs

The discovery of miRNAs just before 2000 added a new and complex dimension to our understanding of the genetic regulation of development. Small RNAs are small noncoding RNA molecules that exert an enormous array of influences on gene expression, mainly at the posttranscriptional level. In vertebrates, small RNAs can be divided into two main groups: those involved in gametogenesis and those that act during embryogenesis. Of those that act during gametogenesis, Piwi-interacting RNAs (piRNAs) are important in spermatogenesis, and endogenous small interfering RNAs (endo-siRNAs) play vital roles in oogenesis. miRNAs are expressed in somatic tissues during embryonic development.

Although small RNAs function through a bewildering array of mechanisms, one major pathway is close to being common (Fig. 4.17). miRNAs often begin as double-stranded molecules with a hairpin loop. Through the activity of an enzyme called Dicer, the miRNA precursor is cleaved, resulting in a single-stranded miRNA, which is then bound to a member of the Argonaute (AGO) protein family. In many cases, the AGO-siRNA complex has RNase activity and is able to disrupt a target RNA molecule enzymatically. In this way specific gene expression is modulated. By applying this principle, developmental geneticists are able to target the disruption of specific genes under investigation by interfering with the mRNAs that these genes produce.

Retinoic Acid

For years, vitamin A (retinol) and its metabolite, retinoic acid, have been known to play very important but equally enigmatic roles in embryonic development. In the 1960s, investigators found that either a severe deficiency or an excess of vitamin A results in a broad spectrum of severe congenital anomalies that can involve the face, eye, hindbrain, limbs, or urogenital system. It was only in the 1990s, when the binding proteins and receptors for the retinoids were characterized and the development of various knockouts was investigated, that specific clues to the function of vitamin A in embryogenesis began to emerge.

Vitamin A enters the body of the embryo as retinol and binds to a retinol-binding protein, which attaches to specific cell surface receptors (Fig. 4.18). Retinol is released from this complex and enters the cytoplasm, where it is bound to cellular retinol-binding protein (CRBP I). In the cytoplasm, the all-trans retinol is enzymatically converted first to all-trans retinaldehyde and then to all-trans retinoic acid, the retinoid with the most potent biological activity (see Fig. 4.18). CRBP and CRABP I (cellular retinoic acid–binding protein) may function to control the amount of retinoids that enters the nucleus. When released from CRABP, retinoic acid enters the nucleus, where it typically binds to a heterodimer consisting of a member of the retinoic acid receptor (RAR) α, β, or γ family and a member of the retinoid X receptor (RXR) α, β, or γ family. This complex of retinoic acid and receptor heterodimer binds to a retinoic acid response element (RARE) on DNA, usually on the enhancer region of a gene, and it acts as a transcription factor, controlling the production of a gene product.

Retinoic acid is produced and used in specific local regions at various times during prenatal and postnatal life. Among its well-defined targets early in development are certain Hox genes (e.g., Hoxb-1); misexpression of these genes caused by either too little or too much retinoic acid can result in serious disturbances in the organization of the hindbrain and pharyngeal neural crest. One of the most spectacular examples of the power of retinoic acid is its ability to cause extra pairs of limbs to form alongside the regenerating tails of amphibians (Fig. 4.19). This is a true example of a homeotic shift in a vertebrate, similar to the formation of double-winged flies or legs instead of antennae in Drosophila (see p. 59).

Developmental Genes and Cancer

Many cancers are caused by mutated genes, and many of these are genes that play a role in normal embryonic development. Two main classes of genes are involved in tumor formation, and each class uses a different mechanism in stimulating tumor formation.

Proto-oncogenes, a class involving a variety of different types of molecules, induce tumor formation through dominant gain-of-function alleles that result in deregulated growth. Through several types of mechanisms, such as single-point mutations, selective amplification, or chromosomal rearrangements, proto-oncogenes can become converted to oncogenes, which are the actual effectors of poorly controlled cellular proliferation. Proto-oncogenes direct the normal formation of molecules including certain growth factors, growth factor receptors, membrane-bound and cytoplasmic signal proteins, and transcription factors.

The other class of genes involved in tumor formation consists of the tumor suppressor genes, which normally function to limit the frequency of cell divisions. Recessive loss-of-function alleles of these genes fail to suppress cell division, thus resulting in uncontrolled divisions in defined populations of cells. A good example of a tumor suppressor gene is Patched, already discussed as the transmembrane receptor for the signaling molecule, shh. Patched normally inhibits the activity of smo. Mutations of Patched eliminate the inhibition of smo and allow uncontrolled downstream activity from smo that stimulates the genome of the affected cell. Such a Patched mutation is the basis for the most common type of cancer, basal cell carcinoma, of the skin. shh itself is involved in tumors of the digestive tract. shh is increased in tumors of the esophagus, stomach, biliary tract, and pancreas, but the hedgehog pathway is not active in cell line tumors from the colon.

Summary