Molecular Basis for Embryonic Development

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Molecular Basis for Embryonic Development

The application of new techniques in molecular biology continues to revolutionize the understanding of the mechanisms underlying both normal and abnormal embryonic development. It is impossible to have a contemporary understanding of embryonic development without integrating fundamental molecular and morphological aspects of embryology. This chapter introduces the most important families of molecules known to direct embryonic development.

One of the most important realizations has been the conservatism of the genes that guide development. Sequencing studies have shown remarkably few changes in the nucleotide bases of many developmentally regulated genes that are represented in species ranging from worms to Drosophila to humans. Because of this phylogenetic conservatism, it has been possible to identify mammalian counterparts of genes that are known from genetic studies to have important developmental functions in other species (Box 4.1).* It is also clear that the same gene may function at different periods of development and in different organs. Such reuse greatly reduces the total number of molecules that are needed to control development. Before and after birth, specific genes may be expressed in normal and abnormal processes. One of the principal themes in contemporary cancer research is the role of mutant forms of developmentally important genes (e.g., proto-oncogenes) in converting normal cells to tumor cells.

Box 4.1 Early Developmental Genetics in Drosophila

Despite the discovery and characterization of many developmentally important genes in mammals, the basic framework for understanding the molecular basis of embryonic development still rests largely on studies of developmental genetics in Drosophila. Although the earliest stages of human development occur under less rigid genetic control than those of Drosophila, an exposure to the fundamental aspects of early Drosophila development nevertheless sets the stage for a deeper understanding of molecular embryogenesis in mammals.

Embryonic development of Drosophila is under tight genetic control. In the earliest stages, the dorsoventral and anteroposterior axes of the embryo are established by the actions of batteries of maternal-effect genes (Fig. 4.1). When these broad parameters have been established, the oval embryo undergoes a series of three sequential steps that result in the segmentation of the entire embryo along its anteroposterior axis. The first step in segmentation, under the control of what are called gap genes, subdivides the embryo into broad regional domains. Loss-of-function gap mutants result in loss of structure, or gaps, in the body pattern several segments in width. In the second step, a group of pair-rule genes is involved in the formation of seven pairs of stripes along the craniocaudal axis of the embryo. The third level in the segmentation process is controlled by the segment-polarity genes, which work at the level of individual segments and are involved in their anteroposterior organization.*

Fig. 4.1 Sequence of genetic control of early development in *Drosophila*.
Within each level of genetic control are listed representative genes.

The segmentation process results in a regular set of subdivisions along the anteroposterior axis of the early Drosophila embryo, but none of the previously mentioned developmental controls imparts specific or regional characteristics to the newly formed segments. This function is relegated to two large clusters of homeotic genes found in the antennapedia complex and the bithorax complex. The specific genes in these two complexes determine the morphogenetic character of the body segments, such as segments bearing antennae, wings, or legs. Mutations of homeotic genes have long been known to produce bizarre malformations in insects, such as extra sets of wings or legs instead of antennae (hence the term antennapedia).

^*In Drosophila, each stripe (segment) is subdivided into anterior and posterior halves. The posterior half of one segment and the anterior half of the next are collectively known as a parasegment. The genetics and developmental aspects of insect parasegments are beyond the scope of this text, but in Chapter 6, when formation of the vertebral column is discussed, a similar set of divisions of the basic body segments in vertebrate embryos is introduced.

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.

Fig. 4.2 Schematic representation of types of developmentally important molecules and their sites of action.

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).

Fig. 4.3 Structure of a typical homeodomain protein.

Fig. 4.4 Schematic representation of classes of homeobox-containing genes also possessing conserved motifs outside the homeodomain.
Names of the different classes of genes are listed on the *left.* The *red boxes* represent the homeobox within each gene class. The other boxes represent conserved motifs specific to each class of genes. (Modified from Duboule D, ed: *Guidebook to the homeobox genes,* Oxford, 1994, Oxford University Press.)

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.

Fig. 4.5 Organization of the human *HOX* complex.
Genes on the 3′ ends of each of the complexes are expressed earlier and more anteriorly than those on the 5′ end (*right*). (Based on Scott MP: *Cell* 71:551-553, 1992.)

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.

Fig. 4.6 Organization of certain homeobox-containing genes of *Drosophila* and mouse and their segmental expression in the body. (Based on DeRobertis EM, Oliver G, Wright CVE: *Sci Am* 263:46-52, 1990. Copyright Patricia J. Wynne.)

Fig. 4.7 Effect of interference of *XlHbox 1* (~Hoxc-6) function on development in *Xenopus*.
A, Normal larva, showing a discrete band *(green)* of *XlHbox 1* expression. B, Caudal expansion of the hindbrain after antibodies to *XlHbox 1* protein are injected into the early embryo. (Based on Wright CV and others: *Cell* 59:81-93, 1989.)

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.

Fig. 4.8 Summary diagram of the members of the *Pax* gene family, showing their location on human chromosomes, sites of expression, and known effects of mutants in human and mouse.
The structures of conserved elements of these genes are schematically represented. CNS, central nervous system; KO, knockout. (Modified from Wehr R, Gruss P: *Int J Dev Biol* 40:369-377, 1996; and Epstein JC: *Trends Cardiovasc Med* 6:255-260, 1996.)

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

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
Activin	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