The Vertebrate Body Plan

The development of the vertebrate body plan has long been a focus of experimental embryology. Many fundamental concepts such as organizers, fields, and morphogens were derived first from observations of vertebrate embryos. The major features of adult vertebrate morphology, including segmented vertebral columns, paired appendages, and skulls, have undergone considerable evolutionary diversification. Therefore, we will focus on the developmental genetics of these major features here, and consider their evolutionary origins and modification in subsequent chapters.

Most vertebrate embryology has focused on a few amphibian (Xenopus, newts), avian (chick, quail), mammalian (mouse), and fish (zebrafish, medaka) species. The early development of these embryos and the mechanisms that orient the primary axes differ considerably, particularly between species with yolky eggs (for example, amphibians, fish) and species that rely upon extraembryonic structures. Nevertheless, all early vertebrate development converges on an embryonic stage in which the head is distinct, a neural tube extends along the dorsal midline above the notochord, and part of the mesoderm is subdivided. At this stage, the major anteroposterior axis (often called the "rostrocaudal" axis) is well defined, the general rostrocaudal domains of major head, trunk, and tail regulatory genes are initially determined, and the secondary fields (for example, limb buds) that will give rise to appendages and other structures are set to emerge. In the following discussion, we will see that the developmental regulatory logic and mechanisms controlling the patterning of vertebrate body axes and cellular fields revisit some already familiar themes mentioned earlier in this chapter.

The rostrocaudal axis: somite formation and segmentation

The mesoderm of vertebrates gives rise to many organs and to the tissues that form major features of vertebrate body architecture. The mesoderm is subdivided into several regions that give rise to different tissues (Fig. 3.17a,b). The lateral plate mesoderm is the source of elements of the circulatory system, the lining of body cavities, and all of the mesodermal components of the limbs (except the muscles). The paraxial mesoderm is subdivided into metameric subunits called somites. The somites provide the framework for the metameric organization of somite-derived tissues, including the axial skeleton (vertebrae and ribs), the dermis and muscles of the back, and the skeletal muscles of the body wall and limbs. Because somite-derived structures such as the vertebrae and ribs have characteristic shapes in different regions of the rostrocaudal axis, the generation and individuation of the somites and their derivatives are fundamental to the evolution and diversification of the vertebrate body plan.

Figure 3.17

Segmentation and somite formation in the vertebrate embryo

(a) The segmentation of the paraxial mesoderm occurs in an anterior to posterior order. (b) The mesoderm of the early vertebrate embryo shown in cross section. (c) Components and targets of the Notch signaling pathway (for example, mouse HES1) are expressed in a dynamic pattern that cycles in the presomitic mesoderm once during the generation of each pair of somites. Source: Part c adapted from Jouve C, Palmeirim I, Henrique D, et al. Development 2000; 127: 1421-1429.

Figure 3.17

Segmentation and somite formation in the vertebrate embryo

(a) The segmentation of the paraxial mesoderm occurs in an anterior to posterior order. (b) The mesoderm of the early vertebrate embryo shown in cross section. (c) Components and targets of the Notch signaling pathway (for example, mouse HES1) are expressed in a dynamic pattern that cycles in the presomitic mesoderm once during the generation of each pair of somites. Source: Part c adapted from Jouve C, Palmeirim I, Henrique D, et al. Development 2000; 127: 1421-1429.

Segmentation of the paraxial mesoderm and somite formation involve a sequential, temporally ordered process that proceeds from the anterior to the posterior of the embryo. New somites are formed from the rostral end of the paraxial mesoderm at regular intervals. In the mouse and the chick, a defined, albeit different number of somites are produced, at roughly 90-minute intervals, whereas in zebrafish a new somite forms every 20 minutes.

Two regulatory systems have been identified that underlie the temporal and spatial regulation of somite formation. One system behaves as a segmentation clock, involving the oscillating expression patterns of genes in the developing somites. The second system translates the clock into a periodic arrangement of segment (somite) boundaries.

The best characterized set of genes whose expression oscillates during somite formation include relatives of the Drosophila hairy and Enhancer of Split gene family (HES/HER genes), a modulator of the Notch receptor protein Lunatic fringe, and the Notch ligand deltaC. These genes are expressed in a dynamic wave that sweeps across the presomitic mesoderm from posterior to anterior once during the formation of each somite (Fig. 3.17c). Oscillating gene expression and somite formation in all vertebrates appears to be regulated by the Notch pathway, although the roles of individual genes vary in different species. The synchronized expression of these genes suggests that they lie downstream of a common cycling activator. The translation of oscillating transcription factor expression into segment boundaries is regulated by the Fgf8 signaling molecule. Fgf8 is expressed in a graded manner in the presomitic mesoderm (PSM) and the level of expression is important for regulating the position of somite boundaries. The integration of these two systems together with antero-posterior patterning information, including Hox genes, creates the unique identities along the vertebrate segmented body plan.

The vertebrate Hox ground plan

The somites of the vertebrate embryo give rise to the major axial structures, including the vertebrae, ribs, and skeletal muscles, as well as the dermis. The vertebrae of the spinal column and their associated processes are of five distinct types: cervical, thoracic, lumbar, sacral, and caudal (Fig. 3.18a). The transitions between these types occur at specific somite positions in each species. Similarly, the forelimb and hindlimb buds, which develop from the unsegmented lateral plate mesoderm, arise at specific axial positions. Therefore, the morphologies of somite derivatives at different axial levels must be genetically regulated.

Patterning along the rostrocaudal axis in all vertebrates is regulated by the Hox genes. Vertebrates have four or more complexes of Hox genes; the mouse, for example, has 39 Hox genes. In general, the anterior boundaries of expression of the Hox genes correlate with the respective locations of the genes within a complex, a principle termed colinearity. Thus the Hoxl genes (which are related to the labial gene) are expressed in the most anterior positions, and the Hox9-13 genes (which are related to the Abd-B gene) are expressed in more posterior positions (see Fig. 2.22b). The anterior boundaries of Hox gene expression are generally sharper than their posterior boundaries and lie in spatial register between the somitic and lateral plate mesoderm. However, they are not in register with Hox gene expression in the neural tube. The tissue-specific boundaries of Hox gene expression indicate that independent genetic regulatory mechanisms control Hox expression in the neural tube.

Patterning of part of the neural tube, the posterior region of the head, and the hindbrain also involves segmentation along the A/P axis. The process of hindbrain segmentation is evolutionarily conserved in vertebrates in terms of the number of subdivisions or rhom-bomeres formed (seven to eight), the neuroanatomical organization of each region, and the pattern of expression of various genes, including transcription factors and signaling proteins that establish rhombomere segmentation, identity, and cell behavior. The best-known regulatory genes involved in this process are the Hoxl through Hox4 genes, various members of which are expressed in and affect the development of discrete rhombomeres (Fig. 3.18a,b).

The same themes pertain to Hox regulation in the vertebrate hindbrain (and elsewhere) as were highlighted for the Drosophila Hox genes:

Mouse b

Hindbrain

Hindbrain kreisler Krox-20

Hoxa-1 Hoxb-1 Hoxa-2 Hoxb-2 Hoxa-3 Hoxb-3 Hoxd-3 Hoxd-4 Hoxb-4 Hoxa-4

c r2

d r2

lateral mesoderm vagal neural crest

Figure 3.18

Regulation of Hox gene expression in the vertebrate hindbrain

(a) The vertebrate hindbrain forms anterior to the spinal cord. (b) The hindbrain is overtly segmented into rhombomeres (r1-r7). (c) Summary of regulatory gene expression in rl—r7. The arrows denote regulatory interactions between two transcription factors (Kreisler and Krox20) and two Hox genes of interest. (d) A schematic of the genetic region around the Hoxa2 and Hoxa3 genes. Specific c/s-regulatory elements (R3 and R5) control the response of Hoxa2 to Krox20 in r3/r5. Note the presence of several other elements controlling Hox expression in other tissues. This diversity of c/s-elements is typical of Hox genes. Source: Modified from Lumsden A, Krumlauf R. Sc/ence 1996; 274: 1109-1115; Manzanares M, Cordes S, Ariza-McNaughton L, et al. Development 1999; 126: 759-769; Nonchev S, et al. Development 1996; 122: 543-554.

lateral mesoderm vagal neural crest

Figure 3.18

Regulation of Hox gene expression in the vertebrate hindbrain

(a) The vertebrate hindbrain forms anterior to the spinal cord. (b) The hindbrain is overtly segmented into rhombomeres (r1-r7). (c) Summary of regulatory gene expression in rl—r7. The arrows denote regulatory interactions between two transcription factors (Kreisler and Krox20) and two Hox genes of interest. (d) A schematic of the genetic region around the Hoxa2 and Hoxa3 genes. Specific c/s-regulatory elements (R3 and R5) control the response of Hoxa2 to Krox20 in r3/r5. Note the presence of several other elements controlling Hox expression in other tissues. This diversity of c/s-elements is typical of Hox genes. Source: Modified from Lumsden A, Krumlauf R. Sc/ence 1996; 274: 1109-1115; Manzanares M, Cordes S, Ariza-McNaughton L, et al. Development 1999; 126: 759-769; Nonchev S, et al. Development 1996; 122: 543-554.

• Numerous independent czs-acting regulatory elements control features of individual Hox gene expressions in different rostrocaudal domains and in different germ layers.

• Both positive and negative regulatory interactions are necessary to define the initial domains of gene expression.

• Autoregulatory and cross-regulatory interactions between Hox genes are necessary to maintain domains of gene expression.

In the developing mouse hindbrain, a few direct transcriptional regulators of Hox gene expression patterns have been identified, including the products of the Krox20 and Kreisler genes, and products of Hox genes themselves. In addition, cz's-regulatory elements have been identified that control rhombomere-specific patterns of Hox gene expression (Fig. 3.18d). For example, expression of the Hoxa2 and Hoxb2 paralogs in rhombomeres 3(r3) and 5(r5) are controlled by distinct elements, independent of those that control expression in the mesoderm and other tissues. The Krox20 protein is specifically expressed in these two rhombomeres and directly regulates the r3/r5 enhancers (Fig. 3.18c). Similarly, expression of the Hoxa3 and Hoxb3 genes in r5/r6 is controlled by discrete elements. The Kreisler protein is specifically expressed in these developing rhombomeres, and it binds to and directly regulates Hoxa3 and Hoxb3 expression through these elements (Fig. 3.18c).

Expression of the Hox genes is coupled to the segmentation clock, providing coordination of somitogenesis and the specification of regional identity. But apart from these rhombomere patterns, not much is known about the transcription factors that position Hox domains along the rostrocaudal axis. In addition, from the cz's-regulatory elements of the Hox complexes, we can infer that the regulatory network likely involves many interactions. When the intergenic regions of a few pairs of Hox genes were analyzed in greater detail, a substantial number of regulatory elements were found interspersed among the Hox genes. Separate cz's-regulatory elements that control gene expression in the lateral mesoderm, neural crest, spinal cord, tail bud, limb buds, and somites have been identified, for instance (Fig. 3.18d). The temporal, spatial, and tissue-specific aspects of each pattern must be regulated by a variety of transcription factors acting through these elements. The different anterior expression boundaries of adjacent genes within complexes could reflect the differential response of cz's-regulatory elements to the same regulator or the specific response of each element to different regulators. Interestingly, some of the czs-acting regulatory elements are shared between pairs of adjacent genes, and this sharing may be a mechanism that preserves the clustering of Hox genes.

Vertebrate limb development

Among the most important structures in vertebrate evolutionary history are the paired pelvic and pectoral appendages. The evolution of the tetrapod limb from the paired fins of fish and the subsequent modification of limb morphologies used in flying, running and jumping, burrowing, and the return to water involved major changes in the anatomy of paired appendages. Coupled with the longstanding research on the experimental embryology of limb buds, immense interest is focused on the developmental genetics of vertebrate limb formation and patterning. For our purposes, there are six major aspects of limb formation and patterning, each of which represents a potential point of divergence between taxa in the functional evolution of limb morphology:

• The positioning of the limb bud along the rostrocaudal axis

• The initial outgrowth of the limb bud

• Establishment and signaling from the limb organizers

• The formation of the limb proximodistal axis and deployment of Hox gene expression in the limb bud

• The specification of major skeletal and cartilage elements

• The regulation of forelimb and hindlimb identity

These developmental processes unfold as a cascade of events that progressively establish the limb field, its two organizers, and many pattern elements (Fig. 3.19).

Position of limb bud initiation along rostrocaudal axis set by Hox, Wnt genes and induced by FGF signaling from flank

Tbxeene and Fgf10 expression in limb mesoderm

AER induced

Eni Wnt 7a Lmx 1

dorsoventral polarity of limb pattern elements

Maintain organizer activities

Organize P/D axis outgrowth

Polarize Hox expression in limb bud and HAND2 (posterior) expression in limb bud mesoderm

ZPA induced

Shh expression anteroposterior polarity of limb pattern elements

Hox expression and function

Chondrogenic condensation

Ihh PTHLH

FGFs BMPs WNTs SOXs

WNTs, GDFs

Figure 3.19

Overview of vertebrate limb formation

The development of the vertebrate limb bud progresses through several stages in which signaling centers and several regulatory genes play key roles in the initiation, outgrowth, and patterning of the limb bud (see text for details).

Bone formation Joint formation

The developmental regulatory logic involved in these processes of limb formation and patterning are analogous to those discussed for insects. The position of the limb field depends on signals whose distribution is regulated along the main body axis. The outgrowth of the limb field involves regulatory hierarchies that establish the anteroposterior and dorsoventral axes of the limb field, and integration of these inputs regulates patterning along the prox-imodistal axis. Superimposed on the potentially equivalent hierarchies in the forelimb and hindlimb fields are selector genes that specifically modify the development of these limbs.

Positioning of the limb buds along the rostrocaudal axis

Limb development begins with the proliferation of mesenchymal cells from within the limb field of the lateral plate mesoderm. As these cells accumulate under the epidermis, a bulge appears—the growing limb bud. This proliferation appears to be under the control of local signals emanating from nearby mesoderm. One strong candidate for the source of this proliferative signal is fibroblast growth factor 10 (FGF10), which is expressed near the site of limb bud initiation and can induce the formation of ectopic limb buds when expressed at novel positions along the rostrocaudal axis. Although Fgf10 is initially expressed throughout the lateral plate mesoderm, it becomes restricted to the presumptive limb areas. Expression in these regions is maintained by a combination of specific Wnt signaling molecules (members of the signaling protein family to which the insect Wg protein belongs) and T-box (Tbx) transcription factors (Fig. 3.20a).

Limbs of different vertebrates arise at different positions, with respect to somite number, along the rostrocaudal axis. Nevertheless, the forelimb buds always arise at the most anterior position of Hoxc6 expression at the transition from the cervical vertebra to the thoracic vertebra. This consistent location suggests that the positioning of the limb bud is determined by both axial coordinates (Hox genes) and local expression of specific Wnt genes.

Outgrowth of the limb bud: dorsoventral and anteroposterior regulatory hierarchies

The early limb bud consists of mesodermal cells and an overlying ectodermal epithelium. As it forms, the mesodermal cells induce the overlying ectoderm to express Fgf8 (Fig. 3.20b) and these cells will eventually form the apical ectodermal ridge (AER), a prominent thickening of cells at the edge of the limb bud where its dorsal and ventral halves meet. The AER organizer is a major signaling center that produces signals such as FGF8 (and other related FGFs), which are necessary for the continued proliferation and differentiation of the growing limb bud. Early loss of the AER, or its signaling functions, prevents outgrowth of the buds and limb formation.

Induction and maintenance of the AER as well as dorsoventral patterning of the limb depends upon the interplay between several signaling pathways in the ventral limb ectoderm (Fig. 3.21). FGF10 signals from the limb mesoderm to the overlying ectoderm and induces Fgf8 expression in the early ventral limb ectoderm, or pre-AER cells. This induction is mediated by the BMP and Wnt signaling pathways (Fig. 3.21b).

These same two signaling pathways also regulate the expression of En-1. En-1 regulates dorsal patterning of the limb, which occurs later during limb bud outgrowth. En-1 represses expression of the Wnt-7a protein, which is then expressed only in the dorsal ectoderm and

Figure 3.20

Regulation of vertebrate limb bud formation

Figure 3.20

Regulation of vertebrate limb bud formation

Dorsal view of a vertebrate forelimb-forming region showing genes involved in (a,b) limb initiation and (c,d) A/P patterning. Rostral is to the top and distal is to the right. (a) The forelimb bud is induced by Wnt2b expression in the chick lateral plate mesoderm. In response to Wnt2b signaling, Tbx5 expression is activated, which maintains Fgf10 expression in the presumptive forelimb region. (b) Fgf10 signals to the overlying ectoderm and, through Wnt3a, induces Fgf8 expression. These Fgf8-expressing cells will give rise to the AER and maintain proliferation of the bud. (c) In the absence of Hedgehog signaling, GLI3 exists in a repressor state (GLI3R). Mutual antagonism of GLI3R and HAND2 prepatterns the limb mesenchyme prior to SHH signaling. (d) Shh is induced in the posterior of the limb bud by HAND2 and restricts GLI3R activity to the anterior mesenchyme. The two signaling sources (ZPA, AER) are maintained by reciprocal interactions involving Shh, Fgfs and the BMP antagonist Gremlin (GRE).

Source: Part a modified from Kawakami Y, Capdevila J, et al. Cell 2001; 104: 891-900. Copyright (2001), reprinted with permission from Elsevier. Part b adapted from te Welscher P, Zuniga A, et al. Science 2002; 298: 827-830. Copyright (2002), reprinted with permission from AAAS.

a dorsal

Wnt-7a ventral

Wnt-7a ventral

Lmx1b

Figure 3.21

AER formation and dorsoventral patterning of the limb bud

En-1

Lmx1b

Fgf8 (AER)

Bmp signaling (via BmpRIA)

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