Periodicity

The important components of somitogenesis (somite formation) are periodicity, epithelialization, specification, and differentiation. The first somites appear in the anterior portion of the trunk, and new somites “bud off” from the rostral end of the paraxial mesoderm at regular intervals (Figures 14.2C,D and 14.3). Somite formation begins as paraxial mesoderm cells become organized into whorls of cells called somitomeres. The somitomeres become compacted and bound together by an epithelium, and eventually separate from the presomitic paraxial mesoderm to form individual somites. Because individual embryos can develop at slightly different rates (as when chick embryos are incubated at slightly different temperatures), the number of somites present is usually the best indicator of how far development has proceeded. The total number of somites formed is characteristic of a species (50 in chicks, 65 in mice, and about 500 in some snakes).

Figure 14.3

Neural tube and somites. Scanning electron micrograph showing well-formed somites and paraxial mesoderm (bottom right) that has not yet separated into distinct somites. A rounding of the paraxial mesoderm into a somitomere can be seen at the lower left, (more...)

Although we do not know much about the mechanisms controlling the periodicity of somite formation, one of the key agents in this process is the hairy gene. What makes the hairy gene so interesting is that it is expressed in a dynamic pattern. First, it is expressed in the caudal portion of each somite, and it persists in these caudal regions for at least 15 hours. Second, hairy is expressed in the presomitic segmental plate or unsegmented mesoderm in a cyclic wavelike manner, cresting every 90 minutes. Its expression is detected first in the caudalmost region of the presomitic mesoderm, and this region of expression moves anteriorly as each somite forms. Eventually, like a wave leaving shells upon a beach, its expression recedes caudally, while the most anterior expression region remains. The caudalmost region of this anterior expression band correlates with the posterior terminus of the next somite to be formed. This process can be seen in Figure 14.4.

Figure 14.4

Somite formation correlates with the expression of the hairy gene. (A) Schematic representation of the posterior portion of an X-somite chick embryo. Somite X is marked. The expression of the hairy gene (purple) is seen in the caudal half of this somite, (more...)

Thus, the expression pattern of the hairy gene correlates with the positioning of the place where a somite will separate from the unsegmented mesoderm. Labeling of cells with diI shows that this wave of hairy expression is not caused by the migration of particular cells that express the hairy gene. Rather, the wavelike pattern of hairy expression is an autonomous property of the presomitic mesoderm. Even if the presomitic mesoderm is separated from the caudalmost mesoderm, the ectoderm, neural tube, notochord, Hensen's node, and lateral plate regions, the dynamic expression of the hairy gene remains (Palmeirim et al. 1997; Jouve et al. 2000).

Separation

The hairy gene encodes a transcription factor (indeed, one that is also used for forming the segmental units of Drosophila—see Chapter 9), but it is not known what its targets are. One set of possible targets (directly or indirectly) are the genes for ephrin and its receptor. The Eph receptor proteins and their ephrin ligands were discussed in Chapter 13 as being able to cause cell-cell repulsion between the posterior somite and migrating neural crest cells. Ephrin and Eph also may be critical for separating the somites. In the zebrafish, the boundary between the most recently separated somite and the presomitic mesoderm forms between ephrinB2 in the posterior of the somite and EphA4 in the most anterior portion of the presomitic mesoderm (Figure 14.5A; Durbin et al. 1998). As somites form, this pattern of gene expression is reiterated caudally. Interfering with this signaling (by injecting embryos with RNA encoding dominant negative Ephs) leads to abnormal somite boundary formation. Eph signaling is thought to mediate cell shape changes, and these could be responsible for separating the presomitic mesoderm at the EphA4-ephrinB2 border. Thus, ephrin-Eph signaling may be responsible for converting the prepattern established by the Hairy protein in the presomitic mesoderm into actual somites.

Figure 14.5

Transition from somitomere to somite. (A) Expression pattern of receptor tyrosine kinase EphA4 (blue) and its ligand, ephrinB2 (red) as somites develop. The somite boundary forms at the junction between the region of ephrin expression on the posterior (more...)

Epithelialization

Several studies in chicks have shown that the conversion from mesenchymal tissue into an epithelial block occurs even before each somite splits off. As seen in Figure 14.3, the cells of the somitomere are randomly organized as a mesenchymal mass, but the synthesis of two extracellular matrix proteins, fibronectin and N-cadherin, links them into arrays that will form tight junctions and generate their own basal laminae (Figure 14.5B; Ostrovsky et al. 1984; Lash and Yamada 1986; Hatta et al. 1987). These extracellular matrix proteins, in turn, may be regulated by the expression of the Paraxis gene. This gene encodes a transcription factor that is also expressed at the rostral (anterior) end of the unsegmented mesoderm of mouse embryos, and which is seen in precisely that region that will form the somite (Figure 14.5C). Injection of antisense oligonucleotides complementary to the Paraxis message produces defects of the paraxial mesoderm, and in the somites of Paraxis-deficient mice, no epithelial structures are formed. These defective somites have segregated from the segmental plate and their cells have differentiated, but they are completely disorganized (Burgess et al. 1995; Barnes et al. 1997). The Paraxis protein is therefore an essential part of the conversion from mesenchyme to epithelium (Burgess et al. 1996; Barnes et al. 1997; Tajbakhsh and Spörle 1998).

Axial specification

Although all the somites look identical, they will form different structures at different positions along the anterior-posterior axis. For instance, the ribs are derived from somites. The somites that form the cervical vertebrae of the neck and the lumbar vertebrae of the abdomen are not capable of forming ribs; ribs are generated only by the somites forming the thoracic vertebrae. Moreover, the specification of the thoracic vertebrae occurs very early in development. If one isolates the region of chick segmental plate that will give rise to a thoracic somite, and transplants this mesoderm into the cervical (neck) region of a younger embryo, the host embryo will develop ribs in its neck. Those ribs will form only on the side where the thoracic mesoderm has been transplanted (Figure 14.6; Kieny et al. 1972; Nowicki and Burke 1999). As discussed in Chapter 11 (see Figure 11.41), the somites are specified in this manner according to the Hox genes they express. Mice that are homozygous for a loss-of-function mutation of Hoxc-8 will convert a lumbar vertebra into an extra ribbed thoracic vertebra (see Figure 11.39).

Figure 14.6

The segmental plate mesoderm is determined as to its position along the anterior-posterior axis before somitogenesis. When segmental plate mesoderm that would ordinarily form thoracic somites is transplanted into a region in a younger embryo (caudal to (more...)

Differentiation within the somite

Somites form (1) the cartilage of the vertebrae and ribs, (2) the muscles of the rib cage, limbs, and back, and (3) the dermis of the dorsal skin. Unlike the early commitment of the mesoderm along the anterior-posterior axis, the commitment of the cells within a somite to their respective fates occurs relatively late, after the somite has already formed. When the somite is first separated from the presomitic mesoderm, any of its cells can become any of the somite-derived structures. However, as the somite matures, its various regions become committed to forming only certain cell types. The ventral medial cells of the somite (those cells located farthest from the back but closest to the neural tube) undergo mitosis, lose their round epithelial characteristics, and become mesenchymal cells again. The portion of the somite that gives rise to these cells is called the sclerotome, and these mesenchymal cells ultimately become the cartilage cells (chondrocytes) of the vertebrae and part (if not all) of each rib (Figures 14.2 and 14.7).

Figure 14.7

Diagram of a transverse section through the trunk of a chick embryo on days 2–4. (A) The 2-day somite can be divided into sclerotome cells and dermamyotome cells. (B) On day 3, the sclerotome cells lose their adhesion to one another and migrate (more...)

Fate mapping with chick-quail chimeras (Ordahl and Le Douarin 1992; Brand-Saberi et al. 1996; Kato and Aoyama 1998) has revealed that the remaining epithelial portion of the somite is arranged into three regions (Figure 14.7). The cells in the two lateral portions of the epithelium (those regions closest to and farthest from the neural tube) are muscle-forming cells. They divide to produce a lower layer of muscle precursor cells, the myoblasts. The resulting double-layered structure is called the dermamyotome, and the lower layer is called the myotome. Those myoblasts formed from the region closest to the neural tube form the epaxial muscles (the deep muscles of the back), while those myoblasts formed in the region farthest from the neural tube produce the hypaxial muscles of the body wall, limbs, and tongue (Figures 14.7 and 14.8; see Christ and Ordahl 1995; Venters et al. 1999). The central region of the dorsal layer of the dermamyotome is called the dermatome, and it generates the mesenchymal connective tissue of the back skin: the dermis. (The dermis of other areas of the body forms from other mesenchymal cells, not from the somites.) The dermamyotome may also produce the distal cartilage of the ribs, its lateral edge producing the most ventral portion of the rib (Figure 14.8; Kato and Aoyama 1998).

Figure 14.8

Myotome derivatives of the mouse embryo. The epaxial muscles form from the region of the dermamyotome closest to the neural tube. The hypaxial muscles form from the region of dermamyotome furthest from the neural tube. The epaxial myotome will form the (more...)

Determination of the sclerotome and dermatome

The specification of the somite is accomplished by the interaction of several tissues. The ventral-medial portion of the somite is induced to become the sclerotome by paracrine factors, especially Sonic hedgehog, secreted from the notochord and the neural tube floor plate (Fan and Tessier-Lavigne 1994; Johnson et al. 1994). If portions of the notochord (or another source of Sonic hedgehog) are transplanted next to other regions of the somite, those regions, too, will become sclerotome cells. Sclerotome cells express a new transcription factor, Pax1, that is required for their differentiation into cartilage and whose presence is necessary for the formation of the vertebrae (Figure 14.9; Smith and Tuan 1996). They also express I-mf, an inhibitor of the myogenic bHLH family of transcription factors that initiate muscle formation (Chen et al. 1996).

Figure 14.9

Model of major postulated interactions in the patterning of the somite. A combination of Wnts (probably Wnt1 and Wnt3a) are induced by BMP4 in the dorsal neural tube. These Wnt proteins, in combination with low concentrations of Sonic hedgehog from the (more...)

The dermatome differentiates in response to another factor secreted by the neural tube, neurotrophin 3 (NT-3). Antibodies that block the activities of NT-3 prevent the conversion of the epithelial dermatome into the loose dermal mesenchyme that migrates beneath the epidermis (Brill et al. 1995).

Determination of the myotome

In similar ways, the myotome is induced by at least two distinct signals. Studies involving transplantation and knockout mice indicate that the epaxial muscle cells coming from the medial portion of the somite are induced by factors from the neural tube, probably Wnt1 and Wnt3a from the dorsal region and low levels of Sonic hedgehog from the ventral region (Münsterberg et al. 1995; Stern et al. 1995; Ikeya and Takada 1998). The hypaxial muscles coming from the lateral edge of the somite are probably induced by a combination of Wnt proteins from the epidermis and bone morphogenetic protein 4 (BMP4) from the lateral plate mesoderm (Cossu et al. 1996a; Pourquié et al. 1996; Dietrich et al. 1998). These factors cause the myotome cells to express particular transcription factors that activate the muscle-specific genes.

In addition to these positive signals, there are inhibitory signals that prevent a signal from affecting an inappropriate group of cells. For example, Sonic hedgehog not only activates sclerotome and myotome development; it also inhibits BMP4 signal from the lateral plate mesoderm from extending medially and ventrally (thus preventing the conversion of sclerotome into muscle) (Watanabe et al. 1998). Similarly, Noggin is produced by the most medial portion of the dermamyotome and prevents BMP4 from giving these cells the migratory characteristics of hypaxial muscle (Marcelle et al. 1997).

And what happens to the notochord, that central mesodermal structure? After it has provided the axial integrity of the early embryo and has induced the formation of the dorsal neural tube, most of it degenerates. Wherever the sclerotome cells have formed a vertebral body, the notochordal cells die. However, in between the vertebrae, the notochordal cells form the tissue of the intervertebral discs, the nuclei pulposi. These are the discs that “slip” in certain back injuries.