SEGMENTATION OF PARAXIAL MESENCHYME

Epiblast cells which ingress through the lateral aspect of the primitive node and the rostral primitive streak (see Fig. 10.3) become committed to a somitic lineage (see p. 184). After passing through the streak the cells retain contact with both the epiblast and hypoblast basal laminae as they migrate and for some time after reaching their destination. The cells form populations of paraxial mesenchyme on each side of the notochord, termed presomitic or unsegmented mesenchyme. Somites will form from cultured presomitic mesenchyme with or without the presence of neural tube tissue or primitive node tissue. As well as specifying somitic lineage, the position of ingression of the epiblast informs the specific destination of the cells. Thus those cells which ingress through the lateral portion of Hensen’s node form the medial halves of the somites, whereas those ingressing through the primitive streak approximately 200 μm caudal to the node produce the lateral halves of the somites. The two somite halves do not appear to intermingle.

The segmentation of the paraxial presomitic mesenchyme occurs as a sequential process along the craniocaudal axis. In some amniote embryos a pair of somites is formed every 90 minutes until the full number is obtained. The molecular pathway for this synchronous segmentation has been termed the segmentation clock. It has been identified as a conserved process in vertebrates from fish to mammals, and is based on the rhythmic production of mRNAs for the transcription of genes related to Notch, a large transmembrane receptor, and a number of transmembrane ligands.

Intrinsically coordinated pulses of mRNA expression appear as a wave within the presomitic mesenchyme as each somite forms. As new cells enter the paraxial mesenchyme caudally they begin phases of up-regulation of the cycling genes followed by downregulation of these genes. During each cycle the most cranial presomitic mesenchyme will segment to form the next somite.

Experimental evidence (from chick embryos) shows that newly formed paraxial mesenchyme cells undergo 12 such cycles before they finally form a somite (Pourquie & Kusumi 2001). Thus from ingression through the primitive streak to segmentation into a somite takes approximately 18 hours. As the somite number varies between vertebrate species, it is likely that the rate of somite formation also varies and may be longer in human embryos. Indeed those vertebrates with elongated bodies and many somites appear to form somites more rapidly than those with shorter bodies, a finding which supports the concept that somite number is controlled in part by species-specific cyclical properties of the presomitic mesenchyme (Richardson et al 1998).

The final determination of somitic boundary formation has not yet been fully elucidated, but seems to require a periodic repression of the Notch pathway genes. The caudal presomitic mesenchyme cells are thought to be maintained in an immature state by their production of FGF8. The cells become competent for segmentation when FGF8 levels drop below a certain threshold. They would then be in close apposition to cells that have segmented (i.e. the next cranial somite).

This area of research is moving rapidly. A detailed critique of the conceptual models of segmentation is given by Stern & Vasiliauskas (2000). An overview of the processes involved in the development of the paraxial mesenchyme, based on the work of Christ et al (2000), is shown in Figure 44.1.

Fig. 44.1 Processes in the development of the paraxial mesenchyme in the avian embryo. Signals are indicated by open arrows; genes are italicized.

(Redrawn with permission from Christ B, Huang R, Wilting J 2000 The development of the avian vertebral column. Anat Embryol 202: 179–94, Springer.)

SOMITOGENESIS

Somitogenesis is a process in which five main stages can be identified (Figs 44.2, 44.3). Compaction and formation of the spherical epithelial somite surrounding free somitocele cells occur prior to segmentation of the paraxial mesenchyme. Once the somite boundaries have been defined there is an epithelial/mesenchymal transition of the ventral and ventromedial walls of the somite to form the sclerotome. This is followed by bilateral movement of these ventral and medial mesenchyme populations around the notochord and the neural tube. The final stage is formation of the epithelial plate of the somite, also termed the dermomyotome, from the remaining somitic epithelium. Segmentation of the paraxial mesenchyme, formation of somites and resultant somitogenetic processes occur in a craniocaudal progression caudal to the otic vesicle from stage 9.

Fig. 44.2 Scanning electron micrograph of a lateral view of an embryo showing the somites. The cranial somites are at the upper border and the more caudal somites are at the lower border. A change in size of the cranially more advanced somites is apparent.

(Photograph by P Collins; printed by S Cox, Electron Microscopy Unit, Southampton General Hospital.)

Fig. 44.3 The stages of somitogenesis. Development occurs in a craniocaudal progression. The more cranially placed somites (at the lower right of the figure) are further developed than those caudally placed (at the upper left of the figure). The stages in somitogenesis are given on the left of the figure; more detailed information is given on the right.

The Golgi apparatus, and actin and α-actinin are all located in the apical region of the epithelial somite cells. Cilia develop on the free surface. The cells are joined by tight junctions (a variety of cell adhesion molecules has been demonstrated in epithelial somites). The basal surface rests on a basal lamina which contains collagen, laminin, fibronectin and cytotactin. Processes from the somite cells pass through this basal lamina to contact the basal laminae of the neural tube and notochord. Dorsoventral patterning of the putative vertebral column is dependent on the interaction of SHH from the notochord/floor plate with Wnt1 and Wnt3a from the roof plate and Wnt6 from the overlying ectoderm.

The epithelial somite undergoes rapid development (Fig. 44.3). The cells of the ventromedial wall which were abutting the neural tube undergo an epithelial/mesenchyme transformation and break apart. The newly formed mesenchymal cells, which are collectively termed the sclerotome, proliferate. The remaining cells of the somite are now termed the epithelial plate of the somite (or dermomyotome). This epithelium is proliferative; it produces the cell lines which will give rise to (nearly) all the striated muscles of the body.

The sclerotomal population has been subdivided in the chick and the fates of the cells followed (Christ, Huang & Scaal 2004) (Fig. 44.4–44.6). The main mass of the sclerotome is termed the central sclerotome, that portion close to the notochord is termed the ventral sclerotome, and the portions adjacent to the dermomyotome are termed dorsal sclerotome and lateral sclerotome. The central sclerotome remains close to the dermomyotome; it will give rise to the pedicles and ventral parts of the neural arches, and the proximal ribs. The ventral sclerotomal cells, which were always lateral to the notochord, proliferate to form an axial cell population within the extracellular matrix of the perinotochordal space, now termed the perinotochordal sheath. The dorsal sclerotomal cells develop relatively late and invade the space between the surface ectoderm and growing neural tube and form the dorsal part of the neural arches. The lateral sclerotomal cells give rise to distal ribs and endothelial cells of blood vessels. Generally there is a dorsolateral expansion of the whole sclerotome rather than the medial migration of a population of sclerotomal cells (Gasser 2006). This can be seen in Figures 44.4–44.6. Sclerotomal cells also give rise to the meninges surrounding the spinal cord, local tendons and ligaments. The somitocele cells which remain mesenchymal throughout somite formation give rise to the vertebral joints, intervertebral discs and the proximal ribs (Christ, Huang & Scaal 2004).

Fig. 44.4 Development of the sclerotome. Scanning electron micrograph of a transverse fracture of a 2 day chick embryo (A) and transverse (B) and longitudinal (C) section through the somites, indicated by dotted line in B. The somites here are in the epithelialization phase of somitogenesis. AO, aorta; DS, dorsal somite; N, notochord; NT, neural tube; SM, somatopleure; SO, somitocele; SP, splanchnopleure; VS, ventral somite; WD, Wolffian duct (mesonephric duct).

(A By courtesy of Dr Heinz Jürgen Jacob, Bochum, Germany)

Fig. 44.5 Development of the sclerotome. Scanning electron micrograph of a transverse fracture of a 3 day chick embryo (A) and transverse (B) and longitudinal (C) section through the developing sclerotome, indicated by dotted line in B. AO, aorta; DM, dermomyotome; N, notochord; NT, neural tube; SC, sclerotome; SM, somatopleure; WD, Wolffian duct (mesonephric duct).

(A By courtesy of Dr Heinz Jürgen Jacob, Bochum, Germany)

Fig. 44.6 Later development of the sclerotome. Transverse (A) and longitudinal (B) section through dotted line indicated in A showing the sclerotomal subdivisions.

The dermomyotome is a proliferative epithelium which produces cells from four borders (Fig. 44.7). Proliferation at the dorsomedial edge produces cells from the cranial to the caudal edge of the dermomyotome; they elongate beneath its basal lamina as they move laterally. Cells similarly proliferate from the cranial and caudal edges of the dermomyotome and these cells also elongate across it. The cells that are produced from these three edges are collectively termed the myotome, and will give rise to skeletal muscle dorsal to the vertebrae, i.e. the epaxial musculature. At limb levels cells de-epithelialize and migrate from the ventrolateral edges of the dermomyotome into the limb bud. Cells produced from this portion of the occipital somites migrate anteriorly to give rise to the intrinsic muscles of the tongue. At interlimb levels a hypaxial proliferation of myogenic cells that will give rise to intercostal and abdominal muscles extends from the ventrolateral edge of the epithelial dermomyotome into the body wall as development proceeds (Scaal & Christ 2004).

Fig. 44.7 A–D, Epaxial and hypaxial muscle origin from the dermomyotome. A, Early dermomyotome proliferation. B, Dermomyotome at interlimb level producing expaxial myoblasts. C, Dermomyotome at limb level. Hypaxial dermomyotome cells de-epithelialize from the ventrolateral edge and migrate into the limb bud. D, Local dermis is formed later by superficial de-epithelialization of dermomyotome cells.

It was thought that the somite gave rise to segmental portions of the dermis of the skin as well as bone and muscle. However, it is now clear that the somitic contribution to the skin from the epithelial plate is limited to de-epithelialization of dermomyotome cells over the epaxial muscles alone, which is a much smaller distribution than the segmental portion of skin usually implied by the term dermatome. The concept that an embryological dermatome, derived from the somite, produces all of the dermis of the skin is therefore outdated.

The regularity of somite formation provides criteria for staging embryos. The staging scheme proposed by Ordahl (1993) will be used in the following account of relative somite development. Ordahl noted that morphogenetic events occur in successive somites at approximately the same rate. The somite most recently formed from the unsegmented mesenchyme is designated as stage I, the next most recent as stage II, etc. After the embryo forms an additional somite, the ages of the previously formed somites increase by one Roman numeral. According to this scheme, compaction occurs at stage I; epithelialization at stages II to III; formation of mesenchymal sclerotome cells from stage V; myotome formation at stage VI; early migration of the ventrolateral lip of the epithelial plate and production of myotome cells are still occurring at stage X.

DEVELOPMENT OF SCLEROTOMES

Sclerotomal populations form from the ventral half of the epithelial somite. An intrasegmental boundary (fissure or cleft, sometimes termed von Ebner’s fissure) that is initially filled with extracellular matrix and a few cells, appears within the sclerotome and divides it into loosely packed cranial and densely packed caudal halves. The epithelial plate, and later the dermomyotome, spans the two half-sclerotomes. The bilateral sclerotomal cell populations migrate towards the notochord and surround it to form the perinotochordal sheath. They undergo a matrix-mediated interaction with the notochord, differentiating chondrogenetically to form the cartilaginous precursor of the vertebral centrum. The perinotochordal sheath transiently expresses type II collagen, and this is believed to initiate type II collagen expression, and thereafter a chondrogenic fate, in those mesenchyme cells which contact it. Each vertebra is formed by the combination of much of the caudal half of one bilateral pair of sclerotomes with much of the cranial half of the next caudal pair of sclerotomes. Their fusion around the notochord produces the blastemal centrum of the vertebra (Figs 44.8 and 44.9). The mesenchyme adjoining the intrasegmental sclerotomic fissure now increases greatly in density to form a well-defined perichordal disc which intervenes between the centra of two adjacent vertebrae and is the future anulus fibrosus of the intervertebral symphysis (‘disc’) (see below).

Fig. 44.8 Formation of vertebrae and intervertebral discs from the mesenchymal sclerotomes. Each vertebra is formed from the cranial half of one bilateral pair of sclerotomes and the caudal half of the next pair of sclerotomes.

Fig. 44.9 Contribution of the somites to the vertebrae. Each somite induces a ventral root to grow out from the spinal cord. When the sclerotomes recombine, the cranial half of the first cervical sclerotome fuses with the occipital sclerotome above contributing to the occipital bone of the skull. The cervical nerves beginning with C1 exit above the corresponding vertebra. Nerve C8 exits below the seventh cervical vertebra (C7), and thereafter nerves arise below their numbered vertebrae.

(By permission from Larsen WJ 1997 Human Embryology, 2nd edn. Edinburgh: Churchill Livingstone.)

The basic pattern of a typical vertebra is initiated by this recombination of caudal and cranial sclerotome halves (Fig. 44.10