Embryology of the Spine

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1 Embryology of the Spine

Gastrulation

The intrauterine process by which the human form develops can be divided into two phases, the embryonic period and the fetal period. The embryonic period lasts from conception to approximately 52 days post gestation. It is a vital period for organogenesis, occurring at a time when the embryo is most prone to external and internal teratogenic insults. The next 7 months encompass the fetal period, a time for tissue specialization and growth.

Immediately following fertilization, the zygote undergoes rapid cell division. Approximately 16 cells make up a ball-like structure called the morula. By the eighth day of gestation, the morula develops two fluid-filled cavities, the primitive yolk sac and the amniotic cyst. The cysts are separated by a double-layer disc of cells. Of these two cell layers, the epiblast lies adjacent to the amniotic sac; it will eventually give rise to all three germ layers during gastrulation, the process by which a two-layer disc becomes a three-layer disc.

Gastrulation begins in the third week of gestation and gives rise to three distinct germ layers, the ectoderm, the mesoderm, and the endoderm. The initial phase of gastrulation begins with formation of the primitive streak, which is sometimes named the primitive groove (Figure 1-1). This midline thickening of the germinal disc terminates in the primitive node. Under control of embryonic growth factors, cells of the epiblast layer migrate inward to form the mesoderm and the endoderm through the process of invagination. Cells migrating farthest from the epiderm and closest to the yolk sac become the endoderm. The remaining epiblast cells will eventually differentiate into the ectoderm (Figure 1-2). The migrating cells that are sandwiched between the endoderm and ectoderm layers will become the mesoderm. Control of these migrations is maintained through various cell-signaling pathways that also contribute to establishment of the body axes in all planes. The signaling pathways, or organizer genes, are secreted by the primitive streak and mesoderm. The cranial direction of the embryonic disc is established by a specialized area of cells, referred to as the anterior visceral endoderm, that expresses genes required for formation of the head and cerebrum. The dorsal-ventral axis is regulated by growth factors in the TGF-β family including bone morphogenic protein-4, fibroblast growth factor, and the sonic hedgehog gene. Control of sidedness is regulated by fibroblast growth factor-8, Nodal, and Lefty-2, all of which are secreted on the left side of the germinal disc. An additional protein, Lefty-1, is secreted to prevent migration of the left sided growth factors across the midline.1

At the cranial end of the primitive streak is a specialized collection of cells, the primitive node. Cells migrating cranially into the primitive node will eventually form the prechordal plate, while those migrating more posterior will fuse with cells in the hypoblastic layer to form the notochordal process. By day 16 or 17 of gestation, the lateral edges of the endoderm continue to invaginate; the two edges will eventually meet and pinch off the notochordal process, forming the definitive notochord. This is the earliest beginning of the bony vertebrae and the remainder of the skeleton. Cell migration continues for approximately 7 days, at which point the primitive streak begins to close in a cranial to caudal direction.

Somite Period

The presence of the notochord induces proliferation of the mesoderm. At approximately 17 days of gestation the mesoderm thickens into two masses, each located directly adjacent to the notochord. This initial layer, termed the paraxial mesoderm, continues to spread laterally to eventually differentiate into three distinct areas, paraxial mesoderm, intermediate mesoderm, and lateral mesoderm. During the somite period, lasting from approximately 19 to 30 days post fertilization, the paraxial mesoderm will develop into segmental bulbs of tissue on either side of the notochord (Figure 1-3). The first pair of somites will appear adjacent to the notochord, and they will continue to develop in a cranial to caudal direction until a total of 42 to 44 pairs of somites appear by the end of the fifth week of gestation. The first 24 somite segments are responsible for the cervical, thoracic, and lumbar spine. Somites 25 through 29 contribute to formation of the sacrum, while pairs 30 through 35 are responsible for coccyx formation. The rest of the 42 to 44 somite pairs disappear through a process of regression, which occurs at approximately 6 weeks of gestation.

The somites continue to differentiate into two distinct tissues. Ventromedial cells develop into the sclerotome, while dorsolateral cells develop into the dermatomyotome. The latter cells will eventually give rise to the integument system and dorsal musculature of the body, while the sclerotome will migrate to surround the notochord and give rise to the vertebral column. Regulation of sclerotome formation is controlled by proteins coded by the sonic hedgehog gene, which is expressed by cells of the notochord. This process of sclerotome migration will begin by the fourth week of gestation. Each sclerotome will be divided by an intersegmental vessel and a loose area of intersegmental mesenchyme. In addition, a pair of myotomes and accompanying segmental nerves will be associated with each sclerotome.

As the process of differentiation continues, each sclerotome will divide into a cranial region of relatively loosely packed cells and a caudal portion of rapidly proliferating and densely packed cells. At this point in spinal development, classic embryology texts describe a phenomenon by which the pace of proliferation is so great that the caudal part of the one sclerotome begins to overgrow into the cranial portion of the adjacent sclerotome and thereby fusing to create a single mass of tissue destined to become the precartilaginous vertebral body. Parke (The Spine, 1999) suggests that this theory of “resegmentation” may not be accurate, and provides compelling evidence toward an alternate route of vertebral body formation. In his summary of the recent evidence, Parke outlines a pathway of spinal development which begins with a uniform layer of axial mesenchyme surrounding the notochord. The sclerotomal organization is still maintained with an intersegmental vessel, a nerve, and a peripheral layer of dermatomyotome associated with each segment. However, the uniform mesenchyme undergoes a period of differentiation by which densities develop within the loose tissues. These dense regions will develop into the intervertebral discs and eventually pinch off the notochord which will be trapped within the dense tissue to become the nucleus pulposus.2 The loose tissues between the discs form the cartilaginous centrum, which is the precursor of the vertebral bodies. The caudal portion of the centrum undergoes rapid proliferation, and cells migrate peripherally to surround the neural tube, forming the membranous neural arches which will serve to protect the neural elements. In total, each bony vertebral segment will consist of five ossification centers, one centrum, two neural arches, and two costal elements.

Ossification of the vertebral bodies occurs around the ninth week of gestation and begins at the thoracolumbar junction. Ossification then proceeds in both cranial and caudal directions, with the caudal segments demonstrating a quicker rate of ossification compared with the cranial segments. Ossification of the posterior arches begins at approximately the same time but begins in the cervical vertebrae and proceeds in a caudal direction. As the two neural arch centers approach midline, they begin to fuse, forming the lamina and spinous process. Fusion of the neural arches first occurs in the lumbar segments during the first year of life and proceeds cranially. Fusion is not completed until ages of 5 to 8 years. The costal ossification centers have a variable role in vertebral body formation. In the cervical spine, these centers have a minimal contribution and may contribute to part of the foramen transversarium. In the thoracic spine, these ossification centers are the precursors of the ribs. In the lumbosacral spine, the costal ossification centers are responsible for formation of the transverse processes and the anterolateral portion of the sacrum.2

Upper Cervical Spine

The upper cervical spine must provide stable support for the cranial vault, and must also position the head and its sensory organs in space. This region has uniquely adapted to the evolutionary requirement of each species. In humans, this area is well suited to support a large cranium while providing approximately ± 80 degrees of lateral rotation and ± 45 degrees of flexion/extension.

A detailed anatomic study of cervical spine anatomy was presented by O’Rahilly and Meyer in a serial time reconstruction of human embryos ranging from 8 to approximately 16 weeks of gestation.3 It is generally believed that the most cranial 4 or 5 pairs of somites are responsible for the occipital-atlas complex. Development of this junction is regulated by growth factors derived from the notochord as it crosses into the cranium. The notochord travels through the middle or slightly anterior portion of each centrum, and up through the future dens at the level of the axis. It then makes an anterior directed turn to enter the skull just above the level of the dens. At this time, each centrum is divided by a thickening of the notochord that will develop into the nucleus pulposus. The true boundary between the spine and cranium is not fully understood, with some authors suggesting that the atlas is a standalone accessory cranial bone with the true head-neck boundary being between the C1 and C2 articulation.

O’Rahilly and Meyer describe the centrum of the axis as being composed of three axial columns which they termed X, Y, and Z. The first and most cranial column, X, will develop an articulation with the anterior tubercle of C1, forming the atlanto-dens joint space.4 By approximately 9 weeks of gestation, the X column, or future dens, is already bounded posteriorly by the transverse ligament and anchored into the occipital condyles by the alar ligamentous complex. Columns Y and Z are separated by the remnants of an intervertebral disc that may persist well into birth. Although it is generally accepted that column Z will form the centrum of the axis, the fate of column Y remains uncertain. Some believe that it is incorporated into the axis centrum, while other embryologists believe, based on reptilian studies, that it is incorporated into the centrum of the atlas. Calcifications in the three columns are readily visible by the time the embryo reached a length of 120 mm; however, fusion will not take place until 6 to 8 years of age. In some instances, the tip of the dens may calcify independently without fusion to the remaining axis; this is termed os odontoideum.

Neural Development

The neural elements likewise form during gastrulation. Under control of growth factors secreted by the prechordal plate, the ectoderm begins to thicken at the cranial end to form the neural plate and the lateral edges of the germinal disc fold to form the neural crests. This process is primary neurulation. As previously discussed, the neural crests meet in the midline to form the neural tube. The tube has two open ends, the cranial and caudal neuropores, both of which communicate with the amniotic cavity. This communication allows for prenatal detection of central nervous system markers in amniotic fluid if neural tube closure has failed to complete. There are likely multiple foci at which neural tube closure initiates and progresses both cranially and caudally in a zipperlike fashion. The cranial neuropore is generally first to close, and final closure is not complete until about 25 days post gestation.1 The caudal neuropore closes approximately 2 days later. Failure of neuropore closure at the cranial end results in anencephaly, a deficiency of skull, scalp, and forebrain. Failure of caudal neuropore closure results in spina bifida. Once closure is completed, the neural tube must separate from the ectoderm. This process is termed dysjunction; premature separation during this step of neurulation may pull primitive mesenchyme tissues inside the developing neural tube, resulting in a lipomeningocele or lipomyelomeningocele.5 Incomplete separation may lead to cutaneous sinuses that communicate with the spinal canal.

Thickenings in the neural tube give rise to the proencephalon (forebrain), mesencephalon (midbrain), and rhombencephalon (hindbrain). A cervical flexure will form connecting the rhombencephalon to the developing spine. The neural tube contains a lumen, the central canal of the spinal cord, which is in continuity with the cerebral ventricles. The neural tube wall consists of rapidly dividing neuroepithelial cells. These cells develop into neuroblasts and form a thick layer called the mantle. While in the mantle layer, the primitive neuroblast cells remain relatively apolar. Neuroblastic differentiation involves transformation of the apolar cells into a bipolar form, with elongation of one end to form the primitive axon and complex specialization of the other end to form the dendrites. At the completion of maturation, this cell will be the neuron. Axons of the neuron will protrude peripherally through the mantle layer and form the marginal layer of the spinal cord. The mantle layer, which contains the cell nuclei, does not undergo myelination and becomes the gray matter of the spinal cord. The axons in the marginal layer will get myelinated and become the white matter.

As the neuroblast proliferates, the spherical neural tube begins to thicken both ventrally and dorsally. The two areas of neuroblast are separated by the sulcus limitans, which prevents cell migration between the two layers. The ventral thickenings will form the basal plate which houses the motor horn cells. The dorsal thickenings will form the alar plate which contains the dorsal sensory neurons. The sympathetic chain is made up of neurons that accumulate in the “intermediate horn,” a small thickening of cells that is found between the alar and basal plates at the level of the thoracic and upper lumbar spine. Neurons of the dorsal sensory horn become known as interneurons or associated neurons. These cells project axons that enter the marginal zone and extend proximally or distally to form communications between afferent and efferent neurons.

The spinal nerves begin to form in the fourth week after gestation. Each nerve is composed of a ventral motor root and a dorsal sensory root. Axons of the ventral motor horn cells project through the marginal zone and coalesce outside of the neural tube into the ventral motor root. These axons will continue to the motor endplates of the muscles formed from its respective sclerotome.

The dorsal sensory root begins its development from neural crest cells, which are of ectodermal origin. These cells migrate laterally during formation of the neural tube, forming the dorsal root ganglia, which contain the cell bodies. Axons project proximally and distally from the ganglia. The proximal axons make up the dorsal sensory roots and enter the neural tube on its dorsal surface into the dorsal horn to communicate with the sensory neurons. The distal axons join with the ventral motor fibers to compose the spinal nerve. They will terminate in the end organs to bring afferent feedback to the central nervous system.

Sacrum and Conus Medullaris Development

Development of the neural structures in the caudal terminus of the spine deserves some special attention. At their respective most distal points, the neural tube and notochord coalesce into an undifferentiated cellular mass that will develop into the coccyx, sacrum, and fifth lumbar vertebrae. This process is the beginning of secondary neurulation. A single canal will form within this mass through a process called canalization. Debate exists in the literature as to whether this newly formed neural tube is initially continuous with the primary neural tube or whether the two coalesce at a later point in development. It is known that the chick embryo develops two distinct neural tubes that anastomose in the sacral region, while in a mouse embryo, the secondary neural tube forms as an extension of the primary neural tube. The pathway of secondary neurulation in humans is not yet elucidated; however, it is known that the distal portion of the tube and central canal will regress in a cephalic direction via a process called retrogressive differentiation. This will give rise to the conus medullaris and will leave behind a thin film of pia mater tissue called the filum terminale.5 Nerve root compression may result when an abnormally thick filum terminale is present (usually greater than 2 mm in diameter).

As retrogressive differentiation continues, the position of the conus medullaris relative to the bony spine continues to change. The conus ascends from the level of the coccyx early in embryogenesis to rest at approximately the L2-3 disc space by the time of birth. Asymmetric rates of growth between the bony spine and the cord result in further caudal migration of the conus during the fetal period so that it comes to its final resting place at L1-2 by a few months after birth. Any final resting position of the cord at or below the L2-3 disc space would imply a tethered cord.

Associated Anomalies

While discussing spinal embryology, it is important to remember that spinal development is not an isolated event. Multiple organ systems are developing in parallel with the spine and often share the same germinal tissue source. Any internal or external insult to the developing embryo may affect other organ systems. The mesoderm is particularly involved in the genesis of several organs. Paraxial mesoderm, the precursor of the centrum and vertebral column, is also responsible for formation of the dermis, skeletal muscle, and the connective tissue of the head.6 The intermediate and lateral mesoderm is responsible for formation of the urogenital, cardiac, and renal systems. In children with known congenital spinal defects, the incidence of associated anomalies has been reported as high as 30% to 60%.7 The most common organ system to be affected is the genitourinary system. Mesoderm tissues that make up the spinal column also contribute to formation of the mesonephros. While it is the medial region of the mesoderm that forms the vertebrae, the ventrolateral region forms the genitourinary organs.8 The cardiopulmonary system is also commonly involved in conjunction with a congenital spinal abnormality. These anomalies may be fatal and should be diagnosed and treated before their associated problems progress. Diagnosis of both congenital spinal defects and associated anomalies may be made on prenatal ultrasound examination.

The timing of insult during fetal development also affects the rate of associated anomalies. Tsou (1980) divided a group of 144 patients with congenital spinal anomalies into two groups: embryonic anomalies, defined as those that occurred in the first 56 days post fertilization, and fetal anomalies, defined as those that occurred from day 57 of gestation to birth.9 They found that the rate of associated defects was 7% in the fetal group as compared with 35% in the embryonic group. Associated orthopedic anomalies included Klippel-Feil syndrome, acetabular dysplasia, clubfoot, congenital short leg, Sprengel deformity, coxa vara, radial clubhand, and thumb aplasia. Nonorthopedic associated anomalies included dextrocardia, hypospadia, microtia, lung aplasia, pulmonary arterial stenosis, imperforate anus, mandibular anomalies, cleft palate, and hemidiaphragm.9

Congenital Spinal Anomalies

Normal spinal development involves coordination between cellular tissues and signaling pathways. Mesenchyme provides the cellular building blocks for the structural tissues of the spine while the notochord provides signaling molecules to organize normal development. Congenital spinal defects may be the result of defects in mesenchymal building blocks, genetic defects in the signaling pathways, or a combination of both. The most commonly used classification system for congenital spinal defects, however, is not based on the etiology of disease but rather on the radiographic morphology. Moe et al. proposed a classification system that breaks congenital spinal defects into three main groups: defects of formation, defects of segmentation, and complex defects of the neural tube.

Defects of Formation

Defects of formation are defined as absence of any structural portion of the vertebral ring. The resultant deformity is a result of the anatomic structure that failed to form properly. The most common morphological result of a failure of formation is a hemivertebra or wedge vertebra. Classification of hemivertebra depends on the presence of growth plates on either side of the body. A fully segmented hemivertebra has growth plates on both sides and is separated by a disc from both the cranial and caudal adjacent vertebral body. A semisegmented hemivertebra only has one growth plate, and thus an intervertebral disc is only found adjacent to either the cranial or caudal segment. A nonsegmented hemivertebra has no active growth plates or discs to separate it from the body above or below. This is a stable situation with minimal potential for increasing deformity with growth. Another stable situation may occur when plasticity of both the cranial and caudal adjacent vertebral bodies allow the adjacent bodies to conform to the shape of a hemivertebra, thus keeping the pedicles in line with the rest of the spine. This stable situation, in which the hemivertebra is referred to as being incarcerated, does not result in a deformity and usually does not require treatment.

Although it is generally agreed that hemivertebrae are the result of the failure of formation, the exact pathophysiology has not yet been elucidated. It is helpful to separate failures of formation as those occurring during the embryonic period and those that occur in the fetal period. During the embryonic stage, most authors propose a theory of “segmental shift,” which occurs during the sclerotomic pairing phase of embryogenesis.9 As the somites join in the midline, it is assumed that each somite is in the same developmental phase as its counterpart across the midline. This development usually proceeds in a predictable pattern from a cranial to caudal direction. Asynchronous development of one somite in a hemimetameric pair may prevent normal midline fusion and result in a caudal shift of the column such that the two contralateral pairs are in a synchronous phase of development. This would leave an isolated out-of-phase hemivertebra without a cross-midline counterpart (Figure 1-4). This segmental shift theory is further supported by the presence of double-balanced hemivertebra where each of the asynchronous hemi-vertebra is found on one side of the midline. The most caudal hemivertebra is commonly found at the lumbosacral junction where there is no further room for compensation from the somite below. Another mechanism for hemivertebra formation may result from a physiologic insult to the somite precursor during the embryonic period. Although midline fusion occurs between corresponding somites, the injured hemimetameric pair may undergo growth retardation of variable severity. Mild growth retardation may result in a hypoplastic hemivertebra in which the growth plates are formed, but the rate of growth is not equal to the opposite side. More severe forms of sclerotome growth retardation may result in a failure of segmentation and will be discussed later.

Insults to the growing spine during the embryonic stage tend to globally affect the vertebral segment, including both posterior and anterior elements. Insults occurring during the fetal period tend to be more specific and only affect a portion of the vertebra, the centrum being the area most commonly afflicted. Centrum hypoplasia and aplasia is described by Tsou as a spectrum of growth retardation that occurs from 2 to 7 months post fertilization during a period of normally rapid vertebral growth.9 A vascular etiology for centrum aplasia and hypoplasia was proposed by Schmorl and Junghanns; however, this has not yet been proven. Identification of these failures of formation is clinically important as they may often result in structural deformity of the spine. Centrum aplasia and posterior hemicentrum have been shown to cause an isolated kyphotic deformity, while wedge vertebra, posterior corner hemivertebra, and a lateral hemicentrum more commonly cause a mixed kyphoscoliotic deformity.6

Defects of Segmentation

Defects in segmentation occur when two or more adjacent vertebrae fail to fully separate resulting in a complete or partial loss of the growth plate. The extent and location of the defect largely determines the resultant deformity. One mechanism of segmentation failure involves a more advanced form of hemimetamer hypoplasia. As cells of the sclerotome undergo their migration, they first feed formation of the centrum, followed by the neural arches. A deficiency in the quantity of sclerotome would first manifest itself as a deficit in the neural arch formation, as they are last to receive the migrating cells. The resultant hypoplasia has a variable amount of penetrance. In the mildest form, only the lamina may be fused, followed by fusion of the facet joints. More severe forms involve fusion of the entire hemivertebra in which the adjacent level lamina, facet joints, and pedicles are fused into a single posterolateral bar.

Segmentation defects may also occur during formation of the intervertebral disc or the adjacent articulations. By the late embryonic period, mesodermal cells have migrated around the notochord and formed dense collections of tissues which will form the annulus fibrosus. In a more common form of segmentation defect, the anterior portion of the annulus undergoes first what Tsou describes as a cartilaginous transformation, followed by osseous metaplasia.9 A bony bar forms between two or more adjacent vertebral bodies as ossification continues into childhood. This anterior tether may result in a severe kyphotic deformity that worsens with continued growth.

Posterior elements are also prone to failures of segmentation. The articulating facet joints form via condensation of mesenchymal tissues that extend in a superior and inferior direction away from the pedicle. Injury to the developing mesenchyme in the neural arches during the later portion of the embryonic period may interrupt normal development of the apophyseal joints. A cartilaginous bridge forms between the superior and inferior articulating processes of two adjacent vertebral segments. This bridge undergoes ossification during early childhood and provides a posterior growth tether. Unilateral involvement would lead to a lordoscoliotic deformity and bilateral bars would lead to a pure lordotic deformity.

Spina Bifida

Derived from the Latin term bifidus, spina bifida literally means a spine split in two. Although the severity of the disease may range from a benign incidental finding on x-ray to severe neurologic damage, the etiology remains the same, a failure of the embryonic vertebral arches to fuse. Causes for this lack of fusion are multifactorial. Mitchell (1997) suggested a weak genetic component by demonstrating an increased risk in siblings of affected children and even further increased risk with multiple affected siblings.10 Environmental factors also play a role in the etiology of spina bifida. Mitchell correlated incidence with time of season, geographic location, ethnicity, race, socioeconomic status, maternal age and parity, and maternal nutritional status, specifically the dietary intake of folic acid and alcohol. Although the mechanism by which folic acid aids in neural tube closure is unknown, the role of folic acid as a substrate in DNA synthesis has been well described. An enzyme called methyl tetra hydroxy folate reductase (MTHFR) is involved in folate metabolism during DNA synthesis. Genetic alterations in this enzyme may lead to decreased enzymatic activity and increase the dietary folate requirements for proper DNA synthesis. As neural tube closure has been shown to begin early in the embryonic period, it is vital to begin folate supplementation as early as possible in the prenatal period and encourage dietary supplementation during family planning.

Spinal bifida occulta, one of the more benign forms of spinal bifida, results from a failure of fusion of the lamina. This relatively common finding has a reported incidence of 10% to 24% in the general population. The disease implies involvement of the posterior arches only and sparing of the cord and meninges from the pathology. Patients typically do not present with any neurological symptoms. Physical exam signs may include skin indentation and/or patches of irregular hair growth in the region of the lower lumbar spine. The most typical diagnosis is made an incidental finding on an x-ray of the lumbar spine. Rarely, associated defects may exist in conjunction with spina bidifa occulta. These may include a tethered cord, distortion of the cord by fibrous bands, syrinx, lipomyelomeningocele, a fatty filum terminale, or diastematomyelia. Collectively, these associated disorders are grouped into a term called occult spinal dystrophism.

Spina bifida cystica refers to a more severe form of spina bifida; it can be broken down into several subgroups based on the degree of the involved tissue layers.6 The first group, spina bifida with meningocele, involves the meninges as well as the posterior arches. A cystic pouch is present within the meninges without involvement of the spinal cord or nerve roots. Patients are typically spared neurologically. Physical exam findings may be similar to those of spina bifida occulta, but also include subcutaneous lipomas and hemangiomas adjacent to the lesion. Spina bifida with myelomeningocele is the next most severe form of spina bifida. This disease results from failure of fusion in the posterior arches with involvement of the spinal cord and meninges. By definition, in spina bifida with myelomeningocele, the neural elements are not exposed to the external environment and are covered by a membranous cerebrospinal fluid–filled sac. This disease typically presents with neurological disorder based on the neurological level of the lesion. Associated anomalies include Arnold-Chiari malformation, hydrocephalus, scoliosis, and kyphosis. The most severe manifestation of spina bifida cystica is myeloschisis. In this severe presentation the neural elements are completely exposed. Neurologic injury is certain and infections are common.