Notochord isolation using laser capture microdissection.

BACKGROUND: Chordoma are malignant tumors of the axial skeleton, which arise from remnants of the notochord. The Notochord (chorda dorsalis) is an essential embryonic structure involved in the development of the nervous system and axial skeleton. Therefore, the notochord seems to be the most biologically relevant control tissue to study chordoma in molecular biology research. Nevertheless, up to now mainly different tissues but not the notochord have been used as control for chordoma, due to difficulty of isolating notochordal tissue. Here, we describe a fast and precise method of isolating notochordal cells. METHODS: Examination of human fetuses, with a gestation of 9, 11 and 13 weeks, using (immuno)histochemical methods was performed. To isolate pure notochord cells for further molecular biology investigation five flash frozen fetuses between 9 and 10 weeks of gestation were dissected by microtome slicing. Thereafter pure notochord cells for further molecular biology investigation where harvested by using laser capture microdissection (LCM). RNA was extracted from these samples and used in quantitative PCR. RESULTS: This study illustrates notochord of embryonic spines in three different stages of gestation (9-11-13 weeks). Immunohistochemical staining with brachyury showed strong staining of the notochord, but also weak staining of the intervertebral disc and vertebral body. LCM of notochord slices and subsequent total RNA extraction resulted in a good yield of total RNA. qPCR analysis of two housekeeping genes confirmed the quality of the RNA. CONCLUSION: LCM is a fast and precise method to isolate notochord and the quality and yield RNA extracted from this tissue is sufficient for qPCR analysis. Therefore early embryo notochord isolated by LCM is suggested to be the gold standard for future research in chordoma development, classification and diagnosis.

Cilia-like structures anchor the amphioxus notochord to its sheath.

Body stiffness is important during undulatory locomotion in fish. In amphioxus, the myosepta play an important role in transmission of muscular forces to the notochord. In order to define the specific supporting role of the notochord in amphioxus during locomotion, the ultrastructure of 10 adult amphioxus specimens was analyzed using transmission electron microscopy. Numerous cilia-like structures were found on the surface of each notochordal cell at the sites of their attachment to the notochordal sheath. Ultrastructurally, these structures consisted of the characteristic arrangement of peripheral and central microtubular doublets and were anchored to the inner layer of the notochordal sheath. Immunohistochemically, a positive reaction to applied dynein and ß-tubulin antibodies characterized the area of the cilia-like structures. We propose that reduced back-and-forth movements of the cilia-like structures might contribute to the flow of the fluid content inside the notochord, thus modulating the stiffness of the amphioxus body during its undulatory locomotion.

Somites, spinal Ganglia, and centra. Enumeration and interrelationships in staged human embryos, and implications for neural tube defects.

Serial sections of 99 human embryos from Carnegie stages 8-23 were investigated and 38 graphic reconstructions were evaluated. At stage 9 somite 1 is of appreciable size and is separated from the otic disc, as also in the next several stages by rhombomeres and pharyngeal arches 3 and 4, thereby differing from the chick. At stage 10 somite 1 begins to differentiate into sclerotome and dermatomyotome. At stage 11 spinal neural crest begins to develop. At stage 12 parts of somites 1-4 are being transformed into the hypoglossal cell cord. It is stressed that the numbers of somites present at stages 9-12 are part of the definition of those stages. At stage 13 dense and loose zones begin to be detectable rostrally in the sclerotomes and also, although out of phase, in the perinotochord. Spinal ganglia begin to develop in phase with the somites. At stages 14-16 the maximum number of somites observed was 38-39 rather than 42-44, as usually given. Moreover, they did not extend to the tapered end of the trunk, which is not a (vertebrated) 'tail'. At stages 17-23 the maximum number of centra was 38-39, including coccygeal vertebrae 4-5. Although most of the somites appear during primary development, all of the spinal ganglia develop during secondary development (stages 13-18). The number of ganglia was at a maximum of 35 at stage 18, but was reduced to 32 already by stage 23. Important points confirmed in this study are that the number of occipital somites in the human is four, and that the level of final closure of the caudal neuropore is future somite 31, which represents approximately future sacral vertebra 2. The interpretation of relevant neural tube defects is discussed in the light of the findings. The ascensus of the conus medullaris during the fetal period is well established, but a concomitant ascent of the situs neuroporicus is proposed here, and has implications for defects that involve tethering of the spinal cord. The main results are integrated in comprehensive graphic representations of the levels and the interrelationships of (a) somites and centra, and (b) somites, neural crest, and spinal ganglia. These may aid in the elucidation of some frequently occurring anomalous conditions.

Axial structures control laterality in the distribution pattern of endothelial cells.

In the midline of the embryo an invisible barrier exists that keeps endothelial cells from migrating to the contralateral side. Interspecific grafting experiments between chick and quail were carried out in order to investigate the role of the axial structures in maintaining this barrier. The quail endothelial cells of the graft were therefore stained with QH1 antibody. In all experimental series quail paraxial mesoderm was used as a source of endothelial cells. First, a quail somite was transplanted either ipsilaterally or contralaterally. The results not only show the existence of laterality in the distribution pattern, but also demonstrate that the laterality does not depend on the origin of the graft but on the environment of the host embryo. Laterality in the distribution pattern of endothelial cells means that the endothelial cells of the two body halves migrate independently and do not change from one side to the other. Single cells do not know whether they are cells from the right or from the left half of the body. In the next series of experiments axial structures were removed in order to modify the barrier. In addition, paraxial mesoderm was exchanged with the corresponding quail tissue in order to determine the migration behaviour of the grafted endothelial cells. The removal of the neural tube does not influence the barrier. After notochordectomy, however, the endothelial cells exhibited a balanced distribution pattern over both halves of the embryo. We concluded that the notochord forms a barrier for endothelial cells that presumably operates on the basis of chemical substances. It is conceivable that our results can explain the lateralization of illnesses of the vascular system, as the Klippel-Trénaunay syndrome or the Sturge-Weber syndrome.

Three-dimensional reconstruction of human embryonic notochords: clue to the pathogenesis of chordoma.

Three-dimensional reconstruction experiments performed on serial sections of human embryos showed that the anatomy of the caudal and rostral ends of the notochord was complex. Forking of the ends, with separate fragments of chordal tissue, was demonstrated and these provide a way by which notochordal cell rests could be left behind in the basicranial and sacral regions when the notochord involutes elsewhere. Assuming the histogenesis of chordomas from notochordal cell rests, this would furnish an explanation for the observed skeletal distribution of chordomas.

Spinal cord-notochord relationship in normal human embryos and in a human embryo with double spinal cord.

Spinal cord-notochord relationship was analyzed histologically and immunohistochemically in normal human conceptuses between the 4-8 developmental weeks and in a 8-week embryo with double spinal cord. In the early 4-week embryo, the gradual closure of the neural tube along the cranio-caudal body axis was paralleled by the differentiation of the median hindge point cells at the ventral midline of the tube and by its temporary close association with the notochord. During the 5th-8th developmental weeks, the neuroepithelium differentiating into three distinct layers was accompanied by a solid, ventromedially positioned notochord. In the abnormal 8-week embryo, the additional spinal cord was located ventrolaterally from the vertebral column. Both spinal cords appeared bilaterally asymmetric, with their floor and roof plates irregularly formed. An abnormally enhanced pattern of neuroepithelial differentiation characterized their dorsal parts. Furthermore, additional spinal nerves and ganglia and an abnormal bony structure were associated with the spinal cord positioned outside the vertebral column. The underlying vertebral bodies were misshaped and contained scattered supernumerary groups of notochord cells. Our investigation underlines the importance of the notochord-neural tube relationship in the morphogenesis of the spinal cord. We suggest that the double spinal cord was induced by the split notochord.

The entire mesodermal mantle behaves as Spemann's organizer in dorsoanterior enhanced Xenopus laevis embryos.

The body plan of Xenopus laevis can be respecified by briefly exposing early cleavage stage embryos to lithium. Such embryos develop exaggerated dorsoanterior structures such as a radial eye and cement gland (K.R. Kao, Y. Masui, and R.P. Elinson, 1986, Nature (London) 322, 371-373). In this paper, we demonstrate that the enhanced dorsoanterior phenotype results from an overcommitment of mesoderm to dorsoanterior mesoderm. Histological and immunohistochemical observations reveal that the embryos have a greatly enlarged notochord with very little muscle tissue. In addition, they develop a radial, beating heart, suggesting that lithium also specifies anterior mesoderm and pharyngeal endoderm. Randomly oriented diametrically opposed marginal zone grafts from lithium-treated embryos, when transplanted into ultraviolet (uv)-irradiated axis-deficient hosts, rescue dorsal axial structures. These transplantation experiments demonstrate that the entire marginal zone of the early gastrula consists of presumptive dorsal mesoderm. Vital dye marking experiments also indicate that the entire marginal zone maps to the prominent proboscis that is composed of chordamesoderm and represents the long axis of the embryo. These results suggest that lithium respecifies the mesoderm of Xenopus laevis embryos so that it differentiates into the Spemann organizer. We suggest that the origin of the dorsoanterior enhanced phenotypes generated by lithium and the dorsoanterior deficient phenotypes generated by uv irradiation are due to relative quantities of organizer. Our evidence demonstrates the existence of a continuum of body plan phenotypes based on this premise.

The first appearance of the neural tube and optic primordium in the human embryo at stage 10.

Thirteen embryos of stage 10 (22 days) were studied in detail and graphic reconstructions of most of them were prepared. The characteristic feature of this stage is 4-12 pairs of somites. Constantly present are the prechordal and notochordal plates (the notochord sensu stricto is not yet apparent), the neurenteric canal or at least its site, the thyroid primordium, probably the mesencephalic and rhombencephalic neural crest and the adenohypophysial primordium. During this stage, the following features appear: terminal notch, optic sulcus, initial formation of neural tube, oropharyngeal membrane, pulmonary primordium, cardiac loop, aortic arches 1-3, intersegmental arteries, and laryngotracheal groove. The primitive streak is still an important feature. Graphic reconstructions have permitted the detection of the telencephalic portion of the forebrain, for the first time at such an early stage. It is proposed that the remainder of the forebrain comprises two subdivisions: D1, which becomes largely the optic primordium during stage 10, and D2, which is the future thalamic region. The optic sulcus is found in D1 but does not extent into D2, as has been claimed in the literature. An indication of invagination of the otic disc appears towards the end of the stage. As compared with the previous stage, the prosencephalon has increased in length, the mesencephalon has remained the same, the rhombencephalon has decreased, and the spinal part of the neural plate has increased fivefold in length. The site of the initial closure of the neural groove is rhombencephalic, upper cervical, or both. The neural plate extends caudally beyond the site of the neurenteric canal. Cytoplasmic inclusions believed to indicate locations of great activity were always detected in the forebrain (especially in the optic primordium), and also in the rhombencephalon, spinal part, and mesencephalon.

Sphenooccipital chordoma presenting as a nasopharyngeal mass. A case report.

While the nasopharynx is most commonly regarded by the otolaryngologist as a primary site of neoplastic involvement, it is also an avenue of spread of base-of-the-skull tumors presenting as bulging nasopharyngeal masses. The temporal sequence of clinical signs and symptoms may reliably predict the origin of a ventrally extending sphenooccipital chordoma seen on a nasopharyngeal examination. This tumor may cause extensive bony erosion of the petrous apex, sphenoid sinus, and clivus and may suggest a more rapidly growing and aggressive tumor type. The extent of the tumor may be accurately determined by conventional tomography, computerized axial tomography, and arteriography. Severl surgical approaches including the infratemporal fossa approach, transoral transpalatal approach and rhinoseptal transphenoidal approach may be appropriately utilized singly or in combination to remove this tumor in whole or part; however, the rhinoseptal transphenoidal approach is emphasized and regarded as the most rational treatment plan for subtotal resection, recognizing the usual futility of an en bloc resection with its associated high morbidity.