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. 2018 Jul 1;439(1):3-18.
doi: 10.1016/j.ydbio.2018.04.005. Epub 2018 Apr 11.

The Role of the Notochord in Amniote Vertebral Column Segmentation

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Free PMC article

The Role of the Notochord in Amniote Vertebral Column Segmentation

Lizzy Ward et al. Dev Biol. .
Free PMC article

Abstract

The vertebral column is segmented, comprising an alternating series of vertebrae and intervertebral discs along the head-tail axis. The vertebrae and outer portion (annulus fibrosus) of the disc are derived from the sclerotome part of the somites, whereas the inner nucleus pulposus of the disc is derived from the notochord. Here we investigate the role of the notochord in vertebral patterning through a series of microsurgical experiments in chick embryos. Ablation of the notochord causes loss of segmentation of vertebral bodies and discs. However, the notochord cannot segment in the absence of the surrounding sclerotome. To test whether the notochord dictates sclerotome segmentation, we grafted an ectopic notochord. We find that the intrinsic segmentation of the sclerotome is dominant over any segmental information the notochord may possess, and no evidence that the chick notochord is intrinsically segmented. We propose that the segmental pattern of vertebral bodies and discs in chick is dictated by the sclerotome, which first signals to the notochord to ensure that the nucleus pulposus develops in register with the somite-derived annulus fibrosus. Later, the notochord is required for maintenance of sclerotome segmentation as the mature vertebral bodies and intervertebral discs form. These results highlight differences in vertebral development between amniotes and teleosts including zebrafish, where the notochord dictates the segmental pattern. The relative importance of the sclerotome and notochord in vertebral patterning has changed significantly during evolution.

Figures

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Fig. 1
Fig. 1
Vertebral body segmentation is lost when the notochord is ablated. A. Schematic showing the notochord ablation procedure. (Above left = dorsal view, below = transverse section; Above right=sagittal view of steps 1–4 of the ablation procedure). B–F. OPT reconstruction of two HH30–32 embryos, six days after notochord ablation and skeletal preparation (black brackets = ablated region). B. First example, whole embryo (head removed). C. First example, ventro-lateral view of vertebral bodies of embryo in B. Zoomed on ablated region. D. Second example, whole embryo (head removed). E. Second example, ventro-lateral view of vertebral bodies of embryo in D. Zoomed on ablated region. (Star = hole/foramen). F. Second example, dorsal view of neural arches of embryo in D. Neural arches show abnormal morphology and disrupted segmentation. G–H. OPT reconstruction of HH30–32 embryo skeletal preparations, six days after a sham notochord ablation (black brackets = operated region; black arrows= position of intervertebral discs). G. Sham ablated embryo, ventral view, zoom on operated region. Vertebral bodies do not fuse, but IVDs are misaligned on either side of the midline. H. Dorsal view of embryo in G, showing misalignment of neural arches in the operated region. (NA= neural arch, VB= vertebral body). I. Bright-field image of embryo immediately after sham ablation operation, showing that the notochord does not sit parallel to the midline after being raised from the endoderm. Inset image shows illustration of this, with notochord in blue (NT=neural tube, S=somite, NC= notochord).
Fig. 2
Fig. 2
An ectopic notochord graft leads to the formation of ectopic sclerotome lateral to the endogenous somites. A. Schematic showing the notochord graft procedure. (Left = quail donor, centre left = sagittal view of steps 1–4 of notochord graft procedure; centre right = chick host with quail notochord grafted lateral to the host somites (step 5); right = transverse section of chick host after grafting). B–D. WMISH for Pax1 (a marker of the sclerotome) in a HH stage 25 embryo, three days after a notochord graft. Ectopic Pax1 expression is seen in the grafted region anterior to the forelimb (B–C; black arrows). No ectopic Pax1 expression is seen on the contralateral side of the embryo (D). E–G. WMISH for Pax1 in a HH stage 25 embryo, three days after a ‘sham’ notochord graft. No ectopic expression is seen in the operated region (E-F) or on the contralateral side of the embryo (G).
Fig. 3
Fig. 3
A notochord graft leads to the formation of ectopic sclerotome with a different segmental periodicity to host sclerotome. A–D. WMISH for Uncx4.1 in HH stage 24–25 embryos three days after a notochord graft. Ectopic Uncx4.1 expression is seen in the region of the notochord graft. A-B. Example one. C–D. Example two. E-H. WMISH for Scleraxis in HH stage 24-25 embryos three days after a notochord graft. Ectopic Scleraxis expression is seen in the region of the notochord graft. E-F. Example one. G-H. Example two. I. Comparison of endogenous and ectopic segment length in Uncx4.1-stained embryos. Ectopic segments are an average of 19% smaller than the adjacent endogenous segments. A pairwise student T-test shows this difference is statistically significant (p < 0.005, n = 14). J. Comparison of endogenous and ectopic segment length in Scleraxis-stained embryos. Ectopic segments are an average of 21% smaller compared to the adjacent endogenous segment. (Black arrows = segments of ectopic expression, Black star = segments of expression that are significantly out of phase with the endogenous expression pattern, ENC= endogenous notochord visible as white stripe extending from A-P along the axis).
Fig. 4
Fig. 4
Ectopic sclerotome is derived from the host. A–E. Notochord graft only. A. Schematic of notochord graft procedure. B. WMISH for sclerotome marker Pax1 (purple) and immunohistochemical stain for the quail-specific marker QCPN (brown) in HH stage 24/25 embryos, three days after a notochord graft. C. Higher magnification of boxed region in B. D. Transverse section of embryo in B shows endogenous and ectopic sclerotome. E. Higher magnification of boxed region in D. Ectopic sclerotome contains no QCPN-positive staining, showing it is derived from the chick host. F–J. Notochord plus somite graft. F. Notochord plus somite graft procedure. G. WMISH for Pax1 and immunohistochemical stain for QCPN in HH stage 24/25 embryos, three days after a notochord and somite graft. H. Higher magnification of boxed region in G. Two rows of ectopic sclerotome can be seen. I. Transverse section of embryo in G showing endogenous sclerotome and two rows of ectopic sclerotome dorsal and ventral to the ectopic quail notochord. J. Higher magnification of boxed region in I. Ectopic sclerotome below the quail notochord graft contains QCPN-positive cells, showing it is derived from the grafted somite. (NT=neural tube, NC=endogenous notochord, ENC=ectopic quail notochord, CES=chick-derived ectopic sclerotome, QES=quail-derived ectopic sclerotome, EndS=endogenous sclerotome).
Fig. 5
Fig. 5
Two potential mechanisms by which ectopic sclerotome segmentation could be compressed. Both models show somites before (left) and after migration of the sclerotome to the notochord. A. Model 1:The notochord contains an intrinsic segmental pattern that instructs the position of the IVD within the sclerotome. The quail notochord is released from tension when excised from the donor embryo, compressing this segmental pattern. This compressed segmental pattern is then translated into the vertebral bodies and IVDs after migration of the sclerotome to the midline. B. Model 2:The notochord contains no intrinsic segmental pattern, but secretes a diffusible attractant causing sclerotome within range to move towards the graft. The pattern of AF and vertebral body precursors is intrinsically determined within the sclerotome. However this pattern is compressed after migration towards the graft due to either competition for space at the midline, or due to the smaller number of cells that have migrated towards the graft compared to the endogenous notochord.
Fig. 6
Fig. 6
Inter-regional notochord grafts suggest that the periodicity of ectopic sclerotome is dependent upon somite size, not the axial region of the notochord. A-D. Schematics showing the four inter-regional notochord grafts carried out (red=grafted quail notochord). A. Cervical notochord grafted to cervical somites (C-C). B. Brachial notochord grafted to cervical somites (B-C). C. Cervical notochord grafted to brachial somites (C-B). D. Brachial notochord grafted to brachial somites (B-B). E-L. Images and quantification of HH stage 24–25 embryos, three days after notochord graft. E, G, I, K. WMISH for Uncx4.1 (purple) shows the segmental pattern of ectopic sclerotome resulting from each graft (black arrows = segments of Uncx4.1 expression; Black stars = segments of expression that are significantly out of phase with the endogenous expression pattern). Letters in top right corner refer to axial region of graft and host. F, H, J, L. Comparison of mean ectopic and endogenous segment length for each graft.
Fig. 7
Fig. 7
A notochord graft leads to expansion of the sclerotome prior to separation of endogenous and ectopic sclerotome. A. WMISH for Pax1 (purple) shows presence of ectopic sclerotome in a HH18 embryo, one day after a notochord graft. Ectopic sclerotome is continuous with the endogenous sclerotome. B. Higher magnification on boxed region of A. Black bracket indicates expanded Pax1 expression. C. Transverse section of embryo one day after a notochord graft (NC), showing expansion of sclerotome into the lateral somite adjacent to the graft. D–E Time-lapse imaging of a developing embryo, in which a quail notochord (NC) has been grafted lateral to the somites and anterior PSM. D. Grafted embryo at 0 h incubation. E. Embryo approximately 7.5 h after the graft. Somites adjacent to the notochord graft (black bracket) expand towards the graft. F. WMISH for somite marker Paraxis (purple) on embryo shown in E, after 8 h incubation. The quail notochord graft (brown) was detected by an immuno-stain for the QCPN quail cell marker. Paraxis expression confirms that somites are expanded towards the graft. G. Quantification of somite area in response to a notochord graft. The mean total area of the four somites closest to a notochord graft, across six embryos, was compared to contralateral somites (control side), after eight hours exposure to a notochord graft. The total area of somites on each side of the embryo is expressed as a percentage of the total area of all eight somites measured per embryo. A paired sample T-test shows that the greater percentage area of somites on the graft side compared to the control side is statistically significiant (t(6) = 3.88, p = 0.008).
Fig. 8
Fig. 8
A notochord graft leads to formation of ectopic cartilage. A–D. OPT reconstructions of skeletal preparations of HH30–33 embryos, six days after a notochord graft. Ectopic cartilage is highlighted in blue. Inset images show bright field images of ectopic cartilage, stained with Alcian Blue. (NA=neural arch; VB=vertebral body, Red arrows= potential segmentation of cartilage) A. Lateral view of whole embryo (head removed) shows ectopic cartilage in grafted region. B. Zoom on boxed region of embryo shown in A. C. Second example of ectopic cartilage. Zoom on ectopic cartilage, which shows a ring-like morphology. D. Third example of embryo showing ectopic cartilage. Zoom on ectopic cartilage. This embryo shows disruption to the morphology of the endogenous vertebrae. (red star=fused vertebral bodies, white star = fused neural arches).
Fig. 9
Fig. 9
Ectopic cartilage resulting from a notochord and neural tube graft. A. Schematic diagram showing notochord and neural tube graft procedure (quail neural tube and notochord shown in red). B. OPT reconstruction of HH33 embryo skeletal preparation, six days after a notochord and neural tube graft. C. Zoom on boxed region of embryo in B. Ectopic cartilage shaded in blue, containing elements of neural arch-like morphology and a hole that resembles a foramen (star). Inset panel shows bright field image of ectopic cartilage, stained with Alcian Blue.
Fig. 10
Fig. 10
PSM ablations suggest the notochord cannot segment in the absence of the surrounding somites. A. Schematic diagram showing the PSM ablation procedure.(Left= dorsal view; right= sagittal view of steps 1–4 of ablation procedure). B–D. WMISH for Tbx6 (a marker of the PSM) in HH11–12 embryos fixed immediately after ablation, demonstrating successful ablation of the PSM from a portion of notochord. C. Transverse sections confirm the complete absence of Tbx6-positive cells in the ablated region (black star=remnants of ink used for contrast during ablation procedure). D. Transverse section shows that the PSM posterior to the ablated region was unaffected. E–G. WMISH for paraxis (marker of the somites) in embryos cultured overnight after PSM ablation, showing that PSM ablation led to a complete absence of somites in the ablated region. F. Transverse section confirming the absence of paraxis-positive cells in the ablated region, with lateral plate mesoderm moving medially to lie adjacent to the notochord. G. Transverse section showing that somites posterior to the ablated region form normally. H–I. Haematoxylin-stained coronal sections through notochord of a six day old embryo, four days after PSM ablation. The length of the ablated region is shown in two sections. H. Section rostral to ablated region. Notochord shows segmental pattern of swellings and constrictions that coincide with dark stripes in surrounding sclerotome which correspond to the future AF region. I. Section spanning ablated region (left) and further caudally (right). The area between black arrowheads shows absence of sclerotome and the notochord shows no signs of segmentation. Immediately anterior and posterior to this, the notochord is surrounded by apparently unsegmented sclerotome. Caudally, normal segmented sclerotome is seen surrounding the notochord and the segmental pattern of swellings and constrictions in the notochord resumes. J–K. Schematic diagram corresponding to sections H-I. NC = notochord, NT = neural tube, US-Scl = unsegmented sclerotome, LPM=lateral plate mesoderm, Black stars = dorsal root ganglia, grey arrows = stripes of high cell density in sclerotome corresponding to future AF.

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