Vertebrate segmentation: from cyclic gene networks to scoliosis

Abstract

One of the most striking features of the human vertebral column is its periodic organization along the anterior-posterior axis. This pattern is established when segments of vertebrates, called somites, bud off at a defined pace from the anterior tip of the embryo's presomitic mesoderm (PSM). To trigger this rhythmic production of somites, three major signaling pathways--Notch, Wnt/β-catenin, and fibroblast growth factor (FGF)--integrate into a molecular network that generates a traveling wave of gene expression along the embryonic axis, called the "segmentation clock." Recent systems approaches have begun identifying specific signaling circuits within the network that set the pace of the oscillations, synchronize gene expression cycles in neighboring cells, and contribute to the robustness and bilateral symmetry of somite formation. These findings establish a new model for vertebrate segmentation and provide a conceptual framework to explain human diseases of the spine, such as congenital scoliosis.

Figure1. The segmentation clock

(A–I) In situ hybridization with c-hairy1 probe showing the different categories of expression patterns in chicken embryos aged of 15 (A, B, and C), 16 (D, E, and F), and 17 (G, H, and I) somites. Anterior to the top. (J) Schematic representation of the correlation between c-hairy1 expression in the PSM with the progression of somite formation. This highly dynamic sequence of c-hairy1 expression in the PSM was observed at all stages of somitogenesis examined, suggesting a cyclic expression of the c-hairy1 mRNA correlated with somite formation. Arrowheads: last formed somite (somite I: SI). From (Palmeirim et al., 1997) (H) Cyclic genes belonging to the Notch and FGF pathways (whose products are indicated in red) oscillate in opposite phase to cyclic genes of the Wnt pathway (blue). A large number of the cyclic genes are involved in negative feedback loops. The basic circuitry of the three signaling pathways is represented. Dashed lines correspond to modes of regulation inferred from work in other systems or based on microarray data.

Figure 2. Synchronization of the presomitic mesoderm cellular oscillators

(A) Smooth transcriptional waves of cyclic gene expression sweeping through the zebrafish PSM (shown in blue). (B) Schematic representation of the PSM cells as coupled phase oscillators. (C) Model of the zebrafish oscillator. The Notch pathway has been proposed to synchronize oscillations by coupling by connecting the zebrafish Her1/Her7 intracellular oscillator to the Notch/DeltaC intercellular loop. The transcription factors Her1/Her7 establish a negative feedback loop controling the periodic repression of DeltaC, allowing the synchronous activation of Notch signaling in neighboring cells. In addition to receiving inputs from Notch signaling, the Her1/Her7 oscillator requires the Her13.2 partner, which is downstream of FGF signaling. The coupling between cellular oscillators provided by the Notch/Delta intercellular loop is thought to confer robustness to the clock synchronized oscillations against developmental noise such as cell proliferation, cell movement or stochastic gene expression.

Figure 3. A model for vertebrate somitogenesis

(A–D) Expression pattern of key components of the segmentation system during somitogenesis in 2-day chicken embryos. (A) Wnt3a, (B) Fgf8, (C) Raldh2, (D) Mesp2 expression by in situ hybridization. (*) last formed somite; The dotted line marks the approximate position of the determination front (I) A segmentation model. Antagonistic gradients of FGF/Wnt signaling (purple) and retinoic acid signaling (green) position the determination front (thick, black line). The periodic signal of the segmentation clock is shown in orange (represented on the left side only). As the embryo extends posteriorly, the determination front moves caudally. Cells that reach the determination front are exposed to the periodic clock signal, initiating the segmentation program and activating simultaneously expression of genes such as Mesp2 (black stripes, represented on the right side only), in a stripe domain that prefigures the future segment. This establishes the segmental pattern of the presumptive somites. Dorsal views, anterior to the top.

Figure 4. Role of Retinoic acid in the control of left-right symmetry of somitogenesis

Retinoic acid signaling activity and NR2F2 expression are asymmetric across the left-right axis in the presomitic mesoderm of early-somite stage embryos. In situ hybridization for mouse Raldh2 (a), mouse NR2F2 (c) and chicken NR2F2 (d). Asymmetric expression is indicated by the white arrowheads. (b) RARE-LacZ mouse embryo indicating the asymmetric RA signaling activity (white arrowheads) (dorsal views). (e-h) Schematic interpretation of the role of RA signaling and Fgf8 in the control of somite bilateral symmetry in early mouse and chicken embryos. Asymmetrical RA signaling in the anterior PSM and somites is shown in blue and antagonizes FGF signaling (orange). Nodal signaling is indicated on the left side (pink). The embryonic side for which Fgf8 acts as a left-right determinant is indicated with the label Fgf8. (e) Wild-type mouse embryo. (f) Rere or Raldh2 mouse mutant. (g) Wild-type chicken embryo. (h) RA-deprived chicken embryo. L (left) and R (right). From (Vilhais-Neto et al., 2010)

Figure 5. Mutation of human orthologues of the segmentation clock genes leads to congenital scoliosis

(A) Radiograph of a patient with Spondylocostal dysostosis (SCDO1) showing the severe axial skeletal malformations associated with a mutation in the DLL3 gene. Courtesy of Peter Turnpenny (B) Radiograph of patient with spondylothoracic dysostosis (STD) illustrating severe vertebral and rib malformations associated with a mutation in the MESP2 gene. Courtesy of Alberto Cornier.