Research

Spatiotemporal Control of Vertebral Segmentation

Spatiotemporal patterns widely occur in biological, chemical, and physical systems. Particularly, embryonic development displays a diverse gamut of repetitive patterns established in many tissues and organs. Branching treelike structures in lungs, kidneys, livers, pancreases, and mammary glands as well as digits and bones in appendages, teeth, and palates are just a few examples. A fascinating instance of repetitive patterning is the sequential segmentation of the paraxial mesoderm into somites along the primary body axis. Somites are segmental structures composed of epithelial cells that encapsulate a core of mesenchymal cells, and they are located as paired tissue blocks on both sides of the caudal neural tube and notochord. Cells located in somites later differentiate into the cells of the vertebral column, skeletal muscle, or dermis. Thus, the somites act as the blueprint for the segmental structure of the vertebral column and additionally influence patterning of associated vasculature and peripheral nerves.

A gene expression oscillator, called the vertebrate segmentation clock (Hes/Her family proteins), controls the periodicity of sequential somite segmentation (McDaniel, Science Advances, 2024). A spatial gradient of phosphorylated, active ERK (pErk) instructs the positions of each segment boundary (Simsek and Özbudak, Cell Reports, 2018). The segmentation clock periodically inhibits and thereby drives oscillatory activity of the pErk gradient. This hierarchical relationship between the segmentation clock and pErk gradient is essential for sequential segmentation of somites (Simsek et al., Nature, 2023).

In our ongoing research, we aim to identify the molecular mechanism by which the segmentation clock inhibits the pErk activity.

Regulatory Mechanism Ensuring Reproducible Pattern Formation Despite Unavoidable Molecular Heterogeneities in Gene Expression and Cell Signaling

Mutations of several genes cause incomplete penetrance and variable expressivity of phenotypes, which are usually attributed to modifier genes or gene-environment interactions. In contrast, we showed stochastic gene expression underlies the variability of somite segmentation defects in embryos mutant for segmentation clock genes her1 or her7. Phenotypic strength is further augmented by low temperature and hypoxia. By performing live imaging of the segmentation clock reporters, we further show that groups of cells with higher oscillation amplitudes successfully form somites while those with lower amplitudes fail to do so. In unfavorable environments, the number of cycles with high amplitude oscillations and the number of successful segmentations proportionally decrease. These results suggest that individual oscillation cycles stochastically fail to pass a threshold amplitude, resulting in segmentation defects in mutants (Keseroglu et al, Nature Communications, 2023). In a preceding publication, we showed that temporally coordinated co-transcription of two segmentation clock genes (her1 or her7) is critical for successful segmentation in zebrafish (Zinani et al., Nature 2021).

Delta/Notch signaling couples the Hes/Her oscillators among close neighboring cells and thereby locally synchronizes clock oscillations in the PSM. Disruption of Delta/ Notch oscillations, either in its loss-of-function mutants or in constitutively overexpressing transgenic embryos, gradually desynchronizes oscillations among the PSM cells and results in segmentation defects after formation of several normal somites (Özbudak and Lewis, 2008).

In our ongoing research, we aim to identify the gene regulatory network features maintaining high amplitude and spatially coordinated expression of the genes involved in different steps of the segmentation process.

Establishment of Rostrocaudal Polarity of Somites

Expression of the segmentation clock genes display kinematic waves along the unsegmented tissues. Though these waves are not necessary for somite segmentation (Simsek et al., Nature, 2023), they regulate establishment of rostrocaudal polarity of somites which is important for proper differentiation of segmented cells.

In our ongoing research, we aim to identify the molecular mechanism by which these kinematic waves are generated.

The Decoding and Execution of Spatiotemporal Information

Somitogenesis involves multiple distinct modular steps (McDaniel, Science Advances, 2024). The spatiotemporal information, encoded by the segmentation clock and the pErk gradient, are integrated to instruct segment boundaries in the middle of the unsegmented tissue. Currently, it is not known how cells decode this spatiotemporal information. Mosaic experiments suggest cells determine segment boundaries non-cell autonomously (Simsek and Özbudak, Cell Reports, 2018). Therefore, the signal decoder machinery likely uses cell membrane proteins. Several membrane proteins, including ephrins, cadherins, and integrins, might play a role in the decoding process.

In our ongoing studies, we investigate the molecular mechanism decoding this segmental commitment in the middle of the unsegmented tissue and executing somite segmentation at the anterior end of the unsegmented tissue.