Spatiotemporal control of pattern formation during somitogenesis.

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 primary body axis, which is conserved in all vertebrates and many arthropods and annelids. In these species, the body axis elongates at the posterior end of the embryo containing an unsegmented tissue. Meanwhile, segments sequentially bud off from the anterior end of the unsegmented tissue, laying down an exquisite repetitive pattern and creating a segmented body plan. In vertebrates, the paraxial mesoderm is sequentially divided into somites. In this review, we will discuss the most prominent models, the most puzzling experimental data, and outstanding questions in vertebrate somite segmentation.

A design logic for sequential segmentation across organisms.

Multitudes of organisms display metameric compartmentalization of their body plan. Segmentation of these compartments happens sequentially in diverse phyla. In several sequentially segmenting species, periodically active molecular clocks and signaling gradients have been found. The clocks are proposed to control the timing of segmentation, while the gradients are proposed to instruct the positions of segment boundaries. However, the identity of the clock and gradient molecules differs across species. Furthermore, sequential segmentation of a basal chordate, Amphioxus, continues at late stages when the small tail bud cell population cannot establish long-range signaling gradients. Thus, it remains to be explained how a conserved morphological trait (i.e., sequential segmentation) is achieved by using different molecules or molecules with different spatial profiles. Here, we first focus on sequential segmentation of somites in vertebrate embryos and then draw parallels with other species. Thereafter, we propose a candidate design principle that has the potential to answer this puzzling question.

Human stem cell models unravel mechanisms of somite segmentation.

In vitro models to study human somitogenesis, the formation of the segmented body plan, have so far been limited.1 Two papers in Nature now report the creation of pluripotent stem cell (PSC)-derived 3D culture systems that recapitulate the formation of somite-like structures and help gain insights into this developmental process.2,3.

Periodic inhibition of Erk activity drives sequential somite segmentation.

Sequential segmentation creates modular body plans of diverse metazoan embryos1-4. Somitogenesis establishes the segmental pattern of the vertebrate body axis. A molecular segmentation clock in the presomitic mesoderm sets the pace of somite formation4. However, how cells are primed to form a segment boundary at a specific location remains unclear. Here we developed precise reporters for the clock and double-phosphorylated Erk (ppErk) gradient in zebrafish. We show that the Her1-Her7 oscillator drives segmental commitment by periodically lowering ppErk, therefore projecting its oscillation onto the ppErk gradient. Pulsatile inhibition of the ppErk gradient can fully substitute for the role of the clock, and kinematic clock waves are dispensable for sequential segmentation. The clock functions upstream of ppErk, which in turn enables neighbouring cells to discretely establish somite boundaries in zebrafish5. Molecularly divergent clocks and morphogen gradients were identified in sequentially segmenting species3,4,6-8. Our findings imply that versatile clocks may establish sequential segmentation in diverse species provided that they inhibit gradients.

Patterning principles of morphogen gradients.

Metazoan embryos develop from a single cell into three-dimensional structured organisms while groups of genetically identical cells attain specialized identities. Cells of the developing embryo both create and accurately interpret morphogen gradients to determine their positions and make specific decisions in response. Here, we first cover intellectual roots of morphogen and positional information concepts. Focusing on animal embryos, we then provide a review of current understanding on how morphogen gradients are established and how their spans are controlled. Lastly, we cover how gradients evolve in time and space during development, and how they encode information to control patterning. In sum, we provide a list of patterning principles for morphogen gradients and review recent advances in quantitative methodologies elucidating information provided by morphogens.

A 3-D Tail Explant Culture to Study Vertebrate Segmentation in Zebrafish.

Vertebrate embryos pattern their major body axis as repetitive somites, the precursors of vertebrae, muscle, and skin. Somites progressively segment from the presomitic mesoderm (PSM) as the tail end of the embryo elongates posteriorly. Somites form with regular periodicity and scale in size. Zebrafish is a popular model organism as it is genetically tractable and has transparent embryos that allow for live imaging. Nevertheless, during somitogenesis, fish embryos are wrapped around a large, rounding yolk. This geometry limits live imaging of PSM tissue in zebrafish embryos, particularly at higher resolutions that require a close objective working distance. Here, we present a flattened 3-D tissue culture method for live imaging of zebrafish tail explants. Tail explants mimic intact embryos by displaying a proportional slowdown of axis elongation and shortening of rostrocaudal somite lengths. We are further able to stall axis elongation speed through explant culture. This, for the first time, enables us to untangle the chemical input of signaling gradients from the mechanistic input of axial elongation. In future studies, this method can be combined with a microfluidic setup to allow time-controlled pharmaceutical perturbations or screening of vertebrate segmentation without any drug penetration concerns.

Regulatory Network of the Scoliosis-Associated Genes Establishes Rostrocaudal Patterning of Somites in Zebrafish.

Gene regulatory networks govern pattern formation and differentiation during embryonic development. Segmentation of somites, precursors of the vertebral column among other tissues, is jointly controlled by temporal signals from the segmentation clock and spatial signals from morphogen gradients. To explore how these temporal and spatial signals are integrated, we combined time-controlled genetic perturbation experiments with computational modeling to reconstruct the core segmentation network in zebrafish. We found that Mesp family transcription factors link the temporal information of the segmentation clock with the spatial action of the fibroblast growth factor signaling gradient to establish rostrocaudal (head to tail) polarity of segmented somites. We further showed that cells gradually commit to patterning by the action of different genes at different spatiotemporal positions. Our study provides a blueprint of the zebrafish segmentation network, which includes evolutionarily conserved genes that are associated with the birth defect congenital scoliosis in humans.

Spatial Fold Change of FGF Signaling Encodes Positional Information for Segmental Determination in Zebrafish.

Signal gradients encode instructive information for numerous decision-making processes during embryonic development. A striking example of precise, scalable tissue-level patterning is the segmentation of somites-the precursors of the vertebral column-during which the fibroblast growth factor (FGF), Wnt, and retinoic acid (RA) pathways establish spatial gradients. Despite decades of studies proposing roles for all three pathways, the dynamic feature of these gradients that encodes instructive information determining segment sizes remained elusive. We developed a non-elongating tail explant system, integrated quantitative measurements with computational modeling, and tested alternative models to show that positional information is encoded solely by spatial fold change (SFC) in FGF signal output. Neighboring cells measure SFC to accurately position the determination front and thus determine segment size. The SFC model successfully recapitulates results of spatiotemporal perturbation experiments on both explants and intact embryos, and it shows that Wnt signaling acts permissively upstream of FGF signaling and that RA gradient is dispensable.

Quantifying spatial and temporal variations of the cell membrane ultra-structure by bimFCS.

It has been long recognized that the cell membrane is heterogeneous on scales ranging from a couple of molecules to micrometers in size and hence diffusion of receptors is length scale dependent. This heterogeneity modulates many cell-membrane-associated processes requiring transient spatiotemporal separation of components. The transient increase in local concentration of interacting signal components enables robust signaling in an otherwise thermally noisy system. Understanding how lipids and proteins self-organize and interact with the cell cortex requires quantifying the motion of the components. Multi-length scale diffusion measurements by single particle tracking, fluorescence correlation spectroscopy (FCS) or related techniques are able to identify components being transiently trapped in nanodomains, from freely moving one and from ones with reduced long-scale diffusion due to interaction with the cell cortex. One particular implementation of multi-length scale diffusion measurements is the combination of FCS with a spatially resolved detector, such as a camera and two-dimensional extended excitation profile. The main advantages of this approach are that all length scales are interrogated simultaneously, uniquely permits quantifying changes to the membrane structure caused by extrenal or internal perturbations. Here, we review how combining total internal reflection microscopy (TIRF) with FC resolves the membrane organization in living cells. We show how to implement the method, which requires only a few seconds of data acquisition to quantify membrane nanodomains, or the spacing of membrane fences caused by the actin cortex. The choice of diffusing fluorescent probe determines which membrane heterogeneity is detected. We review the instrument, sample preparation, experimental and computational requirements to perform such measurements, and discuss the potential and limitations. The discussion includes examples of spatial and temporal comparisons of the membrane structure in response to perturbations demonstrating the complex cell physiology.

Effect of receptor dimerization on membrane lipid raft structure continuously quantified on single cells by camera based fluorescence correlation spectroscopy.

Membrane bound cell signaling is modulated by the membrane ultra-structure, which itself may be affected by signaling. However, measuring the interaction of membrane proteins with membrane structures in intact cells in real-time poses considerable challenges. In this paper we present a non-destructive fluorescence method that quantifies these interactions in single cells, and is able to monitor the same cell continuously to observe small changes. This approach combines total internal fluorescence microscopy with fluorescence correlation spectroscopy to measure the protein's diffusion and molecular concentration in different sized areas simultaneously. It correctly differentiates proteins interacting with membrane fences from proteins interacting with cholesterol-stabilized domains, or lipid rafts. This method detects small perturbations of the membrane ultra-structure or of a protein's tendency to dimerize. Through continuous monitoring of single cells, we demonstrate how dimerization of GPI-anchored proteins increases their association with the structural domains. Using a dual-color approach we study the effect of dimerization of one GPI-anchored protein on another type of GPI-anchored protein expressed in the same cell. Scans over the cell surface reveal a correlation between cholesterol stabilized domains and membrane cytoskeleton.