A segmentation clock patterns cellular differentiation in a bacterial biofilm.

Contrary to multicellular organisms that display segmentation during development, communities of unicellular organisms are believed to be devoid of such sophisticated patterning. Unexpectedly, we find that the gene expression underlying the nitrogen stress response of a developing Bacillus subtilis biofilm becomes organized into a ring-like pattern. Mathematical modeling and genetic probing of the underlying circuit indicate that this patterning is generated by a clock and wavefront mechanism, similar to that driving vertebrate somitogenesis. We experimentally validated this hypothesis by showing that predicted nutrient conditions can even lead to multiple concentric rings, resembling segments. We additionally confirmed that this patterning mechanism is driven by cell-autonomous oscillations. Importantly, we show that the clock and wavefront process also spatially patterns sporulation within the biofilm. Together, these findings reveal a biofilm segmentation clock that organizes cellular differentiation in space and time, thereby challenging the paradigm that such patterning mechanisms are exclusive to plant and animal development.

Oscillatory control of embryonic development.

Proper embryonic development depends on the timely progression of a genetic program. One of the key mechanisms for achieving precise control of developmental timing is to use gene expression oscillations. In this Review, we examine how gene expression oscillations encode temporal information during vertebrate embryonic development by discussing the gene expression oscillations occurring during somitogenesis, neurogenesis, myogenesis and pancreas development. These oscillations play important but varied physiological functions in different contexts. Oscillations control the period of somite formation during somitogenesis, whereas they regulate the proliferation-to-differentiation switch of stem cells and progenitor cells during neurogenesis, myogenesis and pancreas development. We describe the similarities and differences of the expression pattern in space (i.e. whether oscillations are synchronous or asynchronous across neighboring cells) and in time (i.e. different time scales) of mammalian Hes/zebrafish Her genes and their targets in different tissues. We further summarize experimental evidence for the functional role of their oscillations. Finally, we discuss the outstanding questions for future research.

Orchestration of tissue shape changes and gene expression patterns in development.

In development, tissue shape changes and gene expression patterns give rise to morphogenesis. Understanding tissue shape changes requires the analysis of mechanical properties of the tissue such as tissue rigidity, cell influx from neighboring tissues, cell shape changes and cell proliferation. Local and global gene expression patterns can be influenced by neighbor exchange and tissue shape changes. Here we review recent studies on the mechanisms for tissue elongation and its influences on dynamic gene expression patterns by focusing on vertebrate somitogenesis. We first introduce mechanical and biochemical properties of the segmenting tissue that drive tissue elongation. Then, we discuss patterning in the presence of cell mixing, scaling of signaling gradients, and dynamic phase waves of rhythmic gene expression under tissue shape changes. We also highlight the importance of theoretical approaches to address the relation between tissue shape changes and patterning.

Imaging the onset of oscillatory signaling dynamics during mouse embryo gastrulation.

A fundamental requirement for embryonic development is the coordination of signaling activities in space and time. A notable example in vertebrate embryos is found during somitogenesis, where gene expression oscillations linked to the segmentation clock are synchronized across cells in the presomitic mesoderm (PSM) and result in tissue-level wave patterns. To examine their onset during mouse embryo development, we studied the dynamics of the segmentation clock gene Lfng during gastrulation. To this end, we established an imaging setup using selective plane illumination microscopy (SPIM) that enables culture and simultaneous imaging of up to four embryos ('SPIM- for-4'). Using SPIM-for-4, combined with genetically encoded signaling reporters, we detected the onset of Lfng oscillations within newly formed mesoderm at presomite stages. Functionally, we found that initial synchrony and the first ∼6-8 oscillation cycles occurred even when Notch signaling was impaired, revealing similarities to previous findings made in zebrafish embryos. Finally, we show that a spatial period gradient is present at the onset of oscillatory activity, providing a potential mechanism accounting for our observation that wave patterns build up gradually over the first oscillation cycles.

Circadian key component CLOCK/BMAL1 interferes with segmentation clock in mouse embryonic organoids.

In mammals, circadian clocks are strictly suppressed during early embryonic stages, as well as in pluripotent stem cells, by the lack of CLOCK/BMAL1-mediated circadian feedback loops. During ontogenesis, the innate circadian clocks emerge gradually at a late developmental stage, and with these, the circadian temporal order is invested in each cell level throughout a body. Meanwhile, in the early developmental stage, a segmented body plan is essential for an intact developmental process, and somitogenesis is controlled by another cell-autonomous oscillator, the segmentation clock, in the posterior presomitic mesoderm (PSM). In the present study, focusing upon the interaction between circadian key components and the segmentation clock, we investigated the effect of the CLOCK/BMAL1 on the segmentation clock Hes7 oscillation, revealing that the expression of functional CLOCK/BMAL1 severely interferes with the ultradian rhythm of segmentation clock in induced PSM and gastruloids. RNA sequencing analysis implied that the premature expression of CLOCK/BMAL1 affects the Hes7 transcription and its regulatory pathways. These results suggest that the suppression of CLOCK/BMAL1-mediated transcriptional regulation during the somitogenesis may be inevitable for intact mammalian development.

Biological Significance of the Coupling Delay in Synchronized Oscillations.

The significance of the coupling delay, which is the time required for interactions between coupled oscillators, in various oscillatory dynamics has been investigated mathematically for more than three decades, but its biological significance has been revealed only recently. In the segmentation clock, which regulates the periodic formation of somites in embryos, Hes7 expression oscillates synchronously between neighboring presomitic mesoderm (PSM) cells, and this synchronized oscillation is controlled by Notch signaling-mediated coupling between PSM cells. Recent studies have shown that inappropriate coupling delays dampen and desynchronize Hes7 oscillations, as simulated mathematically, leading to the severe fusion of somites and somite-derived tissues such as the vertebrae and ribs. These results indicate the biological significance of the coupling delay in synchronized Hes7 oscillations in the segmentation clock. The recent development of an in vitro PSM-like system will facilitate the detailed analysis of the coupling delay in synchronized oscillations.

The (unusual) heuristic value of Hox gene clusters; a matter of time?

Ever since their first report in 1984, Antennapedia-type homeobox (Hox) genes have been involved in such a series of interesting observations, in particular due to their conserved clustered organization between vertebrates and arthropods, that one may legitimately wonder about the origin of this heuristic value. In this essay, I first consider different examples where Hox gene clusters have been instrumental in providing conceptual advances, taken from various fields of research and mostly involving vertebrate embryos. These examples touch upon our understanding of genomic evolution, the revisiting of 19th century views on the relationships between development and evolution and the building of a new framework to understand long-range and pleiotropic gene regulation during development. I then discuss whether the high value of the Hox gene family, when considered as an epistemic object, is related to its clustered structure (and the absence thereof in some animal species) and, if so, what is it in such particular genetic oddities that made them so generous in providing the scientific community with interesting information.

Patterning and mechanics of somite boundaries in zebrafish embryos.

The body axis of vertebrates is subdivided into repetitive compartments called somites, which give rise primarily to the segmented architecture of the musculoskeletal system in the adult body. Somites form in a sequential and rhythmic manner in embryos and a physical boundary separates each somite from the rest of the unsegmented tissue and adjoining somites. Precise positioning of somite boundaries and determination of boundary cell fate in a select group of cells is thought to be driven by gene expression patterns and morphogen gradients. This pre-patterning step is followed by a mechanical process involving actomyosin activation in boundary cells and formation of an extracellular matrix that results in morphological boundary formation. While genes involved in somite boundary formation have been identified, there are many open questions about the underlying pre-patterning dynamics and mechanics and how these processes are coupled to generate a morphological boundary. Here, focusing on segmentation of zebrafish embryos as a model, we review pre-patterning processes critical for boundary formation and how cytoskeletal activity drives tissue separation. Our outlook is that this system holds exciting new avenues for unearthing general principles of boundary formation in developing embryos.

Information flow in the presence of cell mixing and signaling delays during embryonic development.

Embryonic morphogenesis is organized by an interplay between intercellular signaling and cell movements. Both intercellular signaling and cell movement involve multiple timescales. A key timescale for signaling is the time delay caused by preparation of signaling molecules and integration of received signals into cells' internal state. Movement of cells relative to their neighbors may introduce exchange of positions between cells during signaling. When cells change their relative positions in a tissue, the impact of signaling delays on intercellular signaling increases because the delayed information that cells receive may significantly differ from the present state of the tissue. The time it takes to perform a neighbor exchange sets a timescale of cell mixing that may be important for the outcome of signaling. Here we review recent theoretical work on the interplay of timescales between cell mixing and signaling delays adopting the zebrafish segmentation clock as a model system. We discuss how this interplay can lead to spatial patterns of gene expression that could disrupt the normal formation of segment boundaries in the embryo. The effect of cell mixing and signaling delays highlights the importance of theoretical and experimental frameworks to understand collective cellular behaviors arising from the interplay of multiple timescales in embryonic developmental processes.

Segmentation clock dynamics is strongly synchronized in the forming somite.

During vertebrate somitogenesis an inherent segmentation clock coordinates the spatiotemporal signaling to generate segmented structures that pattern the body axis. Using our experimental and quantitative approach, we study the cell movements and the genetic oscillations of her1 expression level at single-cell resolution simultaneously and scale up to the entire pre-somitic mesoderm (PSM) tissue. From the experimentally determined phases of PSM cellular oscillators, we deduced an in vivo frequency profile gradient along the anterior-posterior PSM axis and inferred precise mathematical relations between spatial cell-level period and tissue-level somitogenesis period. We also confirmed a gradient in the relative velocities of cellular oscillators along the axis. The phase order parameter within an ensemble of oscillators revealed the degree of synchronization in the tailbud and the posterior PSM being only partial, whereas synchronization can be almost complete in the presumptive somite region but with temporal oscillations. Collectively, the degree of synchronization itself, possibly regulated by cell movement and the synchronized temporal phase of the transiently expressed clock protein Her1, can be an additional control mechanism for making precise somite boundaries.

Myc activity is required for maintenance of the neuromesodermal progenitor signalling network and for segmentation clock gene oscillations in mouse.

The Myc transcriptional regulators are implicated in a range of cellular functions, including proliferation, cell cycle progression, metabolism and pluripotency maintenance. Here, we investigated the expression, regulation and function of the Myc family during mouse embryonic axis elongation and segmentation. Expression of both cMyc (Myc - Mouse Genome Informatics) and MycN in the domains in which neuromesodermal progenitors (NMPs) and underlying caudal pre-somitic mesoderm (cPSM) cells reside is coincident with WNT and FGF signals, factors known to maintain progenitors in an undifferentiated state. Pharmacological inhibition of Myc activity downregulates expression of WNT/FGF components. In turn, we find that cMyc expression is WNT, FGF and Notch protein regulated, placing it centrally in the signalling circuit that operates in the tail end that both sustains progenitors and drives maturation of the PSM into somites. Interfering with Myc function in the PSM, where it displays oscillatory expression, delays the timing of segmentation clock oscillations and thus of somite formation. In summary, we identify Myc as a component that links NMP maintenance and PSM maturation during the body axis elongation stages of mouse embryogenesis.

Size-reduced embryos reveal a gradient scaling-based mechanism for zebrafish somite formation.

Little is known about how the sizes of animal tissues are controlled. A prominent example is somite size, which varies widely both within an individual and across species. Despite intense study of the segmentation clock governing the timing of somite generation, how it relates to somite size is poorly understood. Here, we examine somite scaling and find that somite size at specification scales with the length of the presomitic mesoderm (PSM) despite considerable variation in PSM length across developmental stages and in surgically size-reduced embryos. Measurement of clock period, axis elongation speed and clock gene expression patterns demonstrate that existing models fail to explain scaling. We posit a 'clock and scaled gradient' model, in which somite boundaries are set by a dynamically scaling signaling gradient across the PSM. Our model not only explains existing data, but also makes a unique prediction that we confirm experimentally - the formation of periodic 'echoes' in somite size following perturbation of the size of one somite. Our findings demonstrate that gradient scaling plays a central role in both progression and size control of somitogenesis.

ES cell-derived presomitic mesoderm-like tissues for analysis of synchronized oscillations in the segmentation clock.

Somites are periodically formed by segmentation of the anterior parts of the presomitic mesoderm (PSM). In the mouse embryo, this periodicity is controlled by the segmentation clock gene Hes7, which exhibits wave-like oscillatory expression in the PSM. Despite intensive studies, the exact mechanism of such synchronous oscillatory dynamics of Hes7 expression still remains to be analyzed. Detailed analysis of the segmentation clock has been hampered because it requires the use of live embryos, and establishment of an in vitro culture system would facilitate such analyses. Here, we established a simple and efficient method to generate mouse ES cell-derived PSM-like tissues, in which Hes7 expression oscillates like traveling waves. In these tissues, Hes7 oscillation is synchronized between neighboring cells, and the posterior-anterior axis is self-organized as the central-peripheral axis. This method is applicable to chemical-library screening and will facilitate the analysis of the molecular nature of the segmentation clock.

In vitro systems: A new window to the segmentation clock.

Segmental organization of the vertebrate body plan is established by the segmentation clock, a molecular oscillator that controls the periodicity of somite formation. Given the dynamic nature of the segmentation clock, in vivo studies in vertebrate embryos pose technical challenges. As an alternative, simpler models of the segmentation clock based on primary explants and pluripotent stem cells have recently been developed. These ex vivo and in vitro systems enable more quantitative analysis of oscillatory properties and expand the experimental repertoire applicable to the segmentation clock. Crucially, by eliminating the need for model organisms, in vitro models allow us to study the segmentation clock in new species, including our own. The human oscillator was recently recapitulated using induced pluripotent stem cells, providing a window into human development. Certainly, a combination of in vivo and in vitro work holds the most promising potential to unravel the mechanisms behind vertebrate segmentation.

Disruption of somitogenesis by a novel dominant allele of Lfng suggests important roles for protein processing and secretion.

Vertebrate somitogenesis is regulated by a segmentation clock. Clock-linked genes exhibit cyclic expression, with a periodicity matching the rate of somite production. In mice, lunatic fringe (Lfng) expression oscillates, and LFNG protein contributes to periodic repression of Notch signaling. We hypothesized that rapid LFNG turnover could be regulated by protein processing and secretion. Here, we describe a novel Lfng allele (Lfng(RLFNG)), replacing the N-terminal sequences of LFNG, which allow for protein processing and secretion, with the N-terminus of radical fringe (a Golgi-resident protein). This allele is predicted to prevent protein secretion without altering the activity of LFNG, thus increasing the intracellular half-life of the protein. This allele causes dominant skeletal and somite abnormalities that are distinct from those seen in Lfng loss-of-function embryos. Expression of clock-linked genes is perturbed and mature Hes7 transcripts are stabilized in the presomitic mesoderm of mutant mice, suggesting that both transcriptional and post-transcriptional regulation of clock components are perturbed by RLFNG expression. Contrasting phenotypes in the segmentation clock and somite patterning of mutant mice suggest that LFNG protein may have context-dependent effects on Notch activity.

The many roles of Notch signaling during vertebrate somitogenesis.

The embryonic vertebrate body axis contains serially repeated elements, somites, which form sequentially by budding from a posterior tissue called the presomitic mesoderm (PSM). Somites are the embryonic precursors of the vertebrae, ribs and other adult structures. Many inherited human diseases are characterized by dysregulated somitogenesis, resulting in skeletal abnormalities that are evident at birth. Several of these conditions, including some cases of autosomal recessive familial spondylocostal dysostosis (SCDO), arise from mutations in the Notch signaling pathway, which has been demonstrated to be a key player in the regulation of somitogenesis. Here, we review the functional roles of the Notch pathway in vertebrate segmentation, focusing on its activities in a clock that times the formation of somites, as well as in the patterning and production of epithelial somites.

Modelling asymmetric somitogenesis: Deciphering the mechanisms behind species differences.

Somitogenesis is one of the major hallmarks of bilateral symmetry in vertebrates. This symmetry is lost when retinoic acid (RA) signalling is inhibited, allowing the left-right determination pathway to influence somitogenesis. In all three studied vertebrate model species, zebrafish, chicken and mouse, the frequency of somite formation becomes asymmetric, with slower gene expression oscillations driving somitogenesis on the right side. Still, intriguingly, the resulting left-right asymmetric phenotypes differ significantly between these model species. While somitogenesis is generally considered as functionally equivalent among different vertebrates, substantial differences exist in the subset of oscillating genes between different vertebrate species. Variation also appears to exist in the way oscillations cease and somite boundaries become patterned. In addition, in absence of RA, the FGF8 gradient thought to constitute the determination wavefront becomes asymmetric in zebrafish and mouse, extending more anteriorly to the right, while remaining symmetric in chicken. Here we use a computational modelling approach to decipher the causes underlying species differences in asymmetric somitogenesis. Specifically, we investigate to what extent differences can be explained from observed differences in FGF asymmetry and whether differences in somite determination dynamics may also be involved. We demonstrate that a simple clock-and-wavefront model incorporating the observed left-right differences in somitogenesis frequency readily reproduces asymmetric somitogenesis in chicken. However, incorporating asymmetry in FGF signalling was insufficient to robustly reproduce mouse or zebrafish asymmetry phenotypes. In order to explain these phenoptypes we needed to extend the basic model, incorporating species-specific details of the somitogenesis determination mechanism. Our results thus demonstrate that a combination of differences in FGF dynamics and somite determination cause species differences in asymmetric somitogenesis. In addition,they highlight the power of using computational models as well as studying left-right asymmetry to obtain more insight in somitogenesis.

Dynamics of the slowing segmentation clock reveal alternating two-segment periodicity.

The formation of reiterated somites along the vertebrate body axis is controlled by the segmentation clock, a molecular oscillator expressed within presomitic mesoderm (PSM) cells. Although PSM cells oscillate autonomously, they coordinate with neighboring cells to generate a sweeping wave of cyclic gene expression through the PSM that has a periodicity equal to that of somite formation. The velocity of each wave slows as it moves anteriorly through the PSM, although the dynamics of clock slowing have not been well characterized. Here, we investigate segmentation clock dynamics in the anterior PSM in developing zebrafish embryos using an in vivo clock reporter, her1:her1-venus. The her1:her1-venus reporter has single-cell resolution, allowing us to follow segmentation clock oscillations in individual cells in real-time. By retrospectively tracking oscillations of future somite boundary cells, we find that clock reporter signal increases in anterior PSM cells and that the periodicity of reporter oscillations slows to about ∼1.5 times the periodicity in posterior PSM cells. This gradual slowing of the clock in the anterior PSM creates peaks of clock expression that are separated at a two-segment periodicity both spatially and temporally, a phenomenon we observe in single cells and in tissue-wide analyses. These results differ from previous predictions that clock oscillations stop or are stabilized in the anterior PSM. Instead, PSM cells oscillate until they incorporate into somites. Our findings suggest that the segmentation clock may signal somite formation using a phase gradient with a two-somite periodicity.

The kinetics in mathematical models on segmentation clock genes in zebrafish.

Somitogenesis is the process for the development of somites in vertebrate embryos. This process is timely regulated by synchronous oscillatory expression of the segmentation clock genes. Mathematical models expressed by delay equations or ODEs have been proposed to depict the kinetics of these genes in interacting cells. Through mathematical analysis, we investigate the parameter regimes for synchronous oscillations and oscillation-arrested in an ODE model and a model with transcriptional and translational delays, both with Michaelis-Menten type degradations. Comparisons between these regimes for the two models are made. The delay model has larger capacity to accommodate synchronous oscillations. Based on the analysis and numerical computations extended from the analysis, we explore how the periods and amplitudes of the oscillations vary with the degradation rates, synthesis rates, and coupling strength. For typical parameter values, the period and amplitude increase as some synthesis rate or the coupling strength increases in the ODE model. Such variational properties of oscillations depend also on the magnitudes of time delays in delay model. We also illustrate the difference between the dynamics in systems modeled with linear degradation and the ones in systems with Michaelis-Menten type reactions for the degradation. The chief concerns are the connections between the dynamics in these models and the mechanism for the segmentation clocks, and the pertinence of mathematical modeling on somitogenesis in zebrafish.

Oscillatory Control of Notch Signaling in Development.

The Notch effectors Hes1 and Hes7 and the Notch ligand Delta-like1 (Dll1) are expressed in an oscillatory manner during neurogenesis and somitogenesis. These two biological events exhibit different types of oscillations: anti-/out-of-phase oscillation in neural stem cells during neurogenesis and in-phase oscillation in presomitic mesoderm (PSM) cells during somitogenesis. Accelerated or delayed Dll1 expression by shortening or elongating the size of the Dll1 gene, respectively, dampens or quenches Dll1 oscillation at intermediate levels, a phenomenon known as "amplitude/oscillation death" of coupled oscillators. Under this condition, both Hes1 oscillation in neural stem cells and Hes7 oscillation in PSM cells are also dampened. As a result, maintenance of neural stem cells is impaired, leading to microcephaly, while somite segmentation is impaired, leading to severe fusion of somites and their derivatives, such as vertebrae and ribs. Thus, the appropriate timing of Dll1 expression is critical for the oscillatory expression in Notch signaling and normal processes of neurogenesis and somitogenesis. Optogenetic analysis indicated that Dll1 oscillations transfer the oscillatory information between neighboring cells, which may induce anti-/out-of-phase and in-phase oscillations depending on the delay in signaling transmission. These oscillatory dynamics can be described in a unified manner by mathematical modeling.