Controlling human organoid symmetry breaking reveals signaling gradients drive segmentation clock waves.

Axial development of mammals involves coordinated morphogenetic events, including axial elongation, somitogenesis, and neural tube formation. To gain insight into the signals controlling the dynamics of human axial morphogenesis, we generated axially elongating organoids by inducing anteroposterior symmetry breaking of spatially coupled epithelial cysts derived from human pluripotent stem cells. Each organoid was composed of a neural tube flanked by presomitic mesoderm sequentially segmented into somites. Periodic activation of the somite differentiation gene MESP2 coincided in space and time with anteriorly traveling segmentation clock waves in the presomitic mesoderm of the organoids, recapitulating critical aspects of somitogenesis. Timed perturbations demonstrated that FGF and WNT signaling play distinct roles in axial elongation and somitogenesis, and that FGF signaling gradients drive segmentation clock waves. By generating and perturbing organoids that robustly recapitulate the architecture of multiple axial tissues in human embryos, this work offers a means to dissect mechanisms underlying human embryogenesis.

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.

Excitable Dynamics and Yap-Dependent Mechanical Cues Drive the Segmentation Clock.

The periodic segmentation of the vertebrate body axis into somites, and later vertebrae, relies on a genetic oscillator (the segmentation clock) driving the rhythmic activity of signaling pathways in the presomitic mesoderm (PSM). To understand whether oscillations are an intrinsic property of individual cells or represent a population-level phenomenon, we established culture conditions for stable oscillations at the cellular level. This system was used to demonstrate that oscillations are a collective property of PSM cells that can be actively triggered in vitro by a dynamical quorum sensing signal involving Yap and Notch signaling. Manipulation of Yap-dependent mechanical cues is sufficient to predictably switch isolated PSM cells from a quiescent to an oscillatory state in vitro, a behavior reminiscent of excitability in other systems. Together, our work argues that the segmentation clock behaves as an excitable system, introducing a broader paradigm to study such dynamics in vertebrate morphogenesis.

Helix-loop-helix proteins and the advent of cellular diversity: 30 years of discovery.

Helix-loop-helix (HLH) proteins are dimeric transcription factors that control lineage- and developmental-specific gene programs. Genes encoding for HLH proteins arose in unicellular organisms >600 million years ago and then duplicated and diversified from ancestral genes across the metazoan and plant kingdoms to establish multicellularity. Hundreds of HLH proteins have been identified with diverse functions in a wide variety of cell types. HLH proteins orchestrate lineage specification, commitment, self-renewal, proliferation, differentiation, and homing. HLH proteins also regulate circadian clocks, protect against hypoxic stress, promote antigen receptor locus assembly, and program transdifferentiation. HLH proteins deposit or erase epigenetic marks, activate noncoding transcription, and sequester chromatin remodelers across the chromatin landscape to dictate enhancer-promoter communication and somatic recombination. Here the evolution of HLH genes, the structures of HLH domains, and the elaborate activities of HLH proteins in multicellular life are discussed.

Regeneration of the human segmentation clock in somitoids in vitro.

Each vertebrate species appears to have a unique timing mechanism for forming somites along the vertebral column, and the process in human remains poorly understood at the molecular level due to technical and ethical limitations. Here, we report the reconstitution of human segmentation clock by direct reprogramming. We first reprogrammed human urine epithelial cells to a presomitic mesoderm (PSM) state capable of long-term self-renewal and formation of somitoids with an anterior-to-posterior axis. By inserting the RNA reporter Pepper into HES7 and MESP2 loci of these iPSM cells, we show that both transcripts oscillate in the resulting somitoids at ~5 h/cycle. GFP-tagged endogenous HES7 protein moves along the anterior-to-posterior axis during somitoid formation. The geo-sequencing analysis further confirmed anterior-to-posterior polarity and revealed the localized expression of WNT, BMP, FGF, and RA signaling molecules and HOXA-D family members. Our study demonstrates the direct reconstitution of human segmentation clock from somatic cells, which may allow future dissection of the mechanism and components of such a clock and aid regenerative medicine.

Mapping and Functional Dissection of the Rumpless Trait in Piao Chicken Identifies a Causal Loss of Function Mutation in the Novel Gene Rum.

Rumpless chickens exhibit an abnormality in their tail development. The genetics and biology of this trait has been studied for decades to illustrate a broad variation in both the types of inheritance and the severity in the developmental defects of the tail. In this study, we created a backcross pedigree by intercrossing Piao (rumpless) with Xianju (normal) to investigate the genetic mechanisms and molecular basis of the rumpless trait in Piao chicken. Through genome-wide association and linkage analyses, the candidate region was fine-mapped to 798.5 kb (chromosome 2: 86.9 to 87.7 Mb). Whole-genome sequencing analyses identified a single variant, a 4.2 kb deletion, which was completely associated with the rumpless phenotype. Explorations of the expression data identified a novel causative gene, Rum, that produced a long, intronless transcript across the deletion. The expression of Rum is embryo-specific, and it regulates the expression of MSGN1, a key factor in regulating T-box transcription factors required for mesoderm formation and differentiation. These results provide genetic and molecular experimental evidence for a novel mechanism regulating tail development in chicken and report the likely causal mutation for the tail abnormity in the Piao chicken. The novel regulatory gene, Rum, will, due to its role in fundamental embryo development, be of interest for further explorations of a potential role in tail and skeletal development also in other vertebrates.

Anterior expansion and posterior addition to the notochord mechanically coordinate zebrafish embryo axis elongation.

How force generated by the morphogenesis of one tissue impacts the morphogenesis of other tissues to achieve an elongated embryo axis is not well understood. The notochord runs along the length of the somitic compartment and is flanked on either side by somites. Vacuolating notochord cells undergo a constrained expansion, increasing notochord internal pressure and driving its elongation and stiffening. Therefore, the notochord is appropriately positioned to play a role in mechanically elongating the somitic compartment. We used multi-photon cell ablation to remove specific regions of the zebrafish notochord and quantify the impact on axis elongation. We show that anterior expansion generates a force that displaces notochord cells posteriorly relative to adjacent axial tissues, contributing to the elongation of segmented tissue during post-tailbud stages. Unexpanded cells derived from progenitors at the posterior end of the notochord provide resistance to anterior notochord cell expansion, allowing for stress generation along the anterior-posterior axis. Therefore, notochord cell expansion beginning in the anterior, and addition of cells to the posterior notochord, act as temporally coordinated morphogenetic events that shape the zebrafish embryo anterior-posterior axis.

Sculpting with stem cells: how models of embryo development take shape.

During embryogenesis, organisms acquire their shape given boundary conditions that impose geometrical, mechanical and biochemical constraints. A detailed integrative understanding how these morphogenetic information modules pattern and shape the mammalian embryo is still lacking, mostly owing to the inaccessibility of the embryo in vivo for direct observation and manipulation. These impediments are circumvented by the developmental engineering of embryo-like structures (stembryos) from pluripotent stem cells that are easy to access, track, manipulate and scale. Here, we explain how unlocking distinct levels of embryo-like architecture through controlled modulations of the cellular environment enables the identification of minimal sets of mechanical and biochemical inputs necessary to pattern and shape the mammalian embryo. We detail how this can be complemented with precise measurements and manipulations of tissue biochemistry, mechanics and geometry across spatial and temporal scales to provide insights into the mechanochemical feedback loops governing embryo morphogenesis. Finally, we discuss how, even in the absence of active manipulations, stembryos display intrinsic phenotypic variability that can be leveraged to define the constraints that ensure reproducible morphogenesis in vivo.

Lfng and Dll3 cooperate to modulate protein interactions in cis and coordinate oscillatory Notch pathway activation in the segmentation clock.

In mammalian development, oscillatory activation of Notch signaling is required for segmentation clock function during somitogenesis. Notch activity oscillations are synchronized between neighboring cells in the presomitic mesoderm (PSM) and have a period that matches the rate of somite formation. Normal clock function requires cyclic expression of the Lunatic fringe (LFNG) glycosyltransferase, as well as expression of the inhibitory Notch ligand Delta-like 3 (DLL3). How these factors coordinate Notch activation in the clock is not well understood. Recent evidence suggests that LFNG can act in a signal-sending cell to influence Notch activity in the clock, raising the possibility that in this context, glycosylation of Notch pathway proteins by LFNG may affect ligand activity. Here we dissect the genetic interactions of Lfng and Dll3 specifically in the segmentation clock and observe distinctions in the skeletal and clock phenotypes of mutant embryos showing that paradoxically, loss of Dll3 is associated with strong reductions in Notch activity in the caudal PSM. The patterns of Notch activity in the PSM suggest that the loss of Dll3 is epistatic to the loss of Lfng in the segmentation clock, and we present direct evidence for the modification of several DLL1 and DLL3 EGF-repeats by LFNG. We further demonstrate that DLL3 expression in cells co-expressing DLL1 and NOTCH1 can potentiate a cell's signal-sending activity and that this effect is modulated by LFNG, suggesting a mechanism for coordinated regulation of oscillatory Notch activation in the clock by glycosylation and cis-inhibition.

Morphological and temporal variation in early embryogenesis contributes to species divergence in Malawi cichlid fishes.

The cichlid fishes comprise the largest extant vertebrate family and are the quintessential example of rapid "explosive" adaptive radiations and phenotypic diversification. Despite low genetic divergence, East African cichlids harbor a spectacular intra- and interspecific morphological diversity, including the hyper-variable, neural crest (NC)-derived traits such as coloration and craniofacial skeleton. Although the genetic and developmental basis of these phenotypes has been investigated, understanding of when, and specifically how early, in ontogeny species-specific differences emerge, remains limited. Since adult traits often originate during embryonic development, the processes of embryogenesis could serve as a potential source of species-specific variation. Consequently, we designed a staging system by which we compare the features of embryogenesis between three Malawi cichlid species-Astatotilapia calliptera, Tropheops sp. 'mauve' and Rhamphochromis sp. "chilingali"-representing a wide spectrum of variation in pigmentation and craniofacial morphologies. Our results showed fundamental differences in multiple aspects of embryogenesis that could underlie interspecific divergence in adult adaptive traits. First, we identified variation in the somite number and signatures of temporal variation, or heterochrony, in the rates of somite formation. The heterochrony was also evident within and between species throughout ontogeny, up to the juvenile stages. Finally, the identified interspecific differences in the development of pigmentation and craniofacial cartilages, present at the earliest stages of their overt formation, provide compelling evidence that the species-specific trajectories begin divergence during early embryogenesis, potentially during somitogenesis and NC development. Altogether, our results expand our understanding of fundamental cichlid biology and provide new insights into the developmental origins of vertebrate morphological diversity.

Why a Constant Number of Vertebrae? Digital Control of Segmental Identity during Vertebrate Development: The Somite Cycle Controls a Digital, Chromatin-Based Counter That Defines Segmental Identity and Body Plans in Vertebrate Animals.

It is not understood how the numbers and identities of vertebrae are controlled during mammalian development. The remarkable robustness and conservation of segmental numbers may suggest the digital nature of the underlying process. The study proposes a mechanism that allows cells to obtain and store the segmental information in digital form, and to produce a pattern of chromatin accessibility that in turn regulates Hox gene expression specific to the metameric segment. The model requires that a regulatory element be present such that the number of occurrences of the motif between two consecutive Hox genes equals the number of segments under the control of the anterior gene. This is true for the recently discovered hydroxyl radical cleavage 3bp-periodic (HRC3) motif, associated with histone modifications and developmental genes. The finding not only allows the correct prediction of the numbers of segments using only sequence information, but also resolves the 40-year-old enigma of the function of temporal and spatial collinearity of Hox genes. The logic of the mechanism is illustrated in the attached animated video. How different aspects of the proposed mechanism can be tested experimentally is also discussed.

Shaping the zebrafish myotome by intertissue friction and active stress.

Organ formation is an inherently biophysical process, requiring large-scale tissue deformations. Yet, understanding how complex organ shape emerges during development remains a major challenge. During zebrafish embryogenesis, large muscle segments, called myotomes, acquire a characteristic chevron morphology, which is believed to aid swimming. Myotome shape can be altered by perturbing muscle cell differentiation or the interaction between myotomes and surrounding tissues during morphogenesis. To disentangle the mechanisms contributing to shape formation of the myotome, we combine single-cell resolution live imaging with quantitative image analysis and theoretical modeling. We find that, soon after segmentation from the presomitic mesoderm, the future myotome spreads across the underlying tissues. The mechanical coupling between the future myotome and the surrounding tissues appears to spatially vary, effectively resulting in spatially heterogeneous friction. Using a vertex model combined with experimental validation, we show that the interplay of tissue spreading and friction is sufficient to drive the initial phase of chevron shape formation. However, local anisotropic stresses, generated during muscle cell differentiation, are necessary to reach the acute angle of the chevron in wild-type embryos. Finally, tissue plasticity is required for formation and maintenance of the chevron shape, which is mediated by orientated cellular rearrangements. Our work sheds light on how a spatiotemporal sequence of local cellular events can have a nonlocal and irreversible mechanical impact at the tissue scale, leading to robust organ shaping.

Restricted expression of cdc25a in the tailbud is essential for formation of the zebrafish posterior body.

The vertebrate body forms from a multipotent stem cell-like progenitor population that progressively contributes newly differentiated cells to the most posterior end of the embryo. How the progenitor population balances proliferation and other cellular functions is unknown due to the difficulty of analyzing cell division in vivo. Here, we show that proliferation is compartmentalized at the posterior end of the embryo during early zebrafish development by the regulated expression of cdc25a, a key controller of mitotic entry. Through the use of a transgenic line that misexpresses cdc25a, we show that this compartmentalization is critical for the formation of the posterior body. Upon misexpression of cdc25a, several essential T-box transcription factors are abnormally expressed, including Spadetail/Tbx16, which specifically prevents the normal onset of myoD transcription, leading to aberrant muscle formation. Our results demonstrate that compartmentalization of proliferation during early embryogenesis is critical for both extension of the vertebrate body and differentiation of the multipotent posterior progenitor cells to the muscle cell fate.

Unique Features of River Lamprey (Lampetra fluviatilis) Myogenesis.

The river lamprey (L. fluviatilis) is a representative of the ancestral jawless vertebrate group. We performed a histological analysis of trunk muscle fiber differentiation during embryonal, larval, and adult musculature development in this previously unstudied species. Investigation using light, transmission electron (TEM), and confocal microscopy revealed that embryonal and larval musculature differs from adult muscle mass. Here, we present the morphological analysis of L. fluviatilis myogenesis, from unsegmented mesoderm through somite formation, and their differentiation into multinucleated muscle lamellae. Our analysis also revealed the presence of myogenic factors LfPax3/7 and Myf5 in the dermomyotome. In the next stages of development, two types of muscle lamellae can be distinguished: central surrounded by parietal. This pattern is maintained until adulthood, when parietal muscle fibers surround the central muscles on both sides. The two types show different morphological characteristics. Although lampreys are phylogenetically distant from jawed vertebrates, somite morphology, especially dermomyotome function, shows similarity. Here we demonstrate that somitogenesis is a conservative process among all vertebrates. We conclude that river lamprey myogenesis shares features with both ancestral and higher vertebrates.

Pumilio response and AU-rich elements drive rapid decay of Pnrc2-regulated cyclic gene transcripts.

Vertebrate segmentation is regulated by the segmentation clock, a biological oscillator that controls periodic formation of somites, or embryonic segments, which give rise to many mesodermal tissue types. This molecular oscillator generates cyclic gene expression with the same periodicity as somite formation in the presomitic mesoderm (PSM), an area of mesenchymal cells that give rise to mature somites. Molecular components of the clock include the Hes/her family of genes that encode transcriptional repressors, but additional genes cycle. Cyclic gene transcripts are cleared rapidly, and clearance depends upon the pnrc2 (proline-rich nuclear receptor co-activator 2) gene that encodes an mRNA decay adaptor. Previously, we showed that the her1 3'UTR confers instability to otherwise stable transcripts in a Pnrc2-dependent manner, however, the molecular mechanism(s) by which cyclic gene transcripts are cleared remained largely unknown. To identify features of the her1 3'UTR that are critical for Pnrc2-mediated decay, we developed an array of transgenic inducible reporter lines carrying different regions of the 3'UTR. We find that the terminal 179 nucleotides (nts) of the her1 3'UTR are necessary and sufficient to confer rapid instability. Additionally, we show that the 3'UTR of another cyclic gene, deltaC (dlc), also confers Pnrc2-dependent instability. Motif analysis reveals that both her1 and dlc 3'UTRs contain terminally-located Pumilio response elements (PREs) and AU-rich elements (AREs), and we show that the PRE and ARE in the last 179 â€‹nts of the her1 3'UTR drive rapid turnover of reporter mRNA. Finally, we show that mutation of Pnrc2 residues and domains that are known to facilitate interaction of human PNRC2 with decay factors DCP1A and UPF1 reduce the ability of Pnrc2 to restore normal cyclic gene expression in pnrc2 mutant embryos. Our findings suggest that Pnrc2 interacts with decay machinery components and cooperates with Pumilio (Pum) proteins and ARE-binding proteins to promote rapid turnover of cyclic gene transcripts during somitogenesis.

Patterning via local cell-cell interactions in developing systems.

Spatial patterning during embryonic development emerges from the differentiation of progenitor cells that share the same genetic program. One of the main challenges in systems biology is to understand the relationship between gene network and patterning, especially how the cells communicate to coordinate their differentiation. This review aims to describe the principles of pattern formation from local cell-cell interactions mediated by the Notch signalling pathway. Notch mediates signalling via direct cell-cell contact and regulates cell fate decisions in many tissues during embryonic development. Here, I will describe the patterning mechanisms via different Notch ligands and the critical role of Notch oscillations during the segmentation of the vertebrate body, brain development, and blood vessel formation.

RARγ is required for mesodermal gene expression prior to gastrulation in Xenopus.

The developing vertebrate embryo is exquisitely sensitive to retinoic acid (RA) concentration, particularly during anteroposterior patterning. In contrast to Nodal and Wnt signaling, RA was not previously considered to be an instructive signal in mesoderm formation during gastrulation. Here, we show in Xenopus that RARγ is indispensable for the expression of early mesoderm markers and is, therefore, an obligatory factor in mesodermal competence and/or maintenance. We identified several novel targets upregulated by RA receptor signaling in the early gastrula that are expressed in the circumblastoporal ring and linked to mesodermal development. Despite overlapping expression patterns of the genes encoding the RA-synthesizing enzyme Aldh1a2 and the RA-degrading enzyme Cyp26a1, RARγ1 functions as a transcriptional activator in early mesoderm development, suggesting that RA ligand is available to the embryo earlier than previously appreciated. RARγ1 is required for cellular adhesion, as revealed by spontaneous dissociation and depletion of ncam1 mRNA in animal caps harvested from RARγ1 knockdown embryos. RARγ1 knockdown obliterates somite boundaries, and causes loss of Myod protein in the presomitic mesoderm, but ectopic, persistent expression of Myod protein in the trunk. Thus, RARγ1 is required for stabilizing the mesodermal fate, myogenic commitment, somite boundary formation, and terminal skeletal muscle differentiation.

Morphogen-regulated contact-mediated signaling between cells can drive the transitions underlying body segmentation in vertebrates.

We propose a unified mechanism that reproduces the sequence of dynamical transitions observed during somitogenesis, the process of body segmentation during embryonic development, that is invariant across all vertebrate species. This is achieved by combining inter-cellular interactions mediated via receptor-ligand coupling with global spatial heterogeneity introduced through a morphogen gradient known to occur along the anteroposterior axis. Our model reproduces synchronized oscillations in the gene expression in cells at the anterior of the presomitic mesoderm as it grows by adding new cells at its posterior, followed by travelling waves and subsequent arrest of activity, with the eventual appearance of somite-like patterns. This framework integrates a boundary-organized pattern formation mechanism, which uses positional information provided by a morphogen gradient, with the coupling-mediated self-organized emergence of collective dynamics, to explain the processes that lead to segmentation.

Dysbacteriosis-induced LPS elevation disturbs the development of muscle progenitor cells by interfering with retinoic acid signaling.

Whether myogenesis is affected by the maternal gut dysbacteriosis still remains ambiguous. In this study, first we show the elevated level of lipopolysaccharides (LPS) in a gut microbiota dysbiosis mouse model. Second, we demonstrate that the diameter of muscle fibers, limb development, and somitogenesis were inhibited in both the gut microbiota dysbiosis and LPS exposed mice and chicken embryos. These might be due to LPS disturbed the cell survival and key genes which regulate the somitogenesis and myogenesis. RNA sequencing and subsequent validation experiments verified that retinoic acid (RA) signaling perturbation was mainly responsible for the aberrant somite formation and differentiation. Subsequently, we found that LPS-induced reactive oxygen species (ROS generation and antioxidant genes such as Nrf2, AKR1B10) contributed to the above -mentioned interference with RA signaling. These findings highlight that the gut microbiota homeostasis is also involved in regulating the development of muscle progenitor cells during pregnancy.

CDK1 and CDK2 regulate NICD1 turnover and the periodicity of the segmentation clock.

All vertebrates share a segmented body axis. Segments form from the rostral end of the presomitic mesoderm (PSM) with a periodicity that is regulated by the segmentation clock. The segmentation clock is a molecular oscillator that exhibits dynamic clock gene expression across the PSM with a periodicity that matches somite formation. Notch signalling is crucial to this process. Altering Notch intracellular domain (NICD) stability affects both the clock period and somite size. However, the mechanism by which NICD stability is regulated in this context is unclear. We identified a highly conserved site crucial for NICD recognition by the SCF E3 ligase, which targets NICD for degradation. We demonstrate both CDK1 and CDK2 can phosphorylate NICD in the domain where this crucial residue lies and that NICD levels vary in a cell cycle-dependent manner. Inhibiting CDK1 or CDK2 activity increases NICD levels both in vitro and in vivo, leading to a delay of clock gene oscillations and an increase in somite size.