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Funder: Natural Environment Research Council; Id: http://dx.doi.org/10.13039/501100000270, Funder: Wellcome Trust, 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.

In vertebrate embryogenesis, elongation of the posterior body is driven by de novo production of the axial and paraxial mesoderm as well as the neural tube at the posterior end. This process is presumed to depend on the stem cell-like population in the tail bud region, but the details of the gene regulatory network involved are unknown. Previous studies suggested the involvement of pou5f3, an Oct4-type POU gene in zebrafish, in axial elongation. In the present study, we first found that pou5f3 is expressed mainly in the dorsal region of the tail bud immediately after gastrulation, and that this expression is restricted to the posterior-most region of the elongating neural tube during somitogenesis. This pou5f3 expression was complementary to the broad expression of sox3 in the neural tube, and formed a sharp boundary with specific expression of tbxta (orthologue of mammalian T/Brachyury) in the tail bud, implicating pou5f3 in the specification of tail bud-derived cells toward neural differentiation in the spinal cord. When pou5f3 was functionally impaired after gastrulation by induction of a dominant-interfering pou5f3 mutant gene (en-pou5f3), trunk and tail elongation were markedly disturbed at distinct positions along the axis depending on the stage. This finding showed involvement of pou5f3 in de novo generation of the body from the tail bud. Conditional functional abrogation also showed that pou5f3 downregulates mesoderm-forming genes but promotes neural development by activating neurogenesis genes around the tail bud. These results suggest that pou5f3 is involved in formation of the posterior spinal cord.

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.

Fibronectin is essential for somite formation in the vertebrate embryo. Fibronectin matrix assembly starts as cells emerge from the primitive streak and ingress in the unsegmented presomitic mesoderm (PSM). PSM cells undergo cyclic waves of segmentation clock gene expression, followed by Notch-dependent upregulation of meso1 in the rostral PSM which induces somite cleft formation. However, the relevance of the fibronectin matrix for these molecular processes remains unknown. Here, we assessed the role of the PSM fibronectin matrix in the spatio-temporal regulation of chick embryo somitogenesis by perturbing (1) extracellular fibronectin matrix assembly, (2) integrin–fibronectin binding, (3) Rho-associated protein kinase (ROCK) activity and (4) non-muscle myosin II (NM II) function. We found that integrin–fibronectin engagement and NM II activity are required for cell polarization in the nascent somite. All treatments resulted in defective somitic clefts and significantly perturbed meso1 and segmentation clock gene expression in the PSM. Importantly, inhibition of actomyosin-mediated contractility increased the period of hairy1/hes4 oscillations from 90 to 120 min. Together, our work strongly suggests that the fibronectin–integrin–ROCK–NM II axis regulates segmentation clock dynamics and dictates the spatio-temporal localization of somitic clefts. info:eu-repo/semantics/publishedVersion

Somitogenesis is the process by means of which a tissue known as presomitic mesoderm (PSM) is segmented in blocks of cells, called somites, along the anterior-posterior axis of the developing embryo in segmented animals. In vertebrates, somites give rise to axial skeleton, cartilage, tendons, skeletal muscle, and dermis. Somite formation occurs periodically, and this periodicity is driven by a genetic oscillator that operates within PSM cells and is known as the segmentation clock. The correct synchronization of the segmentation clock among PSM cells is essential for somitogenesis to develop normally. When synchronization is disrupted, somites form irregularly and, in consequence, the tissues that originate from them show clear malformations. In this work, based in a model for zebrafish segmentation clock, we investigate by means of a mathematical modeling approach, how PSM-cell synchronization is affected by factors like: the size of PSM-cell networks, the amount of cell-to-cell interactions per PSM cell, the strength of these interactions, and the inherent variability among PSM cells. Interestingly we found that very small PSM-cell networks are unable to synchronize. Moreover, the effect of decreasing the strength of interactions among PSM cells is corrected by increasing the network connectivity-level, and a moderated level of variability among cells can have a positive effect on synchronization, specially in large networks.

Scoliosis is an abnormal bending of the body axis. Truncated vertebrae or a debilitated ability to control the musculature in the back can cause this condition, but in most cases the causative reason for scoliosis is unknown (idiopathic). Using mutants for somite clock genes with mild defects in the vertebral column, we here show that early defects in somitogenesis are not overcome during development and have long lasting and profound consequences for muscle fiber organization, structure and whole muscle volume. These mutants present only mild alterations in the vertebral column, and muscle shortcomings are uncoupled from skeletal defects. None of the mutants presents an overt musculoskeletal phenotype at larval or early adult stages, presumably due to compensatory growth mechanisms. Scoliosis becomes only apparent during aging. We conclude that adult degenerative scoliosis is due to disturbed crosstalk between vertebrae and muscles during early development, resulting in subsequent adult muscle weakness and bending of the body axis.

Pluripotent stem cells (PSCs) are attractive regenerative therapy tools for skeletal tissues. However, a deep understanding of skeletal development is required in order to model this development with PSCs, and for the application of PSCs in clinical settings. Skeletal tissues originate from three types of cell populations: the paraxial mesoderm, lateral plate mesoderm, and neural crest. The paraxial mesoderm gives rise to the sclerotome mainly through somitogenesis. In this process, key developmental processes, including initiation of the segmentation clock, formation of the determination front, and the mesenchymal–epithelial transition, are sequentially coordinated. The sclerotome further forms vertebral columns and contributes to various other tissues, such as tendons, vessels (including the dorsal aorta), and even meninges. To understand the molecular mechanisms underlying these developmental processes, extensive studies have been conducted. These studies have demonstrated that a gradient of activities involving multiple signaling pathways specify the embryonic axis and induce cell-type-specific master transcription factors in a spatiotemporal manner. Moreover, applying the knowledge of mesoderm development, researchers have attempted to recapitulate the in vivo development processes in in vitro settings, using mouse and human PSCs. In this review, we summarize the state-of-the-art understanding of mesoderm development and in vitro modeling of mesoderm development using PSCs. We also discuss future perspectives on the use of PSCs to generate skeletal tissues for basic research and clinical applications., Regenerative medicine: the challenge of building skeletal tissue A deeper understanding of skeletal tissue development and improvements in tissue engineering will help pluripotent stem cell (PSC) therapies to reach their full potential for skeletal repair. The paraxial mesoderm, an embryonic germ layer, is crucial to the formation of healthy axial skeleton. Shoichiro Tani at the University of Tokyo, Japan, and co-workers reviewed current understanding of paraxial mesoderm development and studies involving in vitro PSC skeletal modeling. The formation of the paraxial mesoderm and associated connective tissues comprises multiple stages, and studies in vertebrate embryos have uncovered critical signaling pathways and cellular components important to PSC modeling. Although many individual cellular components can now be modeled, it remains challenging to recreate three-dimensional skeletal tissues. Such an achievement would facilitate a functioning model of bone metabolism, the next step in achieving skeletal regeneration.

The body plan of the mammalian embryo is shaped through the process of gastrulation, an early developmental event that transforms an isotropic group of cells into an ensemble of tissues that is ordered with reference to three orthogonal axes1. Although model organisms have provided much insight into this process, we know very little about gastrulation in humans, owing to the difficulty of obtaining embryos at such early stages of development and the ethical and technical restrictions that limit the feasibility of observing gastrulation ex vivo2. Here we show that human embryonic stem cells can be used to generate gastruloids-three-dimensional multicellular aggregates that differentiate to form derivatives of the three germ layers organized spatiotemporally, without additional extra-embryonic tissues. Human gastruloids undergo elongation along an anteroposterior axis, and we use spatial transcriptomics to show that they exhibit patterned gene expression. This includes a signature of somitogenesis that suggests that 72-h human gastruloids show some features of Carnegie-stage-9 embryos3. Our study represents an experimentally tractable model system to reveal and examine human-specific regulatory processes that occur during axial organization in early development.

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 ​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.

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.

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.

During the development of the vertebrate embryo, segmented structures called somites are periodically formed from the presomitic mesoderm (PSM) and give rise to the vertebral column. While somite formation has been studied in several animal models, it is less clear how well this process is conserved in humans. Recent progress has made it possible to study aspects of human paraxial mesoderm (PM) development such as the human segmentation clockHumans are part of a group of animals called vertebrates, which are all the animals with backbones. Broadly, all vertebrates have a similar body shape with a head at one end and a left and right side that are similar to each other. Although this is not very obvious in humans, vertebrate bodies are derived from pairs of segments arranged from the head to the tail. Each of these segments or somites originates early in embryonic development. Cells from each somite then divide, grow and specialize to form bones such as the vertebrae of the vertebral column, muscles, skin, and other tissues that make up each segment. Studying different animals during embryonic development has provided insights into how somites form and grow, but it is technically difficult to do and only provides an approximate model of how somites develop in humans. Being able to make and study somites using human cells in the lab would help scientists learn more about how somite formation in humans is regulated. Budjan et al. grew human stem cells in the lab as three-dimensional structures called organoids, and used chemical signals similar to the ones produced in the embryo during development to make the cells form somites. Various combinations of signals were tested to find the best way to trigger somite formation. Once the somites formed, Budjan et al. measured them and studied their structure and the genes they used. They found that these lab-grown somites have the same size and structure as natural somites and use many of the same genes. This new organoid model provides a way to study human somite formation and development in the lab for the first time. This can provide insights into the development and evolution of humans and other animals that could then help scientists understand diseases such as the development of abnormal spinal curvature that affects around 1 in 10,000 newborns.

As embryos develop, they are required to generate organised patterns of gene expression across developing tissues. Previous hypotheses used to explain how patterns form in developing tissues have relied on the ideas of positional information and gradients of signalling across the developing tissue, which impart spatial coordinates to individual cells. These coordinates then inform the cell of its respective fate, and hence a pattern of gene expression with spatial organisation is generated. This way of thinking about the problem of pattern formation has been highly successful in explaining the patterning of tissues in vertebrate embryos. However, such an approach begins to fail when challenged with tissues where the cells which make it are highly motile. This thesis will examine the extent to which patterning is robust to cell mixing by disrupting early positional information prior to gastrulation, using whole embryonic explants which undergo widespread cell mixing. These explants elongate, form all three germ layers with spatial organisation and anteroposterior patterned neural tissue. This thesis will then examine the robustness of patterning within the presomitic mesoderm, where a posterior to anterior pattern of gene expression is observed despite high levels of cell rearrangement. Within this tissue, the pattern is observed to accurately scale to the shortening anterior to posterior length and is demonstrated to be highly robust to changes in the degree of cell mixing following experimental perturbation, including in vitro culture of individual cells and pharmacological treatment. Together, this thesis will argue that to understand pattern formation in tissues which are also undergoing extensive cell rearrangement, it is important to consider the gene regulatory network dynamics and how these are influenced by signalling. Rather than taking a positional information “bottom up” type view of cell fate decision making, where signalling gradients inform cells of their position and hence their identity, I will argue for a more holistic approach to understanding development. By considering how “top down” regulation of patterning, mediated via the movement of cells and tissues between domains of signalling, it will be demonstrated that pattern formation can be considered an emergent properly of cells, regulated by signalling as well as cell movements., Vice Chancellor's Scholarship, Cambridge Trust

Vertebrate embryo somitogenesis is the earliest morphological manifestation of the characteristic patterned structure of the adult axial skeleton. Pairs of somites flanking the neural tube are formed periodically during early development, and the molecular mechanisms in temporal control of this early patterning event have been thoroughly studied. The discovery of a molecular Embryo Clock (EC) underlying the periodicity of somite formation shed light on the importance of gene expression dynamics for pattern formation. The EC is now known to be present in all vertebrate organisms studied and this mechanism was also described in limb development and stem cell differentiation. An outstanding question, however, remains unanswered: what sets the different EC paces observed in different organisms and tissues? This review aims to summarize the available knowledge regarding the pace of the EC, its regulation and experimental manipulation and to expose new questions that might help shed light on what is still to unveil. info:eu-repo/semantics/publishedVersion

Ribs are seldom affected by developmental disorders, however, multiple defects in rib structure are observed in the spliceosomal disease cerebro‐costo‐mandibular syndrome (CCMS). These defects include rib gaps, found in the posterior part of the costal shaft in multiple ribs, as well as missing ribs, shortened ribs and abnormal costotransverse articulations, which result in inadequate ventilation at birth and high perinatal mortality. The genetic mechanism of CCMS is a loss‐of‐function mutation in SNRPB, a component of the major spliceosome, and knockdown of this gene in vitro affects the activity of the Wnt/β‐catenin and bone morphogenic protein (BMP) pathways. The aim of the present study was to investigate whether altering these pathways in vivo can recapitulate rib gaps and other rib abnormalities in the model animal. Chick embryos were implanted with beads soaked in Wnt/β‐catenin and BMP pathway modulators during somitogenesis, and incubated until the ribs were formed. Some embryos were harvested in the preceding days for analysis of the chondrogenic marker Sox9, to determine whether pathway modulation affected somite patterning or chondrogenesis. Wnt/β‐catenin inhibition manifested characteristic rib phenotypes seen in CCMS, including rib gaps (P < 0.05) and missing ribs (P < 0.05). BMP pathway activation did not cause rib gaps but yielded missing rib (P < 0.01) and shortened rib phenotypes (P < 0.05). A strong association with vertebral phenotypes was also noted with BMP4 (P < 0.001), including scoliosis (P < 0.05), a feature associated with CCMS. Reduced expression of Sox9 was detected with Wnt/β‐catenin inhibition, indicating that inhibition of chondrogenesis precipitated the rib defects in the presence of Wnt/β‐catenin inhibitors. BMP pathway activators also reduced Sox9 expression, indicating an interruption of somite patterning in the manifestation of rib defects with BMP4. The present study demonstrates that local inhibition of the Wnt/β‐catenin and activation of the BMP pathway can recapitulate rib defects, such as those observed in CCMS. The balance of Wnt/β‐catenin and BMP in the somite is vital for correct rib morphogenesis, and alteration of the activity of these two pathways in CCMS may perturb this balance during somite patterning, leading to the observed rib defects.

Significance Although the circadian clock is essential for regulating the temporal order of physiological functions, circadian oscillation is strictly suppressed in the early-to-mid–stage embryos in mammalian developmental process. The biological significance controlling the suppression of the circadian clock and its delayed emergence in mammalian embryos has been unknown. Here, we show that the premature expression of the functional circadian components CLOCK/BMAL1 in mouse induced presomitic mesoderm and gastruloids can interfere with the segmentation clock Hes7 oscillation and somitogenesis through the Hes7 transcription and its regulatory pathways. This suggests that the CLOCK/BMAL1 function may need to be suppressed during somitogenesis., 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.

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., Summary: In vitro engineering of embryo-like structures (stembryos) from pluripotent stem cells offers unique possibilities to understand how boundary conditions that impose geometrical, mechanical and biochemical constraints pattern and shape the mammalian embryo.

Durante as últimas décadas têm-se estudado com profundidade os mecanismos genéticos que levam ao correto desenvolvimento do embrião vertebrado. Estes mecanismos têm sido estudados especialmente na caraterização da expressão genética nos tecidos e de que modo a sua presença ou ausência se reflete na correta diferenciação de uma célula, no desenvolvimento de um tecido ou órgão e na correta formação do organismo. Contudo para além da localização e intensidade da expressão, uma outra dimensão muito menos explorada é o Tempo. A desregulação temporal da expressão génica pode levar a que os tecidos se diferenciem num momento ou ritmo impróprio, podendo ter consequências graves e levar mesmo à morte do organismo. Um dos processos que evidencia uma periodicidade durante o desenvolvimento, é a somitogénese. À medida que o embrião alonga ao longo do eixo antero-posterior graças à ingressão de células no botão caudal, as células da mesoderme pré-somítica (PSM) anterior formam periodicamente pares de estruturas esféricas transitórias que flanqueiam a notocorda, conhecidas como sómitos. Os sómitos diferenciam-se sendo responsáveis pela formação do esqueleto axial, bem como da musculatura esquelética e a derme das costas. A desregulação temporal da somitogénese, provoca doenças congénitas graves que afetam especialmente a formação das vertebras, como a disostose espondilocostal. Existe um relógio embrionário (EC) subjacente à periodicidade da somitogénese. O EC foi primeiramente descrito por Palmeirim e colegas (1997) no desenvolvimento do embrião de galinha. Este grupo descobriu que o gene hairy1 tem ciclos de expressão na PSM com o mesmo período que a somitogénese, 90 minutos. Posteriormente, este mecanismo foi descoberto noutros organismos e hoje em dia, sabe-se que é conservado em todos os vertebrados. O período EC é específico de cada espécie, sendo, por exemplo, 30 minutos no peixe-zebra, 120 no ratinho e entre 5 e 6 horas no Humano. Sabe-se também que vários genes pertencentes a várias vias de sinalização possuem expressão cíclica. Mais concretamente, pertencentes às vias de Notch, FGF e WNT. Dez anos depois da descoberta do EC, foi descrito que este mecanismo não era exclusivo da PSM. Pascoal e colegas (2007) descobriram que o relógio embrionário estava presente no crescimento do membro da galinha. Mais concretamente, o grupo descreveu que o gene hairy2 tem uma expressão cíclica, apresentando um período de seis horas em vez dos 90 minutos na PSM. O EC é caracterizado por mecanismos de regulação por feedback negativo em que um gene é expresso e traduzido e a proteína vai reprimir a sua própria expressão. Para além disso, o mecanismo é refinado por atrasos em diversas etapas, desde a transcrição, passando pela exportação nuclear do mRNA, pela tradução e terminando na degradação do mRNA e da proteína. Contudo, a regulação do EC, mais propriamente, como é capaz de apresentar diferentes períodos no mesmo organismo, ainda permanece por esclarecer, constituindo a questão central do presente trabalho. Neste trabalho foi explorada a hipótese da regulação da periodicidade do EC em diferentes tecidos ser exercida ao nível das moléculas de RNA. Para abordar esta hipótese, foram aplicadas diversas metodologias experimentais. Em primeiro lugar foram identificados os transcritos produzidos por hairy1 e analisado o seu impacto na segmentação do tronco embrionário (Capítulo 2). Para tal, foi feita uma sobre-expressão de ambos os transcritos identificados na PSM do embrião de galinha. Os nossos resultados indicam que a sobreexpressão do s-hairy1, tal como de hairy1, leva a um atraso no alongamento dos embriões, e a um atraso no início da somitogénese. Neste capítulo, adicionalmente, procurámos aprofundar o conhecimento acerca do relógio embrionário no membro. Estudámos a expressão de hairy1, descobrindo que também cicla com um período de 6 horas no mesênquima distal do membro. A região 3’UTR do mRNA é um dos maiores responsáveis pela regulação do tempo de meia-vida do próprio mRNA. Tendo isso em mente, estudámos os 3’UTRs de três genes centrais do relógio embrionário do peixe-zebra, her1, her7 e deltaC (dlc) (Capítulo 3). No caso de her1 e her7 deletou-se 3’UTR alternativos, contudo os resultados obtidos não revelaram um papel para os 3’UTRs alternativos no funcionamento do EC. No caso do gene dlc, dividiuse o 3’UTR em três regiões de regulação (RR), e procedeu-se à deleção dessas regiões isoladamente, ou em combinação. Quando deletadas RR1, RR2 ou a combinação das duas (RR1&2), não foram detetadas alterações significativas à expressão de dlc. Contudo, quando foram removidas RR3 ou RR2&3 obteve-se uma estabilização da expressão dos genes do EC. Uma avaliação mais aprofundada das deleções RR2 e RR3 revelou que os embriões produziam menos sómitos e mais pequenos, resultado mais notório na deleção RR3. Em ambos os casos também se verificou uma estabilização da expressão de her7. Por último, decidiu-se estudar se os miRNAs podem estar envolvidos na regulação dos períodos do EC (Capítulo 4). Para tal, analisou-se o miRNoma por sequenciação de RNA a partir de três tecidos que apresentam oscilações do EC com diferentes ritmos: PSM determinada (D-PSM), PSM indeterminada (U-PSM) e o domínio distal cíclico do membro superior (DCD). Foram identificados 926 miRNAs conhecidos. Para além disso descobrimos mais 1141 possíveis novos miRNAs em galinha (quase duplicando os que se encontram anotados na base de dados miRBase). Procedemos, posteriormente, a uma análise de expressão diferencial e à validação dos dados por RT-qPCR. Adicionalmente, os miRNAs diferencialmente expressos entre os tecidos foram comparados com os genes negativamente regulados nos mesmos tecidos e procedemos a uma análise de enriquecimento funcional GO e REACTOME. Em suma, utilizando uma nossa estratégia plural para testar a nossa hipótese, levounos a um aprofundar do conhecimentos dos mecanismos que controlam a expressão genética periódica nos tecidos onde o EC opera. Vertebrate embryo body segmentation occurs progressively over time. An embryo clock (EC) characterized by gene expression oscillations underlies somitogenesis periodicity. The EC periodicity is species-specific, i.e., 30 minutes and 90 minutes in zebrafish and chicken, respectively. The EC was also found in chicken forelimb outgrowth, however, with a 6-hour pace. Robust EC gene expression oscillations are built by negative feedback loops, which require tight regulation of mRNA stability. This thesis will present work addressing the hypothesis that alternative transcripts and/or miRNAs may account for different paces of the EC. Different approaches were followed to address this hypothesis: 1) we analyzed the alternative transcripts of the chicken EC hairy1 gene and found that both s-hairy1 and hairy1 overexpression promoted trunk developmental delay. We identified hairy1 gene expression oscillations in the limb bud with a periodicity of 6 hours. 2) Using zebrafish mutants with truncated 3’UTRs in EC mRNAs we found that deletion of the distal portion of the dlc 3’UTR stabilizes EC expression and promotes segmentation alterations. 3) We performed miRNA sequencing (miRNA-Seq) of three chicken embryo tissues: determined PSM (D-PSM), undetermined PSM (U-PSM), and forelimb distal cyclic domain (DCD). We identified 926 known miRNAs and 1141 candidate novel miRNAs not previously described in chicken. Furthermore, a differential expression analysis was conducted, and specific miRNAs were selected for validation and functional analysis. Altogether, by tackling our hypothesis in multiple mechanisms involving RNA regulation, we gave a step further in understanding the temporal control of the EC.

Somitogenesis refers to the segmentation of the paraxial mesoderm, a tissue located on the back of the embryo, into regularly spaced and sized pieces, i.e., the somites. This periodicity is important to assure, for example, the formation of a functional vertebral column and nervous system. Prevailing models of somitogenesis are based on the existence of a genetic regulatory network capable of generating a striped pattern of gene expression, which is subsequently translated into periodic tissue boundaries. An alternative view is that the pre-pattern that guides somitogenesis is not chemical, but of a mechanical origin. A striped pattern of mechanical strain can be formed in physically connected tissues expanding at different rates, as it occurs in the embryo. Here we argue that both molecular and mechanical cues could drive somite periodicity and suggest how they could be integrated.

In vertebrate embryos the presomitic mesoderm becomes progressively segmented into somites at the anterior end while extending along the anterior-posterior axis. A commonly adopted model to explain how this tissue elongates is that of posterior growth, driven in part by the addition of new cells from uncommitted progenitor populations in the tailbud. However, in zebrafish, much of somitogenesis is associated with an absence of overall volume increase, and posterior progenitors do not contribute new cells until the final stages of somitogenesis. Here, we perform a comprehensive 3D morphometric analysis of the paraxial mesoderm and reveal that extension is linked to a volumetric decrease and an increase in cell density. We also find that individual cells decrease in volume over successive somite stages. Live cell tracking confirms that much of this tissue deformation occurs within the presomitic mesoderm progenitor zone and is associated with non-directional rearrangement. Taken together, we propose a compaction-extension mechanism of tissue elongation that highlights the need to better understand the role tissue intrinsic and extrinsic forces in regulating morphogenesis.

The dorsal axial muscles, or epaxial muscles, are a fundamental structure covering the spinal cord and vertebrae, as well as mobilizing the vertebrate trunk. To date, mechanisms underlying the morphogenetic process shaping the epaxial myotome are largely unknown. To address this, we used the medaka zic1/zic4-enhancer mutant Double anal fin (Da), which exhibits ventralized dorsal trunk structures resulting in impaired epaxial myotome morphology and incomplete coverage over the neural tube. In wild type, dorsal dermomyotome (DM) cells, progenitors of myotomal cells, reduce their proliferative activity after somitogenesis and subsequently form unique large protrusions extending dorsally, potentially guiding the epaxial myotome dorsally. In Da, by contrast, DM cells maintain the high proliferative activity and form mainly small protrusions. By combining RNA- and ChIP-sequencing analyses, we revealed direct targets of Zic1 which are specifically expressed in dorsal somites and involved in various aspects of development, such as cell migration, extracellular matrix organization and cell-cell communication. Among these, we identified wnt11r as a crucial factor regulating both cell proliferation and protrusive activity of DM cells. We propose that the dorsal movement of the epaxial myotome is guided by DM cells and that Zic1 empowers this activity via Wnt11r to achieve the neural tube coverage.

International audience; In contrast to mice or zebrafish, trout exhibits post-larval muscle growth through hypertrophy and formation of new myofibers (hyperplasia). The muscle fibers are formed by the fusion of mononucleated cells (myoblasts) regulated by several muscle-specific proteins such as Myomaker or Myomixer. In this work, we identified a unique gene encoding a Myomixer protein of 77 amino acids (aa) in the trout genome. Sequence analysis and phylogenetic tree showed moderate conservation of the overall protein sequence across teleost fish (61% of aa identity between trout and zebrafish Myomixer sequences). Nevertheless, the functionally essential motif, AxLyCxL is perfectly conserved in all studied sequences of vertebrates. Using in situ hybridization, we observed that myomixer was highly expressed in the embryonic myotome, particularly in the hyperplasic area. Moreover, myomixer remained readily expressed in white muscle of juvenile (1 and 20 g) although its expression decreased in mature fish. We also showed that myomixer is up-regulated during muscle regeneration and in vitro myoblasts differentiation. Together, these data indicate that myomixer expression is consistently associated with the formation of new myofibers during somitogenesis, post-larval growth and muscle regeneration in trout.Keywords

Long noncoding RNAs (lncRNAs) have emerged as important regulators of many biological processes, including embryogenesis and development. To provide a systematic analysis of lncRNAs expressed during chicken embryogenesis, we used Iso-Seq and RNA-Seq to identify potential lncRNAs at embryonic stages from d 1 to d 8 of incubation: sequential stages covering gastrulation, somitogenesis, and organogenesis. The data characterized an expanded landscape of lncRNAs, yielding 45,410 distinct lncRNAs (31,282 genes). Amongst these, a set of 13,141 filtered intergenic lncRNAs (lincRNAs) transcribed from 9803 lincRNA gene loci, of which, 66.5% were novel, were further analyzed. These lincRNAs were found to share many characteristics with mammalian lincRNAs, including relatively short lengths, fewer exons, lower expression levels, and stage-specific expression patterns. Functional studies motivated by “guilt-by-association” associated individual lincRNAs with specific GO functions, providing an important resource for future studies of lincRNA function. Most importantly, a weighted gene co-expression network analysis suggested that genes of the brown module were specifically associated with the day 2 stage. LincRNAs within this module were co-expressed with proteins involved in hematopoiesis and lipid metabolism. This study presents the systematic identification of lincRNAs in developing chicken embryos and will serve as a powerful resource for the study of lincRNA functions.

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.

Objective The mammalian Notch ligand DLL1 has essential functions during development. To visualise DLL1 in tissues, for sorting and enrichment of DLL1-expressing cells, and to efficiently purify DLL1 protein complexes we tagged DLL1 in mice with AcGFPHA or Strep/FLAG. Results We generated constructs to express DLL1 that carried C-terminal in-frame an AcGFPHA tag flanked by loxP sites followed by a Strep/FLAG (SF) tag out of frame. Cre-mediated recombination replaced AcGFP-HA by SF. The AcGFPHAstopSF cassette was added to DLL1 for tests in cultured cells and introduced into endogenous DLL1 in mice by homologous recombination. Tagged DLL1 protein was detected by antibodies against GFP and HA or Flag, respectively, both in CHO cells and embryo lysates. In CHO cells the AcGFP fluorophore fused to DLL1 was functional. In vivo AcGFP expression was below the level of detection by direct fluorescence. However, the SF tag allowed us to specifically purify DLL1 complexes from embryo lysates. Homozygous mice expressing AcGFPHA or SF-tagged DLL1 revealed a vertebral column phenotype reminiscent of disturbances in AP polarity during somitogenesis, a process most sensitive to reduced DLL1 function. Thus, even small C-terminal tags can impinge on sensitive developmental processes requiring DLL1 activity.

In vertebrate embryos, a process called somitogenesis lays the foundations of the adult spine. This process involves elongation and segmentation of the paraxial mesoderm to form somites. Although the segmentation aspect of this has been widely studied, the elongation aspect is not well understood. Posterior growth is widely assumed to be the main driver, but there is very little evidence for this ��� particularly in fast-developing species like zebrafish. In this thesis, I present the first long term, multi-scale, 3D characterisation of the zebrafish paraxial mesoderm, and show that this tissue elongates through some form of convergent extension, not through growth. In fact, the tissue is compressed over time, and so decreases in volume. I suggest that these processes may be functionally linked, and thus propose a novel mechanism of ���compression-extension���. Cell tracking, agent-based modelling, and perturbations show that this form of convergent extension does not involve PCP-dependent directional intercalation but, instead, involves convergent flows of cells towards the midline and non-directional intercalation. The cause of compression is not clear, but perturbation experiments suggest that extrinsic forces from the neural tube and TGF�� signalling may be involved. Comparative work in cichlids, chickens, and catsharks suggests that tissue convergence is not unique to zebrafish, and instead is a conserved feature of paraxial mesoderm elongation ��� even in species that undergo high levels of growth during somitogenesis. This suggests that the relative contributions of growth and tissue convergence to the process of paraxial mesoderm elongation have evolved differently across vertebrate lineages, resulting in a spectrum of elongation strategies.

The spine and spinal cord are composed of multiple segments initiated by different embryologic mechanisms and advanced under different systems of control. In humans, the upper central nervous system is formed by primary neurulation, the lower by secondary neurulation, and the intervening segment by junctional neurulation. This article focuses on the distal spine and spinal cord to address their embryogenesis and the molecular derangements that lead to some distal spinal malformations.

Congenital scoliosis (CS) is the result of anomalous vertebrae development, but the pathogenesis of CS remains unclear. Long non‐coding RNAs (lncRNAs) have been implicated in embryo development, but their role in CS remains unknown. In this study, we investigated the role and mechanisms of a specific lncRNA, SULT1C2A, in somitogenesis in a rat model of vitamin A deficiency (VAD)‐induced CS. Bioinformatics analysis and quantitative real‐time PCR (qRT‐PCR) indicated that SULT1C2A expression was down‐regulated in VAD group, accompanied by increased expression of rno‐miR‐466c‐5p but decreased expression of Foxo4 and somitogenesis‐related genes such as Pax1, Nkx3‐2 and Sox9 on gestational day (GD) 9. Luciferase reporter and small interfering RNA (siRNA) assays showed that SULT1C2A functioned as a competing endogenous RNA to inhibit rno‐miR‐466c‐5p expression by direct binding, and rno‐miR‐466c‐5p inhibited Foxo4 expression by binding to its 3′ untranslated region (UTR). The spatiotemporal expression of SULT1C2A, rno‐miR‐466c‐5p and Foxo4 axis was dynamically altered on GDs 3, 8, 11, 15 and 21 as detected by qRT‐PCR and northern blot analyses, with parallel changes in Protein kinase B (AKT) phosphorylation and PI3K expression. Taken together, our findings indicate that SULT1C2A enhanced Foxo4 expression by negatively modulating rno‐miR‐466c‐5p expression via the PI3K‐ATK signalling pathway in the rat model of VAD‐CS. Thus, SULT1C2A may be a potential target for treating CS.

The G protein-coupled receptor APJ/Aplnr has been widely reported to be involved in heart and vascular development and disease, but whether it contributes to organ left-right patterning is largely unknown. Here, we show that in zebrafish, aplnra/b coordinates organ LR patterning in an apela/apln ligand-dependent manner using distinct mechanisms at different stages. During gastrulation and early somitogenesis, aplnra/b loss of function results in heart and liver LR asymmetry defects, accompanied by disturbed KV/cilia morphogenesis and disrupted left-sided Nodal/spaw expression in the LPM. In this process, only aplnra loss of function results in KV/cilia morphogenesis defect. In addition, only apela works as the early endogenous ligand to regulate KV morphogenesis, which then contributes to left-sided Nodal/spaw expression and subsequent organ LR patterning. The aplnra-apela cascade regulates KV morphogenesis by enhancing the expression of foxj1a, but not fgf8 or dnh9, during KV development. At the late somite stage, both aplnra and aplnrb contribute to the expression of lft1 in the trunk midline but do not regulate KV formation, and this role is possibly mediated by both endogenous ligands, apela and apln. In conclusion, our study is the first to identify a role for aplnra/b and their endogenous ligands apela/apln in LR patterning, and it clarifies the distinct roles of aplnra-apela and aplnra/b-apela/apln in orchestrating organ LR patterning.

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.

The direction of visceral organ asymmetry is highly conserved during vertebrate evolution with heart development biased to the left and pancreas and liver development restricted to opposing sides of the midline. Here we show that reversals in visceral organ asymmetry have evolved in Astyanax mexicanus, a teleost species with interfertile surface-dwelling (surface fish) and cave-dwelling (cavefish) forms. Visceral organ asymmetry is conventional in surface fish but some cavefish have evolved reversals in heart, liver, and pancreas development. Corresponding changes in the normally left-sided expression of the Nodal-Pitx2/Lefty signaling system are also present in the cavefish lateral plate mesoderm (LPM). The Nodal antagonists lefty1 (lft1) and lefty2 (lft2), which confine Nodal signaling to the left LPM, are expressed in most surface fish, however, lft2, but not lft1, expression is absent during somitogenesis of most cavefish. Despite this difference, multiple lines of evidence suggested that evolutionary changes in L-R patterning are controlled upstream of Nodal-Pitx2/Lefty signaling. Accordingly, reciprocal hybridization of cavefish and surface fish showed that modifications of heart asymmetry are present in hybrids derived from cavefish mothers but not from surface fish mothers. The results indicate that changes in visceral asymmetry during cavefish evolution are influenced by maternal genetic effects.

Despite the common application in pregnancy at clinical practice, it remains ambiguous whether dexamethasone (Dex) exposure can affect embryonic myogenesis. In this study, firstly we showed that 10-6 M Dex (Cheng et al., 2016; 2017) treatment resulted in abnormal myogenesis in chicken embryos. Secondly, we demonstrated that 10-6 M Dex-induced abnormality of myogenesis resulted from aberrant cell proliferation, as well as from alteration of the differentiation process from the early stage of somitogenesis up to the late stage of myogenesis. The above-mentioned results caused by Dex exposure might be due to the aberrant gene expressions of somite formation (Raldh2, Fgf8, Wnt3a, β-catenin, Slug, Paraxis, N-cadherin) and differentiation (Pax3, MyoD, Wnt3a, Msx1, Shh). Thirdly, RNA sequencing implied the statistically significant differential gene expressions in regulating the myofibril and systemic development, as well as a dramatical alteration of retinoic acid (RA) signaling during somite development in the chicken embryos exposed to Dex. The subsequent validation experiments verified that Dex treatment indeed led to a metabolic change of RA signaling, which was up-regulated and principally mediated by FGF-ERK signaling revealed by means of the combination of chicken embryos and in vitro C2C12 cells. These findings highlight that 10-6 M Dex exposure enhances the risk of abnormal myogenesis through interfering with RA signaling during development.

Arthropod segmentation and vertebrate somitogenesis are leading fields in the experimental and theoretical interrogation of developmental patterning. However, despite the sophistication of current research, basic conceptual issues remain unresolved. These include (1) the mechanistic origins of spatial organisation within the segment addition zone (SAZ); (2) the mechanistic origins of segment polarisation; (3) the mechanistic origins of axial variation; and (4) the evolutionary origins of simultaneous patterning. Here, I explore these problems using coarse-grained models of cross-regulating dynamical processes. In the morphogenetic framework of a row of cells undergoing axial elongation, I simulate interactions between an “oscillator”, a “switch”, and up to three “timers”, successfully reproducing essential patterning behaviours of segmenting systems. By comparing the output of these largely cell-autonomous models to variants that incorporate positional information, I find that scaling relationships, wave patterns, and patterning dynamics all depend on whether the SAZ is regulated by temporal or spatial information. I also identify three mechanisms for polarising oscillator output, all of which functionally implicate the oscillator frequency profile. Finally, I demonstrate significant dynamical and regulatory continuity between sequential and simultaneous modes of segmentation. I discuss these results in the context of the experimental literature.

Summary Somitogenesis is often described using the clock-and-wavefront (CW) model, which does not explain how molecular signaling rearranges the pre-somitic mesoderm (PSM) cells into somites. Our scanning electron microscopy analysis of chicken embryos reveals a caudally-progressing epithelialization front in the dorsal PSM that precedes somite formation. Signs of apical constriction and tissue segmentation appear in this layer 3-4 somite lengths caudal to the last-formed somite. We propose a mechanical instability model in which a steady increase of apical contractility leads to periodic failure of adhesion junctions within the dorsal PSM and positions the future inter-somite boundaries. This model produces spatially periodic segments whose size depends on the speed of the activation front of contraction (F), and the buildup rate of contractility (Λ). The Λ/F ratio determines whether this mechanism produces spatially and temporally regular or irregular segments, and whether segment size increases with the front speed., Graphical abstract, Highlights • Dorsal pre-somitic mesoderm of chicken embryos epithelializes before somite formation • Dorsal epithelium shows signs of apical constriction and early segmentation • A mechanical instability model can reproduce sequential segmentation • A single ratio describes spatial and temporal patterns of segmentation, Poultry Embryology; Mechanical Modeling; Developmental Biology

Background In the past decade, the zebrafish community has widely embraced targeted mutagenesis technologies, resulting in an abundance of mutant lines. While many lines have proven to be useful for investigating gene function, many have also shown no apparent phenotype, or phenotypes not of interest to the originating lab. In order for labs to document and share information about these lines, we have created ZebraShare as a new resource offered within ZFIN. Methods ZebraShare involves a form-based submission process generated by ZFIN. The ZebraShare interface (https://zfin.org/action/zebrashare) can be accessed on ZFIN under “Submit Data”. Users download the Submission Workbook and complete the required fields, then submit the completed workbook with associated images and captions, generating a new ZFIN publication record. ZFIN curators add the submitted phenotype and mutant information to the ZFIN database, provide mapping information about mutations, and cross reference this information across the appropriate ZFIN databases. We present here examples of ZebraShare submissions, including phf21aa, kdm1a, ctnnd1, snu13a, and snu13b mutant lines. Results Users can find ZebraShare submissions by searching ZFIN for specific alleles or line designations, just as for alleles submitted through the normal process. We present several potential examples of submission types to ZebraShare including a phenotypic mutants, mildly phenotypic, and early lethal mutants. Mutants for kdm1a show no apparent skeletal phenotype, and phf21aa mutants show only a mild skeletal phenotype, yet these genes have specific human disease relevance and therefore may be useful for further studies. The p120-catenin encoding gene, ctnnd1, was knocked out to investigate a potential role in brain development or function. The homozygous ctnnd1 mutant disintegrates during early somitogenesis and the heterozygote has localized defects, revealing vital roles in early development. Two snu13 genes were knocked out to investigate a role in muscle formation. The snu13a;snu13b double mutant has an early embryonic lethal phenotype, potentially related to a proposed role in the core splicing complex. In each example, the mutants submitted to ZebraShare display phenotypes that are not ideally suited to their originating lab’s project directions but may be of great relevance to other researchers. Conclusion ZebraShare provides an opportunity for researchers to directly share information about mutant lines within ZFIN, which is widely used by the community as a central database of information about zebrafish lines. Submissions of alleles with a phenotypic or unexpected phenotypes is encouraged to promote collaborations, disseminate lines, reduce redundancy of effort and to promote efficient use of time and resources. We anticipate that as submissions to ZebraShare increase, they will help build an ultimately more complete picture of zebrafish genetics and development.

Shaping functional structures during embryonic development requires both genetic and physical control. During somitogenesis, cell-cell coordination sets up genetic traveling waves in the presomitic mesoderm (PSM) that orchestrate somite formation. While key molecular and genetic aspects of this process are known, the mechanical events required to physically segment somites from the PSM remain unclear. Combining direct mechanical measurements during somite formation, live imaging of cell and tissue structure, and computer simulations, here we show that somites are mechanically sectioned off from the PSM by a large, actomyosin-driven increase in anisotropic stress at the nascent somite-somite boundary. Our results show that this localized increase in stress drives the regional fluidization of the tissue adjacent to the forming somite border, enabling local tissue remodeling and the shaping of the somite. Moreover, we find that active tension fluctuations in the tissue are optimized to mechanically define sharp somite boundaries while minimizing somite morphological defects. Altogether, these results indicate that mechanical changes at the somite-somite border and optimal tension fluctuations in the tissue are essential physical aspects of somite formation.

Periodic segmentation of the presomitic mesoderm of a developing mouse embryo is controlled by a network of signaling pathways. Signaling oscillations and gradients are thought to control the timing and spacing of segment formation, respectively. While the involved signaling pathways have been studied extensively over the last decades, direct evidence for the function of signaling oscillations in controlling somitogenesis has been lacking. To enable the functional investigation of signaling dynamics, microfluidics is a previously established tool for the subtle modulation of these dynamics. With this microfluidics-based entrainment approach endogenous signaling oscillations are synchronized by pulses of pathway modulators. This enables modulation of, for instance, the oscillation period or the phase-relationship between two oscillating pathways. Furthermore, spatial gradients of pathway modulators can be established along the tissue to study how specific changes in the signaling gradients affect somitogenesis. The present protocol is meant to help establish microfluidic approaches for the first-time users of microfluidics. The basic principles and equipment needed to set up a microfluidic system are described, and a chip design is provided, with which a mold for chip generation can conveniently be prepared using a 3D printer. Finally, how to culture primary mouse tissue on a microfluidic chip and how to entrain signaling oscillations to external pulses of pathway modulators are discussed. This microfluidic system can also be adapted to harbor other in vivo and in vitro model systems such as gastruloids and organoids for functional investigation of signaling dynamics and morphogen gradients in other contexts.

In vertebrate embryos the presomitic mesoderm become progressively segmented into somites at the anterior end while extending along the anterior-posterior axis. A commonly adopted model to explain how this tissue elongates is that of posterior growth, driven in part by the addition of new cells from uncommitted progenitor populations in the tailbud. However, in zebrafish, much of somitogenesis is associated with an absence of overall volume increase and posterior progenitors do not contribute new cells until the final stages of somitogenesis. Here, we perform a comprehensive 3D morphometric analysis of the paraxial mesoderm and reveal that extension is linked to a volumetric decrease, compression in both dorsal-ventral and medio-lateral axes, and an increase in cell density. We also find that individual cells decrease in their cell volume over successive somite stages. Live cell tracking confirms that much of this tissue deformation occurs within the presomitic mesoderm progenitor zone and is associated with non-directional rearrangement. Furthermore, unlike the trunk somites that are laid down during gastrulation, tail somites develop from a tissue that can continue to elongate in the absence of functional PCP signalling. Taken together, we propose a compression-extension mechanism of tissue elongation that highlights the need to better understand the role of tissue intrinsic and extrinsic forces play in regulating morphogenesis.

Whether or not the process of somitogenesis and myogenesis is affected by excessive caffeine intake still remains ambiguous. In this study, we first showed that caffeine treatment results in chest wall deformities and simultaneously reduced mRNA expressions of genes involved in myogenesis in the developing chicken embryos. We then used embryo cultures to assess in further detail how caffeine exposure affects the earliest steps of myogenesis, and we demonstrated that the caffeine treatment suppressed somitogenesis of chicken embryos by interfering with the expressions of crucial genes modulating apoptosis, proliferation, and differentiation of myogenic progenitors in differentiating somites. These phenotypes were abrogated by a retinoic acid (RA) antagonist in embryo cultures, even at low caffeine doses in C2C12 cells, implying that excess RA levels are responsible for these phenotypes in cells and possiblyin vivo. These findings highlight that excessive caffeine exposure is negatively involved in regulating the development of myogenic progenitors through interfering with RA signaling. The RA somitogenesis/myogenesis pathway might be directly impacted by caffeine signaling rather than reflecting an indirect effect of the toxicity of excess caffeine dosage.

Rhythmic spatial gene expression patterns termed the segmentation clock regulate vertebrate body axis segmentation during embryogenesis. The integrity of these patterns requires local synchronization between neighboring cells by Delta-Notch signaling and its inhibition results in defective segment boundaries. The oscillating tissue deforms substantially throughout development, but whether such tissue-scale morphogenesis complements local synchronization during pattern generation and segment formation is not understood. Here, we investigate pattern recovery in the zebrafish segmentation clock by washing out a Notch inhibitor, allowing resynchronization at different developmental stages, and analyzing the recovery of normal segments. Although from previous work no defects are expected after recovery, we find that washing out at early stages causes a distinctive intermingling of normal and defective segments, suggesting unexpectedly large fluctuations of synchrony before complete recovery. To investigate this recovery behavior, we develop a new model of the segmentation clock combining key ingredients motivated by prior experimental observations: coupling between neighboring oscillators, a frequency profile, a gradient of cell mixing, tissue length change, and cell advection pattern. This model captures the experimental observation of intermingled normal and defective segments through the formation of persistent phase vortices of the genetic oscillators. Experimentally observed recovery patterns at different developmental stages are predicted by temporal changes of tissue-level properties, such as tissue length and cell advection pattern in the model. These results suggest that segmental pattern recovery occurs at two scales: local pattern formation and transport of these patterns through tissue morphogenesis, highlighting a generic mechanism of pattern dynamics within developing tissues.SIGNIFICANCEInteracting genetic oscillators can generate a coherent rhythm and a tissue-level pattern from an initially desynchronized state. Using experiment and theory we study resynchronization and pattern recovery of the zebrafish segmentation clock, which makes the embryonic body segments. Experimental perturbation of intercellular signaling with an inhibitor results in intermingled normal and defective segments. According to theory, this behavior may be caused by persistent local vortices scattered in the tissue during pattern recovery. Full pattern recovery follows dynamic global properties, such as tissue length and advection pattern, in contrast to other genetic oscillators in a static tissue such as circadian clocks. Our work highlights how dynamics of tissue level properties may couple to biochemical pattern formation in tissues and developing embryos.

Spondylocostal dysostosis (SCDO) is a rare heritable congenital condition, characterized by multiple severe malformations of the vertebrae and ribs. Great advances were made in the last decades at the clinical level, by identifying the genetic mutations underlying the different forms of the disease. These were matched by extraordinary findings in the Developmental Biology field, which elucidated the cellular and molecular mechanisms involved in embryo body segmentation into the precursors of the axial skeleton. Of particular relevance was the discovery of the somitogenesis molecular clock that controls the progression of somite boundary formation over time. An overview of these concepts is presented, including the evidence obtained from animal models on the embryonic origins of the mutant-dependent disease. Evidence of an environmental contribution to the severity of the disease is discussed. Finally, a brief reference is made to emerging in vitro models of human somitogenesis which are being employed to model the molecular and cellular events occurring in SCDO. These represent great promise for understanding this and other human diseases and for the development of more efficient therapeutic approaches. PTDC/BEX-BID/5410/2014, SFRH/BD/146043/2019, UID/BIM/04773/2019 info:eu-repo/semantics/publishedVersion

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., Summary: Targeted multi-photon tissue ablation reveals that coordinated cell expansion and addition to the notochord in zebrafish embryos contributes to the elongation of segmented tissue required for embryo anterior-posterior axis extension.

Summary Somite formation is foundational to creating the vertebrate segmental body plan. Here, we describe three transcriptional trajectories toward somite formation in the early mouse embryo. Precursors of the anterior-most somites ingress through the primitive streak before E7 and migrate anteriorly by E7.5, while a second wave of more posterior somites develops in the vicinity of the streak. Finally, neuromesodermal progenitors (NMPs) are set aside for subsequent trunk somitogenesis. Single-cell profiling of T−/− chimeric embryos shows that the anterior somites develop in the absence of T and suggests a cell-autonomous function of T as a gatekeeper between paraxial mesoderm production and the building of the NMP pool. Moreover, we identify putative regulators of early T-independent somites and challenge the T-Sox2 cross-antagonism model in early NMPs. Our study highlights the concept of molecular flexibility during early cell-type specification, with broad relevance for pluripotent stem cell differentiation and disease modeling., Graphical Abstract, Highlights • Multiple transcriptional trajectories underlie somite emergence • Outlining the in vivo molecular journey toward the anterior T-independent somites • T does not negatively regulate Sox2 to control neural output at E8.5, Guibentif, Griffiths et al. reconstruct three transcriptional journeys from the pluripotent mouse epiblast to anterior somites, posterior somites, and neuromesodermal progenitors (NMPs) present at embryonic day 8.5. Single-cell profiling of T−/−↔WT chimeras validates the molecular journey toward T-independent anterior somites and challenges the notion that NMP differentiation is regulated by T and Sox2 mutual antagonism.

Modular, repetitive structures are a key component of complex multicellular body plans across the tree of life. Typically, these structures are prepatterned by temporal oscillations in gene expression or signaling. Although a clock-and-wavefront mechanism was identified and plant leaf phyllotaxis arises from a Turing-type patterning for vertebrate somitogenesis and arthropod segmentation, the mechanism underlying lateral root patterning has remained elusive. To resolve this enigma, we combined computational modeling with in planta experiments. Intriguingly, auxin oscillations automatically emerge in our model from the interplay between a reflux-loop-generated auxin loading zone and stem-cell-driven growth dynamics generating periodic cell-size variations. In contrast to the clock-and-wavefront mechanism and Turing patterning, the uncovered mechanism predicts both frequency and spacing of lateral-root-forming sites to positively correlate with root meristem growth. We validate this prediction experimentally. Combined, our model and experimental results support that a reflux-and-growth patterning mechanism underlies lateral root priming.

Somitogenesis is a hallmark of vertebrate embryonic development. For years, researchers have been studying this process in a variety of organisms using a wide range of techniques encompassing ex vivo and in vitro approaches. However, most studies still rely on the analysis of two-dimensional (2D) imaging data, which limits proper evaluation of a developmental process like axial extension and somitogenesis involving highly dynamic interactions in a complex 3D space. Here we describe techniques that allow mouse live imaging acquisition, dataset processing, visualization and analysis in 3D and 4D to study the cells (e.g., neuromesodermal progenitors) involved in these developmental processes. We also provide a step-by-step protocol for optical projection tomography and whole-mount immunofluorescence microscopy in mouse embryos (from sample preparation to image acquisition) and show a pipeline that we developed to process and visualize 3D image data. We extend the use of some of these techniques and highlight specific features of different available software (e.g., Fiji/ImageJ, Drishti, Amira and Imaris) that can be used to improve our current understanding of axial extension and somite formation (e.g., 3D reconstructions). Altogether, the techniques here described emphasize the importance of 3D data visualization and analysis in developmental biology, and might help other researchers to better address 3D and 4D image data in the context of vertebrate axial extension and segmentation. Finally, the work also employs novel tools to facilitate teaching vertebrate embryonic development.

Modular body organization is found widely across multicellular organisms, and some of them form repetitive modular structures via the process of segmentation. It's vastly interesting to understand how these regularly repeated structures are robustly generated from the underlying noise in biomolecular interactions. Recent studies from arthropods reveal similarities in segmentation mechanisms with vertebrates, and raise the possibility that the three phylogenetic clades, annelids, arthropods and chordates, might share homology in this process from a bilaterian ancestor. Here, we discuss vertebrate segmentation with particular emphasis on the role of the Notch intercellular signalling pathway. We introduce vertebrate segmentation and Notch signalling, pointing out historical milestones, then describe existing models for the Notch pathway in the synchronization of noisy neighbouring oscillators, and a new role in the modulation of gene expression wave patterns. We ask what functions Notch signalling may have in arthropod segmentation and explore the relationship between Notch-mediated lateral inhibition and synchronization. Finally, we propose open questions and technical challenges to guide future investigations into Notch signalling in segmentation., Highlights • Vertebrate segmentation clocks are different to each other. • Notch has a primary role in synchronization in vertebrates. • Gene expression waves can influence the timing of segment formation. • Synchronization and lateral inhibition circuits are closely related. • Dynamic measurement techniques are needed to dissect clocks.

GATA4/5/6 transcription factors play essential, conserved roles in heart development. How these factors mediate the transition from multipotent mesoderm progenitors to a committed cardiac fate is unclear. To understand how GATA4/5/6 modulate cell fate decisions we labelled, isolated, and performed single-cell gene expression analysis on cells that expressgata5at pre-cardiac time points spanning gastrulation to somitogenesis. We found that most mesendoderm-derived lineages had dynamicgata5/6expression. In the absence of Gata5/6, the population structure of mesendoderm-derived cells was dramatically altered. In addition to the expected absence of cardiac mesoderm, we observed a concomitant expansion of cranial-pharyngeal mesoderm. Functional genetic analyses in zebrafish and the invertebrate chordateCiona, which possess a single GATA4/5/6 homolog, revealed an essential and cell-autonomous role for GATA4/5/6 in promoting cardiac and inhibiting pharyngeal mesoderm identity. Overall, the maintenance and repression of GATA4/5/6 activity plays a critical, evolutionarily conserved role in early development.

The first part of this thesis describes how widely-used tissue dissociation protocols that are often required prior to single-cell RNA sequencing (scRNA-seq) procedures can alter the transcriptome of cells. The second part of this thesis focuses on gastruloids, aggregates of embryonic stem cells that can be used to study mammalian post-implantation development in vitro, and describes how scRNA-seq and spatial transcriptomics technologies can aid in the characterization and development of improvement versions of gastruloid models. Chapter 1 provides an introduction into embryology and stem-cell based in vitro models for embryology, with a focus on gastruloids. This chapter describes the historical context of the gastruloids model, and explains why this model is a useful addition to the toolbox of modern-day embryologists. This chapter also summarizes the advantages and current limitations of the gastruloids system. In addition, this chapter provides a brief introduction into scRNA-seq and spatial transcriptomics technologies. Chapter 2 shows that dissociation procedures that are required for many scRNA-seq experiments can induce a stress response in a subpopulation of these cells. This chapter shows that satellite cells (muscle stem cells) are particularly sensitive to such a dissociation-induced stress response. This chapter highlights that results obtained with scRNA-seq require validation with microscopy, and provides experimental and computational solutions that can be used to remove dissociation-affected subpopulations from scRNA-seq experiments. Chapter 3 provides a detailed single-cell and spatial transcriptomics-based characterization of mouse gastruloids. A detailed comparison with embryos reveals that most embryonic cell types are present in gastruloids, and shows that key markers of somitogenesis are expressed in the correct spatial location. We follow up on these observations in the second part of Chapter 3, and show with live-imaging experiments that the somitogenesis clock is active in mouse gastruloids. We then perform a small drug screening study to perturb the somitogenesis clock in gastruloids. This drug screening reveals how reduced FGF signalling induces a short-tail phenotype in embryos, and exemplifies how gastruloids, which can easily be generated in large numbers, can be used to perform large-scale drug screening procedures. Finally, this chapter describes the discovery that the addition of a small amount of Matrigel can induce the formation of somite-like structures in mouse gastruloids. Chapter 4 describes the development and characterization of the first human version of the gastruloids model. In this chapter, this new human gastruloids system is characterized and compared to mouse gastruloids with spatial transcriptomics. In addition, this chapter describes how various teratogens and inhibitors affect the development of human gastruloids, suggesting that this system can indeed be used to study how environmental factors affect human development. Chapter 5 provides a discussion of the work described in this thesis. This chapter describes alternative tissue dissociation protocols that have been developed by others that followed up on the dissociation-induced stress-response that we discovered in Chapter 2 of this thesis. In addition, Chapter 5 provides an extensive overview of the current challenges, ethical considerations and potential future applications of the (human) gastruloid field.

Endothelial cells appear to emerge from diverse progenitors. However, to which extent their developmental origin contributes to define their cellular and molecular characteristics remains largely unknown. Here, we report that a subset of endothelial cells that emerge from the tailbud possess unique molecular characteristics that set them apart from stereotypical lateral plate mesoderm (LPM)-derived endothelial cells. Lineage tracing shows that these tailbud-derived endothelial cells arise at mid-somitogenesis stages, and surprisingly do not require Npas4l or Etsrp function, indicating that they have distinct spatiotemporal origins and are regulated by distinct molecular mechanisms. Microarray and single cell RNA-seq analyses reveal that somitogenesis- and neurogenesis-associated transcripts are over-represented in these tailbud-derived endothelial cells, suggesting that they possess a unique transcriptomic signature. Taken together, our results further reveal the diversity of endothelial cells with respect to their developmental origin and molecular properties, and provide compelling evidence that the molecular characteristics of endothelial cells may reflect their distinct developmental history.

During vertebrate development, the presomitic mesoderm (PSM) periodically segments into somites, which will form the segmented vertebral column and associated muscle, connective tissue, and dermis. The periodicity of somitogenesis is regulated by a segmentation clock of oscillating Notch activity. Here, we examined mouse mutants lacking onlyFgf4orFgf8, which we previously demonstrated act redundantly to prevent PSM differentiation.Fgf8is not required for somitogenesis, butFgf4mutants display a range of vertebral defects. We analyzedFgf4mutants by quantifying mRNAs fluorescently labeled by hybridization chain reaction within Imaris-based volumetric tissue subsets. These data indicate that FGF4 maintainsHes7levels and normal oscillatory patterns. To support our hypothesis that FGF4 regulates somitogenesis throughHes7, we demonstrate genetic synergy betweenHes7andFgf4, but not withFgf8. Our data indicate thatFgf4is potentially important in a spectrum of human Segmentation Defects of the Vertebrae caused by defective Notch oscillations.