Zebrafish GADD45beta genes are involved in somite segmentation.
ABSTRACT: Somites in vertebrates are periodic segmented structures that give rise to the vertebrae and muscles of body. Somites are generated from presomitic mesoderm (PSM), but it is not fully understood how cellular differentiation and segment formation are achieved in the anterior PSM. We report here that zebrafish gadd45beta1 and gadd45beta2 genes are periodically expressed as paired stripes adjacent to the neural tube in the anterior PSM region where presomitic cells mature. In mammals, it is known that GADD45 (growth arrest and DNA damage) family proteins play a role in cell-cycle control. We found that both knockdown and overexpression of gadd45beta genes caused somite defects with different consequences for marker gene expression. Knockdown of gadd45beta genes with antisense morpholino oligonucleotides caused a broad expansion of mesp-a in the PSM, and both cyclic expression of her1 and segmented expression of MyoD were disorganized. On the other hand, injection of gadd45beta1 or gadd45beta2 suppressed expression of mesp-a and her1 in anterior PSM and MyoD in paraxial mesoderm. These results indicate that regulated expression of gadd45beta genes in the anterior PSM is required for somite segmentation.
Project description:Somitogenesis involves the segmentation of the paraxial mesoderm into units along the anteroposterior axis. Here we show a role for Eph and ephrin signaling in the patterning of presomitic mesoderm and formation of the somites. Ephrin-A-L1 and ephrin-B2 are expressed in an iterative manner in the developing somites and presomitic mesoderm, as is the Eph receptor EphA4. We have examined the role of these proteins by injection of RNA, encoding dominant negative forms of Eph receptors and ephrins. Interruption of Eph signaling leads to abnormal somite boundary formation and reduced or disturbed myoD expression in the myotome. Disruption of Eph family signaling delays the normal down-regulation of her1 and Delta D expression in the anterior presomitic mesoderm and disrupts myogenic differentiation. We suggest that Eph signaling has a key role in the translation of the patterning of presomitic mesoderm into somites.
Project description:The formation of reiterated somites along the vertebrate body axis is controlled by the segmentation clock, a molecular oscillator expressed within presomitic mesoderm (PSM) cells. Although PSM cells oscillate autonomously, they coordinate with neighboring cells to generate a sweeping wave of cyclic gene expression through the PSM that has a periodicity equal to that of somite formation. The velocity of each wave slows as it moves anteriorly through the PSM, although the dynamics of clock slowing have not been well characterized. Here, we investigate segmentation clock dynamics in the anterior PSM in developing zebrafish embryos using an in vivo clock reporter, her1:her1-venus. The her1:her1-venus reporter has single-cell resolution, allowing us to follow segmentation clock oscillations in individual cells in real-time. By retrospectively tracking oscillations of future somite boundary cells, we find that clock reporter signal increases in anterior PSM cells and that the periodicity of reporter oscillations slows to about ?1.5 times the periodicity in posterior PSM cells. This gradual slowing of the clock in the anterior PSM creates peaks of clock expression that are separated at a two-segment periodicity both spatially and temporally, a phenomenon we observe in single cells and in tissue-wide analyses. These results differ from previous predictions that clock oscillations stop or are stabilized in the anterior PSM. Instead, PSM cells oscillate until they incorporate into somites. Our findings suggest that the segmentation clock may signal somite formation using a phase gradient with a two-somite periodicity.
Project description:Somite segmentation depends on a gene expression oscillator or clock in the posterior presomitic mesoderm (PSM) and on read-out machinery in the anterior PSM to convert the pattern of clock phases into a somite pattern. Notch pathway mutations disrupt somitogenesis, and previous studies have suggested that Notch signalling is required both for the oscillations and for the read-out mechanism. By blocking or overactivating the Notch pathway abruptly at different times, we show that Notch signalling has no essential function in the anterior PSM and is required only in the posterior PSM, where it keeps the oscillations of neighbouring cells synchronized. Using a GFP reporter for the oscillator gene her1, we measure the influence of Notch signalling on her1 expression and show by mathematical modelling that this is sufficient for synchronization. Our model, in which intracellular oscillations are generated by delayed autoinhibition of her1 and her7 and synchronized by Notch signalling, explains the observations fully, showing that there are no grounds to invoke any additional role for the Notch pathway in the patterning of somite boundaries in zebrafish.
Project description:Vertebrate segmentation is characterized by the periodic formation of epithelial somites from the mesenchymal presomitic mesoderm (PSM). How the rhythmic signaling pulse delivered by the segmentation clock is translated into the periodic morphogenesis of somites remains poorly understood. Here, we focused on the role of paraxial protocadherin (PAPC/Pcdh8) in this process. We showed that in chicken and mouse embryos, PAPC expression is tightly regulated by the clock and wavefront system in the posterior PSM. We observed that PAPC exhibits a striking complementary pattern to N-cadherin (CDH2), marking the interface of the future somite boundary in the anterior PSM. Gain and loss of function of PAPC in chicken embryos disrupted somite segmentation by altering the CDH2-dependent epithelialization of PSM cells. Our data suggest that clathrin-mediated endocytosis is increased in PAPC-expressing cells, subsequently affecting CDH2 internalization in the anterior compartment of the future somite. This in turn generates a differential adhesion interface, allowing formation of the acellular fissure that defines the somite boundary. Thus, periodic expression of PAPC in the anterior PSM triggers rhythmic endocytosis of CDH2, allowing for segmental de-adhesion and individualization of somites.
Project description:The segmented body plan of vertebrates is prefigured by reiterated embryonic mesodermal structures called somites. In the mouse embryo, timely somite formation from the presomitic mesoderm (PSM) is controlled by the "segmentation clock," a molecular oscillator that triggers progressive waves of Notch activity throughout the PSM. Notch clock activity is suppressed in the posterior PSM by FGF signaling until it crosses a determination front at which its net activity is sufficiently high to effect segmentation. Here, Notch and Wnt signaling directs somite anterior/posterior (A/P) polarity specification and boundary formation via regulation of the segmentation effector gene Mesoderm posterior 2. How Notch and Wnt signaling becomes coordinated at this front is incompletely defined. Here we show that the activity of the cAMP responsive element binding protein (CREB) family of transcription factors exhibits Wnt3a-dependent oscillatory behavior near the determination front and is in unison with Notch activity. Inhibition of CREB family in the mesoderm causes defects in somite segmentation and a loss in somite posterior polarity leading to fusions of vertebrae and ribs. Among the CREB family downstream genes, several are known to be regulated by Wnt3a. Of those, we show that the CREB family occupies a conserved binding site in the promoter region of Delta-like 1, encoding a Notch ligand, in the anterior PSM as a mechanism to specify posterior identity of somites. Together, these data support that the CREB family acts at the determination front to modulate Wnt signaling and strengthen Notch signaling as a means to orchestrate cells for somite segmentation and anterior/posterior patterning.
Project description:BACKGROUND: The vertebrate adult axial skeleton, trunk and limb skeletal muscles and dermis of the back all arise from early embryonic structures called somites. Somites are symmetrically positioned flanking the embryo axial structures (neural tube and notochord) and are periodically formed in a anterior-posterior direction from the presomitic mesoderm. The time required to form a somite pair is constant and species-specific. This extraordinary periodicity is proposed to depend on an underlying somitogenesis molecular clock, firstly evidenced by the cyclic expression of the chick hairy1 gene in the unsegmented presomitic mesoderm with a 90 min periodicity, corresponding to the time required to form a somite pair in the chick embryo. The number of hairy1 oscillations at any given moment is proposed to provide the cell with both temporal and positional information along the embryo's anterior-posterior axis. Nevertheless, how this is accomplished and what biological processes are involved is still unknown. Aiming at understanding the molecular events triggered by the somitogenesis clock Hairy1 protein, we have employed the yeast two-hybrid system to identify Hairy1 interaction partners. RESULTS: Sap18, an adaptor molecule of the Sin3/HDAC transcriptional repressor complex, was found to interact with the C-terminal portion of the Hairy1 protein in a yeast two-hybrid assay and the Hairy1/Sap18 interaction was independently confirmed by co-immunoprecipitation experiments. We have characterized the expression patterns of both sap18 and sin3a genes during chick embryo development, using in situ hybridization experiments. We found that both sap18 and sin3a expression patterns co-localize in vivo with hairy1 expression domains in chick rostral presomitic mesoderm and caudal region of somites. CONCLUSION: Hairy1 belongs to the hairy-enhancer-of-split family of transcriptional repressor proteins. Our results indicate that during chick somitogenesis Hairy1 may mediate gene transcriptional repression by recruiting the Sin3/HDAC complex, through a direct interaction with the Sap18 adaptor molecule. Moreover, since sap18 and sin3a are not expressed in the PSM territory where hairy1 presents cyclic expression, our study strongly points to different roles for Hairy1 throughout the PSM and in the prospective somite and caudal region of already formed somites.
Project description:Bipotent axial stem cells residing in the caudal epiblast during late gastrulation generate neuroectodermal and presomitic mesodermal progeny that coordinate somitogenesis with neural tube formation, but the mechanism that controls these two fates is not fully understood. Retinoic acid (RA) restricts the anterior extent of caudal fibroblast growth factor 8 (Fgf8) expression in both mesoderm and neural plate to control somitogenesis and neurogenesis, however it remains unclear where RA acts to control the spatial expression of caudal Fgf8. Here, we found that mouse Raldh2-/- embryos, lacking RA synthesis and displaying a consistent small somite defect, exhibited abnormal expression of key markers of axial stem cell progeny, with decreased Sox2+ and Sox1+ neuroectodermal progeny and increased Tbx6+ presomitic mesodermal progeny. The Raldh2-/- small somite defect was rescued by treatment with an FGF receptor antagonist. Rdh10 mutants, with a less severe RA synthesis defect, were found to exhibit a small somite defect and anterior expansion of caudal Fgf8 expression only for somites 1-6, with normal somite size and Fgf8 expression thereafter. Rdh10 mutants were found to lack RA activity during the early phase when somites are small, but at the 6-somite stage RA activity was detected in neural plate although not in presomitic mesoderm. Expression of a dominant-negative RA receptor in mesoderm eliminated RA activity in presomitic mesoderm but did not affect somitogenesis. Thus, RA activity in the neural plate is sufficient to prevent anterior expansion of caudal Fgf8 expression associated with a small somite defect. Our studies provide evidence that RA restriction of Fgf8 expression in undifferentiated neural progenitors stimulates neurogenesis while also restricting the anterior extent of the mesodermal Fgf8 mRNA gradient that controls somite size, providing new insight into the mechanism that coordinates somitogenesis with neurogenesis.
Project description:Somitogenesis is the earliest sign of segmentation in the developing vertebrate embryo. This process starts very early, soon after gastrulation has initiated and proceeds in an anterior-to-posterior direction during body axis elongation. It is widely accepted that somitogenesis is controlled by a molecular oscillator with the same periodicity as somite formation. This periodic mechanism is repeated a specific number of times until the embryo acquires a defined specie-specific final number of somites at the end of the process of axis elongation. This final number of somites varies widely between vertebrate species. How termination of the process of somitogenesis is determined is still unknown.Here we show that during development there is an imbalance between the speed of somite formation and growth of the presomitic mesoderm (PSM)/tail bud. This decrease in the PSM size of the chick embryo is not due to an acceleration of the speed of somite formation because it remains constant until the last stages of somitogenesis, when it slows down. When the chick embryo reaches its final number of somites at stage HH 24-25 there is still some remaining unsegmented PSM in which expression of components of the somitogenesis oscillator is no longer dynamic. Finally, we identify a change in expression of retinoic acid regulating factors in the tail bud at late stages of somitogenesis, such that in the chick embryo there is a pronounced onset of Raldh2 expression while in the mouse embryo the expression of the RA inhibitor Cyp26A1 is downregulated.Our results show that the chick somitogenesis oscillator is arrested before all paraxial mesoderm is segmented into somites. In addition, endogenous retinoic acid is probably also involved in the termination of the process of segmentation, and in tail growth in general.
Project description:One of the most striking features of the human vertebral column is its periodic organization along the anterior-posterior axis. This pattern is established when segments of vertebrates, called somites, bud off at a defined pace from the anterior tip of the embryo's presomitic mesoderm (PSM). To trigger this rhythmic production of somites, three major signaling pathways--Notch, Wnt/?-catenin, and fibroblast growth factor (FGF)--integrate into a molecular network that generates a traveling wave of gene expression along the embryonic axis, called the "segmentation clock." Recent systems approaches have begun identifying specific signaling circuits within the network that set the pace of the oscillations, synchronize gene expression cycles in neighboring cells, and contribute to the robustness and bilateral symmetry of somite formation. These findings establish a new model for vertebrate segmentation and provide a conceptual framework to explain human diseases of the spine, such as congenital scoliosis.
Project description:The number of vertebrae is defined strictly for a given species and depends on the number of somites, which are the earliest metameric structures that form in development. Somites are formed by sequential segmentation. The periodicity of somite segmentation is orchestrated by the synchronous oscillation of gene expression in the presomitic mesoderm (PSM), termed the "somite segmentation clock," in which Notch signaling plays a crucial role. Here we show that the clock period is sensitive to Notch activity, which is fine-tuned by its feedback regulator, Notch-regulated ankyrin repeat protein (Nrarp), and that Nrarp is essential for forming the proper number and morphology of axial skeleton components. Null-mutant mice for Nrarp have fewer vertebrae and have defective morphologies. Notch activity is enhanced in the PSM of the Nrarp(-/-) embryo, where the ~2-h segmentation period is extended by 5 min, thereby forming fewer somites and their resultant vertebrae. Reduced Notch activity partially rescues the Nrarp(-/-) phenotype in the number of somites, but not in morphology. Therefore we propose that the period of the somite segmentation clock is sensitive to Notch activity and that Nrarp plays essential roles in the morphology of vertebrae and ribs.