By exploiting the cell-cycle-dependent proteolysis of two ubiquitination oscillators, human Cdt1 and geminin, which are the direct substrates of SCF Skp2 and APC Cdh1 complexes, respectively, Fucci technique labels mammalian cell nuclei in G 1 and S/G2/M phases with different colors. Transgenic mice expressing these G 1 and S/G2/M markers offer a powerful means to investigate the coordination of the cell cycle with morphogenetic processes. We attempted to introduce these markers into zebrafish embryos to take advantage of their favorable optical properties. However, although the fundamental mechanisms for cell-cycle control appear to be well conserved among species, the G 1 marker based on the SCF Skp2 -mediated degradation of human Cdt1 did not work in fish cells, probably because the marker was not ubiquitinated properly by a fish E3 ligase complex. We describe here the generation of a Fucci derivative using zebrafish homologs of Cdt1 and geminin, which provides sweeping views of cell proliferation in whole fish embryos. Remarkably, we discovered two anterior-toposterior waves of cell-cycle transitions, G 1/S and M/G1, in the differentiating notochord. Our study demonstrates the effectiveness of using the Cul4 Ddb1 -mediated Cdt1 degradation pathway common to all metazoans for the development of a G 1 marker that works in the nonmammalian animal model. cell cycle ͉ fluorescent protein ͉ imaging ͉ ubiquitination E ukaryotic cells ensure tight regulation of cell division by maintaining close control over the levels of cell-cycle proteins. For example, Cdt1 and geminin have opposite effects on DNA replication during S phase, and their levels fluctuate accordingly throughout the cell cycle (1, 2). Cdt1 levels are highest in G 1 phase just before DNA replication and decrease as cells transition into S phase, whereas geminin levels rise during S phase and fall during G 1 phase. Cells control Cdt1 and geminin activity at the protein level by ubiquitination, which precisely targets unwanted proteins for destruction.We harnessed the regulation of cell-cycle-dependent ubiquitination to develop a genetically encoded indicator for cell-cycle progression: Fucci ( fluorescent ubiquitination-based cell cycle indicator) (3). The original Fucci probe was generated by fusing mKO2 (monomeric Kusabira Orange2) and mAG (monomeric Azami Green) to the ubiquitination domains of human Cdt1 (hCdt1) and human geminin (hGem): hCdt1(30/120) and hGem(1/110), respectively. These two chimeric proteins, mKO2-hCdt1(30/120) and mAG-hGem(1/110) ( Fig. 1 A and B), accumulate reciprocally in the nuclei of transfected mammalian cells during the cell cycle, labeling nuclei of G 1 phase cells orange and those in S/G 2 /M phase green. Thus, these proteins function as effective G 1 and S/G 2 /M markers. We also developed a S/G 2 /M marker, mAG-hGem(1/60), which accumulates in both the nucleus and cytoplasm (4) and reveals the morphology of individual cells that have undergone DNA replication. This permits cell proliferation to be monitored along with t...

The vertebral column exhibits segmentation and regionalization along the antero-posterior axis. During embryogenesis, the rhythmic production of the precursors of the vertebrae, the somites, imposes a segmented aspect to the spine, whereas the spine's regional differentiation is controlled by Hox genes. Here we show that in the paraxial mesoderm, Hoxb genes are first activated in a temporal collinear fashion in precursors located in the epiblast lateral to the primitive streak. Our data suggest that collinear activation of Hoxb genes regulates the flux of cells from the epiblast to the streak and thus directly controls the establishment of the genes' characteristic nested expression domains in the somites. This suggests that establishment of the spatial co-linearity in the embryo is directly controlled by the Hox genes themselves.

In vertebrate development, the body plan is determined by primordial morphogen gradients that suffuse the embryo. Retinoic acid (RA) is an important morphogen involved in patterning the anterior-posterior axis of structures, including the hindbrain and paraxial mesoderm. RA diffuses over long distances, and its activity is spatially restricted by synthesizing and degrading enzymes. However, gradients of endogenous morphogens in live embryos have not been directly observed; indeed, their existence, distribution and requirement for correct patterning remain controversial. Here we report a family of genetically encoded indicators for RA that we have termed GEPRAs (genetically encoded probes for RA). Using the principle of fluorescence resonance energy transfer we engineered the ligand-binding domains of RA receptors to incorporate cyan-emitting and yellow-emitting fluorescent proteins as fluorescence resonance energy transfer donor and acceptor, respectively, for the reliable detection of ambient free RA. We created three GEPRAs with different affinities for RA, enabling the quantitative measurement of physiological RA concentrations. Live imaging of zebrafish embryos at the gastrula and somitogenesis stages revealed a linear concentration gradient of endogenous RA in a two-tailed source-sink arrangement across the embryo. Modelling of the observed linear RA gradient suggests that the rate of RA diffusion exceeds the spatiotemporal dynamics of embryogenesis, resulting in stability to perturbation. Furthermore, we used GEPRAs in combination with genetic and pharmacological perturbations to resolve competing hypotheses on the structure of the RA gradient during hindbrain formation and somitogenesis. Live imaging of endogenous concentration gradients across embryonic development will allow the precise assignment of molecular mechanisms to developmental dynamics and will accelerate the application of approaches based on morphogen gradients to tissue engineering and regenerative medicine.

In vertebrates, the total number of vertebrae is precisely defined. Vertebrae derive from embryonic somites that are continuously produced posteriorly from the presomitic mesoderm (PSM) during body formation. We show that in the chicken embryo, activation of posterior Hox genes (paralogs 9–13) in the tail-bud correlates with the slowing down of axis elongation. Our data indicate that a subset of progressively more posterior Hox genes, which are collinearly activated in vertebral precursors, repress Wnt activity with increasing strength. This leads to a graded repression of the Brachyury/T transcription factor, reducing mesoderm ingression and slowing down the elongation process. Due to the continuation of somite formation, this mechanism leads to the progressive reduction of PSM size. This ultimately brings the retinoic acid (RA)-producing segmented region in close vicinity to the tail bud, potentially accounting for the termination of segmentation and axis elongation.DOI: http://dx.doi.org/10.7554/eLife.04379.001

Vertebrae display distinct morphological features at different levels of the body axis. Links between collinear Hox gene activation and the progressive mode of body axis elongation have provided a fascinating blueprint of the mechanisms for establishing these morphological identities. In this review, we first discuss the regulation and possible role of collinear Hox gene activation during body formation and then highlight the direct role of Hox genes in controlling cellular movements during gastrulation, therefore contributing to body formation. Additional related research aspects, such as imaging of chromatin regulation, roles of micro RNAs and evolutional findings are also discussed.

Chemokines are characterized by the homing activity of leukocytes to targeted inflammation sites. Recent research indicates that chemokines play more divergent roles in various phases of pathogenesis as well as immune reactions. The chemokine receptor, CCR1, and its ligands are thought to be involved in inflammatory bone destruction, but their physiological roles in the bone metabolism in vivo have not yet been elucidated. In the present study, we investigated the roles of CCR1 in bone metabolism using CCR1-deficient mice. Chemokines are initially identified as small cytokines that direct the homing of circulating leukocytes into sites of inflammation (1). Chemokines are now recognized to be major factors in inflammation and immune development as well as tumor growth, angiogenesis, and osteolysis. Chemokine receptors are expressed in a well organized spatiotemporal manner in various types of leukocytes, including lymphocytes, granulocytes, and macrophages. They facilitate the recruitment of these cells into inflammatory sites during the appropriate phase of inflammation.Recent findings indicate that chemokine receptors, including CCR1 7 and its related chemokines, CCL3 and CCL9, are involved in the pathogenesis of a variety of diseases. In particular, CCL3 (also called MIP-1␣), a major pro-inflammatory chemokine produced at inflammatory sites, appears to play a crucial role in pathological osteoclastogenesis (2, 3). In osteolytic bone inflammation (e.g. rheumatoid arthritis-associated bone destruction), CCL3 induces ectopic osteoclastogenesis (4) * This work was supported in part by Grant H19-nano-012 from the Ministry of Health, Labor and Welfare (to K. Y.) and by a research fellowship from the Japan Society for the Promotion of Science for Young Scientists (2007Scientists ( -2009 ( The abbreviations used are: CCR, C-C chemokine receptor; M-CSF, macrophage-colony stimulation factor; BALP, bone-specific alkaline phosphatase; CCL, C-C chemokine ligand; MCP-1, macrophage chemoattractant protein-1; MIP-1, macrophage inflammatory protein-1; CT, computed tomography; PTX, pertussis toxin from Bordetella pertussis; RANK, receptor activator of NF-B; RANKL, receptor activator of NF-B ligand; RANTES, regulated upon activation normal T expression and secreted; TRAP, tartrate-resistant acid phosphatase; NTx, N-telopeptides.

Summary The vertebrate spine exhibits two striking characteristics. The first one is the periodic arrangement of its elements – the vertebrae – along the antero-posterior axis. This segmented organization is the result of somitogenesis, which takes place during organogenesis. The segmentation machinery involves a molecular oscillator – the segmentation clock – which delivers a periodic signal controlling somite production. During embryonic axis elongation, this signal is displaced posteriorly by a system of traveling signaling gradients – the wavefront – which depends on the Wnt, FGF and retinoic acid pathways. The other characteristic feature of the spine is the subdivision of groups of vertebrae into anatomical domains, such as the cervical, thoracic, lumbar, sacral and caudal regions. This axial regionalization is controlled by a set of transcription factors called Hox genes. Hox genes exhibit nested expression domains in the somites which reflect their linear arrangement along the chromosomes– a property termed colinearity. The colinear disposition of Hox genes expression domains provides a blueprint for the regionalization of the future vertebral territories of the spine. In amniotes, Hox genes are activated in the somite precursors of the epiblast in a temporal colinear sequence and they were proposed to control their progressive ingression into the nascent paraxial mesoderm. Consequently, the positioning of the expression domains of Hox genes along the antero-posterior axis is largely controlled by the timing of Hox activation during gastrulation. Positioning of the somitic Hox domains is subsequently refined through a cross talk with the segmentation machinery in the presomitic mesoderm. In this review, we focus on our current understanding of the embryonic mechanisms that establish vertebral identities during vertebrate development.

The skeletal muscles and axial skeleton of vertebrates derive from the embryonic paraxial mesoderm. In amniotes, paraxial mesoderm is formed bilaterally to the nerve cord as a result of primitive streak and tail-bud regression during body axis formation. In chick and mouse embryos, paraxial mesoderm was proposed to derive from a population of resident cells located in the regressing primitive streak and tail bud. In contrast, in lower vertebrates, paraxial mesoderm is formed as a result of the continuation of ingression movements of gastrulation. Here, we reinvestigate paraxial mesoderm formation in the chicken embryo and demonstrate that these two modes are concomitantly at work to set up the paraxial mesoderm. Although the medial part of somites derives from stem cells resident in the primitive streak/tail bud, the lateral part derives from continuous ingression of epiblastic material. Our fate mapping further shows that the paraxial mesoderm territory in the epiblast is regionalized along the anteroposterior axis as in lower vertebrates. These observations suggest that the mechanisms responsible for paraxial mesoderm formation are largely conserved across vertebrates.vertebrate ͉ epiblast ͉ primitive streak ͉ tail bud ͉ body axis formation