Xenopus tropicalis has been developed as a model organism for developmental biology, providing a system offering both modern genetics and classical embryology. Recently, the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated (CRISPR/Cas) system for genome modification has provided an additional tool for Xenopus researchers to achieve simple and efficient targeted mutagenesis. Here, we provide insights into experimental design and procedures permitting successful application of this technique to Xenopus researchers, and offer a general strategy for performing loss-of-function assays in F0 and subsequently F1 embryos.
Nodal/TGF? signaling regulates diverse biological responses. By combining RNA-seq on Foxh1 and Nodal signaling loss-of-function embryos with ChIP-seq of Foxh1 and Smad2/3, we report a comprehensive genome-wide interaction between Foxh1 and Smad2/3 in mediating Nodal signaling during vertebrate mesendoderm development. This study significantly increases the total number of Nodal target genes regulated by Foxh1 and Smad2/3, and reinforces the notion that Foxh1-Smad2/3-mediated Nodal signaling directly coordinates the expression of a cohort of genes involved in the control of gene transcription, signaling pathway modulation and tissue morphogenesis during gastrulation. We also show that Foxh1 may function independently of Nodal signaling, in addition to its role as a transcription factor mediating Nodal signaling via Smad2/3. Finally, we propose an evolutionarily conserved interaction between Foxh1 and PouV, a mechanism observed in Pou5f1-mediated regulation of pluripotency in human embryonic stem and epiblast cells.
Bone morphogenetic protein (BMP) signaling plays an essential role in early tooth development, evidenced by disruption of BMP signaling leading to an early arrested tooth development. Despite being a central mediator of BMP canonical signaling pathway, inactivation of Smad4 in dental mesenchyme does not result in early developmental defects. In the current study, we investigated the mechanism of receptor-activated Smads (R-Smads) and Smad4 in the regulation of the odontogenic gene Msx1 expression in the dental mesenchyme. We showed that the canonical BMP signaling is not operating in the early developing tooth, as assessed by failed activation of the BRE-Gal transgenic allele and the absence of phospho-(p)Smad1/5/8-Smad4 complexes. The absence of pSmad1/5/8-Smad4 complex appeared to be the consequence of saturation of Smad4 by pSmad2/3 in the dental mesenchyme as knockdown of Smad2/3 or overexpression of Smad4 led to the formation of pSmad1/5/8-Smad4 complexes and activation of canonical BMP signaling in dental mesenchymal cells. We showed that Smad1/5 but not Smad4 are required for BMP-induced expression of Msx1 in dental mesenchymal cells. We further presented evidence that in the absence of Smad4, BMPs are still able to induce pSmad1/5/8 nuclear translocation and their binding to the Msx1 promoter directly in dental mesenchymal cells. Our results demonstrate the functional operation of an atypical canonical BMP signaling (Smad4-independent and Smad1/5/8-dependent) pathway in the dental mesenchyme during early odontogenesis, which may have general implication in the development of other organs.
Head specification by the head-selector gene, orthodenticle (otx), is highly conserved among bilaterian lineages. However, the molecular mechanisms by which Otx and other transcription factors (TFs) interact with the genome to direct head formation are largely unknown. Here we employ ChIP-seq and RNA-seq approaches in Xenopus tropicalis gastrulae and find that occupancy of the corepressor, TLE/Groucho, is a better indicator of tissue-specific cis-regulatory modules (CRMs) than the coactivator p300, during early embryonic stages. On the basis of TLE binding and comprehensive CRM profiling, we define two distinct types of Otx2- and TLE-occupied CRMs. Using these devices, Otx2 and other head organizer TFs (for example, Lim1/Lhx1 (activator) or Goosecoid (repressor)) are able to upregulate or downregulate a large battery of target genes in the head organizer. An underlying principle is that Otx marks target genes for head specification to be regulated positively or negatively by partner TFs through specific types of CRMs.
Ag-Pd-Au-Cu alloys have been used widely for dental prosthetic applications. Significant enhancement of the mechanical properties of the Ag-20Pd-12Au-14.5Cu alloy as a result of the precipitation of the ?' phase through high-temperature solution treatment (ST), which is different from conventional aging treatment in these alloys, has been reported. The relationship between the unique hardening behavior and precipitation of the ?' phase in Ag-20Pd-12Au-xCu alloys (x=6.5, 13, 14.5, 17, and 20mass%) subjected to the high-temperature ST at 1123K for 3.6ks was investigated in this study. Unique hardening behavior after the high-temperature ST also occurs in Ag-20Pd-12Au-xCu alloys (x=13, 17, and 20) with precipitation of the ?' phase. However, hardening is not observed and the ?' phase does not precipitate in the Ag-20Pd-12Au-6.5Cu alloy after the same ST. The tensile strength and 0.2% proof stress also increase in Ag-20Pd-12Au-xCu alloys (x=13, 14.5, 17, and 20) after the high-temperature ST. In addition, these values after the high-temperature ST increase with increasing Cu content in Ag-20Pd-12Au-xCu alloys (x=14.5, 17, and 20). The formation process of the ?' phase can be explained in terms of diffusion of Ag and Cu atoms and precipitation of the ?' phase. Clarification of the relationship between hardening and precipitation of the ?' phase via high-temperature ST is expected to help the development of more effective heat treatments for hardening in Ag-20Pd-12Au-xCu alloys.
The age-hardening behavior of the dental-casting Ag-20Pd-12Au-14.5Cu alloy subjected to aging treatment at around 673K is well known, and this hardening has been widely employed in various applications. To date, the age-hardening of this alloy has been explained to attribute to the precipitation of a ? phase, which is a B2-type ordered CuPd phase or PdCuxZn1-x phase. In this study, results obtained from microstructural observations using a transmission electron microscopy and a scanning transmission electron microscopy revealed that a fine L10-type ordered ?' phase precipitated in the matrix and a coarse-structure region (consisting of Ag- and Cu-rich regions) appeared after aging treatment at 673K and contributed to increase in hardness. The microstructure of the coarse ? phase, which existed before aging treatment, did not change by aging treatment. Thus, it is concluded that the fine ?' phase precipitated by aging treatment contributed more to increase in hardness than the coarse-structure region and coarse ? phase.
Anodic oxide nanostructures (nanopores and nanotubes) were fabricated on a biomedical ?-type titanium alloy, Ti-29Nb-13Ta-4.6Zr alloy (TNTZ), by anodization in order to improve the adhesive strength of a medical polymer, segmented polyurethane (SPU), to TNTZ. TNTZ was anodized in 1.0M H3PO4 solution with 0.5 mass% NaF using a direct-current power supply at a voltage of 20V. A nanoporous structure is formed on TNTZ in the first stage of anodization, and the formation of a nanotube structure occurs subsequently beneath the nanoporous structure. The nanostructures formed on TNTZ by anodization for less than 3,600s exhibit higher adhesive strengths than those formed at longer anodization times. The adhesive strength of the SPU coating on the nanoporous structure formed on top of TNTZ by anodization for 1,200s improves by 144% compared to that of the SPU coating on as-polished TNTZ with a mirror surface. The adhesive strength of the SPU coating on the nanotube structure formed on TNTZ by anodization for 3,600s increases by 50%. These improvements in the adhesive strength of SPU are the result of an anchor effect introduced by the nanostructures formed by anodization. Fracture occurs at the interface of the nanoporous structure and the SPU coating layer. In contrast, in the case that SPU coating has been performed on the nanotube structure, fracture occurs inside the nanotubes.
The springback behavior of Ti-12Cr and Ti-29Nb-13Ta-4.6Zr (TNTZ) during deformation by bending was investigated; and the microstructures of the non-deformed and deformed parts of both alloys were systematically examined to clarify the relationship between microstructure and springback behavior. For the deformed Ti-12Cr alloy, deformation-induced ?-phase transformation occurs in both the areas of compression and tension within the deformed part, which increases the Young?s modulus. With the deformed TNTZ alloy, deformation-induced ?-phase transformation is observed in the area of compression within the deformed part; while a deformation-induced ?? martensite transformation occurs in the area under tension, which is likely to be associated with the pseudoelasticity of TNTZ. Among these two alloys, Ti-12Cr exhibits a smaller springback and a much greater bending strength when compared with TNTZ; making Ti-12Cr the more advantageous for spinal fixation applications.
During embryogenesis, signaling molecules produced by one cell population direct gene regulatory changes in neighboring cells and influence their developmental fates and spatial organization. One of the earliest events in the development of the vertebrate embryo is the establishment of three germ layers, consisting of the ectoderm, mesoderm and endoderm. Attempts to measure gene expression in vivo in different germ layers and cell types are typically complicated by the heterogeneity of cell types within biological samples (i.e., embryos), as the responses of individual cell types are intermingled into an aggregate observation of heterogeneous cell types. Here, we propose a novel method to elucidate gene regulatory circuits from these aggregate measurements in embryos of the frog Xenopus tropicalis using gene network inference algorithms and then test the ability of the inferred networks to predict spatial gene expression patterns.
Dental Ag-20Pd-12Au-14.5Cu alloys exhibit a unique hardening behavior, which the mechanical strengths enhance significantly which enhances the mechanical strength significantly after high-temperature (1123K) solution treatment without aging treatment. The mechanism of the unique hardening is not clear. The contribution of two precipitates (?' and ? phases) to the unique hardening behavior in the as-solutionized Ag-20Pd-12Au-14.5Cu alloys was investigated. In addition, the chemical composition of the ?' phase was investigated. The fine ?' phase densely precipitates in a matrix. The ?' phase (semi-coherent precipitate), which causes lattice strain, contributes greatly to the unique hardening behavior. On the other hand, the coarse ? phase sparsely precipitates in the matrix. The contribution of the ? phase (incoherent precipitate), which does not cause lattice strain, is small. The chemical composition of the ?' phase was determined. This study reveals that the fine ?' phase precipitated by high-temperature solution treatment leads to the unique hardening behavior in dental Ag-20Pd-12Au-14.5Cu alloys in the viewpoints of the lattice strain contrast and interface coherency. It is expected to make the heat treatment process more practical for hardening. The determined chemical composition of ?' phase would be helpful to study an unknown formation process of ?' phase.
In order to meet the requirements of the patients and surgeons simultaneously for spinal fixation applications, a novel biomedical alloy with a changeable Youngs modulus, that is, with a low Youngs modulus to prevent the stress-shielding effect for patients and a high Youngs modulus to suppress springback for surgeons, was developed. In this study, the chromium and oxygen contents in ternary Ti(11, 12mass%)Cr-(0.2, 0.4, 0.6mass%)O alloys were optimized in order to achieve a changeable Youngs modulus via deformation-induced ?-phase transformation with good mechanical properties. The Youngs moduli of all the examined alloys increase after cold rolling, which is attributed to the deformation-induced ?-phase transformation. This transformation is suppressed by oxygen but enhanced with lower chromium content, which is related to the ?(bcc)-lattice stability. Among the examined alloys, the Ti-11Cr-0.2O alloy shows a low Youngs modulus of less than 80GPa in the solution-treated (ST) condition and a high Youngs modulus of more than 90GPa in the cold rolled (CR) condition. The Ti-11Cr-0.2O alloy also exhibits a high tensile strength, above 1000MPa, with an acceptable elongation of ~12% in the ST condition. Furthermore, the Ti-11Cr-0.2O alloy exhibits minimal springback. This value of springback is the closest to that of Ti64 ELI alloy among the compared alloys. Therefore, the Ti-11Cr-0.2O alloy, which has a good balance between large changeable Youngs modulus, high tensile strength, good plasticity, and minimal springback, is considered to be a potential candidate for spinal fixation applications.
For spinal-fixation applications, implants should have a high Youngs modulus to reduce springback during operations, though a low Youngs modulus is required to prevent stress shielding for patients after surgeries. In the present study, Ti-29Nb-13Ta-4.6Zr alloy (TNTZ) with a low Youngs modulus was modified by adding Cr to obtain a higher deformation-induced Youngs modulus in order to satisfy these contradictory requirements. Two newly designed alloys, TNTZ-8Ti-2Cr and TNTZ-16Ti-4Cr, possess more stable ? phases than TNTZ. These alloys consist of single ? phases and exhibit relatively low Youngs moduli of <65GPa after solution treatment. However, after cold rolling, they exhibit higher Youngs moduli owing to a deformation-induced ?-phase transformation. These modified TNTZ alloys show significantly less springback than the original TNTZ alloy based on tensile and bending loading-unloading tests. Thus, the Cr-added TNTZ alloys are beneficial for spinal-fixation applications.
Bone morphogenetic proteins (BMPs) play crucial roles in craniofacial development but little is known about their interactions with other signals, such as Endothelin 1 (Edn1) and Jagged/Notch, which pattern the dorsal-ventral (DV) axis of the pharyngeal arches. Here, we use transgenic zebrafish to monitor and perturb BMP signaling during arch formation. With a BMP-responsive transgene, Tg(Bre:GFP), we show active BMP signaling in neural crest (NC)-derived skeletal precursors of the ventral arches, and in surrounding epithelia. Loss-of-function studies using a heat shock-inducible, dominant-negative BMP receptor 1a [Tg(hs70I:dnBmpr1a-GFP)] to bypass early roles show that BMP signaling is required for ventral arch development just after NC migration, the same stages at which we detect Tg(Bre:GFP). Inhibition of BMP signaling at these stages reduces expression of the ventral signal Edn1, as well as ventral-specific genes such as hand2 and dlx6a in the arches, and expands expression of the dorsal signal jag1b. This results in a loss or reduction of ventral and intermediate skeletal elements and a mis-shapen dorsal arch skeleton. Conversely, ectopic BMP causes dorsal expansion of ventral-specific gene expression and corresponding reductions/transformations of dorsal cartilages. Soon after NC migration, BMP is required to induce Edn1 and overexpression of either signal partially rescues ventral skeletal defects in embryos deficient for the other. However, once arch primordia are established the effects of BMPs become restricted to more ventral and anterior (palate) domains, which do not depend on Edn1. This suggests that BMPs act upstream and in parallel to Edn1 to promote ventral fates in the arches during early DV patterning, but later acquire distinct roles that further subdivide the identities of NC cells to pattern the craniofacial skeleton.
The basal chordate amphioxus resembles vertebrates in having a dorsal, hollow nerve cord, a notochord and somites. However, it lacks extensive gene duplications, and its embryos are small and gastrulate by simple invagination. Here we demonstrate that Nodal/Vg1 signaling acts from early cleavage through the gastrula stage to specify and maintain dorsal/anterior development while, starting at the early gastrula stage, BMP signaling promotes ventral/posterior identity. Knockdown and gain-of-function experiments show that these pathways act in opposition to one another. Signaling by these pathways is modulated by dorsally and/or anteriorly expressed genes including Chordin, Cerberus, and Blimp1. Overexpression and/or reporter assays in Xenopus demonstrate that the functions of these proteins are conserved between amphioxus and vertebrates. Thus, a fundamental genetic mechanism for axial patterning involving opposing Nodal and BMP signaling is present in amphioxus and probably also in the common ancestor of amphioxus and vertebrates or even earlier in deuterostome evolution.
The bone morphogenetic protein (BMP) signaling pathway is a conserved and evolutionarily ancient regulatory module affecting a large variety of cellular behaviors. The evolutionary flexibility in using BMP responses presumably arose by co-option of a canonical BMP signaling cascade to regulate the transcription of diverse batteries of target genes. This begs the question of how seemingly interchangeable BMP signaling components elicit widely different outputs in different cell types, an important issue in the context of understanding how BMP signaling integrates with gene regulatory networks to control development. Because a molecular understanding of how BMP signaling activates different batteries of target genes is an essential prerequisite to comprehending the roles of BMPs in regulating cellular responses, here we review the current knowledge of how BMP-regulated target genes are selected by the signal transduction machinery. We highlight recent studies suggesting the evolutionary conservation of BMP target gene regulation signaling by Schnurri family zinc finger proteins. Developmental Dynamics 238:1321-1331, 2009. (c) 2009 Wiley-Liss, Inc.
During embryogenesis, the pancreas develops from separate dorsal and ventral buds, which fuse to form the mature pancreas. Little is known about the functional differences between these two buds or the relative contribution of cells derived from each region to the pancreas after fusion. To follow the fate of dorsal or ventral bud derived cells in the pancreas after fusion, we produced chimeric Elas-GFP transgenic/wild-type embryos in which either dorsal or ventral pancreatic bud cells expressed GFP. We found that ventral pancreatic cells migrate extensively into the dorsal pancreas after fusion, whereas the converse does not occur. Moreover, we found that annular pancreatic tissue is composed exclusively of ventral pancreas-derived cells. To identify ventral pancreas-specific genes that may play a role in pancreatic bud fusion, we isolated individual dorsal and ventral pancreatic buds, prior to fusion, from NF38/39 Xenopus laevis tadpoles and compared their gene expression profiles (NF refers to the specific stage of Xenopus development). As a result of this screen, we have identified several new ventral pancreas-specific genes, all of which are expressed in the same location within the ventral pancreas at the junction where the two ventral pancreatic buds fuse. Morpholino-mediated knockdown of one of these ventral-specific genes, transmembrane 4 superfamily member 3 (tm4sf3), inhibited dorsal-ventral pancreatic bud fusion, as well as acinar cell differentiation. Conversely, overexpression of tm4sf3 promoted development of annular pancreas. Our results are the first to define molecular and behavioral differences between the dorsal and ventral pancreas, and suggest an unexpected role for the ventral pancreas in pancreatic bud fusion.
The FLRT family of transmembrane proteins has been implicated in the regulation of FGF signalling, neurite outgrowth, homotypic cell sorting and cadherin-mediated adhesion. In an expression screen we identified the Netrin receptors Unc5B and Unc5D as high-affinity FLRT3 interactors. Upon overexpression, Unc5B phenocopies FLRT3 and both proteins synergize in inducing cell deadhesion in Xenopus embryos. Morpholino knock-downs of Unc5B and FLRT3 synergistically affect Xenopus development and induce morphogenetic defects. The small GTPase Rnd1, which transmits FLRT3 deadhesion activity, physically and functionally interacts with Unc5B, and mediates its effect on cell adhesion. The results suggest that FLRT3, Unc5B and Rnd1 proteins interact to modulate cell adhesion in early Xenopus development.
The pancreas is both an exocrine and endocrine endodermal organ involved in digestion and glucose homeostasis. During embryogenesis, the anlagen of the pancreas arise from dorsal and ventral evaginations of the foregut that later fuse to form a single organ. To better understand the molecular genetics of early pancreas development, we sought to isolate markers that are uniquely expressed in this tissue. Microarray analysis was performed comparing dissected pancreatic buds, liver buds, and the stomach region of tadpole stage Xenopus embryos. A total of 912 genes were found to be differentially expressed between these organs during early stages of organogenesis. K-means clustering analysis predicted 120 of these genes to be specifically enriched in the pancreas. Of these, we report on the novel expression patterns of 24 genes. Our analyses implicate the involvement of previously unsuspected signaling pathways during early pancreas development. Developmental Dynamics 238:1455-1466, 2009. (c) 2009 Wiley-Liss, Inc.
Transcription of eukaryotic genes is an exceedingly sophisticated and complicated process, orchestrated by layers of control mechanisms involving a myriad of transcription factors and DNA control sequences, with both groups subject to multiple modifications. The availability of various recent genomic approaches has provided previously unforeseen opportunities to examine the cis-regulatory landscape of the entire genome, resulting in the identification of a potentially overwhelming number of enhancers and novel enhancer functions. In this review, we focus on the activities of enhancers in metazoans and discuss how they serve to regulate gene expression during early development.
Hydroxyapatite (HAp) films were deposited on a ?-type titanium alloy, Ti-29Nb-13Ta-4.6Zr (TNTZ), by metal organic chemical vapor deposition (MOCVD) in order to improve its hard-tissue compatibility. The surface morphologies of TNTZ substrates were changed by acid treatments and mechanical polishing prior to the HAp film deposition. The adhesive strength of the HAp films formed on TNTZ substrates treated with an HF solution increased to twice that of the HAp film deposited on a TNTZ substrate with a mirror-like finish. Complex microstructures with deeply etched grain boundaries, formed on the TNTZ substrates after immersion in the HF solution, were responsible for the increase in the adhesive strength of the HAp film caused by an interlocking effect. The HAp films on TNTZ substrates treated with a H(2)SO(4) solution exhibited lower adhesive strength than HAp films on TNTZ substrates treated with HF solution, regardless of the surface roughness of the substrates. Additionally, acid treatments using HNO(3) and H(2)O(2) solutions did not change the surface morphologies of the TNTZ substrates. The complex microstructures with deeply etched grain boundaries and nanosized asperities formed on the TNTZ substrates are important factors in the improvement of the adhesive strengths of HAp films deposited on TNTZ substrates.
Insights into Bone morphogenetic protein (Bmp) functions during forebrain development have been limited by a lack of Bmp signaling readouts. Here we used a novel Bmp signaling reporter ("BRE-gal" mice) to study Bmp signaling in the dorsal telencephalon. At early stages, BRE-gal expression was restricted to the dorsal telencephalic midline. At later stages, strong BRE-gal expression occurred in neurons of the marginal zone and dentate gyrus. Comparisons to nuclear phospho-Smad1/5/8 (pSmad) and Msx1 indicated that BRE-gal expression occurred exclusively in neural cells with high-level Bmp signaling. BRE-gal responsiveness to Bmps was confirmed in reporter-negative cortical cells cultured with Bmp4, and both in vivo and in vitro, BRE-gal expression was switch-like, or ultrasensitive. In the early dorsal telencephalon, BRE-gal expression negatively correlated with the cortical selector gene Lhx2, indicating a BRE-gal expression border that coincides with the cortex-hem boundary. However, in Lhx2 null chimeras, neither BRE-gal nor nuclear pSmad increases were observed in ectopic hem cells. These findings establish BRE-gal as an ultrasensitive reporter of Bmp signaling in the dorsal telencephalon, imply that hem fate can be specified at different Bmp signaling intensities, and suggest that Lhx2 primarily regulates the responses to--rather than the intensity of--Bmp signaling in dorsal telencephalic cells.
Cellular responses to Bmp ligands are regulated at multiple levels, both extracellularly and intracellularly. Therefore, the presence of these growth factors is not an accurate indicator of Bmp signaling activity. While a common approach to detect Bmp signaling activity is to determine the presence of phosphorylated forms of Smad1, 5 and 8 by immunostaining, this approach is time consuming and not quantitative. In order to provide a simpler readout system to examine the presence of Bmp signaling in developing animals, we developed BRE-gal mouse embryonic stem cells and a transgenic mouse line that specifically respond to Bmp ligand stimulation. Our reporter identifies specific transcriptional responses that are mediated by Smad1 and Smad4 with the Schnurri transcription factor complex binding to a conserved Bmp-Responsive Element (BRE), originally identified among Drosophila, Xenopus and human Bmp targets. Our BRE-gal mES cells specifically respond to Bmp ligands at concentrations as low as 5 ng/ml; and BRE-gal reporter mice, derived from the BRE-gal mES cells, show dynamic activity in many cellular sites, including extraembryonic structures and mammary glands, thereby making this a useful scientific tool.
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