The Ribosome Biogenesis Factor Nol11 Is Required for Optimal rDNA Transcription and Craniofacial Development in Xenopus

The production of ribosomes is ubiquitous and fundamental to life. As such, it is surprising that defects in ribosome biogenesis underlie a growing number of symptomatically distinct inherited disorders, collectively called ribosomopathies. We previously determined that the nucleolar protein, NOL11, is essential for optimal pre-rRNA transcription and processing in human tissue culture cells. However, the role of NOL11 in the development of a multicellular organism remains unknown. Here, we reveal a critical function for NOL11 in vertebrate ribosome biogenesis and craniofacial development. Nol11 is strongly expressed in the developing cranial neural crest (CNC) of both amphibians and mammals, and knockdown of Xenopus nol11 results in impaired pre-rRNA transcription and processing, increased apoptosis, and abnormal development of the craniofacial cartilages. Inhibition of p53 rescues this skeletal phenotype, but not the underlying ribosome biogenesis defect, demonstrating an evolutionarily conserved control mechanism through which ribosome-impaired craniofacial cells are removed. Excessive activation of this mechanism impairs craniofacial development. Together, our findings reveal a novel requirement for Nol11 in craniofacial development, present the first frog model of a ribosomopathy, and provide further insight into the clinically important relationship between specific ribosome biogenesis proteins and craniofacial cell survival

Loading rate effect on the fracture behaviour of highstrength concrete

This research deals with the sensitivity of eight types of performancedesigned high-strength concrete to the loading rate. Variations in the composition of the concrete produce the desired performance, for instance having null shrinkage or being able to be pumped at elevated heights without segregation, but they also produce variations in the fracture properties that are reported in this paper. We performed tests at five loading rates spanning six orders of magnitude in the displacement rate, from 1.74 × 10-5 mm/s to 17.4 mm/s. Load-displacement curves show that their peak is higher as the displacement rate increases, whereas the corresponding displacement is almost constant. Fracture energy also increases, but only for loading rates higher than 0.01 mm/s. We use a formula based on a cohesive law with a viscous term to study the results. The correlation of the formula to the experimental results is good and it allows us to obtain the theoretical value for the fracture energy under strictly static conditions. In addition, both the fracture energy and the characteristic length of the concretes used in the study diminish as the compressive strength of their aggregates increases

Exon capture and bulk segregant analysis: rapid discovery of causative mutations using high-throughput sequencing

<p>Abstract</p> <p>Background</p> <p>Exome sequencing has transformed human genetic analysis and may do the same for other vertebrate model systems. However, a major challenge is sifting through the large number of sequence variants to identify the causative mutation for a given phenotype. In models like <it>Xenopus tropicalis</it>, an incomplete and occasionally incorrect genome assembly compounds this problem. To facilitate cloning of <it>X. tropicalis</it> mutants identified in forward genetic screens, we sought to combine bulk segregant analysis and exome sequencing into a single step.</p> <p>Results</p> <p>Here we report the first use of exon capture sequencing to identify mutations in a non-mammalian, vertebrate model. We demonstrate that bulk segregant analysis coupled with exon capture sequencing is not only able to identify causative mutations but can also generate linkage information, facilitate the assembly of scaffolds, identify misassembles, and discover thousands of SNPs for fine mapping.</p> <p>Conclusion</p> <p>Exon capture sequencing and bulk segregant analysis is a rapid, inexpensive method to clone mutants identified in forward genetic screens. With sufficient meioses, this method can be generalized to any model system with a genome assembly, polished or unpolished, and in the latter case, it also provides many critical genomic resources.</p

RAPGEF5 regulates nuclear translocation of β-catenin

SUMMARYCanonical Wnt signaling coordinates many critical aspects of embryonic development, while dysregulated Wnt signaling contributes to common diseases, including congenital malformations and cancer. The nuclear localization of β-catenin is the defining step in pathway activation. However, despite intensive investigation, the mechanisms regulating β-catenin nuclear transport remain undefined. In a patient with congenital heart disease and heterotaxy, a disorder of left-right patterning, we previously identified the guanine nucleotide exchange factor, RAPGEF5. Here, we demonstrate that RAPGEF5 regulates left-right patterning via Wnt signaling. In particular, RAPGEF5, regulates the nuclear translocation of β-catenin independently of both β-catenin cytoplasmic stabilization and the importin β1/Ran mediated transport system. We propose a model whereby RAPGEF5 activates the nuclear GTPases, Rap1/2, to facilitate the nuclear transport of β-catenin, defining a parallel nuclear transport pathway to Ran. Our results suggest new targets for modulating Wnt signaling in disease states.</jats:p

Expression of <i>nol11</i> during vertebrate development.

<p>A) Wild type <i>nol11</i> expression pattern during <i>Xenopus tropicalis</i> development. Note the strong expression in developing neural folds (NF) and the presumptive CNC at stages 16 and 22 (lateral [left] and dorsal [right] views presented). Expression is strongly associated with the migrating and differentiating CNC at subsequent stages, and is also detected in the region of the ventral blood islands (BI) and isthmus (Is) at stage 28. BA, branchial arch; Ht, heart; Lv, liver region; Op, optic placode. B) and C) Anterior transverse dissections showing expression of <i>nol11</i> in neural folds and premigratory CNC of stage 14 and 16 embryos respectively. Plane of dissection is represented by the red dotted line marked c in A. D) Horizontal dissection (shown as dotted red line marked d in A) of <i>nol11</i> expression in the branchial arches of stage 28 <i>Xenopus</i> embryos. E) <i>nol11</i> expression in E8.5, E9.5 and E10.5 wild type mouse embryos. At E8.5 expression is strongly detected in the neural folds. Transcripts are associated with CNC positive regions at both E9.5 and E10.5. BA2, 2^nd branchial arch; FNP, frontonasal prominence; Ht, heart; mdBA1, mandibular BA1; mxBA1, maxillary BA1; Op, optic placode; Ot, otic placode; T, trigeminal region.</p

Nol11 depletion impairs rDNA transcription and pre-rRNA processing in <i>X</i>. <i>tropicalis</i>.

<p>A) Scheme of pre-rRNA processing pathways in <i>X tropicalis</i>. The pre-rRNA is transcribed by RNAPI as a 40S polycistronic precursor. Several cleavages are required to separate the mature rRNAs. The locations of oligonucleotide probes used for northern blots are indicated by lettered lines (a, c) and the cleavage sites indicated. This scheme was adapted from [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005018#pgen.1005018.ref071" target="_blank">71</a>–<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005018#pgen.1005018.ref075" target="_blank">75</a>]. B) Morpholino (MO) depletion of Nol11 impairs pre-rRNA transcription at stage 28. The northern blot was hybridized with probe a (Fig. 5A) and with a probe to the 7SL RNA as a loading control (lower panel). Bands were quantified and analysed by RAMP ([<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005018#pgen.1005018.ref060" target="_blank">60</a>]; <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005018#pgen.1005018.s006" target="_blank">S6A,B Fig</a>) C) Morpholino (MO) depletion of Nol11 impairs pre-rRNA transcription and processing. The northern blot was hybridized with probe c (Fig. 5A) and with a probe to the 7SL RNA as a loading control (lower panel). Bands were quantified and analysed by RAMP ([<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005018#pgen.1005018.ref060" target="_blank">60</a>]; <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005018#pgen.1005018.s006" target="_blank">S6C,D, E, F Fig</a>). D) Depletion of Nol11 leads to increased p53 levels. The expression of p53 from control and <i>nol11</i> depleted embryos was analysed by western blot with anti-p53 antibodies. GAPDH levels were used as a loading control. Values for p53 expression normalized to GAPDH are represented in the bar graph. E) MO-resistant human NOL11 (hNOL11) mRNA but not p53 depletion rescues pre-rRNA levels. Embryos injected as shown by + and—in the figure at stages 22 and 28. The pre-rRNAs were visualized with probe a on a northern blot; hybridization to the 7SL RNA was used as a loading control.</p

The nol11 craniofacial phenotype.

<p>A) Gross morphology and cartilage staining of UC, <i>nol11</i> whole embryo, <i>nol11</i> one-sided knockdowns and CMO one-sided knockdown embryos. Note the reduced cartilage size and abnormal morphology in <i>nol11</i> morphants (red arrowheads) while CMO injected embryos are unaffected. B) Craniofacial cartilage size is significantly reduced in <i>nol11</i> but not CMO morphants. C) Co-injection of human NOL11 RNA can rescue the cartilage phenotype in approximately 75% of treated embryos. Cartilage staining of an RNA rescued embryo; <i>nol11</i> MO was injected at the one cell stage and human <i>NOL11</i> RNA was injected into one cell at the two cell stage. Green arrowheads highlight rescued side.</p