Evaluation of time profile reconstruction from complex two-color microarray designs

<p>Abstract</p> <p>Background</p> <p>As an alternative to the frequently used "reference design" for two-channel microarrays, other designs have been proposed. These designs have been shown to be more profitable from a theoretical point of view (more replicates of the conditions of interest for the same number of arrays). However, the interpretation of the measurements is less straightforward and a reconstruction method is needed to convert the observed ratios into the genuine profile of interest (e.g. a time profile). The potential advantages of using these alternative designs thus largely depend on the success of the profile reconstruction. Therefore, we compared to what extent different linear models agree with each other in reconstructing expression ratios and corresponding time profiles from a complex design.</p> <p>Results</p> <p>On average the correlation between the estimated ratios was high, and all methods agreed with each other in predicting the same profile, especially for genes of which the expression profile showed a large variance across the different time points. Assessing the similarity in profile shape, it appears that, the more similar the underlying principles of the methods (model and input data), the more similar their results. Methods with a dye effect seemed more robust against array failure. The influence of a different normalization was not drastic and independent of the method used.</p> <p>Conclusion</p> <p>Including a dye effect such as in the methods lmbr_dye, anovaFix and anovaMix compensates for residual dye related inconsistencies in the data and renders the results more robust against array failure. Including random effects requires more parameters to be estimated and is only advised when a design is used with a sufficient number of replicates. Because of this, we believe lmbr_dye, anovaFix and anovaMix are most appropriate for practical use.</p

Spatiotemporal lipid profiling during early embryo development of Xenopus laevis using dynamic Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) Imaging

Time-of-Flight secondary ion mass spectrometry (ToF-SIMS) imaging has been used for the direct analysis of single intact Xenopus laevis (X. laevis) embryo surfaces, locating multiple lipids during fertilisation and the early embryo development stages with sub-cellular lateral resolution (~4 Microns). The method avoids the complicated sample preparation for lipid analysis of the embryos, which requires selective chemical extraction of a pool of samples and chromatographic separation, while preserving the spatial distribution of biological species. The results show ToF-SIMS is capable of profiling multiple components (e.g., glycerophosphocholine, sphingomyelin, cholesterol, vitamin E, diacylglycerol, triacylglycerol) in a single X. laevis embryo. We observe lipid remodelling during fertilisation and early embryo development via time course sampling. The study also reveals the lipid distribution on the gametes fusion site. The methodology used in the study opens the possibility of studying developmental biology using high resolution imaging MS and of understanding the functional role of the biological molecules

Foxm1 regulates neural progenitor fate during spinal cord regeneration

From Wiley via Jisc Publications RouterHistory: received 2020-05-20, rev-recd 2021-06-24, accepted 2021-07-01, pub-electronic 2021-08-24Article version: VoRPublication status: PublishedFunder: Wellcome Trust; Grant(s): 205894/Z/17/ZFunder: Biotechnology and Biological Sciences Research Council Research Training Support; Id: http://dx.doi.org/10.13039/501100000268; Grant(s): BB/M011208/1Funder: UKRI|Medical Research Council (MRC); Id: http://dx.doi.org/10.13039/501100000265; Grant(s): MR/M008908/1Funder: Wellcome Trust (ISSF fund)Abstract: Xenopus tadpoles have the ability to regenerate their tails upon amputation. Although some of the molecular and cellular mechanisms that globally regulate tail regeneration have been characterised, tissue‐specific response to injury remains poorly understood. Using a combination of bulk and single‐cell RNA sequencing on isolated spinal cords before and after amputation, we identify a number of genes specifically expressed in the spinal cord during regeneration. We show that Foxm1, a transcription factor known to promote proliferation, is essential for spinal cord regeneration. Surprisingly, Foxm1 does not control the cell cycle length of neural progenitors but regulates their fate after division. In foxm1−/− tadpoles, we observe a reduction in the number of neurons in the regenerating spinal cord, suggesting that neuronal differentiation is necessary for the regenerative process. Altogether, our data uncover a spinal cord‐specific response to injury and reveal a new role for neuronal differentiation during regeneration

Analysis of neural progenitors from embryogenesis to juvenile adult in Xenopus laevis reveals biphasic neurogenesis and continuous lengthening of the cell cycle.

Xenopus laevis is a prominent model system for studying neural development, but our understanding of the long-term temporal dynamics of neurogenesis remains incomplete. Here, we present the first continuous description of neurogenesis in X. laevis, covering the entire period of development from the specification of neural ectoderm during gastrulation to juvenile frog. We have used molecular markers to identify progenitors and neurons, short-term bromodeoxyuridine (BrdU) incorporation to map the generation of newborn neurons and dual pulse S-phase labelling to characterise changes in their cell cycle length. Our study revealed the persistence of Sox3-positive progenitor cells from the earliest stages of neural development through to the juvenile adult. Two periods of intense neuronal generation were observed, confirming the existence of primary and secondary waves of neurogenesis, punctuated by a period of quiescence before metamorphosis and culminating in another period of quiescence in the young adult. Analysis of multiple parameters indicates that neural progenitors alternate between global phases of differentiation and amplification and that, regardless of their behaviour, their cell cycle lengthens monotonically during development, at least at the population level

aPKC Phosphorylates p27Xic1, Providing a Mechanistic Link between Apicobasal Polarity and Cell-Cycle Control

SummaryDuring the development of the nervous system, apicobasally polarized stem cells are characterized by a shorter cell cycle than nonpolar progenitors, leading to a lower differentiation potential of these cells. However, how polarization might be directly linked to the kinetics of the cell cycle is not understood. Here, we report that apicobasally polarized neuroepithelial cells in Xenopus laevis have a shorter cell cycle than nonpolar progenitors, consistent with mammalian systems. We show that the apically localized serine/threonine kinase aPKC directly phosphorylates an N-terminal site of the cell-cycle inhibitor p27Xic1 and reduces its ability to inhibit the cyclin-dependent kinase 2 (Cdk2), leading to shortening of G1 and S phases. Overexpression of activated aPKC blocks the neuronal differentiation-promoting activity of p27Xic1. These findings provide a direct mechanistic link between apicobasal polarity and the cell cycle, which may explain how proliferation is favored over differentiation in polarized neural stem cells

Cloning and Characterization of Three Xenopus Slug Promoters Reveal Direct Regulation by Lef/\uce\ub2-Catenin Signaling

In amphibians and birds, one of the first steps of neu- ral crest cell (NCC) determination is expression of the transcription factor Slug. This marker has been used to demonstrate that BMP and Wnt molecules play a major role in NCC induction. However, it is unknown whether Slug expression is directly or indirectly regulated by these signals. We report here the cloning and character- ization of three Xenopus Slug promoters: that of the Xenopus tropicalis slug gene and those of two Xenopus laevis Slug pseudoalleles. Although the three genes en- code proteins with almost identical amino acid se- quences and are expressed with similar spatiotemporal patterns, their 5 -flanking regions are quite different. A striking difference is a deletion in the X. tropicalis gene located precisely at the transcription initiation site that results in the X. tropicalis promoter being inefficient in X. laevis. Additionally, we identified two regions com- mon to the three promoters that are necessary and suf- ficient to drive specific expression in NCCs. Interest- ingly, one of the common regulatory regions presents a functional Lef/ -catenin-binding site necessary for spe- cific expression. As the Lef -catenin complex is a down- stream effector of Wnt signaling, these results suggest that Xenopus Slug is a direct target of NCC determina- tion signals

Foxm1 regulates neural progenitor fate during spinal cord regeneration

Xenopus tadpoles have the ability to regenerate their tails upon amputation. Although some of the molecular and cellular mechanisms that globally regulate tail regeneration have been characterised, tissue‐specific response to injury remains poorly understood. Using a combination of bulk and single‐cell RNA sequencing on isolated spinal cords before and after amputation, we identify a number of genes specifically expressed in the spinal cord during regeneration. We show that Foxm1, a transcription factor known to promote proliferation, is essential for spinal cord regeneration. Surprisingly, Foxm1 does not control the cell cycle length of neural progenitors but regulates their fate after division. In foxm1 (−/−) tadpoles, we observe a reduction in the number of neurons in the regenerating spinal cord, suggesting that neuronal differentiation is necessary for the regenerative process. Altogether, our data uncover a spinal cord‐specific response to injury and reveal a new role for neuronal differentiation during regeneration

Spatiotemporal lipid profiling during early embryo development of Xenopus laevis using dynamic ToF-SIMS imaging

Time-of-flight secondary ion mass spectrometry (ToF-SIMS) imaging has been used for the direct analysis of single intact Xenopus laevis embryo surfaces, locating multiple lipids during fertilization and the early embryo development stages with subcellular lateral resolution (∼4 μm). The method avoids the complicated sample preparation for lipid analysis of the embryos, which requires selective chemical extraction of a pool of samples and chromatographic separation, while preserving the spatial distribution of biological species. The results show ToF-SIMS is capable of profiling multiple components (e.g., glycerophosphocholine, SM, cholesterol, vitamin E, diacylglycerol, and triacylglycerol) in a single X. laevis embryo. We observe lipid remodeling during fertilization and early embryo development via time course sampling. The study also reveals the lipid distribution on the gamete fusion site. The methodology used in the study opens the possibility of studying developmental biology using high resolution imaging MS and of understanding the functional role of the biological molecules