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Systematic functional profiling of the gene set that directs embryonic development is an important challenge. To tackle this challenge, we used 4D imaging of C. elegans embryogenesis to capture the effects of 500 gene knockdowns and developed an automated approach to compare developmental phenotypes. The automated approach quantifies features-including germ layer cell numbers, tissue position, and tissue shape-to generate temporal curves whose parameterization yields numerical phenotypic signatures. In conjunction with a new similarity metric that operates across phenotypic space, these signatures enabled the generation of ranked lists of genes predicted to have similar functions, accessible in the PhenoBank web portal, for ∼25% of essential development genes. The approach identified new gene and pathway relationships in cell fate specification and morphogenesis and highlighted the utilization of specialized energy generation pathways during embryogenesis. Collectively, the effort establishes the foundation for comprehensive analysis of the gene set that builds a multicellular organism., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

This study tackles the individual and joint effect of alpha -tocopherol and hydroxytyrosol acetate on the oxidation of sunflower oil submitted to accelerated storage conditions at intermediate temperature, in order to deepen the understanding of antioxidant-prooxidant behaviour. This was accomplished by 1 H Nuclear Magnetic Resonance. For this purpose, the evolution of the degradation of both the main components of the oil and the aforementioned added compounds was monitored by this technique throughout the storage time. Furthermore, the formation of a very large number of oxylipins and the evolution of their concentration up to a very advanced stage of oil oxidation, as well as the occurrence of lipolysis, were also simultaneously studied. The results obtained show very clearly and thoroughly that in the oxidation process of the oil enriched in binary mixtures, interactions occur between alpha -tocopherol and hydroxytyrosol acetate that notably reduce the antioxidant effect of the latter compound with the corresponding negative consequences that this entails. The methodology used here has proved to be very efficient to evaluate the antioxidant power of mixtures of compounds.

Sunflower oil samples, both unenriched and enriched with four different concentrations of hydroxytyrosol acetate, were subjected to accelerated storage at 70 °C until a very advanced oxidation stage and the process was monitored by 1 H NMR spectroscopy. The aim of the study is to know the effect that the presence of this antioxidant has on the oxidation process of sunflower oil under the aforementioned conditions, as well as on the formation and evolution of the concentration of a significant number of oxylipins. The oxidation process was studied globally by monitoring, during storage time, the degradation of both the linoleic acyl group of sunflower oil, which is the main component of sunflower oil, and the added hydroxytyrosol acetate. Simultaneously, the identification of up to twenty-six different types of oxylipins formed in the oxidation process and the monitoring of the evolution of their concentration over the storage time were carried out. In this way, essential information about the effect that hydroxytyrosol acetate provokes on the oxidation of this oil rich in omega-6 polyunsaturated acyl groups, has been obtained. It has also been shown that the enrichment of sunflower oil with this antioxidant under the conditions tested does not prevent the oxidation process but slows it down, affecting the entire oxidation process.

Lipid oxidation causes food degradation and the formation of toxic compounds. Therefore, the addition to foods of compounds able to avoid, delay or minimize this degradative process is a commonly used strategy. Nevertheless, neither the identity of most of the formed compounds in this complex process nor the way in which their formation is affected by the strategy used are well known. In this context, the effect the temperature increase and the enrichment level in alpha-tocopherol on the evolution of the walnut oil oxidation, as a model of an oil rich in polyunsaturated omega-6 acyl groups, submitted to storage conditions, are tackled by 1 H NMR. The study has allowed knowing the degradation kinetic of both the oil acyl groups and alpha-tocopherol, the identification of a very high number of oxylipins and the kinetic of their formation. The temperature increase accelerates the formation of all oxylipins, favouring the formation of hydroperoxy conjugated E , E -dienes and related derivatives versus that of the Z , E -isomers. The enrichment in alpha-tocopherol accelerates the formation of hydroperoxy conjugated Z , E -dienes and related derivatives, and delays in relation to the formation of the former that of the E , E -isomers and related derivatives, hindering, to a certain extent, the formation of the latter in line with the enrichment level.

C. elegans is the premier system for the systematic analysis of cell fate specification and morphogenetic events during embryonic development. One challenge is that embryogenesis dynamically unfolds over a period of about 13 h; this half day-long timescale has constrained the scope of experiments by limiting the number of embryos that can be imaged. Here, we describe a semi-high-throughput protocol that allows for the simultaneous 3D time-lapse imaging of development in 80-100 embryos at moderate time resolution, from up to 14 different conditions, in a single overnight run. The protocol is straightforward and can be implemented by any laboratory with access to a microscope with point visiting capacity. The utility of this protocol is demonstrated by using it to image two custom-built strains expressing fluorescent markers optimized to visualize key aspects of germ-layer specification and morphogenesis. To analyze the data, a custom program that crops individual embryos out of a broader field of view in all channels, z-steps, and timepoints and saves the sequences for each embryo into a separate tiff stack was built. The program, which includes a user-friendly graphical user interface (GUI), streamlines data processing by isolating, pre-processing, and uniformly orienting individual embryos in preparation for visualization or automated analysis. Also supplied is an ImageJ macro that compiles individual embryo data into a multi-panel file that displays maximum intensity fluorescence projection and brightfield images for each embryo at each time point. The protocols and tools described herein were validated by using them to characterize embryonic development following knock-down of 40 previously described developmental genes; this analysis visualized previously annotated developmental phenotypes and revealed new ones. In summary, this work details a semi-high-throughput imaging method coupled with a cropping program and ImageJ visualization tool that, when combined with strains expressing informative fluorescent markers, greatly accelerates experiments to analyze embryonic development.

Undergraduate research experiences are excellent opportunities to engage students in science alongside experienced scientists, but at large institutions, it is challenging to accommodate all students. To address and engage a larger number of students, we developed a modular laboratory course based on the course-based undergraduate research experiences model. This new course was integrated with the scientific aims of a research laboratory studying the cellular and molecular mechanisms underlying tissue regeneration in planarians. In this course, students were asked to identify genes with roles in planarian biology. Students analyzed and cloned an assigned gene, determined its expression pattern in situ and examined its function in regeneration. Additionally, we developed critical thinking and scientific communication skills by incorporating activities focused on critical concepts. Students obtained high quality primary data and were successful in completing and mastering the course learning outcomes. They benefitted by developing basic research skills, learning to perform, trouble-shooting experiments, reading and critically analyzing primary literature, and using the information to defend and explain their experimental results. Through this course, students also increased their confidence and ability to perform independent scientific research. The course was designed to make it accessible to the community to implement and adapt as appropriate in diverse institutions. © 2019 International Union of Biochemistry and Molecular Biology, 47(5):547-559, 2019., (© 2019 International Union of Biochemistry and Molecular Biology.)

The Caenorhabditis elegans embryo is an important model for analyzing mechanisms of cell fate specification and tissue morphogenesis. Sophisticated lineage-tracing approaches for analyzing embryogenesis have been developed but are labor intensive and do not naturally integrate morphogenetic readouts. To enable the rapid classification of developmental phenotypes, we developed a high-content method that employs two custom strains: a Germ Layer strain that expresses nuclear markers in the ectoderm, mesoderm and endoderm/pharynx; and a Morphogenesis strain that expresses markers labeling epidermal cell junctions and the neuronal cell surface. We describe a procedure that allows simultaneous live imaging of development in 80-100 embryos and provide a custom program that generates cropped, oriented image stacks of individual embryos to facilitate analysis. We demonstrate the utility of our method by perturbing 40 previously characterized developmental genes in variants of the two strains containing RNAi-sensitizing mutations. The resulting datasets yielded distinct, reproducible signature phenotypes for a broad spectrum of genes that are involved in cell fate specification and morphogenesis. In addition, our analysis provides new in vivo evidence for MBK-2 function in mesoderm fate specification and LET-381 function in elongation., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Non-centrosomal microtubule arrays assemble in differentiated tissues to perform mechanical and transport-based functions. In this study, we identify Caenorhabditis elegans NOCA-1 as a protein with homology to vertebrate ninein. NOCA-1 contributes to the assembly of non-centrosomal microtubule arrays in multiple tissues. In the larval epidermis, NOCA-1 functions redundantly with the minus end protection factor Patronin/PTRN-1 to assemble a circumferential microtubule array essential for worm growth and morphogenesis. Controlled degradation of a γ-tubulin complex subunit in this tissue revealed that γ-tubulin acts with NOCA-1 in parallel to Patronin/PTRN-1. In the germline, NOCA-1 and γ-tubulin co-localize at the cell surface, and inhibiting either leads to a microtubule assembly defect. γ-tubulin targets independently of NOCA-1, but NOCA-1 targeting requires γ-tubulin when a non-essential putatively palmitoylated cysteine is mutated. These results show that NOCA-1 acts with γ-tubulin to assemble non-centrosomal arrays in multiple tissues and highlight functional overlap between the ninein and Patronin protein families.

The centrosome organizes microtubule arrays within animal cells and comprises two centrioles surrounded by an amorphous protein mass called the pericentriolar material (PCM). Despite the importance of centrosomes as microtubule-organizing centers, the mechanism and regulation of PCM assembly are not well understood. In Caenorhabditis elegans, PCM assembly requires the coiled-coil protein SPD-5. We found that recombinant SPD-5 could polymerize to form micrometer-sized porous networks in vitro. Network assembly was accelerated by two conserved regulators that control PCM assembly in vivo, Polo-like kinase-1 and SPD-2/Cep192. Only the assembled SPD-5 networks, and not unassembled SPD-5 protein, functioned as a scaffold for other PCM proteins. Thus, PCM size and binding capacity emerge from the regulated polymerization of one coiled-coil protein to form a porous network., (Copyright © 2015, American Association for the Advancement of Science.)

A subset of transcription factors classified as neural crest 'specifiers' are also core epithelial-mesenchymal transition regulatory factors, both in the neural crest and in tumour progression. The bHLH factor Twist is among the least well studied of these factors. Here we demonstrate that Twist is required for cranial neural crest formation and fate determination in Xenopus. We further show that Twist function in the neural crest is dependent upon its carboxy-terminal WR domain. The WR domain mediates physical interactions between Twist and other core epithelial-mesenchymal transition factors, including Snail1 and Snail2, which are essential for proper function. Interaction with Snail1/2, and Twist function more generally, is regulated by GSK-3-β-mediated phosphorylation of conserved sites in the WR domain. Together, these findings elucidate a mechanism for coordinated control of a group of structurally diverse factors that function as a regulatory unit in both developmental and pathological epithelial-mesenchymal transitions.

The neural crest (NC) is a population of multipotent stem cell-like progenitors that arise at the neural plate border in vertebrates and migrate extensively before giving rise to diverse derivatives. A number of components of the neural crest gene regulatory network (NC-GRN) are used reiteratively to control multiple steps in the development of these cells. It is therefore important to understand the mechanisms that control the distinct function of reiteratively used factors in different cellular contexts, and an important strategy for doing so is to identify and characterize the regulatory factors they interact with. Here we report that the LIM adaptor protein, LMO4, is a Slug/Snail interacting protein that is essential for NC development. LMO4 is expressed in NC forming regions of the embryo, as well as in the central nervous system and the cranial placodes. LMO4 is necessary for normal NC development as morpholino-mediated knockdown of this factor leads to loss of NC precursor formation at the neural plate border. Misexpression of LMO4 leads to ectopic expression of some neural crest markers, but a reduction in the expression of others. LMO4 binds directly to Slug and Snail, but not to other components of the NC-GRN and can modulate Slug-mediated neural crest induction, suggesting a mechanistic link between these factors. Together these findings implicate LMO4 as a critical component of the NC-GRN and shed new light on the control of Snail family repressors., (Copyright © 2011 Elsevier Inc. All rights reserved.)