Deciphering animal development through proteomics: requirements and prospects

In recent years proteomic techniques have started to become very useful tools in a variety of model systems of developmental biology. Applications cover many different aspects of development, including the characterization of changes in the proteome during early embryonic stages. During early animal development the embryo becomes patterned through the temporally and spatially controlled activation of distinct sets of genes. Patterning information is then translated, from gastrulation onwards, into regional specific morphogenetic cell and tissue movements that give the embryo its characteristic shape. On the molecular level, patterning is the outcome of intercellular communication via signaling molecules and the local activation or repression of transcription factors. Genetic approaches have been used very successfully to elucidate the processes behind these events. Morphogenetic movements, on the other hand, have to be orchestrated through regional changes in the mechanical properties of cells. The molecular mechanisms that govern these changes have remained much more elusive, at least in part due to the fact that they are more under translational/posttranslational control than patterning events. However, recent studies indicate that proteomic approaches can provide the means to finally unravel the mechanisms that link patterning to the generation of embryonic form. To intensify research in this direction will require close collaboration between proteome scientists and developmental researchers. It is with this aim in mind that we first give an outline of the classical questions of patterning and morphogenesis. We then summarize the proteomic approaches that have been applied in developmental model systems and describe the pioneering studies that have been done to study morphogenesis. Finally we discuss current and future strategies that will allow characterizing the changes in the embryonic proteome and ultimately lead to a deeper understanding of the cellular mechanisms that govern the generation of embryonic form

β-Catenin controls cell sorting at the notochord–somite boundary independently of cadherin-mediated adhesion

In Xenopus laevis, patterning of the trunk mesoderm into the dorsal notochord and lateral somites depends on differential regulation of Wnt–β-catenin signaling. To study the cellular requirements for the physical separation of these tissues, we manipulated β-catenin activity in individual cells that were scattered within the trunk mesoderm. We found that high activity led to efficient cell sorting from the notochord to the somites, whereas reduced activity led to sorting in the opposite direction. Analysis of individual cells overexpressing β-catenin revealed that these cells were unable to establish stable contacts with notochord cells but could freely cross the boundary to integrate within the somitic tissue. Interference with cadherin-mediated adhesion disrupted tissue architecture, but it did not affect sorting and boundary formation. Based on these results, we propose that the boundary itself is the result of cell-autonomous changes in contact behavior that do not rely on differences in absolute levels of adhesion

The tumor-associated EpCAM regulates morphogenetic movements through intracellular signaling.

Epithelial cell adhesion molecule (EpCAM) is best known as a tumor-associated protein highly expressed in carcinomas. The function of this cell surface protein during embryonic development and its potential role in cancer are still poorly understood. We identified EpCAM in a gain-of-function screen for inducers of abnormal tissue mixing during gastrulation. Elevated EpCAM levels in either the ectoderm or the mesoderm confer "invasive" properties to cells in both populations. We found that this phenotype represents an "overstimulation" of an essential activity of EpCAM in controlling cell movements during embryonic development. Surprisingly, this property is independent of the putative adhesive function of EpCAM, and rather relies on a novel signaling function that operates through down-regulation of PKC activity. We show that inhibition of novel PKCs accounts entirely for the invasive phenotype induced by abnormally high levels of EpCAM as well as for its normal function in regulating cell rearrangement during early development

Antibody Characterization Report for Optineurin (2023)

<p>This report presents a guide to selecting high-quality commercial antibodies against Optineurin by immunoblot (Western blot), immunoprecipitation and immunofluorescence, using a knockout based validation approach. The research displayed in this study can be considered a subsequent study following the initial Optineurin report published to the YCharOS community in 2021 (DOI:10.5281/zenodo.4730992). Antibodies ab213556**, 60293-1-Ig*, 702766** and 711879**, previously characterized in the initial report, were retested in all three applications, employing our revised standardized protocols. Additionally, new recombinant antibodies were subject to characterization.</p><p>This study was funded in part by Genome Québec's Genomics Integration Program, awarded to the research laboratory of Peter S. McPherson.This target was nominated by the ALS-Reproducible Antibody Platform (ALS-RAP), established as a public-private partnership by three prominent ALS charities - the ALS Association (USA), the Motor Neurone Disease Association (UK), and the ALS Society of Canada.</p><p> </p&gt

Antibody Characterization Report for TATA-binding protein associated factor 2N (TAF15)

<p>In this report, five commercial antibodies against TATA-binding protein associated factor 2N (TAF15) were characterized for use Western Blot, immunoprecipitation, and immunofluorescence, using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. </p><p>This work is part of the ALS-Reproducible Antibody Platform (ALS-RAP), established as a public-private partnership by three prominent ALS charities - the ALS Association (USA), the Motor Neurone Disease Association (UK), and the ALS Society of Canada.</p&gt

Antibody Characterization Report for Heterogeneous nuclear ribonucleoproteins A2/B1 (hnRNP A2/B1)

<p>This report presents a guide to selecting high-quality commercial antibodies against Heterogeneous nuclear ribonucleoproteins A2/B1 (hnRNP A2/B1) by immunoblot (Western blot), immunoprecipitation and immunofluorescence, using a knockout based validation approach.</p><p>This study was funded in part by the ALS-Reproducible Antibody Platform (ALS-RAP), established as a public-private partnership by three prominent ALS charities - the ALS Association (USA), the Motor Neurone Disease Association (UK), and the ALS Society of Canada.</p&gt

An Optimized Workflow to Generate and Characterize iPSC-Derived Motor Neuron (MN) Spheroids

A multitude of in vitro models based on induced pluripotent stem cell (iPSC)-derived motor neurons (MNs) have been developed to investigate the underlying causes of selective MN degeneration in motor neuron diseases (MNDs). For instance, spheroids are simple 3D models that have the potential to be generated in large numbers that can be used across different assays. In this study, we generated MN spheroids and developed a workflow to analyze them. To start, the morphological profiling of the spheroids was achieved by developing a pipeline to obtain measurements of their size and shape. Next, we confirmed the expression of different MN markers at the transcript and protein levels by qPCR and immunocytochemistry of tissue-cleared samples, respectively. Finally, we assessed the capacity of the MN spheroids to display functional activity in the form of action potentials and bursts using a microelectrode array approach. Although most of the cells displayed an MN identity, we also characterized the presence of other cell types, namely interneurons and oligodendrocytes, which share the same neural progenitor pool with MNs. In summary, we successfully developed an MN 3D model, and we optimized a workflow that can be applied to perform its morphological, gene expression, protein, and functional profiling over time