Developmental Biology


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0 Q&A 440 Views Feb 5, 2024

Vertebrate embryogenesis is a highly dynamic process involving coordinated cell and tissue movements that generate the final embryonic body plan. Many of these movements are difficult to image at high resolution because they occur deep within the embryo along the midline, causing light scattering and requiring longer working distances. Here, we present an explant-based method to image transverse cross sections of living zebrafish embryos. This method allows for the capture of all cell movements at high-resolution throughout the embryonic trunk, including hard-to-image deep tissues. This technique offers an alternative to expensive or computationally difficult microscopy methods.

Key features

• Generates intact zebrafish explants with minimal tissue disturbance.

• Allows for live imaging of deep tissues normally obscured by common confocal microscopy techniques.

• Immobilizes tissues for extended periods required for time-lapse imaging.

• Utilizes readily available reagents and tools, which can minimize the time and cost of the procedure.

Graphical overview

0 Q&A 845 Views Jan 20, 2024

All living organisms require the division of a cell into daughter cells for their growth and maintenance. During cell division, both genetic and cytoplasmic contents are equally distributed between the two daughter cells. At the end of cell division, cytoplasmic contents and the plasma membrane are physically separated between the two daughter cells via a process known as cytokinesis. Hundreds of proteins and lipids involved in the cytokinetic process have been identified; however, much less is known about the mechanisms by which these molecules regulate cytokinesis, being therefore an intense area of current research. Male meiotic cytokinesis in Drosophila melanogaster testes has been shown to be an excellent model to study cytokinesis in vivo. Currently, several excellent protocols are available to study cytokinesis in Drosophila testes. However, improved methods are required to study cytokinesis under in vitro and ex vivo conditions. Here, we demonstrate a simple method to perform live imaging on individual spermatocyte cysts isolated from adult testes. We evaluate amenability of this in vitro method for treatment with pharmacological agents. We show that cytokinesis is strongly inhibited upon treatment with Dynasore, a dynamin inhibitor known to block clathrin-mediated endocytosis. In addition, we also demonstrate an ex vivo method to perform live imaging on whole mount adult testes on gas permeable membrane chambers. We believe the protocols described here are valuable tools to study cytokinetic mechanisms under various genetic and treatment conditions.

Key features

• In vitro method to study male meiotic cytokinesis in dissected spermatocyte cysts.

• In vitro method allows acute treatment with various pharmacological agents to study cytokinesis.

• Ex vivo method to image male meiosis cytokinesis in intact adult testes.

• Requires 15–60 min to set up and could be imaged up to 6–12 h.

Graphical overview

In vitro and ex vivo live imaging of male meiotic cytokinesis in adult Drosophila testes

0 Q&A 1205 Views Dec 20, 2023

The African killifish Nothobranchius furzeri is an attractive research organism for regeneration- and aging-related studies due to its remarkably short generation time and rapid aging. Dynamic changes in cell proliferation are an essential biological process involved in development, regeneration, and aging. Quantifying the dynamics of cell proliferation in these contexts facilitates the elucidation of the attendant underlying mechanisms. Whole-mount and cryosectioning sample preparation are the preferred approaches to investigate the distribution of cellular structures, cell–cell communication, and spatial gene expression within tissues. Using African killifish caudal fin regeneration as an example, we describe an efficient and detailed protocol to investigate cell proliferation dynamics in both space and time during caudal fin regeneration. The quantification of cell proliferation was achieved through high-resolution immunofluorescence of the proliferation marker Phospho-Histone H3 (H3P). We focused on the characterization of epithelial and mesenchymal proliferation in three-dimensional space at two regeneration time points. Our protocol provides a reliable tool for comparing cell proliferation under different biological contexts.

Key features

• Elaborates in detail the method used by Wang et al. (2020) to quantify whole-organ mitotic events during tail fin regeneration in vertebrates.

• Enables proliferation analysis of millimeter-sized homeostatic and regenerating tissues.

• Three-day alternative method to whole mount using cryosections.

• Allows automatic quantification using ImageJ macros and R scripts.

Graphical overview

0 Q&A 336 Views Dec 5, 2023

The hypothalamus is an evolutionarily ancient part of the vertebrate ventral forebrain that integrates the dialogue between environment, peripheral body, and brain to centrally govern an array of physiologies and behaviours. Characterizing the mechanisms that control hypothalamic development illuminates both hypothalamic organization and function. Critical to the ability to unravel such mechanisms is the skill to isolate hypothalamic tissue, enabling both its acute analysis and its analysis after explant and culture. Tissue explants, in which cells develop in a manner analogous to their in vivo counterparts, are a highly effective tool to investigate the extrinsic signals and tissue-intrinsic self-organising features that drive hypothalamic development. The hypothalamus, however, is induced and patterned at neural tube stages of development, when the tissue is difficult to isolate, and its resident cells complex to define. No single molecular marker distinguishes early hypothalamic progenitor subsets from other cell types in the neural tube, and so their accurate dissection requires the simultaneous analysis of multiple proteins or mRNAs, techniques that were previously limited by antibody availability or were arduous to perform. Here, we overcome these challenges. We describe methodologies to precisely isolate early hypothalamic tissue from the embryonic chick at three distinct patterning stages and to culture hypothalamic explants in three-dimensional gels. We then describe optimised protocols for the analysis of embryos, isolated embryonic tissue, or cultured hypothalamic explants by multiplex hybridisation chain reaction. These methods can be applied to other vertebrates, including mouse, and to other tissue types.

Key features

• Detailed protocols for enzymatic isolation of embryonic chick hypothalamus at three patterning stages; methods can be extended to other vertebrates and tissues.

• Brief methodologies for three-dimensional culture of hypothalamic tissue explants.

• Optimised protocols for multiplex hybridisation chain reaction for analysis of embryos, isolated embryonic tissues, or explants.

Graphical overview

0 Q&A 426 Views Dec 5, 2023

Neovascular diseases of the retina, such as diabetic retinopathy (DR) and age-related macular degeneration (AMD), are proliferative retinopathies involving the growth of new blood vessels on the retina, which in turn causes impairment and potential loss of vision. A drawback of conventional angiogenesis assays is that they are not representative of the angiogenic processes in the retina. In the retina, the new blood vessels grow (from pre-existing blood vessels) and migrate into a non-perfused region of the eye including the inner limiting membrane of the retina and the vitreous, both of which contribute to vision loss. The Matrigel Duplex Assay (MDA) measures the migration of angiogenic capillaries from a primary Matrigel layer to a secondary Matrigel layer, which resembles the pathological angiogenesis in AMD and DR. The methodology of MDA is comprised of two steps. In the first step, the human retinal microvascular endothelial cells (HRMECs) are mixed with phenol red–containing Matrigel (in a 1:1 ratio) and seeded in the center of an 8-well chamber slide. After 24 h, a second layer of phenol red–free Matrigel is overlaid over the first layer. Over the course of the next 24 h, the HRMECs invade from the primary Matrigel layer to the secondary layer. Subsequently, the angiogenic sprouts are visualized by brightfield phase contrast microscopy and quantified by ImageJ software. The present manuscript measures the angiogenesis-inhibitory activity of the Src kinase inhibitor PP2 in primary HRMECs using the MDA. The MDA may be used for multiple applications like screening anti-angiogenic drugs, measuring the pro-angiogenic activity of growth factors, and elucidating signaling pathways underlying retinal angiogenesis in normal and disease states.

Graphical overview

0 Q&A 391 Views Oct 20, 2023

The interaction of RNA with specific RNA-binding proteins (RBP) leads to the establishment of complex regulatory networks through which gene expression is controlled. Careful consideration should be given to the exact environment where a given RNA/RBP interplay occurs, as the functional responses might depend on the type of organism as well as the specific cellular or subcellular contexts. This requisite becomes particularly crucial for the study of long non-coding RNAs (lncRNA), as a consequence of their peculiar tissue-specificity and timely regulated expression. The functional characterization of lncRNAs has traditionally relied on the use of established cell lines that, although useful, are unable to fully recapitulate the complexity of a tissue or organ. Here, we detail an optimized protocol, with comments and tips, to identify the RNA interactome of given RBPs by performing cross-linking immunoprecipitation (CLIP) from mouse embryonal hearts. We tested the efficiency of this protocol on the murine pCharme, a muscle-specific lncRNA interacting with Matrin3 (MATR3) and forming RNA-enriched condensates of biological significance in the nucleus.

Key features

• The protocol refines previous methods of cardiac extracts preparation to use for CLIP assays.

• The protocol allows the quantitative RNA-seq analysis of transcripts interacting with selected proteins.

• Depending on the embryonal stage, a high number of hearts can be required as starting material.

• The steps are adaptable to other tissues and biochemical assays.

Graphical overview

Identification of RNA/protein interactions from developing hearts

0 Q&A 270 Views Oct 5, 2023

Fertilized teleost fish eggs are a complex formation, in which dividing cells arelocated in a small point in the entire volume of eggs. Isolating embryonic cellscan be considered a necessary step in the research of developmentalpeculiarities of fish cells at the earliest stages of embryogenesis beforeembryo formation. The main advantages of the offered protocol are rapidisolation, no enzymes, and overall low cost compared to other protocols. Theprotocol is suitable for the isolation of embryonic cells from medium-sized eggsat the stages of blastula or gastrula, for studies in a variety of applications(e.g., microscopy, flow cytometry, and other methods). Fertilized nelma eggs(Stenodus leucichthys nelma) are used in the protocol as a model.

Key features

• Fast and cheap isolation of cells from fish eggs at early stages (blastula orgastrula).

• Applicable for most of the methods for cell study (any staining, microscopy, flowcytometry, etc.).

• Can be applied to other teleost fish eggs with medium egg diameter of 3–4mm.

Graphical overview

0 Q&A 610 Views Sep 5, 2023

Congenital heart disease (CHD) is often associated with myogenic defects. During heart development, cardiomyocyte growth requires essential cues from extrinsic factors such as insulin-like growth factor 2 (IGF-2). To determine whether and how growth factors account for embryonic cardiomyocyte proliferation, isolation followed by culturing of embryonic cardiomyocytes can be utilized as a useful tool for heart developmental studies. Current protocols for isolating cardiomyocytes from the heart do not include a cardiomyocyte-specific reporter to distinguish cardiomyocytes from other cell types. To optimize visualization of cardiomyocyte proliferation, our protocol utilizes a Tnnt2-promoter-driven H2B-GFP knock-in mouse model (TNNT2H2B-GFP/+) for in vitro visualization of nuclear-tagged cardiomyocyte-specific fluorescence. A cardiomyocyte-specific genetic reporter paired with an effective proliferation assay improves the reproducibility of mechanistic studies by increasing the accuracy of cell identification, proliferated cell counting, and cardiomyocyte tracking.

Key features

• This protocol refines previous methods of cardiomyocyte isolation to specifically target embryonic cardiomyocytes.

• UsesH2B-GFP/+cardiomyocyte reporters as identified by Yan et al. (2016).

• Traces cell proliferation with Phospho-Histone 3 (p-H3) assay.

• Has applications in assessing the role of growth factors in cardiomyocyte proliferation.

Graphical overview

0 Q&A 585 Views Sep 5, 2023

The centrosome governs many pan-cellular processes including cell division, migration, and cilium formation. However, very little is known about its cell type-specific protein composition and the sub-organellar domains where these protein interactions take place. Here, we outline a protocol for the spatial interrogation of the centrosome proteome in human cells, such as those differentiated from induced pluripotent stem cells (iPSCs), through co-immunoprecipitation of protein complexes around selected baits that are known to reside at different structural parts of the centrosome, followed by mass spectrometry. The protocol describes expansion and differentiation of human iPSCs to dorsal forebrain neural progenitors and cortical projection neurons, harvesting and lysis of cells for protein isolation, co-immunoprecipitation with antibodies against selected bait proteins, preparation for mass spectrometry, processing the mass spectrometry output files using MaxQuant software, and statistical analysis using Perseus software to identify the enriched proteins by each bait. Given the large number of cells needed for the isolation of centrosome proteins, this protocol can be scaled up or down by modifying the number of bait proteins and can also be carried out in batches. It can potentially be adapted for other cell types, organelles, and species as well.

Graphical overview

An overview of the protocol for analyzing the spatial protein composition of the centrosome in human induced pluripotent stem cell (iPSC)-derived neural cells. ① Human iPSCs are expanded, which serve as the starting cell population for the neural induction (Sections A, B, and C in Procedure). ② Neurons are induced and differentiated for 40 days (Section D in Procedure), in at least four biological replicates. ③ Total protein is isolated either at 15th or 40th day of differentiation, for neural stem cells and neurons, respectively (Sections E and F in Procedure). ④ Selected bait proteins are immunoprecipitated using the respective antibodies (Sections G and H in Procedure). ⑤ Co-immunoprecipitated samples are analyzed with mass spectrometry (Section I in Procedure). ⑥ Mass spectrometry output (.RAW) files are processed using MaxQuant software to calculate intensities (Section A in Data analysis). ⑦ The resulting data are pre-processed, filtered, and statistically analyzed using Perseus and R software (Sections B and C in Data analysis) ⑧ Further analysis is done using software or web tools such as Cytoscape or STRING to gain biological insights (Sections D and E in Data analysis).

0 Q&A 490 Views Aug 20, 2023

Synapses are specialized structures that enable neuronal communication, which is essential for brain function and development. Alterations in synaptic proteins have been linked to various neurological and neuropsychiatric disorders. Therefore, manipulating synaptic proteins in vivo can provide insight into the molecular mechanisms underlying these disorders and aid in developing new therapeutic strategies. Previous methods such as constitutive knock-out animals are limited by developmental compensation and off-target effects. The current approach outlines procedures for age-dependent molecular manipulations in mice using helper-dependent adenovirus viral vectors (HdAd) at distinct developmental time points. Using stereotactic injection of HdAds in both newborn and juvenile mice, we demonstrate the versatility of this method to express Cre recombinase in globular bushy cells of juvenile Rac1fl/fl mice to ablate presynaptic Rac1 and study its role in synaptic transmission. Separately, we overexpress CaV2 α1 subunits at two distinct developmental time points to elucidate the mechanisms that determine presynaptic CaV2 channel abundance and preference. This method presents a reliable, cost-effective, and minimally invasive approach for controlling gene expression in specific regions of the mouse brain and will be a powerful tool to decipher brain function in health and disease.

Key features

• Virus-mediated genetic perturbation in neonatal and young adult mice.

• Stereotaxic injection allows targeting of brain structures at different developmental stages to study the impact of genetic perturbation throughout the development.

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