Developmental Biology


Protocols in Current Issue
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0 Q&A 1125 Views Jun 5, 2022

Live labelling of active transcription sites is critical to our understanding of transcriptional dynamics. In the most widely used method, RNA sequence MS2 repeats are added to the transcript of interest, on which fluorescently tagged Major Coat Protein binds, and labels transcription sites and transcripts. Here we describe another strategy, using the Argonaute protein NRDE-3, repurposed as an RNA-programmable RNA binding protein. We label active transcription sites in C. elegans embryos and larvae, without editing the gene of interest. NRDE-3 is programmed by feeding nematodes with double-stranded RNA matching the target gene. This method does not require genome editing and is inexpensive and fast to apply to many different genes.

Graphical abstract:

1 Q&A 1462 Views Apr 5, 2022

Craniofacial anomalies (CFA) are a diverse group of deformities, which affect the growth of the head and face. Dysregulation of cranial neural crest cell (NCC) migration, proliferation, differentiation, and/or cell fate specification have been reported to contribute to CFA. Understanding of the mechanisms through which cranial NCCs contribute for craniofacial development may lead to identifying meaningful clinical targets for the prevention and treatment of CFA. Isolation and culture of cranial NCCs in vitro facilitates screening and analyses of molecular cellular mechanisms of cranial NCCs implicated in craniofacial development. Here, we present a method for the isolation and culture of cranial NCCs harvested from the first branchial arch at early embryonic stages. Morphology of isolated cranial NCCs was similar to O9-1 cells, a cell line for neural crest stem cells. Moreover, cranial NCCs isolated from a transgenic mouse line with enhanced bone morphogenetic protein (BMP) signaling in NCCs showed an increase in their chondrogenic differentiation capacity, suggesting maintenance of their in vivo differentiation potentials observed in vitro. Taken together, our established method is useful to visualize cellular behaviors of cranial NCCs.

0 Q&A 2454 Views Feb 20, 2022

All eukaryotic cells are equipped with transmembrane lipid transporters, which are key players in membrane lipid asymmetry, vesicular trafficking, and membrane fusion. The link between mutations in these transporters and disease in humans highlights their essential role in cell homeostasis. Yet, many key features of their activities, their substrate specificity, and their regulation remain to be elucidated. Here, we describe an optimized quantitative flow cytometry-based lipid uptake assay utilizing nitrobenzoxadiazolyl (NBD) fluorescent lipids to study lipid internalization in mammalian cell lines, which allows characterizing lipid transporter activities at the plasma membrane. This approach allows for a rapid analysis of large cell populations, thereby greatly reducing sampling variability. The protocol can be applied to study a wide range of mammalian cell lines, to test the impact of gene knockouts on lipid internalization at the plasma membrane, and to uncover the dynamics of lipid transport at the plasma membrane.

Graphic abstract:

Internalization of NBD-labeled lipids from the plasma membrane of CHO-K1 cells.

0 Q&A 2208 Views Jan 5, 2022

During development, cells must quickly switch from one cell state to the next to execute precise and timely differentiation. One method to ensure fast transitions in cell states is by controlling gene expression at the post-transcriptional level through action of RNA-binding proteins on mRNAs. The ability to accurately identify the RNA targets of RNA-binding proteins at specific stages is key to understanding the functional role of RNA-binding proteins during development. Here we describe an adapted formaldehyde RNA immunoprecipitation (fRIP) protocol to identify the in vivo RNA targets of a cytoplasmic RNA-binding protein, YTHDC2, from testis, during the first wave of spermatogenesis, at the stage when germ cells are shutting off the proliferative program and initiating terminal differentiation (Bailey et al., 2017). This protocol enables quick and efficient identification of endogenous RNAs bound to an RNA-binding protein, and facilitates the monitoring of stage-specific changes during development.

0 Q&A 2562 Views Dec 20, 2021

Adipocytes exhibit different morphological and functional characteristics, depending on their anatomical location, developmental origin, and stimulus. While white adipocytes tend to accumulate energy as triglycerides, brown and beige adipocytes tend to direct carbon sources to fuel thermogenesis. White and beige adipocytes originate from common progenitor cells, which are distinct from brown adipocyte precursors. Having a method to study white vs. beige vs. brown adipocyte differentiation may help to unveil the mechanisms driving distinct adipogenic programs. Preadipocytes can be cultured and differentiated in vitro using a combination of compounds to stimulate adipogenesis. Here, we describe and compare protocols designed to stimulate adipocyte differentiation and induce brown/beige-like or white-like characteristics in differentiating adipocytes. The protocols consist in exposing murine preadipocytes to pharmacological stimuli aimed at triggering adipogenesis and inducing (or not) a thermogenic gene expression program. After 8 days of differentiation with a pro-browning cocktail, immortalized preadipocytes isolated from interscapular brown fat (9B cells) or inguinal white fat (9W cells) from the same mouse expressed higher levels of brown/beige adipocyte markers (e.g., Ucp1) and pan-adipocyte differentiation markers (e.g., Pparg, Cebpa and aP2) when compared to the same cells differentiated with a cocktail that lacked brown/beige adipogenic inducers (i.e., rosiglitazone, T3, and indomethacin). Consistent with a higher thermogenic potential of brown vs. beige adipocytes, differentiated 9B cells expressed higher Ucp1 levels than differentiated 9W cells. This simple protocol may help researchers to understand mechanisms of adipogenesis and how adipocytes become thermogenic.

0 Q&A 1773 Views Nov 20, 2021

Regionalized distribution of genes plays crucial roles in the formation of the spatial pattern in tissues and embryos during development. In situ hybridization has been one of the most widely used methods to screen, identify, and validate the spatial distribution of genes in tissues and embryos, due to its relative simplicity and low cost. However, acquisition of high-quality hybridization signals remains a challenge while maintaining good tissue morphology, especially for small tissues such as early post-implantation mouse embryos. In this protocol, we present a detailed RNA in situ hybridization protocol suitable for wholemount early post-implantation mouse embryos and other small tissue samples. This protocol uses digoxigenin (DIG) labeled riboprobes to hybridize with target transcripts, alkaline phosphatase-conjugated anti-DIG antibodies to recognize DIG-labeled nucleotides, and nitroblue tetrazolium (NBT)/5-bromo-4-chloro-3-indolyl-phosphate (BCIP) chromogenic substrates for color development. Specific steps and notes on riboprobe preparation, embryo collection, probe hybridization, and color development are all included in the following protocol.

Graphic abstract:

Overview of Wholemount in situ Hybridization in Early Mouse Embryos.

0 Q&A 2800 Views Nov 5, 2021

Extracellular vesicles (EVs) are thought to mediate intercellular communication through the delivery of cargo proteins and RNA to target cells. The uptake of EVs is often followed visually using lipophilic-dyes or fluorescently-tagged proteins to label membrane constituents that are then internalized into recipient cells (Christianson et al., 2013; De Jong et al., 2019). However, these methods do not probe the exposure of EV cargo to intracellular compartments, such as the cytoplasm and nucleus, where protein or RNA molecules could elicit functional changes in recipient cells. In this protocol, we employ an EV cargo protein-APEX fusion to detect proximity interactions with recipient cell cytoplasmic/nuclear targets. This approach results in the biotinylation of proteins in close contact with the reporter fusion and thus permits profiling of biotinylated proteins affinity purified on immobilized streptavidin beads.

Graphic abstract:

Schematic showing three steps of APEX-mediated proximity labeling of proteins in cells targeted by EVs.

0 Q&A 2628 Views Nov 5, 2021

Blood cells have a limited lifespan and are replenished by a small number of hematopoietic stem and progenitor cells (HSPCs). Adult vertebrate hematopoiesis occurs in the bone marrow, liver, and spleen, rendering a comprehensive analysis of the entire HSPC pool nearly impossible. The Drosophila blood system is well studied and has developmental, molecular, and functional parallels with that of vertebrates. Unlike vertebrates, post-embryonic hematopoiesis in Drosophila is essentially restricted to the larval lymph gland (LG), a multi-lobed organ that flanks the dorsal vessel. Because the anterior-most or primary lobes of the LG are easy to dissect out, their cellular and molecular characteristics have been studied in considerable detail. The 2-3 pairs of posterior lobes are more delicate and fragile and have largely been ignored. However, posterior lobes harbor a significant blood progenitor pool, and several hematopoietic mutants show differences in phenotype between the anterior and posterior lobes. Hence, a comprehensive analysis of the LG is important for a thorough understanding of Drosophila hematopoiesis. Most studies focus on isolating the primary lobes by methods that generally dislodge and damage other lobes. To obtain preparations of the whole LG, including intact posterior lobes, here we provide a detailed protocol for larval fillet dissection. This allows accessing and analyzing complete LG lobes, along with dorsal vessel and pericardial cells. We demonstrate that tissue architecture and integrity is maintained and provide methods for quantitative analysis. This protocol can be used to quickly and effectively isolate complete LGs from first instar larval to pupal stages and can be implemented with ease.

0 Q&A 2638 Views Sep 5, 2021

Recent popularization of next-generation sequencing enables conducting easy transcriptome analysis. Nevertheless, substantial RNA isolation work prior to RNA sequencing, as well as the high cost involved, still makes the routine use of large-scale transcriptome analysis difficult. For example, conventional phenol-chloroform RNA extraction cannot be easily applied to hundreds of samples. Therefore, we developed Direct-TRI, a new cost-effective and high throughput RNA-extraction method that uses a commercial guanidine-phenol-based RNA extraction reagent and a 96-well silica column plate. We applied Direct-TRI to zebrafish whole larvae and juvenile samples and obtained comparable RNA qualities by several different homogenization methods such as vortexing, manual homogenizing, and freezing/crushing. Direct-TRI enabled the extraction of 192 RNA samples in an hour with a cost of less than a dollar per sample. Direct-TRI is useful for large-scale transcriptome studies, manipulating hundreds of zebrafish individuals, and may be used with other animal samples.

0 Q&A 2285 Views Aug 20, 2021

Recent advances in single-cell RNA-sequencing (scRNA-seq) technologies provide unprecedented opportunities to identify new cell types and characterize cell states. One of the most important requirements for performing scRNA-seq is to obtain high-quality single cells in suspension. Recently, we used this approach to characterize Drosophila blood cells (hemocytes). Here, we provide a detailed protocol for obtaining single hemocytes in suspension, which can be used for microfluidics-based scRNA-seq platforms. This protocol involves the simple bleeding of third instar larvae and the subsequent purification of the hemolymph using either Optiprep-based gradient centrifugation or traditional centrifugation methods to obtain single hemocytes of high quality for scRNA-seq. Importantly, this method for single-hemocyte preparation is straightforward and reproducible, with negligible issues associated with cell viability as the entire procedure involves no enzymatic dissociation.

Graphic abstract:

Workflow for the preparation of Drosophila larval blood cells in suspension. Hemocytes (blood cells) of the sessile and circulatory compartments of larvae are derived by simple bleeding and purification using gradient centrifugation. Blood cells are counted and subsequently encapsulated by microfluidics-based scRNA-seq platforms. Blood cells represented in the schematic are derived from third instar larvae of the genotype Hemolectin-GAL4.Delta, UAS-2xEGFP (BDSC stock #30140).

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