发育生物学

Cell-type specific transcriptional programs underlie the development and maintenance of organs. Not only distinct cell types within a tissue, even cells with supposedly identical cell fates show a high degree of transcriptional heterogeneity. Inevitable, low cell numbers are a major hurdle to study transcriptomes of pure cell populations. Here we describe DigiTAG, a high-throughput method that combines transposase fragmentation and molecular barcoding to retrieve high quality transcriptome data of rare cell types in Drosophila melanogaster. The protocol showcases how DigiTAG can be used to analyse the transcriptome of rare neural stem cells (type II neuroblasts) of Drosophila larval brains, but can also be utilized for other cell types or model systems.

The brown alga Ectocarpus has a haploid-diploid life cycle that involves alternation between two multicellular generations, the sporophyte and the gametophyte. Life cycle generation is not determined by ploidy but by a genetic system that includes two different three amino acid loop extension homeodomain transcription factors called OUROBOROS and SAMSARA. In addition, sporophytes have been shown to secrete a diffusible factor into the medium that can induce gametophyte initial cells to switch from the gametophyte to the sporophyte developmental program. The protocol presented here describes how to produce sporophyte-conditioned medium containing the diffusible sporophyte-inducing factor and how to assay for activity of the factor using a meio-spore-based bioassay. The protocol, which describes how several steps of these procedures can be optimised, will represent a useful tool for future work aimed at characterising the diffusible factor and investigating its mode of action.

The acrosome reaction is a highly regulated exocytotic event that primes spermatozoa for successful fertilization. Upon induction, acrosomal exocytosis proceeds via a wave of vesiculation that radiates across the sperm head, destabilizing the acrosomal vesicle and resulting in the release of the acrosomal contents. Having shed their acrosome, spermatozoa are then capable of penetrating the outer vestments of the oocyte and initiating fertilization. Accordingly, the failure of spermatozoa to complete an acrosome reaction represents a relatively common etiology in male infertility patients, and the ability to induce acrosomal exocytosis has found clinical utility in the evaluation of sperm fertilizing capacity. Here, we firstly describe protocols for driving the capacitation of human spermatozoa in vitro using chemically defined media in order to prime the cells for completion of acrosomal exocytosis. We then describe methodology routinely used for the induction of acrosomal exocytosis incorporating either a physiological agonist (i.e., the steroidal hormone, progesterone) or pharmacological reagent (i.e., the divalent cation ionophore, A23187). Finally, we describe the application of histochemical and immunofluorescence techniques that can be applied to study the completion of the acrosome reaction. Such protocols have important diagnostic utility for sperm function testing in both clinical and andrological research laboratories.

Mesenchymal stem cells have the ability to differentiate into multiple lineages, including adipocytes, osteoblasts and chondrocytes. Mesenchymal stem cells can be induced to differentiate into chondrocytes in extracellular matrices, such as alginate or collagen gel. Mesenchymal stem cells in a cell pellet or micromass culture can be also induced to form cartilages in a defined medium containing chondrogenic cytokines, such as transforming growth factor-β (TGF-β). Here, we describe a simple method to form cartilage by seeding mesenchymal cells derived from limb-bud cells at high cell density. First, we dissected the limb buds from embryonic mice (embryonic day 12.5) and digested them with enzymes (dispase and collagenase). After filtration using a cell strainer, we seeded the cells at high density. Unlike other methods, the method described here is simple and does not require the use of specialized equipment, expensive materials or complex reagents.

Immunocytochemistry of cultured cells is a common and effective technique for determining compositions and localizations of proteins within cellular structures. However, traditional cultured cell fixation and staining protocols are not effective in preserving cultured cell cytonemes, long specialized filopodia that are dedicated to morphogen transport. As a result, limited mechanistic interrogation has been performed to assess their regulation. We developed a fixation protocol for cultured cells that preserves cytonemes, which allows for immunofluorescent analysis of endogenous and over-expressed proteins localizing to the delicate cellular structures.