A clonal analysis of spinal cord development in the zebrafish

 We describe the results of a clonal analysis of spinal cord development in the zebrafish. The data were obtained from embryos in which fluorescent lineage tracer was injected into single cells in the neural plate at the two-somite stage. Injected animals were allowed to survive until either 4 days or 2 weeks postfertilization. In other experiments, bromodeoxy uridine (BrdU) was injected intraperitoneally at 30 h postfertilization (hpf) after lineage tracer injection in the neural plate at the two-somite stage, and the embryos fixed at 38 hpf. We restricted our experiments to the thoracic region of the spinal cord. Our experiments were aimed at answering questions regarding the proliferative abilities of neuroepithelial cells during embryonic development (as deduced from the size of the clones), the modes of cell division (as deduced from the uptake of BrdU into clone cells), positional differences in the proliferation of cells within the neural plate itself, the cellular composition of the clones, and cell dispersion (deduced from the regional distribution of clone cells). Received: 30 December 1994 / Accepted: 9 March 1997     

Primary culture of Caenorhabditis elegans developing embryo cells for electrophysiological, cell biological and molecular studies

Cell culture is an invaluable tool for investigation of basic biological processes. However, technical hurdles including low cell yield, poor cell differentiation and poor attachment to the growth substrate have limited the use of this tool for studies of the genetic model organism Caenorhabditis elegans. This protocol describes a method for the large-scale culture of C. elegans embryo cells. We also describe methods for in vitro RNA interference, fluorescence-activated cell sorting of embryo cells and imaging of cultured cells for patch-clamp electrophysiology studies. Developing embryos are isolated from gravid adult worms. After eggshell removal by enzymatic digestion, embryo cells are dissociated and plated onto glass substrates. Isolated cells terminally differentiate within 24 h. Analysis of gene expression patterns and cell-type frequency suggests that in vitro embryo cell cultures recapitulate the developmental characteristics of L1 larvae. Cultured embryo cells are well suited for physiological analysis as well as molecular and cell biological studies. The embryo cell isolation protocol can be completed in 5-6 h.     

High-throughput fluorescence-based isolation of live C. elegans larvae

For the nematode Caenorhabditis elegans, automated selection of animals of specific genotypes from a mixed pool has become essential for genetic interaction or chemical screens. To date, such selection has been accomplished using specialized instruments. However, access to such dedicated equipment is not common. Here we describe live animal fluorescence-activated cell sorting (laFACS), a protocol for automatic selection of live first larval stage (L1) animals using a standard FACS system. We show that FACS can be used for the precise identification of GFP-expressing and non-GFP-expressing subpopulations and can accomplish high-speed sorting of live animals. We have routinely collected 100,000 or more homozygotes from a mixed starting population within 2 h, and with greater than 99% purity. The sorted animals continue to develop normally, making this protocol ideally suited for the isolation of terminal mutants for use in genetic interaction or chemical genetic screens.     

An acid phosphatase as a biochemical marker for intestinal development in the nematode Caenorhabditis elegans.

We describe an acid phosphatase enzyme (EC 3.1.3.2) that is localized to the intestine of the nematode Caenorhabditis elegans and that should serve as a convenient biochemical marker for gut differentiation. In adult worms, acid phosphatase activity is located along the edge of the gut lumen in the vicinity of the intestinal brush border. All but the anterior six cells of the intestine stain for phosphatase activity; the nonstaining cells all descend from the Ea(l/r)(a/p)a cells. Acid phosphatase activity is low in oocytes and early embryos but increases substantially when embryos reach late morphogenesis stage; this increase corresponds to the appearance of a major band of acid phosphatase activity detectable on isoelectric focusing gels. We designate this band as the product of the pho-1 gene. The pattern of acid phosphatase expression in several embryonic mutants suggests that pho-1 expression in the developing intestine is lineage autonomous. We induced an isoelectric focusing variant in the pho-1 enzyme and used this to map the pho-1 locus about 1.5 map units to the left of center of chromosome II. We purified the pho-1 enzyme to homogeneity (6500-fold purification; 4% recovery of activity); the pho-1 acid phosphatase is a homodimeric glycoprotein with a subunit molecular weight of 55,000 Da. This paper establishes a new experimental system with which to investigate the molecular basis of lineage-specific gene expression during C. elegans development.     

Spatially and temporally controlled electroporation of early chick embryos

The introduction of in ovo electroporation a decade ago has helped the chick embryo to become a powerful system to study gene regulation and function during development. Although this is a simple procedure for embryos of 2-d incubation, earlier stages (from laying to early neurulation, 0-1 d) present special challenges. Here we describe a robust and reproducible protocol for electroporation of expression vectors and morpholino oligonucleotides into the epiblast of embryos from soon after laying (stage XI) to stages 6-7 (early neurulation), with precise spatial and temporal control. Within 3 h, about 12 embryos can be electroporated and set up for culture by the New technique; the effects of morpholinos can be assessed immediately after electroporation, and robust overexpression from plasmid DNA is seen 2-3 h after electroporation. These techniques can be used for time-lapse imaging, gain- and loss-of-function experiments and studying gene regulatory elements in living embryos.     

Quantitative Analysis of Protein Expression to Study Lineage Specification in Mouse Preimplantation Embryos

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specificationin mouse preimplantation embryos. The procedures necessary for embryo collection, immunofluorescence, imaging on a confocal microscope, and image segmentation and analysis are described. This method allows quantitation of the expression of multiple nuclear markers and the spatial (XYZ) coordinates of all cells in the embryo. It takes advantage of MINS, an image segmentation software tool specifically developed for the analysis of confocal images of preimplantation embryos and embryonic stem cell (ESC) colonies. MINS carries out unsupervised nuclear segmentation across the X, Y and Z dimensions, and produces information on cell position in three-dimensional space, as well as nuclear fluorescence levels for all channels with minimal user input. While this protocol has been optimized for the analysis of images of preimplantation stage mouse embryos, it can easily be adapted to the analysis of any other samples exhibiting a good signal-to-noise ratio and where high nuclear density poses a hurdle to image segmentation (e.g., expression analysis of embryonic stem cell (ESC) colonies, differentiating cells in culture, embryos of other species or stages, etc.).     

Genome-wide RNAi screening in Caenorhabditis elegans

In Caenorhabditis elegans, introduction of double-stranded RNA (dsRNA) results in the specific inactivation of an endogenous gene with corresponding sequence; this technique is known as RNA interference (RNAi). It has previously been shown that RNAi can be performed by direct microinjection of dsRNA into adult hermaphrodite worms, by soaking worms in a solution of dsRNA, or by feeding worms Escherichia coli expressing target-gene dsRNA. We have developed a simple optimized protocol exploiting this third mode of dsRNA introduction, RNAi by feeding, which allows rapid and effective analysis of gene function in C. elegans. Furthermore, we have constructed a library of bacterial strains corresponding to roughly 86% of the estimated 19,000 predicted genes in C. elegans, and we have used it to perform genome-wide analyses of gene function. This library is publicly available, reusable resource allowing for rapid large-scale RNAi experiments. We have used this library to perform genome-wide analyses of gene function in C. elegans. Here, we describe the protocols used for bacterial library construction and for high-throughput screening in C. elegans using RNAi by feeding.     

Muscle differentiation in normal and cleavage-arrested mutant embryos of Caenorhabditis elegans

The differentiation of body-wall muscle cells was studied in the nematode Caenorhabditis elegans. Specific antibodies to myosin and paramyosin, major protein constituents of differentiated muscle, react with mesodermal cells in wild-type embryos towards the end of the first half of embryogenesis. Immunoreactive cells (2–16) first appear in embryos with 400–450 of the 550 cells present at hatching. Such embryos have developed at 25.5°C for $\text{3–4}\text{1}\text{2}$ hr beyond the two-cell stage. As development proceeds, a maximum of 81 immunoreactive cells forms four columns running anterior-posterior. Each column is composed of two lines of tightly opposed round cells, which then elongate into spindle-shaped cells. Mutant embryos in which cleavage arrests prematurely also generate cells that produce myosin and paramyosin. The initiation of muscle differentiation appears to be independent of the number of cell or nuclear divisions within a lineage or of the proliferation of other cells. These results suggest that the biosynthesis of muscle-specific proteins by nematode embryonic muscle cells is regulated by mechanisms intrinsic to these cells.     

Basic Caenorhabditis elegans methods: synchronization and observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has since been used extensively as a model organism. C. elegans possesses key attributes such as simplicity, transparency and short life cycle that have made it a suitable experimental system for fundamental biological studies for many years. Discoveries in this nematode have broad implications because many cellular and molecular processes that control animal development are evolutionary conserved. C. elegans life cycle goes through an embryonic stage and four larval stages before animals reach adulthood. Development can take 2 to 4 days depending on the temperature. In each of the stages several characteristic traits can be observed. The knowledge of its complete cell lineage together with the deep annotation of its genome turn this nematode into a great model in fields as diverse as the neurobiology, aging, stem cell biology and germ line biology. An additional feature that makes C. elegans an attractive model to work with is the possibility of obtaining populations of worms synchronized at a specific stage through a relatively easy protocol. The ease of maintaining and propagating this nematode added to the possibility of synchronization provide a powerful tool to obtain large amounts of worms, which can be used for a wide variety of small or high-throughput experiments such as RNAi screens, microarrays, massive sequencing, immunoblot or in situ hybridization, among others. Because of its transparency, C. elegans structures can be distinguished under the microscope using Differential Interference Contrast microscopy, also known as Nomarski microscopy. The use of a fluorescent DNA binder, DAPI (4',6-diamidino-2-phenylindole), for instance, can lead to the specific identification and localization of individual cells, as well as subcellular structures/defects associated to them.     

Analysis of synaptic transmission in Caenorhabditis elegans using an aldicarb-sensitivity assay

Caenorhabditis elegans has emerged as a powerful model system for studying the biology of the synapse. Here we describe a widely used assay for synaptic transmission at the C. elegans neuromuscular junction. This protocol monitors the sensitivity of C. elegans to the paralyzing affects of an acetylcholinesterase inhibitor, aldicarb. Briefly, adult worms are incubated in the presence of aldicarb and scored for the time-course of aldicarb-induced paralysis. Animals harboring mutations in genes that affect synaptic transmission generally exhibit a change in their sensitivity to aldicarb (either increased sensitivity for enhancements in synaptic transmission or decreased sensitivity for blockage in synaptic transmission). This technique provides a simple assay for the accurate comparative analysis of synaptic transmission in multiple C. elegans strains. The protocol described can be performed relatively quickly and is a practical alternative to other techniques used to study synaptic transmission. This protocol can also be modified to follow the paralytic effects with other pharmacological reagents. The assay can be performed in about 3-6 hours depending on the severity of synaptic transmission defects.     

Control of cell-cycle timing in early embryos of Caenorhabditis elegans

A technique has been developed for extruding either substantial amounts of cytoplasm without nuclei or individual nuclei with small amounts of cytoplasm from early embryos of C. elegans after perforating the eggshell with a laser microbeam. This technique, in conjunction with laser-induced cell fusion, has allowed the altering of nuclear/cytoplasmic ratios and the exposing of the nucleus of one cell to cytoplasm from another. Using these approaches the roles of nuclei and cytoplasm in determining the different cell-cycle periods of the several blastomere lineages in early embryos have been examined. It was found that nuclei in a common cytoplasm divide synchronously; enucleated blastomeres retain a cycling period characteristic of their lineage; cycling period is not substantially affected by changes in the ratio of nuclear to cytoplasmic volumes or the DNA content per cell; the period of a cell from one lineage can be substantially altered by introduction of cytoplasm from a cell of another lineage with a different period; and short-term effects of foreign cytoplasm on the timing of the subsequent mitosis differ depending on position of the donor cell in the cell cycle. These results are discussed in connection with models for the action of cytoplasmic factors in controlling cell-cycle timing.     

Archenteron-Forming Capacity in Blastomeres Isolated from Eight-Cell Stage Embryos of the Starfish, Asterina pectinifera

Starfish blastomeres are reported to be totipotent up to the 8-cell stage. We reinvestigated the development of blastomeres of 8-cell stage embryos with a regular cubic shape consisting of two tiers of 4 blastomeres. On dissociation of the embryo by disrupting the fertilization membrane at the 8-cell stage, each of the 4 blastomeres of the vegetal hemisphere gave rise to an embryo that gastrulated, whereas blastomeres from the animal hemisphere did not. By injection of a cell lineage tracer into blastomeres of 8-cell stage embryos, we found that only those of the vegetal hemisphere formed cells constituting the archenteron. Next, we compressed 4-cell stage embryos along the animal-vegetal axis so that all the blastomeres in the 8-cell stage were in a single layer. When these 8 blastomeres were then dissociated, an average of 7 of them developed into gastrulae. By cell lineage analysis, all the blastomeres in single-layered embryos at the 8-cell stage were shown to have the capacity to form cells constituting an archenteron. Taken together, these findings indicate that the fate to form the archenteron is specified by a cytoplasmic factor(s) localized at the vegetal hemisphere, and that isolated blastomeres that have inherited this factor develop into gastrulae.