发育生物学

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

Rodent cages equipped with access to a voluntary running wheel are commonly used to study the effects of aerobic physical activity on physiology and behavior. Notable discoveries in exercise neurobiology, including the key role of brain-derived neurotrophic factor (BDNF) in neural plasticity and cognition, have been made using rodents housed with voluntary running wheels. A major advantage of using home-cage running wheels over treadmills is the elimination of stress potentially associated with forced running. In addition, voluntary wheel running may simulate a more natural running pattern in laboratory mice. Singly housing mice with voluntary running wheels is traditionally employed to obtain exact quantitation of the distance ran; however, social isolation stress is often ignored to obtain precise running distances. Moreover, voluntary exercise studies in adolescent mice must consider the neurodevelopmental implications of isolation stress. In this protocol, we wean 21-day-old mouse pups directly into running wheel-equipped cages and pair-house them to reduce the impact of social isolation and other developmentally specific factors that could adversely affect their behavior or development. Individual running distances are obtained from each mouse in the cage using a radio-frequency identification (RFID) ear tag and a hidden antenna placed directly under the running wheel. We have demonstrated that voluntary running during a specific juvenile-adolescent developmental period can improve hippocampal memory when tested during adolescence (Ivy et al., 2020). Individual exercise tracking of group-housed mice can enable future studies to precisely correlate the amount of exercise with readouts such as cell-specific gene expression, epigenetic mechanisms, serum biomarkers, and behavior, in an intra-individual manner.

Graphic abstract:

Figure 1. Illustration of the dual RFID and Vital View system for individual mouse running in a pair-housed cage

Circulation of cerebrospinal fluid (CSF) plays an important role during development. In zebrafish embryo, the flow of CSF has been found to be bidirectional in the central canal of the spinal cord. In order to compare conditions and genetic mutants across each other, we recently automated the quantification of the velocity profile of exogenous fluorescent particles in the CSF. We demonstrated that the beating of motile and tilted cilia localized on the ventral side of the central canal was sufficient to generate locally such bidirectionality. Our approach can easily be extended to characterize CSF flow in various genetic mutants. We provide here a detailed protocol and a user interface program to quantify CSF dynamics. In order to interpret potential changes in CSF flow profiles, we provide additional tools to measure the central canal diameter, characterize cilia dynamics and compare experimental data with our theoretical model in order to estimate the impact of cilia in generating a volume force in the central canal. Our approach can also be of use for measuring particle velocity in vivo and modeling flow in diverse biological solutions.

The quantitative measurement of water flow-induced swimming of fish species using a swimmill is a powerful method to evaluate motor ability of individual fish. Zebrafish is a commonly used vertebrate that enables the study of morphological, physiological and behavioral characteristics associated with genes. We here established a reproducible method that allows to measure the body length and the critical swimming speed of adult zebrafish using a swimmill.

The collagen contraction assay is an in vitro, three-dimensional method to determine the factor(s) affecting the contractile behavior of activated cells such as fibroblasts in either physiological or pathological scenarios. The collagen lattices/hydrogels are seeded with fibroblasts to mimic the interactions between these cells and their surrounding extracellular matrix proteins in the connective tissue. This method is an important platform to assess components as potential therapeutic targets to prevent pathologies such as fibrosis, which are manifestations of hyperactivated fibroblasts. We have described a basic version of this collagen contraction assay, which is amenable to customization using different cell types under diverse experimental conditions.

Here we describe a simple method to measure larval muscle contraction and locomotion behavior. The method enables the user to acquire data, without the necessity of using expensive equipment (Rotstein et al., 2018). To measure contraction and locomotion behaviour, single larvae are positioned at the center of a humidified Petri dish. Larval movement is recorded over time using the movie function of a consumer digital camera. Subsequently, videos are analyzed using ImageJ (Rueden et al., 2017) for distance measurements and counting of contractions. Data are represented as box or scatter plots using GraphPad Prism (^©GraphPad Software).

Cilia are microtubule-based hair-like projections found in organisms, ranging from protozoa to mammals. This protocol provides methods for immunofluorescence staining of cilia in cultured cells and zebrafish embryos.

Olfaction is a well-studied sensory mechanism in Caenorhabditis elegans (C. elegans). The nematodes respond to a wide range of chemicals by either attraction, repulsion or a mixture thereof (Bargmann et al., 1993). We have used olfaction to characterize behavioural and molecular circadian rhythms in C. elegans. The circadian clock is a biological oscillator that provides an endogenous temporal structure that approximately matches the 24-hour periodicity in the environment (due to the rotational movement of the Earth). Circadian rhythms are present in most organisms from cyanobacteria to humans and they typically regulate sensory functions among many other processes. Olfaction is under circadian control in many animals (Granados-Fuentes et al., 2006; Granados-Fuentes et al., 2011; Tanoue et al., 2008; Krishnan et al., 1999). This protocol was designed to allow the assessment of olfaction for a population of worms within a short time interval, in the same plate where the worms grew (to avoid washing steps that may disturb the rhythms), and in the presence of food.

In Xenopus, the left-right axis is established following an extracellular vectorial leftward flow driven by monocilia at the gastrocoel roof plate (GRP) during late gastrulation / neurulation (Schweickert et al., 2007). As the GRP lies inside the developing archenteron, imaging of flow is challenging. Here we present the detailed procedure to visualize leftward flow in Xenopus laevis embryos.

Analyzing the swimming ability of 2 days post fertilization zebrafish embryos can be a useful technique to study neuromuscular function. Here is a protocol for determining the time it takes for zebrafish embryos to swim a predetermined distance.