Methods for Characterizing Cell Morphology and Protein Localization During Development and Regeneration

Animals are highly complex systems composed of hundreds of different cell types organized into dozens of distinct tissues and organs. Studying how groups of cells interact with one another and their environment during embryonic development not only gives us a better understanding of how adult bodies are constructed but can also reveal the molecular and cellular underpinnings of diseases and birth defects. Certain model systems are better suited to addressing specific biological questions than others, and scientists employ a wide variety of species and experimental techniques to probe the full diversity of organismal biology. For example, a given developmental biology experiment might involve genetic engineering, animal dissections, molecular biology, biochemistry, and high-resolution microscopy. Such methods can be challenging to reproduce based on written descriptions and figures alone, and therefore, it is important to document them in video form to foster the dissemination of the proper technique and analysis methods.

The relationship between developmental mechanisms and regeneration, particularly in the nervous system, is an area of intense interest, and the zebrafish, Danio rerio, has become a key model in this field due to its lifelong growth and regenerative capabilities. Notably, there is a complex interplay between cells in the retina and the optic tectum, and this interplay is necessary for establishing proper connections as new neurons are added to the eyes and brain¹. Hagen et al. describe a technique for characterizing how neural growth and development are affected by denervation following eye removal in living zebrafish embryos². They describe in detail how to 1) perform eye-removal surgery, 2) culture larvae following the surgery, 3) fix, dissect, and store the brain samples, 4) perform immunofluorescence, and 5) properly mount and image the samples. This technique can be used to study a wide variety of neurodevelopmental processes in fixed tissues, and it can also be modified for experiments in live embryos.

Neural crest cells (NCCs) represent an important developmental lineage that gives rise to pigment cells, cartilage, bone, smooth muscle, neurons, and glia, and these cells are implicated in many types of birth defects³. In addition to their impressive pluripotency, NCCs also undergo an epithelial-to-mesenchymal transition followed by extensive migration during development, thus making them a fascinating paradigm for understanding the interplay between cell fate and morphology⁴. Chick embryos are a powerful system for studying NCC biology, because NCCs can be visualized in vivo and cultured ex vivo, thus allowing for many types of molecular treatments that are difficult with other systems. Jacques-Fricke et al. describe a flexible method for culturing cranial neural folds to generate primary NCC cultures on fibronectin-coated coverslips, which can then be followed by fixed-imaging or live-imaging experiments⁵. They describe how to 1) incubate and isolate the embryos, 2) dissect and plate the neural folds, 3) fix and stain the NCCs, and 4) quantify and analyze the NCC morphology.

The basement membrane—an extracellular matrix generated along the basal surface of epithelial cells—is critical for the establishment of tissue morphology and organ function across animals⁶. The Drosophila follicular epithelium, which surrounds the forming egg, is an excellent model for studying how basement membranes are formed, as it secretes all the major conserved components of the basement membrane⁷, and the matrix is oriented toward the “outside” of the egg chamber, thus facilitating microscopy-based analyses⁸. Shah and Devergne describe the use one of the most popular types of super-resolution microscopy—Airyscan—to examine how vesicular trafficking affects basement membrane formation⁹. They show how to 1) collect and fix Drosophila ovaries, 2) perform immunofluorescence, 3) properly mount the samples, and 4) capture high-quality super-resolution datasets for three-dimensional analysis. We note that this article includes an extremely useful and concise tutorial on the proper acquisition of non-saturated, three-dimensional confocal datasets, which should be of general interest to anyone wishing to quantify fluorescence images.

While microscope-based super-resolution technologies (e.g., STED, SIM, and Airyscan)¹⁰ are becoming increasingly commonplace, they are more expensive than standard confocal microscopes and are not available to all researchers. An alternative technique for obtaining super-resolution images is expansion microscopy (ExM), which involves the physical enlargement of a biological sample in three dimensions by embedding and binding it to a swellable hydrogel¹¹. The expanded samples can then be visualized using a standard confocal microscope to generate images with a lateral resolution on the order of tens of nanometers, rivaling other types of super-resolution microscopy¹². Our group describes how to implement ExM in whole-mount Drosophila embryos to reveal subcellular features of the actomyosin cytoskeleton and mitochondrial networks that are not detectable with standard confocal microscopy¹³. We describe how to 1) select properly staged embryos, 2) perform immunofluorescence, 3) prepare and mount the embryos for expansion, 3) digest and expand the embryos, and 4) collect super-resolution datasets. This technique should be modifiable for a wide range of biological specimens on the order of 1 mm, making it accessible to a wide range of biology labs.

Developmental biology is, by necessity, a highly inventive and cross-disciplinary field that draws on techniques from diverse scientific areas to quantify cell biology as it occurs in the animal. Here, we highlight four developmental biology methods in the zebrafish, chick, and fruit fly that feature both classical and cutting-edge techniques to probe cell morphology and subcellular protein localization during animal development. Currently, the field is focused on merging quantitative analyses of cell morphology and behavior with transcriptomic and proteomic experiments to reveal the full diversity of molecules and biomechanical forces that control animal development.

The authors have nothing to disclose.

We would like to acknowledge generous funding from the National Institute of General Medical Science (NIGMS), part of the National Institutes of Health (NIH), which facilitated the generation of this collection and editorial. The Paré lab is funded by an AREA grant (1R15GM143729-01) to Dr. Paré, and we are also part of the Arkansas Integrative Metabolic Research Center (1P20GM139768-01 5743), an NIH Center of Biomedical Research Excellence.