Developmental Biology

X-ray computed microtomography (µCT) is a powerful tool to reveal the 3D structure of tissues and organs. Compared with the traditional sectioning, staining, and microscopy image acquisition, it allows a better understanding of the morphology and a precise morphometric analysis. Here, we describe a method for 3D visualization and morphometric analysis by µCT scanning of the embryonic heart of iodine-stained E15.5 mouse embryos.

Visualization of cell structure with fluorescent dye for characterizing cell size, shape, and arrangement is a common method to study tissue morphology and morphogenesis. In order to observe shoot apical meristem (SAM) in Arabidopsis thaliana by laser scanning confocal microscopy, we modified the pseudo-Schiff propidium iodide staining method by adding a series solution treatment to stain the deep cells. The advantage of this method is mainly reflected by the direct observation of the clearly bounded cell arrangement and the typical three-layer cells in SAM without the traditional tissue slicing.

The developing cerebral cortex of mammals is generated from nascent pyramidal neurons, which radially migrate from their birthplace in the ventral part of the neural tube to the cortical surface. Subtle aberrations in this process may cause significant changes in cortical structure and lead to developmental neurological disorders. During pyramidal neuron migration, we recently showed that the migrating neuron, which bypasses its last preceding neuron, is critical for its proper positioning and contributes to cerebral cortex thickness. Studying this process requires an imaging system with single-cell resolution and a prolonged observation window. Therefore, we built a system to maintain an organotypic brain slice on the stage of a Leica SP5 confocal microscope, which facilitated high-resolution imaging over a 12-hour time-lapse observation period of cellular events during neuron migration. Here, we share our protocol along with guidelines for overcoming difficulties during the setup. This protocol facilitates the observation of, but is not limited to, neurodevelopmental and pathological processes occurring during neuron migration.

Adult stem cells not only maintain tissue homeostasis but are also critical for tissue regeneration during injury. Skeletal stem cells are multipotent stem cells that can even generate bones and cartilage upon transplantation to an ectopic site. This tissue generation process requires essential stem cell characteristics including self-renewal, engraftment, proliferation, and differentiation in the microenvironment. Our research team has successfully characterized and isolated skeletal stem cells (SSCs) from the cranial suture called suture stem cells (SuSCs), which are responsible for craniofacial bone development, homeostasis, and injury-induced repair. To assess their stemness features, we have demonstrated the use of kidney capsule transplantation for an in vivo clonal expansion study. The results show bone formation at a single-cell level, thus permitting a faithful assessment of stem cell numbers at the ectopic site. The sensitivity in assessing stem cell presence permits using kidney capsule transplantation to determine stem cell frequency by limiting dilution assay. Here, we described detailed protocols for kidney capsule transplantation and limiting dilution assay. These methods are extremely valuable both for the evaluation of skeletogenic ability and the determination of stem cell frequency.

Successful advancement in the treatment of diabetes mellitus is not possible without well-established methodology for beta cell mass calculation. Here, we offer the protocol to assess beta cell mass during embryonic development in the mouse. The described protocol has detailed steps on how to process extremely small embryonic pancreatic tissue, cut it on the cryostat, and stain tissue slides for microscopic analysis. The method does not require usage of confocal microscopy and takes advantage of enhanced automated image analysis with proprietary as well as open-source software packages.

Advances in imaging technology offer new opportunities in developmental biology. To observe the development of internal structures, microtome cross-sectioning followed by H&E staining on glass slides is a common procedure; however, this technique can be destructive, and artifacts can be introduced during the process. In this protocol, we describe a less invasive procedure with which we can stain insect samples and obtain reconstructed three-dimensional images using micro-computed tomography, or micro-CT (µCT). Specifically, we utilize the fungus-farming ambrosia beetle species Euwallacea validus to observe the morphology of mycangia, a critical internal organ with which beetles transport fungal symbionts. Not only this protocol is ideal to observe mycangia, our staining/scanning procedure can also be applied to observe other delicate tissues and small organs in arthropods.

Graphical abstract

Currently, there are several in vitro protocols that focus on directing human induced pluripotent stem cell (hiPSC) differentiation into either the cardiac or pulmonary lineage. However, these systemsprotocols are unable to recapitulate the critical exchange of signals and cells between the heart and lungs during early development. To address this gap, here we describe a protocol to co-differentiate cardiac and pulmonary progenitors within a single hiPSC culture by temporal specific modulation of Wnt and Nodal signaling. Subsequently, human cardio-pulmonary micro-tissues (μTs) can be generated by culturing the co-induced cardiac and pulmonary progenitors in 3D suspension culture. Anticipated results include expedited alveolarization in the presence of cardiac cells, and segregation of the cardiac and pulmonary μTs in the absence of exogenous Wnt signaling. This protocol can be used to model cardiac and pulmonary co-development, with potential applications in drug testing, and as a platform for expediting the maturation of pulmonary cells for lung tissue engineering.

Aging and neuronal deterioration constitute important risk factors for the development of neuronal-related diseases, such as different dementia. The nematode Caenorhabditis elegans has emerged as a popular model system for studying neurodegeneration diseases, due to its complete neuronal connectivity map. DiI is a red fluorescent dye that can fill the worm amphid neurons and enables the visualization of their neurodegeneration over time. This protocol provides an efficient, fast, and safe method to stain worm amphid neurons to highlight the chemosensory structures of live nematodes.

In this study, we present a detailed protocol for live imaging and quantitative analysis of floral meristem development in Aquilegia coerulea, a member of the buttercup family (Ranunculaceae). Using confocal microscopy and the image analysis software MorphoGraphX, we were able to examine the cellular growth dynamics during floral organ primordia initiation, and the transition from floral meristem proliferation to termination. This protocol provides a powerful tool to study the development of the meristem and floral organ primordia, and should be easily adaptable to many plant lineages, including other emerging model systems. It will allow researchers to explore questions outside the scope of common model systems.

In the Japanese rhinoceros beetle Trypoxylus dichotomus, various candidate genes required for a specific phenotype of interest are listed by next-generation sequencing analysis. Their functions were investigated using RNA interference (RNAi) method, the only gene function analysis tool for T. dichotomus developed so far. The summarized procedure for the RNAi method used for T. dichotomus is to synthesize double-stranded RNA (dsRNA), and inject it in larvae or pupae of T. dichotomus. Although some dedicated materials or equipment are generally required to inject dsRNA in insects, the advantage of the protocol described here is that it is possible to inject dsRNA in T. dichotomus with one syringe.