Neuroscience

In this protocol, we focused on analyzing internal branches of Drosophila class IV neurons. These neurons are characterized by their highly branched axons and dendrites and intricately tile the larval body. As Drosophila larvae progress through developmental stages, the dendritic arbors of Class IV neurons undergo notable transformations. As Drosophila larvae develop, their Class IV dendritic arbors grow. In the initial 24 h after egg laying (AEL), the dendrites are smaller than segments. During the subsequent 24 h of the first instar larval stage, dendritic arbors outpace segment growth, achieving tiling. After 48 h, arbors and segments grow concurrently. Epidermal cells near Class IV dendrites expand in proportion to segment growth. This observation suggested that Class IV cells might grow via branch dilation—uniformly elongating branches, akin to Class I cells [1,2]. To understand whether the class IV complex arbor structure is formed by dilation or simply from growing tips, we developed this protocol to introduce a systematic approach for quantitatively assessing the growth dynamics of internal branches.

Heterochrony refers to changes in the timing of developmental events, and it is precisely regulated in the organisms by the heterochronic genes such as C. elegans lin-4 and let-7. Mutations in these genes cause precocious or retarded development of certain cell lineages. With well-defined cell lineages, C. elegans is one of the best model systems to study heterochronic genes, since the subtle changes in the development of cell lineages can be easily identified. Among the different cell types in C. elegans, hypodermal seam cells and their lineages are well known to be maintained by lin-14, whose expression level is regulated by two miRNA genes, lin-4 and let-7, at the larval stages. Therefore, analyzing the heterochronic phenotype of hypodermal seam cells in C. elegans could yield detailed insights into the status of the miRNA pathway. Here we describe the assay protocol to analyze the heterochronic phenotypes of C. elegans hypodermal seam cells, which can be used as a reliable method to study the miRNA pathway.