Developmental Biology

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.

Recent popularization of next-generation sequencing enables conducting easy transcriptome analysis. Nevertheless, substantial RNA isolation work prior to RNA sequencing, as well as the high cost involved, still makes the routine use of large-scale transcriptome analysis difficult. For example, conventional phenol-chloroform RNA extraction cannot be easily applied to hundreds of samples. Therefore, we developed Direct-TRI, a new cost-effective and high throughput RNA-extraction method that uses a commercial guanidine-phenol-based RNA extraction reagent and a 96-well silica column plate. We applied Direct-TRI to zebrafish whole larvae and juvenile samples and obtained comparable RNA qualities by several different homogenization methods such as vortexing, manual homogenizing, and freezing/crushing. Direct-TRI enabled the extraction of 192 RNA samples in an hour with a cost of less than a dollar per sample. Direct-TRI is useful for large-scale transcriptome studies, manipulating hundreds of zebrafish individuals, and may be used with other animal samples.

The Drosophila melanogaster trachea is a branched network of rigid chitin-lined tubes that ramify throughout the body and functions as the fly’s respiratory organ. Small openings at the ends of the tracheal tubes allow gas exchange to occur by diffusion between internal tissues and the exterior environment. Tracheal tubes are lined by a single layer of epithelial cells, which secrete chitin and control tube morphology and size. Studies of tracheal development in Drosophila embryos have elucidated fundamental mechanisms of tube morphogenesis and maintenance in vivo, and identified major signaling pathways that regulate these processes (Manning and Krasnow, 1993; Affolter and Shilo, 2000; Zuo et al., 2013; Kerman et al., 2006; Schottenfeld et al., 2010). In recent years, there has been growing interest in the trachea during metamorphosis, when tracheal branches that had served as the respiratory organ in the larva decays and is repaired or replaced by new tracheal tissue arising from committed tracheal progenitor cells, or mature tracheal cells de-differentiated to a progenitor state (Manning and Krasnow, 1993; Sato and Kornberg, 2002; Guha et al., 2008; Guha, and Kornberg, 2005; Weaver and Krasnow, 2008; Pitsouli and Perrimon, 2010; Chen and Krasnow, 2014) forming the adult tracheal by the end of the process. The ongoing decay and tissue formation models aspects of tissue repair and regeneration in other organisms, and has been used to understand how progenitor cells divide and differentiate (Pitsouli and Perrimon, 2010; Pitsouli and Perrimon, 2013), and how they grow out of their niche to replace decaying tissue (Chen and Krasnow, 2014). Here, we present a protocol to dissect, fix, and immunostain tracheal tissue in Drosophila larvae and pupae undergoing metamorphosis. This protocol can be used to immunostain proteins expressed in tracheal tissue, or to amplify signals from weakly expressed fluorescent reporters (as shown in Figure 6). With the appropriate antibodies and genetic reporters, this protocol can be used to visualize decaying larval trachea and the progenitor cells that replace them in a time-course analysis, as well as determine expression of proteins in these cells that may play a role in tissue decay and replacement.