Continuous imaging of live tissues provides clear temporal sequence of biological events. The Drosophila imaginal discs have been popular experimental subjects for the study of a wide variety of biological phenomena, but long term culture that allows normal development has not been satisfactory. Here we report a culture method that can sustain normal development for 18 hours and allows live imaging. The method is validated in multiple discs and for cell proliferation, differentiation and migration. However, it does not support disc growth and cannot support cell proliferation for more than 7 to 12 hr. We monitored the cellular behavior of retinal basal glia in the developing eye disc and found that distinct glia type has distinct properties of proliferation and migration. The live imaging provided direct proof that wrapping glia differentiated from existing glia after migrating to the anterior front, and unexpectedly found that they undergo endoreplication before wrapping axons, and their nuclei migrate up and down along the axons. UV-induced specific labeling of a single carpet glia also showed that the two carpet glia membrane do not overlap and suggests a tiling or repulsion mechanism between the two cells. These findings demonstrated the usefulness of an ex vivo culture method and live imaging.

Photoreceptor (PR) axons project from the retina to the optic lobe in brain and form a precise retinotopic map in the Drosophila visual system. Yet the role of retinal basal glia in the retinotopic map formation is not previously known. We examined the formation of the retinotopic map by marking single PR pairs and following their axonal projections. In addition to confirming previous studies that the spatial information is preserved from the retina to the optic stalk and then to the optic lamina, we found that the young PR R3/4 axons transiently overshoot and then retract to their final destination, the lamina plexus. We then examined the process of wrapping glia (WG) membrane extension in the eye disc and showed that the WG membrane extensions also follow the retinotopic map. We show that the WG is important for the proper spatial distribution of PR axons in the optic stalk and lamina, suggesting an active role of wrapping glia in the retinotopic map formation.

Live imaging provides the ability to continuously track dynamic cellular and developmental processes in real time. Drosophila larval imaginal discs have been used to study many biological processes, such as cell proliferation, differentiation, growth, migration, apoptosis, competition, cell-cell signaling, and compartmental boundary formation. However, methods for the long-term ex vivo culture and live imaging of the imaginal discs have not been satisfactory, despite many efforts. Recently, we developed a method for the long-term ex vivo culture and live imaging of imaginal discs for up to 18 h. In addition to using a high insulin concentration in the culture medium, a low-melting agarose was also used to embed the disc to prevent it from drifting during the imaging period. This report uses the eye-antennal discs as an example. Photoreceptor R3/4-specific mδ0.5-Ga4 expression was followed to demonstrate that photoreceptor differentiation and ommatidial rotation can be observed during a 10 h live imaging period. This is a detailed protocol describing this simple method.

The retinal basal glia (RBG) is a group of glia that migrates from the optic stalk into the third instar larval eye disc while the photoreceptor cells (PR) are differentiating. The RBGs are grouped into three major classes based on molecular and morphological characteristics: surface glia (SG), wrapping glia (WG) and carpet glia (CG). The SGs migrate and divide. The WGs are postmitotic and wraps PR axons. The CGs have giant nucleus and extensive membrane extension that each covers half of the eye disc. In this study, we used lineage tracing methods to determine the lineage relationships among these glia subtypes and the temporal profile of the lineage decisions for RBG development. We found that the CG lineage segregated from the other RBG very early in the embryonic stage. It has been proposed that the SGs migrate under the CG membrane, which prevented SGs from contacting with the PR axons lying above the CG membrane. Upon passing the front of the CG membrane, which is slightly behind the morphogenetic furrow that marks the front of PR differentiation, the migrating SG contact the nascent PR axon, which in turn release FGF to induce SGs’ differentiation into WG. Interestingly, we found that SGs are equally distributed apical and basal to the CG membrane, so that the apical SGs are not prevented from contacting PR axons by CG membrane. Clonal analysis reveals that the apical and basal RBG are derived from distinct lineages determined before they enter the eye disc. Moreover, the basal SG lack the competence to respond to FGFR signaling, preventing its differentiation into WG. Our findings suggest that this novel glia-to-glia differentiation is both dependent on early lineage decision and on a yet unidentified regulatory mechanism, which can provide spatiotemporal coordination of WG differentiation with the progressive differentiation of photoreceptor neurons.

Huntington’s disease (HD) belongs to protein misfolding disorders associated with polyglutamine (polyQ)-rich mutant huntingtin (mHtt) protein inclusions. Currently, it is indicated that the aggregation of polyQ-rich mHtt participates in neuronal toxicity and dysfunction. Here, we designed and synthesized a polyglutamine-specific gold nanoparticle (AuNP) complex, which specifically targeted mHtt and alleviated its toxicity. The polyglutamine-specific AuNPs were prepared by decorating the surface of AuNPs with an amphiphilic peptide (JLD1) consisting of both polyglutamine-binding sequences and negatively charged sequences. By applying the polyQ aggregation model system, we demonstrated that AuNPs–JLD1 dissociated the fibrillary aggregates from the polyQ peptide and reduced its β-sheet content in a concentration-dependent manner. By further integrating polyethyleneimine (PEI) onto AuNPs–JLD1, we generated a complex (AuNPs–JLD1–PEI). We showed that this complex could penetrate cells, bind to cytosolic mHtt proteins, dissociate mHtt inclusions, reduce mHtt oligomers, and ameliorate mHtt-induced toxicity. AuNPs–JLD1–PEI was also able to be transported to the brain and improved the functional deterioration in the HD Drosophila larva model. Our results revealed the feasibility of combining AuNPs, JLD1s, and cell-penetrating polymers against mHtt protein aggregation and oligomerization, which hinted on the early therapeutic strategies against HD.