Tzumin Lee

The mosaic analysis with repressible cell markers (MARCM) technique allows for lineage tracing in Drosophila. Here, the authors report an improvement on this technique, twin-spot MARCM, which permits high-resolution lineage tracing of both sister clones. A comprehensive understanding of the brain requires the analysis of individual neurons. We used twin-spot mosaic analysis with repressible cell markers (twin-spot MARCM) to trace cell lineages at high resolution by independently labeling paired sister clones. We determined patterns of neurogenesis and the influences of lineage on neuron-type specification. Notably, neural progenitors were able to yield intermediate precursors that create one, two or more neurons. Furthermore, neurons acquired stereotyped projections according to their temporal position in various brain sublineages. Twin-spot MARCM also permitted birth dating of mutant clones, enabling us to detect a single temporal fate that required chinmo in a sublineage of six Drosophila central complex neurons. In sum, twin-spot MARCM can reveal the developmental origins of neurons and the mechanisms that underlie cell fate.

Drosophila Down syndrome cell adhesion molecule(Dscam) can be variably spliced to encode 152,064 distinct single-pass transmembrane proteins. In addition to 19,008 possible ectodomains and two alternative transmembrane segments, it may carry endodomains containing or lacking exons 19 and 23. Here, we determine the role of Dscam endodomain diversity in neural development. Dscam with full-length endodomain is largely restricted to embryogenesis. In contrast, most Dscams lack exons 19 and 23 at postembryonic stages. As implicated from the expression patterns, removal ofDscamexon 19-containing variants disrupts wiring of embryonic neurons while silencing ofDscamtranscripts lacking exon 19 or exon 23 effectively blocks postembryonic neuronal morphogenesis. Furthermore, compared with exon 19-containing Dscam, transgenic Dscam without exon 19 is more efficiently targeted to neurites and more potently suppresses axon bifurcation inDscammutant neurons. In sum, Dscam with or without exon 19 in its endodomain is used to govern different stage-specific neuronal morphogenetic processes, possibly due to differences in protein targeting.

Glial cells exist throughout the nervous system, and play essential roles in various aspects of neural development and function. Distinct types of glia may govern diverse glial functions. To determine the roles of glia requires systematic characterization of glia diversity and development. In the adultDrosophilacentral brain, we identify five different types of glia based on its location, morphology, marker expression, and development. Perineurial and subperineurial glia reside in two separate single-cell layers on the brain surface, cortex glia form a glial mesh in the brain cortex where neuronal cell bodies reside, while ensheathing and astrocyte-like glia enwrap and infiltrate into neuropils, respectively. Clonal analysis reveals that distinct glial types derive from different precursors, and that most adult perineurial, ensheathing, and astrocyte-like glia are produced after embryogenesis. Notably, perineurial glial cells are made locally on the brain surface without the involvement ofgcm(glial cell missing). In contrast, the widespread ensheathing and astrocyte-like glia derive from specific brain regions in agcm-dependent manner. This study documents glia diversity in the adult fly brain and demonstrates involvement of different developmental programs in the derivation of distinct types of glia. It lays an essential foundation for studying glia development and function in theDrosophilabrain.

Many membrane proteins, including Drosophila Dscam, are enriched in dendrites or axons within neurons. However, little is known about how the differential distribution is established and maintained. Here we investigated the mechanisms underlying the dendritic targeting of Dscam[TM1]. Through forward genetic mosaic screens and by silencing specific genes via targeted RNAi, we found that several genes, encoding various components of the dynein-dynactin complex, are required for restricting Dscam[TM1] to the mushroom body dendrites. In contrast, compromising dynein/dynactin function did not affect dendritic targeting of two other dendritic markers, Nod and Rdl. Tracing newly synthesized Dscam[TM1] further revealed that compromising dynein/dynactin function did not affect the initial dendritic targeting of Dscam[TM1], but disrupted the maintenance of its restriction to dendrites. The results of this study suggest multiple mechanisms of dendritic protein targeting. Notably, dynein-dynactin plays a role in excluding dendritic Dscam, but not Rdl, from axons by retrograde transport.

The antennal lobe (AL) is the primary structure in the Drosophila brain that relays odor information from the antennae to higher brain centers. The characterization of uniglomerular projection neurons (PNs) and some local interneurons has facilitated our understanding of olfaction; however, many other AL neurons remain unidentified. Because neuron types are mostly specified by lineage and temporal origins, we use the MARCM techniques with a set of enhancer-trap GAL4 lines to perform systematical lineage analysis to characterize neuron morphologies, lineage origin and birth timing in the three AL neuron lineages that contain GAL4-GH146-positive PNs: anterodorsal, lateral and ventral lineages. The results show that the anterodorsal lineage is composed of pure uniglomerular PNs that project through the inner antennocerebral tract. The ventral lineage produces uniglomerular and multiglomerular PNs that project through the middle antennocerebral tract. The lateral lineage generates multiple types of neurons, including uniglomeurlar PNs, diverse atypical PNs, various types of AL local interneurons and the neurons that make no connection within the ALs. Specific neuron types in all three lineages are produced in specific time windows, although multiple neuron types in the lateral lineage are made simultaneously. These systematic cell lineage analyses have not only filled gaps in the olfactory map, but have also exemplified additional strategies used in the brain to increase neuronal diversity.

Drosophila Dscam isoforms are derived from two alternative transmembrane/juxtamembrane domains (TMs) in addition to thousands of ectodomain variants. Using a microRNA-based RNA interference technology, we selectively knocked down different subsets of Dscams containing either the exon 17.1- or exon 17.2-encoding TM. Eliminating Dscam[TM1] reduced Dscam expression but minimally affected postembryonic axonal morphogenesis. In contrast, depleting Dscam[TM2] blocked axon arborization. Further removal of Dscam[TM1] enhanced the loss-of-Dscam[TM2] axonal phenotypes. However, Dscam[TM1] primarily regulates dendritic development, as evidenced by the observations that removing Dscam[TM1] alone impeded elaboration of dendrites and that transgenic Dscam[TM1], but not Dscam[TM2], effectively rescued Dscam mutant dendritic phenotypes in mosaic organisms. These distinct Dscam functions can be attributed to the juxtamembrane regions of TMs that govern dendritic versus axonal targeting of Dscam as well. Together, we suggest that specific Drosophila Dscam juxtamembrane variants control dendritic elaboration and axonal arborization.

The molecular mechanisms underlying remodeling of neural networks remain largely unknown. In Drosophila, widespread neural remodeling occurs during metamorphosis, and is regulated by ecdysone. Kruppel‐homolog 1 (Kr‐h1) is a zinc finger transcription factor known to play a role in orchestrating ecdysone‐regulated transcriptional pathways and, furthermore, implicated in governing axon morphogenesis. Interestingly, in honey bee workers, neural expression of the Apis mellifera homolog of Kr‐h1 is enhanced during their transition to foraging behavior when there is increased neurite outgrowth, branching, and synapse formation. Here, we assessed the role(s) of KR‐H1 in Drosophila neuronal remodeling and morphology. We characterized the effect of Kr‐h1 expression on neuronal morphology through Drosophila larval, pupal, and adult stages. Increased expression of Kr‐h1 led to reduced branching in individual neurons and gross morphological changes in the mushroom bodies (MBs), while knocking down Kr‐h1 did not produce any obvious changes in neural morphology. Drosophila Kr‐h1 is normally expressed when MB neurons do not undergo active morphogenesis, suggesting that it may play a role in inhibiting morphogenesis. Further, loss of endogenous KR‐H1 enhanced the neuronal morphogenesis that is otherwise delayed due to defective TGF‐β signaling. However, loss of KR‐H1 alone did not affect neuronal morphogenesis. In addition, Kr‐h1 expression remains strongly linked to ecdysone‐regulated pathways: Kr‐h1 expression is regulated by usp, which dimerizes to the ecdysone receptor, and Kr‐h1 expression is essential for proper patterning of the ecdysone receptor isoforms in the late larval central nervous system. Thus, although KR‐H1 has a potential for modulating neuronal morphogenesis, it appears physiologically involved in coordinating general ecdysone signaling. © 2007 Wiley Periodicals, Inc. Develop Neurobiol, 2007

The intermingling of larval functional neurons with adult‐specific neurons during metamorphosis contributes to the development of the adult Drosophila brain. To better understand this process, we characterized the development of a dorsal cluster (DC) of Atonal‐positive neurons that are born at early larval stages but do not undergo extensive morphogenesis until pupal formation. We found that Baboon(Babo)/dSmad2‐mediated TGF‐β signaling, known to be essential for remodeling of larval functional neurons, is also indispensable for proper morphogenesis of these adult‐specific neurons. Mosaic analysis reveals slowed development of mutant DC neurons, as evidenced by delays in both neuronal morphogenesis and atonal expression. We observe similar phenomena in other adult‐specific neurons. We further demonstrate that Babo/dSmad2 operates autonomously in individual neurons and specifically during the late larval stage. Our results suggest that Babo/dSmad2 signaling prior to metamorphosis may be widely required to prepare neurons for the dynamic environment present during metamorphosis.