Dietmar Schmucker

Ultra-deep profiling of alternatively spliced Drosophila Dscam isoforms by circularization-assisted multi-segment sequencing

The Drosophila melanogaster gene Dscam (Down syndrome cell adhesion molecule) can generate thousands of different ectodomains via mutual exclusive splicing of three large exon clusters. The isoform diversity plays a profound role in both neuronal wiring and pathogen recognition. However, the isoform expression pattern at the global level remained unexplored. Here, we developed a novel method that allows for direct quantification of the alternatively spliced exon combinations from over hundreds of millions of Dscam transcripts in one sequencing run. With unprecedented sequencing depth, we detected a total of 18 496 isoforms, out of 19 008 theoretically possible combinations. Importantly, we demonstrated that alternative splicing between different clusters is independent. Moreover, the isoforms were expressed across a broad dynamic range, with significant bias in cell/tissue and developmental stage‐specific patterns. Hitherto underappreciated, such bias can dramatically reduce the ability of neurons to display unique surface receptor codes. Therefore, the seemingly excessive diversity encoded in the Dscam locus might nevertheless be essential for a robust self and non‐self discrimination in neurons.

Cell-intrinsic requirement of Dscam1 isoform diversity for axon collateral formation

Wiring the developing insect brain Developmental brain wiring requires a complex set of cellular interactions often orchestrated by a large number of surface receptors. Thousands of Dscam1 receptor isoforms function as “surface tags” endowing neurons with unique molecular identities. These isoforms are important for neuronal self-recognition and dendrite self-avoidance. He et al. report that the diversity of Dscam1 isoforms is also essential for complex axonal branching of sensory neurons in developing fruit flies. Science, this issue p. 1182 Different versions of a membrane receptor control axonal growth and the extent of growth cone sprouting in flies. The isoform diversity of the Drosophila Dscam1 receptor is important for neuronal self-recognition and self-avoidance. A canonical model suggests that homophilic binding of identical Dscam1 receptor isoforms on sister dendrites ensures self-avoidance even when only a single isoform is expressed. We detected a cell-intrinsic function of Dscam1 that requires the coexpression of multiple isoforms. Manipulation of the Dscam1 isoform pool in single neurons caused severe disruption of collateral formation of mechanosensory axons. Changes in isoform abundance led to dominant dosage-sensitive inhibition of branching. We propose that the ratio of matching to nonmatching isoforms within a cell influences the Dscam1-mediated signaling strength, which in turn controls axon growth and growth cone sprouting. Cell-intrinsic use of surface receptor diversity may be of general importance in regulating axonal branching during brain wiring.

Mutual inhibition among postmitotic neurons regulates robustness of brain wiring in Drosophila

Brain connectivity maps display a delicate balance between individual variation and stereotypy, suggesting the existence of dedicated mechanisms that simultaneously permit and limit individual variation. We show that during the development of the Drosophila central nervous system, mutual inhibition among groups of neighboring postmitotic neurons during development regulates the robustness of axon target choice in a nondeterministic neuronal circuit. Specifically, neighboring postmitotic neurons communicate through Notch signaling during axonal targeting, to ensure balanced alternative axon target choices without a corresponding change in cell fate. Loss of Notch in postmitotic neurons modulates an axons target choice. However, because neighboring axons respond by choosing the complementary target, the stereotyped connectivity pattern is preserved. In contrast, loss of Notch in clones of neighboring postmitotic neurons results in erroneous coinnervation by multiple axons. Our observations establish mutual inhibition of axonal target choice as a robustness mechanism for brain wiring and unveil a novel cell fate independent function for canonical Notch signaling. DOI: http://dx.doi.org/10.7554/eLife.00337.001

Investigating CNS synaptogenesis at single-synapse resolution by combining reverse genetics with correlative light and electron microscopy

Determining direct synaptic connections of specific neurons in the central nervous system (CNS) is a major technical challenge in neuroscience. As a corollary, molecular pathways controlling developmental synaptogenesis in vivo remain difficult to address. Here, we present genetic tools for efficient and versatile labeling of organelles, cytoskeletal components and proteins at single-neuron and single-synapse resolution in Drosophila mechanosensory (ms) neurons. We extended the imaging analysis to the ultrastructural level by developing a protocol for correlative light and 3D electron microscopy (3D CLEM). We show that in ms neurons, synaptic puncta revealed by genetically encoded markers serve as a reliable indicator of individual active zones. Block-face scanning electron microscopy analysis of ms axons revealed T-bar-shaped dense bodies and other characteristic ultrastructural features of CNS synapses. For a mechanistic analysis, we directly combined the single-neuron labeling approach with cell-specific gene disruption techniques. In proof-of-principle experiments we found evidence for a highly similar requirement for the scaffolding molecule Liprin-α and its interactors Lar and DSyd-1 (RhoGAP100F) in synaptic vesicle recruitment. This suggests that these important synapse regulators might serve a shared role at presynaptic sites within the CNS. In principle, our CLEM approach is broadly applicable to the developmental and ultrastructural analysis of any cell type that can be targeted with genetically encoded markers. Summary: Genetic tools, and 3D correlative light and electron microscopy allow the dissection of the mechanisms governing synaptogenesis at single-cell resolution in the Drosophila CNS.

Axonal wiring in neural development: Target-independent mechanisms help to establish precision and complexity

The connectivity patterns of many neural circuits are highly ordered and often impressively complex. The intricate order and complexity of neuronal wiring remain not only a challenge for questions related to circuit functions but also for our understanding of how they develop with such an apparent precision. The chemotropic guidance of the growing axon by target‐derived cues represents a central paradigm for how neurons get connected with the correct target cells. However, many studies reveal a remarkable variety of important target‐independent wiring mechanisms. These mechanisms include axonal sorting, axonal tiling, growth cone polarization, as well as cell‐intrinsic mechanisms underlying growth cone sprouting, and neurite branching. Our review focuses on target independent wiring mechanisms and in particular on recent progress emerging from studies on three different sensory systems: olfactory, visual, and somatosensory. We discuss molecular mechanisms that operate during axon‐axon interactions or constitute axon‐intrinsic functions and outline how they complement the well‐known target‐dependent wiring mechanisms.

Quantitative profiling of Drosophila melanogaster Dscam1 isoforms reveals no changes in splicing after bacterial exposure

The hypervariable Dscam1 (Down syndrome cell adhesion molecule 1) gene can produce thousands of different ectodomain isoforms via mutually exclusive alternative splicing. Dscam1 appears to be involved in the immune response of some insects and crustaceans. It has been proposed that the diverse isoforms may be involved in the recognition of, or the defence against, diverse parasite epitopes, although evidence to support this is sparse. A prediction that can be generated from this hypothesis is that the gene expression of specific exons and/or isoforms is influenced by exposure to an immune elicitor. To test this hypothesis, we for the first time, use a long read RNA sequencing method to directly investigate the Dscam1 splicing pattern after exposing adult Drosophila melanogaster and a S2 cell line to live Escherichia coli. After bacterial exposure both models showed increased expression of immune-related genes, indicating that the immune system had been activated. However there were no changes in total Dscam1 mRNA expression. RNA sequencing further showed that there were no significant changes in individual exon expression and no changes in isoform splicing patterns in response to bacterial exposure. Therefore our studies do not support a change of D. melanogaster Dscam1 isoform diversity in response to live E. coli. Nevertheless, in future this approach could be used to identify potentially immune-related Dscam1 splicing regulation in other host species or in response to other pathogens.

Role of self-avoidance in neuronal wiring.

• Self-avoidance. Neuronal self-avoidance represent a repulsion system, which is required for preventing aberrant overlap of sister neurites.

Regulation of Drosophila Brain Wiring by Neuropil Interactions via a Slit-Robo-RPTP Signaling Complex

Summary The axonal wiring molecule Slit and its Round-About (Robo) receptors are conserved regulators of nerve cord patterning. Robo receptors also contribute to wiring brain circuits. Whether molecular mechanisms regulating these signals are modified to fit more complex brain wiring processes is unclear. We investigated the role of Slit and Robo receptors in wiring Drosophila higher-order brain circuits and identified differences in the cellular and molecular mechanisms of Robo/Slit function. First, we find that signaling by Robo receptors in the brain is regulated by the Receptor Protein Tyrosine Phosphatase RPTP69d. RPTP69d increases membrane availability of Robo3 without affecting its phosphorylation state. Second, we detect no midline localization of Slit during brain development. Instead, Slit is enriched in the mushroom body, a neuronal structure covering large areas of the brain. Thus, a divergent molecular mechanism regulates neuronal circuit wiring in the Drosophila brain, partly in response to signals from the mushroom body.

A Fat-Facets-Dscam1-JNK Pathway Enhances Axonal Growth in Development and after Injury

Injury to the adult central nervous systems (CNS) can result in severe long-term disability because damaged CNS connections fail to regenerate after trauma. Identification of regulators that enhance the intrinsic growth capacity of severed axons is a first step to restore function. Here, we conducted a gain-of-function genetic screen in Drosophila to identify strong inducers of axonal growth after injury. We focus on a novel axis the Down Syndrome Cell Adhesion Molecule (Dscam1), the de-ubiquitinating enzyme Fat Facets (Faf)/Usp9x and the Jun N-Terminal Kinase (JNK) pathway transcription factor Kayak (Kay)/Fos. Genetic and biochemical analyses link these genes in a common signaling pathway whereby Faf stabilizes Dscam1 protein levels, by acting on the 3′-UTR of its mRNA, and Dscam1 acts upstream of the growth-promoting JNK signal. The mammalian homolog of Faf, Usp9x/FAM, shares both the regenerative and Dscam1 stabilizing activities, suggesting a conserved mechanism.

Characterization of a Single Genomic Locus Encoding the Clustered Protocadherin Receptor Diversity in Xenopus tropicalis.

Clustered protocadherins (cPcdhs) constitute the largest subgroup of the cadherin superfamily, and in mammals are grouped into clusters of α-, β-, and γ-types. Tens of tandemly arranged paralogous Pcdh genes of the Pcdh clusters generate a substantial diversity of receptor isoforms. cPcdhs are known to have important roles in neuronal development, and genetic alterations of cPcdhs have been found to be associated with several neurological diseases. Here, we present a first characterization of cPcdhs in Xenopus tropicalis. We determined and annotated all cPcdh isoforms, revealing that they are present in a single chromosomal locus. We validated a total of 96 isoforms, which we show are organized in three distinct clusters. The X. tropicalis cPcdh locus is composed of one α- and two distinct γ-Pcdh clusters (pcdh-γ1 and pcdh-γ2). Bioinformatics analyses assisted by genomic BAC clone sequencing showed that the X. tropicalis α- and γ-Pcdhs are conserved at the cluster level, but, unlike mammals, X. tropicalis does not contain a β-Pcdh cluster. In contrast, the number of γ-Pcdh isoforms has expanded, possibly due to lineage-specific gene duplications. Interestingly, the number of X. tropicalis α-Pcdhs is identical between X. tropicalis and mouse. Moreover, we find highly conserved as well as novel promoter elements potentially involved in regulating the cluster-specific expression of cPcdh isoforms. This study provides important information for the understanding of the evolutionary history of cPcdh genes and future mechanistic studies. It provides an annotated X. tropicalis cPcdh genomic map and a first molecular characterization essential for functional and comparative studies.