Olivier Urwyler

Neuronal morphogenesis, integration into circuits, and remodeling of synaptic connections occur in temporally and spatially defined steps. Accordingly, the expression of proteins and specific protein isoforms that contribute to these processes must be controlled quantitatively in time and space. A wide variety of post-transcriptional regulatory mechanisms, which act on pre-mRNA and mRNA molecules contribute to this control. They are thereby critically involved in physiological and pathophysiological nervous system development, function, and maintenance. Here, we review recent findings on how mRNA metabolism contributes to neuronal development, from neural stem cell maintenance to synapse specification, with a particular focus on axon growth, guidance, branching, and synapse formation. We emphasize the role of RNA-binding proteins, and highlight their emerging roles in the poorly understood molecular processes of RNA editing, alternative polyadenylation, and temporal control of splicing, while also discussing alternative splicing, RNA localization, and local translation. We illustrate with the example of the evolutionary conserved Musashi protein family how individual RNA-binding proteins are, on the one hand, acting in different processes of RNA metabolism, and, on the other hand, impacting multiple steps in neuronal development and circuit formation. Finally, we provide links to diseases that have been associated with the malfunction of RNA-binding proteins and disrupted post-transcriptional regulation.

Compartmentalization of axons: Neurons can spread branches far and wide across the central nervous system. One neuron can simultaneously contact several other neurons, amplifying network complexity. Studying the Drosophila brain, Urwyler et al. found that not all axon branches are the same, even within one neuron (see the Perspective by Falkner and Scheiffele). A membrane-anchored phosphatase functions within a subset of axon branches to regulate synapse formation. Thus, the pattern of synapses on one neuron is not random but rather is spatially restricted according to subcompartments in the axon. Science , this issue p. eaau9952 ; see also p. 437

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.

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

Summary: Morphogenesis in multicellular organisms requires the careful coordination of cytoskeletal elements, dynamic regulation of cell adhesion and extensive cell migration. sosie (sie) is a novel gene required in various morphogenesis processes in Drosophila oogenesis. Lack of sie interferes with normal egg chamber packaging, maintenance of epithelial integrity and control of follicle cell migration, indicating that sie is involved in controlling epithelial integrity and cell migration. For these functions sie is required both in the germ line and in the soma. Consistent with this, Sosie localizes to plasma membranes in the germ line and in the somatic follicle cells and is predicted to present an EGF-like domain on the extracellular side. Two positively charged residues, C-terminal to the predicted transmembrane domain (on the cytoplasmic side), are required for normal plasma membrane localization of Sosie. Because sie also contributes to normal cortical localization of βH-Spectrin, it appears that cortical βH-Spectrin mediates some of the functions of sosie. sie also interacts with the genes coding for the actin organizers Filamin and Profilin and, in the absence of sie function, F-actin is less well organized and nurse cells frequently fuse.

The trimeric CAK complex functions in cell cycle control by phosphorylating and activating Cdks while TFIIH-linked CAK functions in transcription. CAK also associates into a tetramer with Xpd, and our analysis of young Drosophila embryos that do not require transcription now suggests a cell cycle function for this interaction. xpd is essential for the coordination and rapid progression of the mitotic divisions during the late nuclear division cycles. Lack of Xpd also causes defects in the dynamics of the mitotic spindle and chromosomal instability as seen in the failure to segregate chromosomes properly during ana- and telophase. These defects appear to be also nucleotide excision repair (NER)–independent. In the absence of Xpd, misrouted spindle microtubules attach to chromosomes of neighboring mitotic figures, removing them from their normal location and causing multipolar spindles and aneuploidy. Lack of Xpd also causes changes in the dynamics of subcellular and temporal distribution of the CAK component Cdk7 and local mitotic kinase activity. xpd thus functions normally to re-localize Cdk7(CAK) to different subcellular compartments, apparently removing it from its cell cycle substrate, the mitotic Cdk. This work proves that the multitask protein Xpd also plays an essential role in cell cycle regulation that appears to be independent of transcription or NER. Xpd dynamically localizes Cdk7/CAK to and away from subcellular substrates, thereby controlling local mitotic kinase activity. Possibly through this activity, xpd controls spindle dynamics and chromosome segregation in our model system. This novel role of xpd should also lead to new insights into the understanding of the neurological and cancer aspects of the human XPD disease phenotypes. Mutations in human xpd cause three different syndromes—XP (xeroderma pigmentosum), TTD (trichothiodystrophy), and CS (Cockayne syndrome)—and various different phenotypes, such as sun-induced hyperpigmentation of the skin, cutaneous abnormalities, neuronal degeneration, and developmental retardation. In addition, while some mutations cause a highly elevated cancer risk, others do not. The multitask protein Xpd functions in transcription, nucleotide excision repair (NER), and in cell cycle regulation. In a situation where transcription is not required and NER not induced, we specifically analyzed the cell cycle function of Xpd in Drosophila. In this situation Xpd locally controls the dynamic localization of Cdk7, the catalytic subunit of the Cdk activating kinase (CAK) to and away from its cellular targets, thereby regulating mitotic kinase activity and mitotic exit. Xpd also controls spindle dynamics to prevent formation of multipolar and promiscuous spindles and aneuploidy. Through multitask proteins like Xpd and Cdk7 cells regulate different cellular pathways in a coordinated fashion. In addition to the basic research relevance, the newly gained knowledge about the cell cycle function of Xpd and its control of spindle dynamics is also relevant for human xpd patients because it shows a possible pathway that could lead to highly increased cancer risk and neurological defects.

Protein phosphorylation is involved in posttranslational control of essentially all biological processes. Using mass spectrometry, recent analyses of whole phosphoproteomes led to the identification of numerous new phosphorylation sites. However, the function of most of these sites remained unknown. We chose the Drosophila Bicaudal-D protein to estimate the importance of individual phosphorylation events. Being involved in different cellular processes, BicD is required for oocyte determination, for RNA transport during oogenesis and embryogenesis, and for photoreceptor nuclei migration in the developing eye. The numerous roles of BicD and the available evidence for functional importance of BicD phosphorylation led us to identify eight phosphorylation sites of BicD, and we tested a total of 14 identified and suspected phosphoserine residues for their functional importance in vivo in flies. Surprisingly, all these serines turned out to be dispensable for providing sufficient basal BicD activity for normal growth and development. However, in a genetically sensitized background where the BicD^A40V protein variant provides only partial activity, serine 103 substitutions are not neutral anymore, but show surprising differences. The S103D substitution completely inactivates the protein, whereas S103A behaves neutral, and the S103F substitution, isolated in a genetic screen, restores BicD^A40V function. Our results suggest that many BicD phosphorylation events may either be fortuitous or play a modulating function as shown for Ser¹⁰³. Remarkably, amongst the Drosophila serines we found phosphorylated, Ser¹⁰³ is the only one that is fully conserved in mammalian BicD.