Deep Learning Decodes Multi-layered Roles of Polycomb Repressor Complex 2 During Mouse CNS Development

A prominent aspect of most, if not all, central nervous systems (CNSs) is that anterior regions (brain) are larger than posterior ones (spinal cord). Studies in Drosophila and mouse have revealed that the Polycomb Repressor Complex 2 (PRC2) acts by several mechanisms to promote anterior CNS expansion. However, it is unclear if PRC2 acts directly and/or indirectly upon key downstream genes, what the full spectrum of PRC2 action is during embryonic CNS development and how PRC2 integrates with the epigenetic landscape. We removed PRC2 function from the developing mouse CNS, by mutating the key gene Eed, and generated spatio-temporal transcriptomic data. We developed a bioinformatics workflow that incorporates standard statistical analyses with machine learning to integrate the transcriptomic response to PRC2 inactivation with epigenetic information from ENCODE. This multi-variate analysis corroborates the central involvement of PRC2 in anterior CNS expansion, and reveals layered regulation via PRC2. These findings uncover a differential logic for the role of PRC2 upon functionally distinct gene categories that drive CNS anterior expansion. To support the analysis of emerging multi-modal datasets, we provide a novel bioinformatics package that can disentangle regulatory underpinnings of heterogeneous biological processes.

FGFR/Heartless and Smog interact synergistically to negatively regulate Fog mediated G-protein coupled receptor signaling in the Drosophila nervous system

Folded gastrulation (Fog) is a secreted ligand that signals through the G-protein-coupled receptors Mist and Smog and the G-protein Concertina to activate downstream effectors to elicit cell-shape change during gastrulation. In the embryonic central nervous system (CNS), Fog has roles in axon guidance and glial morphogenesis. However, the elements of the pathway as well as mechanisms required for transducing the signal in this context have not been determined. We find that while Concertina is essential for Fog signaling, Mist is dispensable and Smog, surprisingly, functions as a negative regulator of the pathway in the CNS. Interestingly Heartless, a fibroblast growth factor receptor, also functions as a negative regulator. Furthermore, both Heartless and Smog interact in a synergistic manner to regulate Fog signaling. Our results thus identify Heartless and Smog as part of a common regulatory pathway that functions to restrict Fog signaling in the embryonic CNS and highlights the context-specific role for Fog receptors during development.

Proteolytic cleavage of Slit by the Tolkin protease converts an axon repulsion cue to an axon growth cue in vivo

ABSTRACTSlit is a secreted protein that has a canonical function of repelling growing axons from the CNS midline. The full-length Slit (Slit-FL) is cleaved into Slit-N and Slit-C fragments, which have potentially distinct functions via different receptors. Here, we report that the BMP-1/Tolloid family metalloprotease Tolkin (Tok) is responsible for Slit proteolysis in vivo and in vitro. In Drosophilatok mutants lacking Slit cleavage, midline repulsion of axons occurs normally, confirming that Slit-FL is sufficient to repel axons. However, longitudinal axon guidance is highly disrupted in tok mutants and can be rescued by midline expression of Slit-N, suggesting that Slit is the primary substrate for Tok in the embryonic CNS. Transgenic restoration of Slit-N or Slit-C does not repel axons in Slit-null flies. Slit-FL and Slit-N are both biologically active cues with distinct axon guidance functions in vivo. Slit signaling is used in diverse biological processes; therefore, differentiating between Slit-FL and Slit fragments will be essential for evaluating Slit function in broader contexts.

FGFR/Heartless and Smog interact synergistically to negatively regulate Fog mediated GPCR signaling

G-protein coupled receptor (GPCR) signaling triggered by Folded gastrulation (Fog) is one of the pathways known to regulate glial organization and morphogenesis in the embryonic CNS in Drosophila. Fog is best known for its role in epithelial morphogenesis during gastrulation. Here, the signaling pathway includes GPCRs Mist and Smog and the G-Protein Concertina (Cta) which activate downstream effectors to bring about cytoskeletal changes essential for cell shape changeIn this study, we identify molecular players that mediate and serve as important regulators of Fog signaling in the embryonic CNS. We find that while Cta is essential for Fog signaling neither receptors, Mist nor Smog mediates signaling in the CNS. On the contrary, we find that Smog functions as a negative regulator of the pathway. Surprisingly, Heartless which encodes a fibroblast growth factor receptor, also functions as a negative regulator of Fog signaling. Further, we find that both heartless and smog interact in a synergistic manner to regulate Fog signaling.This study thus identifies novel regulators of Fog signaling that may play an important role in fine-tuning the pathway to control cell morphogenesis. It also suggests the likelihood of there being multiple receptors for Fog that mediate and regulate signaling in a context specific manner.Author SummaryIn Drosophila, Folded gastrulation (Fog) functions as ligand that signals via GPCRs to regulate cell shape during gastrulation -one of the earliest events in embryogenesis. Here, Fog signals via receptors Mist and Smog to activate the G-protein Concertina to elicit change in cell shape. In the embryonic central nervous system (CNS) this pathway regulates shape and organization of glia important for functions such as insulation of neurons and synapses.The mechanism of Fog signal transduction in the CNS and its regulation is not well understood. We have sought to address these questions in our study. We find that Concertina is an essential factor for Fog signaling in the CNS but interestingly Mist is not. In contrast, Smog functions as a negative regulator such that loss of Smog enhances Fog signaling. A similar role is played by the receptor tyrosine kinase-Heartless. Interestingly, we find that Smog and Heartless interact as part of a common genetic network to regulate Fog signaling. Our results thus provide novel insights into the regulation of Fog signaling and shed light on how signaling can be fine-tuned in a context dependent manner to control cell shape change which plays a critical role during development and organ formation.

Drosophila Embryonic CNS Development: Neurogenesis, Gliogenesis, Cell Fate, and Differentiation

The Drosophila embryonic central nervous system (CNS) is a complex organ consisting of ∼15,000 neurons and glia that is generated in ∼1 day of development. For the past 40 years, Drosophila developmental neuroscientists have described each step of CNS development in precise molecular genetic detail. This has led to an understanding of how an intricate nervous system emerges from a single cell. These studies have also provided important, new concepts in developmental biology, and provided an essential model for understanding similar processes in other organisms. In this article, the key genes that guide Drosophila CNS development and how they function is reviewed. Features of CNS development covered in this review are neurogenesis, gliogenesis, cell fate specification, and differentiation.