Emerging Roles of Single-Cell Multi-Omics in Studying Developmental Temporal Patterning.

The complexity of brain structure and function is rooted in the precise spatial and temporal regulation of selective developmental events. During neurogenesis, both vertebrates and invertebrates generate a wide variety of specialized cell types through the expansion and specification of a restricted set of neuronal progenitors. Temporal patterning of neural progenitors rests on fine regulation between cell-intrinsic and cell-extrinsic mechanisms. The rapid emergence of high-throughput single-cell technologies combined with elaborate computational analysis has started to provide us with unprecedented biological insights related to temporal patterning in the developing central nervous system (CNS). Here, we present an overview of recent advances in Drosophila and vertebrates, focusing both on cell-intrinsic mechanisms and environmental influences. We then describe the various multi-omics approaches that have strongly contributed to our current understanding and discuss perspectives on the various -omics approaches that hold great potential for the future of temporal patterning research

Lipid droplet availability affects neural stem/progenitor cell metabolism and proliferation.

Neural stem/progenitor cells (NSPCs) generate new neurons throughout adulthood. However, the underlying regulatory processes are still not fully understood. Lipid metabolism plays an important role in regulating NSPC activity: build-up of lipids is crucial for NSPC proliferation, whereas break-down of lipids has been shown to regulate NSPC quiescence. Despite their central role for cellular lipid metabolism, the role of lipid droplets (LDs), the lipid storing organelles, in NSPCs remains underexplored. Here we show that LDs are highly abundant in adult mouse NSPCs, and that LD accumulation is significantly altered upon fate changes such as quiescence and differentiation. NSPC proliferation is influenced by the number of LDs, inhibition of LD build-up, breakdown or usage, and the asymmetric inheritance of LDs during mitosis. Furthermore, high LD-containing NSPCs have increased metabolic activity and capacity, but do not suffer from increased oxidative damage. Together, these data indicate an instructive role for LDs in driving NSPC behaviour

Absence of RNA-binding protein FXR2P prevents prolonged phase of kainate-induced seizures.

Status epilepticus (SE) is a condition in which seizures are not self-terminating and thereby pose a serious threat to the patient's life. The molecular mechanisms underlying SE are likely heterogeneous and not well understood. Here, we reveal a role for the RNA-binding protein Fragile X-Related Protein 2 (FXR2P) in SE. Fxr2 KO mice display reduced sensitivity specifically to kainic acid-induced SE. Immunoprecipitation of FXR2P coupled to next-generation sequencing of associated mRNAs shows that FXR2P targets are enriched in genes that encode glutamatergic post-synaptic components. Of note, the FXR2P target transcriptome has a significant overlap with epilepsy and SE risk genes. In addition, Fxr2 KO mice fail to show sustained ERK1/2 phosphorylation induced by KA and present reduced burst activity in the hippocampus. Taken together, our findings show that the absence of FXR2P decreases the expression of glutamatergic proteins, and this decrease might prevent self-sustained seizures

Specialized astrocytes mediate glutamatergic gliotransmission in the CNS

Multimodal astrocyte–neuron communications govern brain circuitry assembly and function1. For example, through rapid glutamate release, astrocytes can control excitability, plasticity and synchronous activity2,3 of synaptic networks, while also contributing to their dysregulation in neuropsychiatric conditions4–7. For astrocytes to communicate through fast focal glutamate release, they should possess an apparatus for Ca2+-dependent exocytosis similar to neurons8–10. However, the existence of this mechanism has been questioned11–13 owing to inconsistent data14–17 and a lack of direct supporting evidence. Here we revisited the astrocyte glutamate exocytosis hypothesis by considering the emerging molecular heterogeneity of astrocytes18–21 and using molecular, bioinformatic and imaging approaches, together with cell-specific genetic tools that interfere with glutamate exocytosis in vivo. By analysing existing single-cell RNA-sequencing databases and our patch-seq data, we identified nine molecularly distinct clusters of hippocampal astrocytes, among which we found a notable subpopulation that selectively expressed synaptic-like glutamate-release machinery and localized to discrete hippocampal sites. Using GluSnFR-based glutamate imaging22 in situ and in vivo, we identified a corresponding astrocyte subgroup that responds reliably to astrocyte-selective stimulations with subsecond glutamate release events at spatially precise hotspots, which were suppressed by astrocyte-targeted deletion of vesicular glutamate transporter 1 (VGLUT1). Furthermore, deletion of this transporter or its isoform VGLUT2 revealed specific contributions of glutamatergic astrocytes in cortico-hippocampal and nigrostriatal circuits during normal behaviour and pathological processes. By uncovering this atypical subpopulation of specialized astrocytes in the adult brain, we provide insights into the complex roles of astrocytes in central nervous system (CNS) physiology and diseases, and identify a potential therapeutic target

Interactions between Connected Half-Sarcomeres Produce Emergent Mechanical Behavior in a Mathematical Model of Muscle

Most reductionist theories of muscle attribute a fiber's mechanical properties to the scaled behavior of a single half-sarcomere. Mathematical models of this type can explain many of the known mechanical properties of muscle but have to incorporate a passive mechanical component that becomes ∼300% stiffer in activating conditions to reproduce the force response elicited by stretching a fast mammalian muscle fiber. The available experimental data suggests that titin filaments, which are the mostly likely source of the passive component, become at most ∼30% stiffer in saturating Ca2+ solutions. The work described in this manuscript used computer modeling to test an alternative systems theory that attributes the stretch response of a mammalian fiber to the composite behavior of a collection of half-sarcomeres. The principal finding was that the stretch response of a chemically permeabilized rabbit psoas fiber could be reproduced with a framework consisting of 300 half-sarcomeres arranged in 6 parallel myofibrils without requiring titin filaments to stiffen in activating solutions. Ablation of inter-myofibrillar links in the computer simulations lowered isometric force values and lowered energy absorption during a stretch. This computed behavior mimics effects previously observed in experiments using muscles from desmin-deficient mice in which the connections between Z-disks in adjacent myofibrils are presumably compromised. The current simulations suggest that muscle fibers exhibit emergent properties that reflect interactions between half-sarcomeres and are not properties of a single half-sarcomere in isolation. It is therefore likely that full quantitative understanding of a fiber's mechanical properties requires detailed analysis of a complete fiber system and cannot be achieved by focusing solely on the properties of a single half-sarcomere

New insights into CNS development from multiomics approaches.

Our understanding of the central nervous system (CNS) development has been strongly enhanced by the recent progress of single-cell multiomics approaches. Certainly, the multiplex profiling of individual cell epigenomes and transcriptomes together with dynamic lineage tracing systems brings encouraging new perspectives and prompts a paradigm shift in neuroscience developmental research. In this review, we outline the latest multiomics -based findings in CNS development, from the early CNS patterning to the regional specification of the CNS along anterior-posterior axis (forebrain, midbrain, hindbrain and spinal cord). Overall, multiomics development has substantially impacted current knowledge and has challenged our classical models for embryonic CNS development. Integrating all these newly generated -omics databases represents the next step to overcome challenges in understanding developmental diseases

A neural substrate for negative affect dictates female parental behavior.

Parental behaviors secure the well-being of newborns and concomitantly limit negative affective states in adults, which emerge when coping with neonatal distress becomes challenging. Whether negative-affect-related neuronal circuits orchestrate parental actions is unknown. Here, we identify parental signatures in lateral habenula neurons receiving bed nucleus of stria terminalis innervation ( <sup>BNST</sup> LHb). We find that LHb neurons of virgin female mice increase their activity following pup distress vocalization and are necessary for pup-call-driven aversive behaviors. LHb activity rises during pup retrieval, a behavior worsened by LHb inactivation. Intersectional cell identification and transcriptional profiling associate <sup>BNST</sup> LHb cells to parenting and outline a gene expression in female virgins similar to that in mothers but different from that in non-parental virgin male mice. Finally, tracking and manipulating <sup>BNST</sup> LHb cell activity demonstrates their specificity for encoding negative affect and pup retrieval. Thus, a negative affect neural circuit processes newborn distress signals and may limit them by guiding female parenting