The HUSH complex controls brain architecture and protocadherin fidelity

Fine-tuning of neural connectivity is important for cerebral functions and brain evolution. Protocadherins provide barcodes for neuronal identity as well as synapse formation and expansion of protocadherin cluster genes has been linked to advanced cognitive functions. The tightly controlled stochastic and combinatorial expression of the different protocadherin isoforms in individual neurons provides the molecular basis for neuronal diversity, neuronal network complexity and function of the vertebrate brain. How protocadherins are epigenetically controlled has not yet been fully elucidated. Here we show that the HUSH (human silencing hub) complex containing H3K9me3 binding protein M-phase phosphoprotein 8 (MPP8) and Microrchidia CW-type zinc finger protein 2 (MORC2), critically controls the fidelity of protocadherin expression. MPP8 and MORC2A are highly expressed in the murine brain and exclusively found in neurons. Genetic inactivation of Mphosph8 (coding for MPP8) or Morc2a in the nervous system of mice leads to increased brain size, altered brain architecture, and behavioral changes. Mechanistically, MPP8 and MORC2A precisely and selectively suppress the repetitive-like protocadherin gene cluster on mouse chromosome 18 in a H3K9me3-dependent manner, thereby affecting synapse formation. Moreover, we demonstrate that individual MPHOSPH8- or MORC2-deficient neurons in human cerebral organoids express increased numbers of clustered protocadherin isoforms. Our data identify the HUSH complex, previously linked to silencing of repetitive transposable elements, as a key epigenetic regulator of protocadherin expression in the nervous system and thereby brain development and neuronal individuality in mice and humans.

Modular Derivation and Unbiased Single-cell Analysis of Regional Human Hindbrain And Spinal Neurons Enables Discovery of Nuanced Transcriptomic Patterns along Developmental Axes

Our inability to derive the vast neuronal diversity of the posterior central nervous system (pCNS) using human pluripotent stem cells (hPSCs) poses a major impediment to understanding human neurodevelopment and disease in the hindbrain and spinal cord. Here we establish a modular differentiation paradigm that recapitulates patterning along both the rostrocaudal (R/C) and dorsoventral (D/V) axes of the pCNS, enabling derivation of any neuronal phenotype with discrete regional specificity. First, neuromesodermal progenitors (NMPs) with discrete Hox profiles are efficiently converted to pCNS progenitors (pCNSPs). Then by tuning D/V signaling, pCNSPs are directed to ventral Shh-dependent MNs (MNs) and locomotor interneurons (INs) or dorsal TGF-β-dependent proprioceptive INs and TGF-β-independent sensory INs. We applied D/V protocols to NMPs spanning the R/C axis for expansive single-cell RNA-sequencing (scRNAseq) analysis. By implementing a novel computational pipeline comprising sparse non-negative matrix factorization, consensus clustering, and combinatorial gene expression pattern identification, we detect hundreds of transcriptional markers within region-specific neuronal phenotypes, enabling discovery of gene expression patterns along the developmental axes. These findings highlight the potential of these resources to advance a mechanistic understanding of pCNS development, expand the potential and accuracy of in vitro models, and inform novel regenerative therapeutic strategies.

An in vivo Calcium Imaging Approach for the Identification of Cell-Type Specific Patterns in the Developing Cortex

Neuronal activity profoundly shapes the maturation of developing neurons. However, technical limitations have hampered the ability to capture the progression of activity patterns in genetically defined neuronal populations. This task is particularly daunting given the substantial diversity of pyramidal cells and interneurons in the neocortex. A hallmark in the development of this neuronal diversity is the participation in network activity that regulates circuit assembly. Here, we describe detailed methodology on imaging neuronal cohorts longitudinally throughout postnatal stages in the mouse somatosensory cortex. To capture neuronal activity, we expressed the genetically encoded calcium sensor GCaMP6s in three distinct interneuron populations, the 5HT3aR-expressing layer 1 (L1) interneurons, SST interneurons, and VIP interneurons. We performed cranial window surgeries as early as postnatal day (P) 5 and imaged the same cohort of neurons in un-anesthetized mice from P6 to P36. This Longitudinal two-photon imaging preparation allows the activity of single neurons to be tracked throughout development as well as plasticity induced by sensory experience and learning, opening up avenues of research to answer fundamental questions in neural development in vivo.

The Temporal Mechanisms Guiding Interneuron Differentiation in the Spinal Cord

Neurogenesis timing is an essential developmental mechanism for neuronal diversity and organization throughout the central nervous system. In the mouse spinal cord, growing evidence is beginning to reveal that neurogenesis timing acts in tandem with spatial molecular controls to diversify molecularly and functionally distinct post-mitotic interneuron subpopulations. Particularly, in some cases, this temporal ordering of interneuron differentiation has been shown to instruct specific sensorimotor circuit wirings. In zebrafish, in vivo preparations have revealed that sequential neurogenesis waves of interneurons and motor neurons form speed-dependent locomotor circuits throughout the spinal cord and brainstem. In the present review, we discuss temporal principals of interneuron diversity taken from both mouse and zebrafish systems highlighting how each can lend illuminating insights to the other. Moving forward, it is important to combine the collective knowledge from different systems to eventually understand how temporally regulated subpopulation function differentially across speed- and/or state-dependent sensorimotor movement tasks.

Single-cell genomics reveals region-specific developmental trajectories underlying neuronal diversity in the prenatal human hypothalamus

The hypothalamus is critically important for regulating most autonomic, metabolic, and behavioral functions essential for life and species propagation, yet a comprehensive understanding of neuronal subtypes and their development in the human brain is lacking. Here, we characterized the prenatal human hypothalamus by sequencing the transcriptomes of 45,574 single-cells from 12 embryos, spanning gestational weeks 4 through 25. These cells describe a temporal trajectory from proliferative stem cell populations to maturing neurons and glia, including 38 distinct excitatory and inhibitory neuronal subtypes. Merging these data with paired samples from the cortex and ganglionic eminences (GE) revealed two distinct neurogenesis pathways, one shared between GE and hypothalamus and a second unique to cortex. Gene regulatory network modeling predicted that these distinct maturation trajectories involve the activation of region- and cell type-specific transcription factor networks. These results provide the first comprehensive transcriptomic view of human hypothalamus development at cellular resolution.

A comprehensive series of temporal transcription factors in the fly visual system

The brain consists of thousands of different neuronal types that are generated through multiple divisions of neuronal stem cells. These stem cells have the capacity to generate different neuronal types at different stages of their development. In Drosophila, this temporal patterning is driven by the successive expression of temporal transcription factors (tTFs). While a number of tTFs are known in different animals and across various parts of the nervous system, these have been mostly identified by informed guesses and antibody availability. We used single-cell mRNA sequencing to identify the complete series of tTFs that specify most Drosophila medulla neurons in the optic lobe. We tested the genetic interactions among these tTFs. While we verify the general principle that tTFs regulate the progression of the series by activating the next tTFs in the series and repressing the previous ones, we also identify more complex regulations. Two of the tTFs, Eyeless and Dichaete, act as hubs integrating the input of several upstream tTFs before allowing the series to progress and in turn regulating the expression of several downstream tTFs. Moreover, we show that tTFs not only specify neuronal identity by controlling the expression of cell type-specific genes. Finally, we describe the very first steps of neuronal differentiation and find that terminal differentiation genes, such as neurotransmitter-related genes, are present as transcripts, but not as proteins, in immature larval neurons days before they are being used in functioning neurons; we show that these mechanisms are conserved in humans. Our results offer a comprehensive description of a temporal series of tTFs in a neuronal system, offering mechanistic insights into the regulation of the progression of the series and the regulation of neuronal diversity. This represents a proof-of-principle for the use of single-cell mRNA sequencing for the comparison of temporal patterning across phyla that can lead to an understanding of how the human brain develops and how it has evolved.