Variant in NHLRC2 leads to increased hnRNP C2 in developing neurons and the hippocampus of a mouse model of FINCA disease.

BACKGROUND: FINCA disease is a pediatric cerebropulmonary disease caused by variants in the NHL repeat-containing 2 (NHLRC2) gene. Neurological symptoms are among the first manifestations of FINCA disease, but the consequences of NHLRC2 deficiency in the central nervous system are currently unexplored. METHODS: The orthologous mouse gene is essential for development, and its complete loss leads to early embryonic lethality. In the current study, we used CRISPR/Cas9 to generate an Nhlrc2 knockin (KI) mouse line, harboring the FINCA patient missense mutation (c.442G > T, p.Asp148Tyr). A FINCA mouse model, resembling the compound heterozygote genotype of FINCA patients, was obtained by crossing the KI and Nhlrc2 knockout mouse lines. To reveal NHLRC2-interacting proteins in developing neurons, we compared cortical neuronal precursor cells of E13.5 FINCA and wild-type mouse embryos by two-dimensional difference gel electrophoresis. RESULTS: Despite the significant decrease in NHLRC2, the mice did not develop severe early onset multiorgan disease in either sex. We discovered 19 altered proteins in FINCA neuronal precursor cells; several of which are involved in vesicular transport pathways and actin dynamics which have been previously reported in other cell types including human to have an association with dysfunctional NHLRC2. Interestingly, isoform C2 of hnRNP C1/C2 was significantly increased in both developing neurons and the hippocampus of adult female FINCA mice, connecting NHLRC2 dysfunction with accumulation of RNA binding protein. CONCLUSIONS: We describe here the first NHLRC2-deficient mouse model to overcome embryonic lethality, enabling further studies on predisposing and causative mechanisms behind FINCA disease. Our novel findings suggest that disrupted RNA metabolism may contribute to the neurodegeneration observed in FINCA patients.

Mass Generation, Neuron Labeling, and 3D Imaging of Minibrains.

Minibrain is a 3D brain in vitro spheroid model, composed of a mixed population of neurons and glial cells, generated from human iPSC derived neural stem cells. Despite the advances in human 3D in vitro models such as aggregates, spheroids and organoids, there is a lack of labeling and imaging methodologies to characterize these models. In this study, we present a step-by-step methodology to generate human minibrain nurseries and novel strategies to subsequently label projection neurons, perform immunohistochemistry and 3D imaging of the minibrains at large multiplexable scales. To visualize projection neurons, we adapt viral transduction and to visualize the organization of cell types we implement immunohistochemistry. To facilitate 3D imaging of minibrains, we present here pipelines and accessories for one step mounting and clearing suitable for confocal microscopy. The pipelines are specifically designed in such a way that the assays can be multiplexed with ease for large-scale screenings using minibrains and other organoid models. Using the pipeline, we present (i) dendrite morphometric properties obtained from 3D neuron morphology reconstructions, (ii) diversity in neuron morphology, and (iii) quantified distribution of progenitors and POU3F2 positive neurons in human minibrains.

Dynamic regulation of chromatin accessibility by pluripotency transcription factors across the cell cycle.

The pioneer activity of transcription factors allows for opening of inaccessible regulatory elements and has been extensively studied in the context of cellular differentiation and reprogramming. In contrast, the function of pioneer activity in self-renewing cell divisions and across the cell cycle is poorly understood. Here we assessed the interplay between OCT4 and SOX2 in controlling chromatin accessibility of mouse embryonic stem cells. We found that OCT4 and SOX2 operate in a largely independent manner even at co-occupied sites, and that their cooperative binding is mostly mediated indirectly through regulation of chromatin accessibility. Controlled protein degradation strategies revealed that the uninterrupted presence of OCT4 is required for post-mitotic re-establishment and interphase maintenance of chromatin accessibility, and that highly OCT4-bound enhancers are particularly vulnerable to transient loss of OCT4 expression. Our study sheds light on the constant pioneer activity required to maintain the dynamic pluripotency regulatory landscape in an accessible state.

Endogenous fluctuations of OCT4 and SOX2 bias pluripotent cell fate decisions.

SOX2 and OCT4 are pioneer transcription factors playing a key role in embryonic stem (ES) cell self-renewal and differentiation. How temporal fluctuations in their expression levels bias lineage commitment is unknown. Here, we generated knock-in reporter fusion ES cell lines allowing to monitor endogenous SOX2 and OCT4 protein fluctuations in living cells and to determine their impact on mesendodermal and neuroectodermal commitment. We found that small differences in SOX2 and OCT4 levels impact cell fate commitment in G1 but not in S phase. Elevated SOX2 levels modestly increased neuroectodermal commitment and decreased mesendodermal commitment upon directed differentiation. In contrast, elevated OCT4 levels strongly biased ES cells towards both neuroectodermal and mesendodermal fates in undirected differentiation. Using ATAC-seq on ES cells gated for different endogenous SOX2 and OCT4 levels, we found that high OCT4 levels increased chromatin accessibility at differentiation-associated enhancers. This suggests that small endogenous fluctuations of pioneer transcription factors can bias cell fate decisions by concentration-dependent priming of differentiation-associated enhancers.

In vivo pulse labeling of isochronic cohorts of cells in the central nervous system using FlashTag.

The tracing of neuronal cell lineages is critical to our understanding of cellular diversity in the CNS. This protocol describes a fluorescence birth-dating technique to label, track and isolate isochronic cohorts of newborn cells in the CNS in vivo in mouse embryos. Injection of carboxyfluorescein esters (CFSEs) into the cerebral ventricle allows pulse labeling of mitotic (M phase) ventricular zone (VZ) progenitors and their progeny across the CNS, a procedure we termed FlashTag. Specificity for M-phase apical progenitors is a result of the somata of these cells transiently contacting the ventricular wall during this cell-cycle phase, exposing them to CFSE injected into the cerebrospinal fluid. Using this approach, the developmental trajectory of progenitors and their daughter neurons can be tracked. Labeled cells can be imaged ex vivo or in fixed tissue, targeted for electrophysiological experiments or isolated using FACS for cell culture or (single-cell) RNA sequencing. Multiple embryos can be labeled within 30 min. The dye is retained for several weeks, allowing labeled cells to be identified postnatally. This protocol describes the labeling procedure using in utero injection, the isolation of live cells using FACS and the processing of labeled tissue for immunohistochemistry.

Coupling progenitor and neuronal diversity in the developing neocortex.

The adult neocortex is composed of several types of glutamatergic neurons, which are sequentially born from progenitors during development. The extent and nature of progenitor diversity, and how it relates to neuronal diversity, is still poorly understood. In this review, we discuss key features of neocortical progenitors across several species, including their morphological properties, cell cycling behaviour and molecular signatures, and how these features relate to the competence of these cells to generate distinct types of progenies.

Sequential transcriptional waves direct the differentiation of newborn neurons in the mouse neocortex.

During corticogenesis, excitatory neurons are born from progenitors located in the ventricular zone (VZ), from where they migrate to assemble into circuits. How neuronal identity is dynamically specified upon progenitor division is unknown. Here, we study this process using a high-temporal-resolution technology allowing fluorescent tagging of isochronic cohorts of newborn VZ cells. By combining this in vivo approach with single-cell transcriptomics in mice, we identify and functionally characterize neuron-specific primordial transcriptional programs as they dynamically unfold. Our results reveal early transcriptional waves that instruct the sequence and pace of neuronal differentiation events, guiding newborn neurons toward their final fate, and contribute to a road map for the reverse engineering of specific classes of cortical neurons from undifferentiated cells.

Migration Speed of Cajal-Retzius Cells Modulated by Vesicular Trafficking Controls the Size of Higher-Order Cortical Areas.

In the neocortex, higher-order areas are essential to integrate sensory-motor information and have expanded in size during evolution. How higher-order areas are specified, however, remains largely unknown. Here, we show that the migration and distribution of early-born neurons, the Cajal-Retzius cells (CRs), controls the size of higher-order areas in the mouse somatosensory, auditory, and visual cortex. Using live imaging, genetics, and in silico modeling, we show that subtype-specific differences in the onset, speed, and directionality of CR migration determine their differential invasion of the developing cortical surface. CR migration speed is cell autonomously modulated by vesicle-associated membrane protein 3 (VAMP3), a classically non-neuronal mediator of endosomal recycling. Increasing CR migration speed alters their distribution in the developing cerebral cortex and leads to an expansion of postnatal higher-order areas and congruent rewiring of thalamo-cortical input. Our findings thus identify novel roles for neuronal migration and VAMP3-dependent vesicular trafficking in cortical wiring.

Asymmetric cell division of granule neuron progenitors in the external granule layer of the mouse cerebellum.

The plane of division of granule neuron progenitors (GNPs) was analysed with respect to the pial surface in P0 to P14 cerebellum and the results showed that there was a significant bias towards the plane of cell division being parallel to pial surface across this developmental window. In addition, the distribution of ß-Catenin in anaphase cells was analysed, which showed that there was a significant asymmetry in the distribution of ß-Catenin in dividing GNPs. Further, inhibition of Sonic Hedgehog (Shh) signalling had an effect on plane of cell division. Asymmetric distribution of ß-Catenin was shown to occur towards the source of a localized extracellular cue.

Retinal input directs the recruitment of inhibitory interneurons into thalamic visual circuits.

Inhibitory interneurons (INs) critically control the excitability and plasticity of neuronal networks, but whether activity can direct INs into specific circuits during development is unknown. Here, we report that in the dorsal lateral geniculate nucleus (dLGN), which relays retinal input to the cortex, circuit activity is required for the migration, molecular differentiation, and functional integration of INs. We first characterize the prenatal origin and molecular identity of dLGN INs, revealing their recruitment from an Otx2(+) neuronal pool located in the adjacent ventral LGN. Using time-lapse and electrophysiological recordings, together with genetic and pharmacological perturbation of retinal waves, we show that retinal activity directs the navigation and circuit incorporation of dLGN INs during the first postnatal week, thereby regulating the inhibition of thalamocortical circuits. These findings identify an input-dependent mechanism regulating IN migration and circuit inhibition, which may account for the progressive recruitment of INs into expanding excitatory circuits during evolution.

Expression of Sonic hedgehog during cell proliferation in the human cerebellum.

The regulation of cell proliferation in the external granular layer (EGL) of the developing cerebellum is important for its normal patterning. An important signal that regulates EGL cell proliferation is Sonic hedgehog (Shh). Shh is secreted by the Purkinje cells (PC) and has a mitogenic effect on the granule cell precursors of the EGL. Deregulation of Shh signaling has been associated with abnormal development, and been implicated in medulloblastomas, which are tumors that arise from the cerebellum. Given the importance of the Shh pathway in cerebellum development and disease, there has been no systematic study of its expression pattern during human cerebellum development. In this study, we describe the expression pattern of Shh, its receptor patched, smoothened, and its effectors that belong to the Gli family of transcription factors, during normal human cerebellum development from 10 weeks of gestational age, and in medulloblastomas that represents a case of abnormal cell proliferation in the cerebellum. This expression pattern is compared to equivalent stages in the normal development of cerebellum in mouse, as well as in tumors. Important differences between human and mouse that reflect differences in the normal developmental program between the 2 species are observed. First, in humans there appears to be a stage of Shh signaling within the EGL, when the PC are not yet the source of Shh. Second, unlike in the postnatal mouse cerebellum, expression of Shh in the PC in the postnatal human cerebellum is downregulated. Finally, medulloblastomas in the human but not in patched heterozygote mouse express Shh. These results highlight cross-species differences in the regulation of the Shh signaling pathway.