Biology subjects

Arenas, E.

Publications and source records attributed to Arenas, E..

13 recordsLinked to original sources

bioRxiv2026

SorCS2 controls Wnt signaling homeostasis during neurodevelopment by regulating Ror2 activity and trafficking

Wnt signaling orchestrates key developmental processes, yet mechanisms restricting its spatial and temporal activity remain incompletely understood. Here we identify SorCS2, a VPS10p-domain sorting receptor, as a conserved regulator of Wnt signaling homeostasis. Integrating proteomics, cell biology, embryology, and vertebrate models, we show that SorCS2 interacts with the noncanonical Wnt receptor Ror2 and controls its trafficking, Wnt5a-induced internalization, and lysosomal degradation. In mouse dopaminergic progenitors, SorCS2 deficiency enhances responsiveness to canonical and noncanonical Wnt ligands, increases Ror2 phosphorylation, and amplifies downstream signaling of both Wnt signaling cascades. Loss of SorCS2 further disrupts the ligand-receptor feedback loop, leading to elevated Ror2, Wnt5a, and Wnt1 expression, accelerated proliferation and neurogenesis, and increased expression of Tcf4, a gene implicated in neuropsychiatric disorders. Across vertebrate systems, SorCS2-dependent control of Ror2 signaling is required for anterior-posterior axis elongation, convergent extension, somitogenesis, and brain morphogenesis. These findings define a SorCS2-dependent sorting mechanism that fine-tunes morphogen receptor signaling during vertebrate neurodevelopment.

developmental biology↗

bioRxiv2025

Development of a protocol utilizing single-cell analysis for the differentiation of human iPSCs into SOX6+ midbrain dopaminergic neurons.

Parkinsons disease (PD) is characterized by the progressive loss of midbrain dopaminergic (mDA) neurons, leading to severe debilitating motor impairment. Recent single-cell analyses have revealed that specific mDA subpopulations, such as SOX6 AGTR1 neurons, are most vulnerable to degeneration. Current human pluripotent stem cell differentiation protocols fail to selectively generate these subtypes. Here, we describe a multiomic-guided strategy that enriches SOX6+ mDA neurons by combining enhancing Sonic Hedgehog agonism with prolonged Wnt activation. This approach accelerates floor plate specification, increases expression of mDA developmental markers, and substantially increases the yield of SOX6 mature mDA neurons compared to prior attempts. Following intrastriatal transplantation into hemiparkinsonian mice, these cells restored motor function in approximately 4 months and generated grafts containing SOX6 A9-like neurons. Our work thus establishes a reproducible differentiation platform for generating the PD-susceptible mDA subtype, providing a foundation for precision disease modelling and subtype-targeted cell replacement therapies.

cell biology↗

bioRxiv2025

Prevention of Transgene Silencing During Human PluripotentStem Cell Differentiation

While high and stable transgene expression can be achieved in undifferentiated pluripotent stem cells, conventional transgene expression systems are often silenced upon differentiation. Silencing occurs with both randomly integrated transgenes, introduced via transposase or lentiviral methods, and with transgenes targeted to specific genomic sites, including at commonly used safe harbor loci. The challenge to robustly express experimental transgenes in differentiated pluripotent stem cells is a major bottleneck in the field for applications such as CRISPR screening. Here, we conducted a comparative analysis to systematically evaluate the impact of various promoters, transcriptional regulatory elements, insulators, and genomic integration sites on transgene silencing during neuronal differentiation. Our findings reveal that specific combinations of promoters and transcriptional stability elements are able to prevent transgene silencing during differentiation, whereas chromatin insulators had less impact on silencing and three novel safe harbor integration sites performed similarly to the CLYBL locus. Guided by these insights we developed the PiggyBac vector TK4, which showed complete resistance to transgene silencing across various neuronal and microglial differentiation protocols from six different pluripotent stem cell lines, as independently confirmed by seven different laboratories. This construct will be highly useful for assays requiring stable transgene expression during differentiation, and holds the potential for broad applications in various research fields.

neuroscience↗

bioRxiv2024

Midbrain Dopaminergic Neuron Development is Regulated by Two Molecularly Distinct Subtypes of Radial Glia Cells

Understanding midbrain dopaminergic (mDA) neuron development is key to advancing cell replacement therapies for Parkinsons disease (PD). Recent single-cell RNA-sequencing (scRNA-seq) studies have identified different subtypes of transient radial glia (Rgl) cell types in the developing mouse and human ventral midbrain. However, their individual functions and their impact on mDA neuron development are unclear. Here we analyze the transcriptome of endogenous mouse and human ventral midbrain Rgl and assess the function of key midbrain floor plate Rgl factors in human stem cells during mDA neuron differentiation. We find that Rgl1 is defined by a neurogenic network centered on Arntl, and that this transcription factor regulates human mDA neurogenesis. Conversely, the transcriptome of Rgl3 is dominated by signaling and extracellular matrix molecules that control different aspects of human mDA neuron development. Thus, our results suggest a function of Rgl1 as a mDA progenitor, and of Rgl3 as a signaling mDA niche cell. Moreover, using human stem cells we demonstrate that new knowledge of cell-type specific intrinsic and extrinsic developmental factors can readily be applied to improve the generation of cells with therapeutic interest, such as human mDA neurons for PD.

neuroscience↗

bioRxiv2022

Comprehensive cell atlas of the first-trimester developing human brain

The adult human brain likely comprises more than a thousand kinds of neurons, and an unknown number of glial cell types, but how cellular diversity arises during early brain development is not known. Here, in order to reveal the precise sequence of events during early brain development, we used single-cell RNA sequencing and spatial transcriptomics to uncover cell states and trajectories in human brains at 5 - 14 post-conceptional weeks (p.c.w.). We identified twelve major classes and over 600 distinct cell states, which mapped to precise spatial anatomical domains at 5 p.c.w. We uncovered detailed differentiation trajectories of the human forebrain, and a surprisingly large number of region-specific glioblasts maturing into distinct pre-astrocytes and pre-oligodendrocyte precursor cells (pre-OPCs). Our findings reveal the emergence of cell types during the critical first trimester of human brain development.

developmental biology↗

bioRxiv2022

Transcriptomic diversity of cell types across the adult human brain

The human brain directs a wide range of complex behaviors ranging from fine motor skills to abstract intelligence and emotion. However, the diversity of cell types that support these skills has not been fully described. Here we used high-throughput single-nucleus RNA sequencing to systematically survey cells across the entire adult human brain in three postmortem donors. We sampled over three million nuclei from approximately 100 dissections across the forebrain, midbrain, and hindbrain. Our analysis identified 461 clusters and 3313 subclusters organized largely according to developmental origins. We found area-specific cortical neurons, as well as an unexpectedly high diversity of midbrain and hindbrain neurons. Astrocytes also exhibited regional diversity at multiple scales, comprising subtypes specific to the telencephalon and to more precise anatomical locations. Oligodendrocyte precursors comprised two distinct major types specific to the telencephalon and to the rest of the brain. Together, these findings demonstrate the unique cellular composition of the telencephalon with respect to all major brain cell types. As the first single-cell transcriptomic census of the entire human brain, we provide a resource for understanding the molecular diversity of the human brain in health and disease.

neuroscience↗

bioRxiv2022

Single cell transcriptomics reveals correct developmental dynamics and high-quality midbrain cell types by improved hESC differentiation.

Stem cell technologies provide new opportunities for modeling cells in the healthy and diseased states and for regenerative medicine. In both cases developmental knowledge as well as the quality and molecular properties of the cells are essential for their future application. In this study we identify developmental factors important for the differentiation of human embryonic stem cells (hESCs) into midbrain dopaminergic (mDA) neurons. We found that Laminin-511, and dual canonical and non-canonical WNT activation followed by GSK3{beta} inhibition plus FGF8b, improved midbrain patterning. In addition, mDA neurogenesis and differentiation was enhanced by activation of liver X receptors and inhibition of fibroblast growth factor signaling. Moreover, single-cell RNA-sequencing analysis revealed a developmental dynamics similar to that of the endogenous human ventral midbrain and the emergence of high quality molecularly-defined midbrain cell types, including mDA neurons that become functional. Thus, our study identifies novel factors important for human midbrain development and opens the door for a future application of molecularly-defined hESC-derived midbrain cell types in Parkinsons disease.

neuroscience↗

bioRxiv2022

Early pre-neural serotonin modulates balance of late monoamines and behavioral patterns in fish model system

The presence of serotonergic system during early pre-neural development is enigmatic and conserved amongst all studied invertebrate and vertebrate animals. We took advantage of zebrafish model system to address what is the role of early serotonin before first neurons form. Unexpectedly, we experimentally revealed the existence of delayed developmental neurogenic and behavioral effects resulting from the manipulations of pre-neural (zygote, blastula and gastrula) serotonergic system. In particular, the delayed effects included differences in the synthesis of serotonin in early serotonergic neurons in the central nervous system as well as in behavioral alterations after habituation in zebrafish larvae. These effects appeared as highly specific and did not coincide with any major abnormalities. The same manipulations of the serotonergic system at neural developmental stages did not show such effects, which confirms that early effects of serotonergic system manipulation are not based on retained serotonin in embryonic cells. Accordingly, gene expression analysis demonstrated specific changes only in response to the elevation of early pre-neural serotonin, which included the delayed and pre-mature onsets of different gene expression programs. Taken together, our results introduce a novel function of early pre-neural serotonergic system in a vertebrate embryo - tuning and fine control of specific mechanisms at later neural developmental stages that result in a mild variation of a behavioral adaptive spectrum.

developmental biology↗

bioRxiv2022

Increasing computational protein design literacy through cohort-based learning for undergraduate students

Undergraduate research experiences can improve student success in graduate education and STEM careers. During the COVID-19 pandemic, undergraduate researchers at our institution and many others lost their work-study research positions due to interruption of in-person research activities. This imposed a financial burden on the students and eliminated an important learning opportunity. To address these challenges, we created a paid, fully-remote, cohort-based research curriculum in computational protein design. Our curriculum used existing protein design methods as a platform to first educate and train undergraduate students and then to test research hypotheses. In the first phase, students learned computational methods to assess the stability of designed protein assemblies. In the second phase, students used a larger dataset to identify factors that could improve the accuracy of current protein design algorithms. This cohort-based program created valuable new research opportunities for undergraduates at our institute and enhanced the undergraduates feeling of connection with the lab. Students learned transferable and useful skills such as literature review, programming basics, data analysis, hypothesis testing, and scientific communication. Our program provides a model of structured computational research training opportunities for undergraduate researchers in any field for organizations looking to expand educational access. Graphical Abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

scientific communication and education↗

bioRxiv2020

MEIS-WNT5A axis regulates development of 4th ventricle choroid plexus

The choroid plexus (ChP) produces cerebrospinal fluid and forms a critical barrier between the brain and the circulation. While the ChP forms in each brain ventricle, it adopts a different shape in each one and remarkably little is known about the mechanisms underlying its development. Here, we show that epithelial WNT5A is critical for determining fourth ventricle (4V) ChP morphogenesis and size. Systemic Wnt5a knockout, or forced WNT5A overexpression beginning at E10.5, profoundly reduced the size and development of ChP in all ventricles. However, conditional deletion of Wnt5a expression in Foxj1-expressing epithelial cells affected only the branched, villous morphology of the 4V ChP. We found that WNT5A was enriched in epithelial cells localized to the distal tips of 4V ChP villi, where WNT5A acted locally to activate non-canonical Wnt signaling via Ror1/Ror2 receptors. During 4V ChP development, MEIS1 bound to the proximal Wnt5a promoter, and gain- and loss-of-function approaches demonstrated that MEIS1 regulated Wnt5a expression. Collectively, our findings demonstrate a dual function of WNT5A in ChP development and identify MEIS1 and MEIS2 as upstream regulators of Wnt5a in the 4V ChP epithelium.

developmental biology↗

bioRxiv2019

Genetic Identification of Cell Types Underlying Brain Complex Traits Yields Novel Insights Into the Etiology of Parkinson's Disease

Genome-wide association studies (GWAS) have discovered hundreds of loci associated with complex brain disorders, and provide the best current insights into the etiology of these idiopathic traits. However, it remains unclear in which cell types these variants may be active, which is essential for understanding disease etiology and for disease modelling. Here we integrate GWAS results with single-cell transcriptomic data from the entire nervous system to systematically identify cell types underlying psychiatric disorders, neurological conditions, and other brain complex traits. We show that psychiatric disorders are predominantly associated with excitatory neurons from the cortex/hippocampus, medium spiny neurons from the striatum, diverse sets of midbrain neurons, and inhibitory neurons from the cortex/hippocampus. Cognitive traits were generally associated with similar cell types but their associations were driven by different genes. Neurological disorders were associated with different cell types, consistent with other lines of evidence. Notably, we found that Parkinsons disease is not only genetically associated with dopaminergic neurons but also with serotonergic neurons and cells from the oligodendrocyte lineage. Using post-mortem brain transcriptomic data, we confirmed alterations in these cells, even at the earliest stages of disease progression. Altogether, our study provides a solid framework for understanding the cellular basis of complex brain disorders and reveals a new unexpected role of oligodendrocytes in Parkinsons disease.

genomics↗

bioRxiv2018

Molecular architecture of the mouse nervous system

The mammalian nervous system executes complex behaviors controlled by specialised, precisely positioned and interacting cell types. Here, we used RNA sequencing of half a million single cells to create a detailed census of cell types in the mouse nervous system. We mapped cell types spatially and derived a hierarchical, data-driven taxonomy. Neurons were the most diverse, and were grouped by developmental anatomical units, and by the expression of neurotransmitters and neuropeptides. Neuronal diversity was driven by genes encoding cell identity, synaptic connectivity, neurotransmission and membrane conductance. We discovered several distinct, regionally restricted, astrocytes types, which obeyed developmental boundaries and correlated with the spatial distribution of key glutamate and glycine neurotransmitters. In contrast, oligodendrocytes showed a loss of regional identity, followed by a secondary diversification. The resource presented here lays a solid foundation for understanding the molecular architecture of the mammalian nervous system, and enables genetic manipulation of specific cell types.

neuroscience↗

bioRxiv2017

Molecular analysis of the midbrain dopaminergic niche during neurogenesis

Midbrain dopaminergic (mDA) neurons degenerate in Parkinsons disease and are one of the main targets for cell replacement therapies. However, a comprehensive view of the signals and cell types contributing to mDA neurogenesis is not yet available. By analyzing the transcriptome of the mouse ventral midbrain at a tissue and single-cell level during mDA neurogenesis we found that three recently identified radial glia types 1-3 (Rgl1-3) contribute to different key aspects of mDA neurogenesis. While Rgl3 expressed most extracellular matrix components and multiple ligands for various pathways controlling mDA neuron development, such as Wnt and Shh, Rgl1-2 expressed most receptors. Moreover, we found that specific transcription factor networks explain the transcriptome and suggest a function for each individual radial glia. A network controlling neurogenesis was found in Rgl1, progenitor maintenance in Rgl2 and the secretion of factors forming the mDA niche by Rgl3. Our results thus uncover a broad repertoire of developmental signals expressed by each midbrain cell type during mDA neurogenesis. Cells identified for their emerging importance are Rgl3, a niche cell type, and Rgl1, a neurogenic progenitor that expresses ARNTL, a transcription factor that we find is required for mDA neurogenesis.

systems biology↗