Biology subjects

Kee, N.

Publications and source records attributed to Kee, N..

5 recordsLinked to original sources

bioRxiv2024

Sox9 and Nfi transcription factors regulate the timing of neurogenesis and ependymal maturation in dopamine progenitors

Correct timing of neurogenesis is critical for both generating the correct number and subtypes of glia and neurons in the embryo, as well as preventing tumours and the depletion of stem cell pools in the adults. Here we analyse how the midbrain dopamine neuron (mDA) progenitors transition into cell cycle arrest (G0) and begin to mature into ependymal cells. The comparison of mDA progenitors from different embryonic stages revealed the upregulation of Nfi and Sox9 transcription factors during development. Their conditional inactivation in the early embryonic midbrain leads to delayed G0 entry and ependymal maturation, reduced gliogenesis, and increased generation of neurons, including mDA neurons. In contrast, their inactivation in late embryogenesis does not result in mitotic re-entry, suggesting that these factors are necessary for the G0 induction, but not for its maintenance. Our characterisation of mDA-progenitor-derived adult ependymal cells by single-cell RNA sequencing and histology show that they both retain several progenitor features but also secrete neuropeptides and contact neighbouring cells and blood vessels, indicating that these cells may form a novel part of the circumventricular organ system.

developmental biology↗

bioRxiv2024

Posterior specification of multi-lineage axial assembloids from human pluripotent stem cells.

Elongation of the posterior body axis is driven by multi-potent neuromesodermal progenitors (NMPs), which both self-renew and simultaneously generate neural tube, neural crest, and presomitc mesoderm lineages at successive anterior posterior (A-P) levels. The ensuing diversification of these three NMP lineages is remarkably extensive, and also essential for an immense range of clinically important adult posterior body tissues. Here, we describe a human pluripotent stem cell protocol that successfully specifies authentic NMPs using a cocktail of seven factors (7F). 7F-NMPs express requisite markers, exhibit co-linear HOX activation, and can be purposely specified into each of the three NMP daughter lineages, demonstrating genuine multi-potency. 3D assembly of neural tube, neural crest, and presomitic mesoderm spheroids followed by long-term floating culture derives mature, multi-compartment Posterior Axial Assembloids, or PAXAs. PAXAs constitute a complex heterogeneous tissue containing spinal motor neurons and interneurons, central and peripheral glia, connective tissues, muscle satellite cells and contractile muscle fibres. Together, 7F-NMP and PAXA protocols establish a versatile in vitro platform to model mechanisms of human posterior body axis development, and for the study of a wide range of human diseases.

cell biology↗

bioRxiv2020

LCM-seq identifies robust markers of vulnerable and resistant human midbrain dopamine neurons

Defining transcriptional profiles of substantia nigra pars compacta (SNc) and ventral tegmental area (VTA) dopamine neurons in human is critical to understanding their differential vulnerability in Parkinson Disease. However, reported marker profiles for these neuron populations are derived predominantly from rodents, utilize small sample sizes and display extensive variability between studies. Here, we map selective expression profiles of dopamine neurons in an extensive collection of human SNc and VTA using laser capture microdissection coupled with Smart-seq2 RNA sequencing (LCM-seq). By applying a bootstrapping strategy as sample input to DESeq2, we identify 33 differentially expressed SNc- or VTA-specific markers and we also compute the minimal cohort size required to identify differentially expressed genes (DEGs) that are concordant regardless of cohort size. Among the identified DEGs, ZCCHC12, CDH13 and SERPINE2, are minimally required to distinguish SNc or VTA dopamine neurons in both human and mouse. In summary, our study identifies novel markers, besides previously identified ones, which will be instrumentsal for future studies aiming to modulate dopamine neuron resilience as well as validate cell identity of stem cell-derived dopamine neurons.

neuroscience↗

bioRxiv2018

Spatial transcriptomics and in silico random pooling identify novel dopamine neuron subtype markers

Defining transcriptional profiles of substantia nigra pars compacta (SNc) and ventral tegmental area (VTA) dopamine neurons is critical to understanding their differential vulnerability in Parkinsons Disease (PD). Here, we determine transcriptomes of human SNc and VTA dopamine neurons using LCM-seq on a large sample cohort. We apply a bootstrapping strategy as sample input to DESeq2 and identify 33 stably differentially expressed genes (DEGs) between these two subpopulations. We also compute a minimal sample size for identification of stable DEGs, which highlights why previous reported profiles from small sample sizes display extensive variability. Network analysis reveal gene interactions unique to each subpopulation and highlight differences in regulation of mitochondrial stability, apoptosis, neuronal survival, cytoskeleton regulation, extracellular matrix modulation and well as synapse integrity, which could explain the relative resilience of VTA dopamine neurons. Analysis of PD tissues showed that while identified stable DEGs can distinguish the subpopulations also in disease, the SNc markers SLIT1 and ATP2A3 were downregulated and thus appears to be biomarkers of disease. In summary, our study identifies human SNc and VTA marker profiles, which will be instrumental for studies aiming to modulate dopamine neuron resilience and to validate cell identity of stem cell-derived dopamine neurons.

neuroscience↗

bioRxiv2018

Axon-seq decodes the motor axon transcriptome and its modulation in response to ALS

Spinal motor axons traverse large distances to innervate target muscles, thus requiring local control of cellular events for proper functioning. To interrogate axon-specific processes we developed Axon-seq, a refined method incorporating microfluidics, RNA-seq and bioinformatic-QC. We show that the axonal transcriptome is distinct from somas and contains fewer genes. We identified 3,500-5,000 transcripts in mouse and human stem cell-derived spinal motor axons, most of which are required for oxidative energy production and ribogenesis. Axons contained transcription factor mRNAs, e.g. Ybx1, with implications for local functions. As motor axons degenerate in amyotrophic lateral sclerosis (ALS), we investigated their response to the SOD1G93A mutation, identifying 121 ALS-dysregulated transcripts. Several of these are implicated in axonal function, including Nrp1, Dbn1 and Nek1, a known ALS-causing gene. In conclusion, Axon-seq provides an improved method for RNA-seq of axons, increasing our understanding of peripheral axon biology and identifying novel therapeutic targets in motor neuron disease.

neuroscience↗