Neural Fate Research Articles

Reprogrammed human lateral ganglionic eminence precursors generate striatal neurons and restore motor function in a rat model of Huntington’s disease

BackgroundHuntington’s disease (HD) is a genetic neurological disorder predominantly characterised by the progressive loss of GABAergic medium spiny neurons in the striatum resulting in motor dysfunction. One potential strategy for the treatment of HD is the development of cell replacement therapies to restore neuronal circuitry and function by the replacement of lost neurons. We propose the generation of lineage-specific human lateral ganglionic eminence precursors (hiLGEP) using direct reprogramming technology provides a novel and clinically viable cell source for cell replacement therapy for HD.MethodshiLGEPs were derived by direct reprogramming of adult human dermal fibroblasts (aHDFs) using chemically modified mRNA (cmRNA) and a defined reprogramming medium. hiLGEPs were differentiated in vitro using an optimised striatal differentiation medium. Acquisition of a striatal precursor and neural cell fate was assessed through gene expression and immunocytochemical analysis of key markers. hiLGEP-derived striatal neuron functionality in vitro was demonstrated by calcium imaging using Cal-520. To investigate the ability for hiLGEP to survive, differentiate and functionally integrate in vivo, we transplanted hiLGEPs into the striatum of quinolinic acid (QA)-lesioned rats and performed behavioural assessment using the cylinder test over the course of 14 weeks. Survival and differentiation of hiLGEPs was assessed at 8 and 14-weeks post-transplant by immunohistochemical analysis.ResultsWe demonstrate the capability to generate hiLGEPs from aHDFs using cmRNA encoding the pro-neural genes SOX2 and PAX6, combined with a reprogramming medium containing Gö6983, Y-27,632, N-2 and Activin A. hiLGEPs generated functional DARPP32 + neurons following 14 days of culture in BrainPhys™ media supplemented with dorsomorphin and Activin A. We investigated the ability for hiLGEPs to survive transplantation, differentiate to medium spiny-like striatal neurons and improve motor function in the QA lesion rat model of HD. Fourteen weeks after transplantation, we observed STEM121 + neurons co-expressing MAP2, DARPP32, GAD65/67, or GABA. Rats transplanted with hiLGEPs also demonstrated reduction in motor function impairment as determined by spontaneous exploratory forelimb use when compared to saline transplanted animals.ConclusionThis study provides proof-of-concept and demonstrates for the first time that aHDFs can be directly reprogrammed to hiLGEPs which survive transplantation, undergo neuronal differentiation to generate medium spiny-like striatal neurons, and reduce functional impairment in the QA lesion rat model of HD.

Extramacrochaetae regulates Notch signaling in the Drosophila eye through non-apoptotic caspase activity.

Many cell fate decisions are determined transcriptionally. Accordingly, some fate specification is prevented by Inhibitor of DNA-binding (Id) proteins that interfere with DNA binding by master regulatory transcription factors. We show that the Drosophila Id protein Extra macrochaetae (Emc) also affects developmental decisions by regulating caspase activity. Emc, which prevents proneural bHLH transcription factors from specifying neural cell fate, also prevents homodimerization of another bHLH protein, Daughterless (Da), and thereby maintains expression of the Death-Associated Inhibitor of Apoptosis (diap1) gene. Accordingly, we found that multiple effects of emc mutations on cell growth and on eye development were all caused by activation of caspases. These effects included acceleration of the morphogenetic furrow, failure of R7 photoreceptor cell specification, and delayed differentiation of non-neuronal cone cells. Within emc mutant clones, Notch signaling was elevated in the morphogenetic furrow, increasing morphogenetic furrow speed. This was associated with caspase-dependent increase in levels of Delta protein, the transmembrane ligand for Notch. Posterior to the morphogenetic furrow, elevated Delta cis-inhibited Notch signaling that was required for R7 specification and cone cell differentiation. Growth inhibition of emc mutant clones in wing imaginal discs also depended on caspases. Thus, emc mutations reveal the importance of restraining caspase activity even in non-apoptotic cells to prevent abnormal development, in the Drosophila eye through effects on Notch signaling.

Extramacrochaetae regulates Notch signaling in the Drosophila eye through non-apoptotic caspase activity

Many cell fate decisions are determined transcriptionally. Accordingly, some fate specification is prevented by Inhibitor of DNA-binding (Id) proteins that interfere with DNA binding by master regulatory transcription factors. We show that the Drosophila Id protein Extra macrochaetae (Emc) also affects developmental decisions by regulating caspase activity. Emc, which prevents proneural bHLH transcription factors from specifying neural cell fate, also prevents homodimerization of another bHLH protein, Daughterless (Da), and thereby maintains expression of the Death-Associated Inhibitor of Apoptosis (diap1) gene. Accordingly, we found that multiple effects of emc mutations on cell growth and on eye development were all caused by activation of caspases. These effects included acceleration of the morphogenetic furrow, failure of R7 photoreceptor cell specification, and delayed differentiation of non-neuronal cone cells. Within emc mutant clones, Notch signaling was elevated in the morphogenetic furrow, increasing morphogenetic furrow speed. This was associated with caspase-dependent increase in levels of Delta protein, the transmembrane ligand for Notch. Posterior to the morphogenetic furrow, elevated Delta cis-inhibited Notch signaling that was required for R7 specification and cone cell differentiation. Growth inhibition of emc mutant clones in wing imaginal discs also depended on caspases. Thus, emc mutations reveal the importance of restraining caspase activity even in non-apoptotic cells to prevent abnormal development, in the Drosophila eye through effects on Notch signaling.

LPS-induced neuroinflammation induces changes in the transcriptional profile of members of the CoRest repressive complex in the hippocampus.

The action of Corepressor for Element Silencing Transcription Factor 1 (CoREST) is primarily related to neural fate decisions. However, the molecular mechanisms linking neuroinflammation to the histone modifying complex remain unclear. CoREST is a hub for several cofactors that play important roles in epigenetic remodeling and transcriptional regulation. It allows us to question their functions during the inflammatory response in the Central Nervous System. The impact of LPS-induced neuroinflammation on the transcriptional epigenetic control of members with CoRest repressive complex in the hippocampus was investigated. Characterizing the basal transcriptional profile in the hippocampus of members with CoREST repressive complex showed that the Rcor3 is the most expressed gene, and the Rcor2 is the least expressed one. It was also demonstrated that the levels of Lsd1, CoREST1 (Rcor1), and CoREST2 (Rcor2) transcripts increased in the hippocampus after LPS i.p. administration, while for CoREST3 (Rcor3), no significant difference was observed. A significant increase was noticed in the percentages of the 5-meC mark (Hypermethylation) for the Rcor1 and Lsd1 genes with a positive Pearson correlation between methylation and expression. However, the correlation was directly proportional, ruling out DNA methylation as the main mechanism in transcriptional control.

Mechanical confinement matters: Unveiling the effect of two-photon polymerized 2.5D and 3D microarchitectures on neuronal YAP expression and neurite outgrowth

The effect of mechanical cues on cellular behaviour has been reported in multiple studies so far, and a specific aspect of interest is the role of mechanotransductive proteins in neuronal development. Among these, yes-associated protein (YAP) is responsible for multiple functions in neuronal development such as neuronal progenitor cells migration and differentiation while myocardin-related transcription factor A (MRTFA) facilitates neurite outgrowth and axonal pathfinding. Both proteins have indirectly intertwined fates via their signalling pathways. There is little literature investigating the roles of YAP and MRTFA in vitro concerning neurite outgrowth in mechanically confined microenvironments. Moreover, our understanding of their relationship in immature neurons cultured within engineered confined microenvironments is still lacking. In this study, we fabricated, via two-photon polymerization (2PP), 2.5D microgrooves and 3D polymeric microchannels, with a diameter range from 5 to 30 μm. We cultured SH-SY5Y cells and differentiated them into immature neuron-like cells on both 2.5D and 3D microstructures to investigate the effect of mechanical confinement on cell morphology and protein expression. In 2.5D microgrooves, both YAP and MRTFA nuclear/cytoplasmic (N/C) ratios exhibited maxima in the 10 μm grooves indicating a strong relation with mechanical-stress-inducing confinement. In 3D microchannels, both proteins’ N/C ratio exhibited minima in presence of 5 or 10 μm channels, a behaviour that was opposite to the ones observed in the 2.5D microgrooves and that indicates how the geometry and mechanical confinement of 3D microenvironments are unique compared to 2.5D ones due to focal adhesion, actin, and nuclear polarization. Further, especially in presence of 2.5D microgrooves, cells featured an inversely proportional relationship between YAP N/C ratio and the average neurite length. Finally, we also cultured human induced pluripotent stem cells (hiPSCs) and differentiated them into cortical neurons on the microstructures for up to 2 weeks. Interestingly, YAP and MRTFA N/C ratios also showed a maximum around the 10 μm 2.5D microgrooves, indicating the physiological relevance of our study. Our results elucidate the possible differences induced by 2.5D and 3D confining microenvironments in neuronal development and paves the way for understanding the intricate interplay between mechanotransductive proteins and their effect on neural cell fate within engineered cell microenvironments.

Extramacrochaetae regulates Notch signaling in the Drosophila eye through non-apoptotic caspase activity.

Many cell fate decisions are determined transcriptionally. Accordingly, some fate specification is prevented by Inhibitor of DNA binding (Id) proteins that interfere with DNA binding by master regulatory transcription factors. We show that the Drosophila Id protein Extra macrochaetae (Emc) also affects developmental decisions by regulating caspase activity. Emc, which prevents proneural bHLH transcription factors from specifying neural cell fate, also prevents homodimerization of another bHLH protein, Daughterless (Da), and thereby maintains expression of the Death-Associated Inhibitor of Apoptosis (diap1) gene. Accordingly, we found that multiple effects of emc mutations on cell growth and on eye development were all caused by activation of caspases. These effects included acceleration of the morphogenetic furrow, failure of R7 photoreceptor cell specification, and delayed differentiation of non-neuronal cone cells. Within emc mutant clones, Notch signaling was elevated in the morphogenetic furrow, increasing morphogenetic furrow speed. This was associated with caspase-dependent increase in levels of Delta protein, the transmembrane ligand for Notch. Posterior to the morphogenetic furrow, elevated Delta cis-inhibited Notch signaling that was required for R7 specification and cone cell differentiation. Growth inhibition of emc mutant clones in wing imaginal discs also depended on caspases. Thus, emc mutations reveal the importance of restraining caspase activity even in non-apoptotic cells to prevent abnormal development, in the Drosophila eye through effects on Notch signaling.

An attractor state zone precedes neural crest fate in melanoma initiation.

The field cancerization theory suggests that a group of cells containing oncogenic mutations are predisposed to transformation1, 2. We previously identified single cells in BRAF V600E ;p53 -/- zebrafish that reactivate an embryonic neural crest state before initiating melanoma3-5. Here we show that single cells reactivate the neural crest fate from within large fields of adjacent abnormal melanocytes, which we term the "cancer precursor zone." These cancer precursor zone melanocytes have an aberrant morphology, dysplastic nuclei, and altered gene expression. Using single cell RNA-seq and ATAC-seq, we defined a distinct transcriptional cell attractor state for cancer precursor zones and validated the stage-specific gene expression initiation signatures in human melanoma. We identify the cancer precursor zone driver, ID1, which binds to TCF12 and inhibits downstream targets important for the maintenance of melanocyte morphology and cell cycle control. Examination of patient samples revealed precursor melanocytes expressing ID1, often surrounding invasive melanoma, indicating a role for ID1 in early melanomagenesis. This work reveals a surprising field effect of melanoma initiation in vivo in which tumors arise from within a zone of morphologically distinct, but clinically covert, precursors with altered transcriptional fate. Our studies identify novel targets that could improve early diagnosis and prevention of melanoma.

Heterozygous variants in USP25 cause genetic generalized epilepsy.

USP25 encodes ubiquitin-specific protease 25, a key member of the deubiquitinating enzyme family that is involved in neural fate determination. Although abnormal expression in Down's syndrome was reported previously, the specific role of USP25 in human diseases has not been defined. In this study, we performed trio-based whole exome sequencing in a cohort of 319 cases (families) with generalized epilepsy of unknown aetiology. Five heterozygous USP25 variants, including two de novo and three co-segregated variants, were determined in eight individuals affected by generalized seizures and/or febrile seizures from five unrelated families. The frequency of USP25 variants showed a significantly high aggregation in this cohort compared with the East Asian population and all populations in the gnomAD database. The mean age at onset of febrile and afebrile seizures were 10 months (infancy) and 11.8 years (juvenile), respectively. The patients achieved seizure freedom, except that one had occasional nocturnal seizures at the last follow-up. Two patients exhibited intellectual disability. Usp25 was expressed ubiquitously in mouse brain with two peaks, on embryonic Days 14-16 and postnatal Day 21, respectively. In human brain, likewise, USP25 is expressed in the fetus/early childhood stage and with a second peak at ∼12-20 years old, consistent with the seizure onset age in patients during infancy and in juveniles. To investigate the functional impact of USP25 deficiency in vivo, we established Usp25 knockout mice, which showed increased seizure susceptibility compared with wild-type mice in a pentylenetetrazol-induced seizure test. To explore the impact of USP25 variants, we used multiple functional detections. In HEK293 T cells, the variant associated with a severe phenotype (p.Gln889Ter) led to a significant reduction of mRNA and protein expressions but formed stable truncated dimers with an increment of deubiquitinating enzyme activities and abnormal cellular aggregations, indicating a gain-of-function effect. The p.Gln889Ter and p.Leu1045del variants increased neuronal excitability in mouse brain, with a higher firing ability in p.Gln889Ter. These functional impairments align with the severity of the observed phenotypes, suggesting a genotype-phenotype correlation. Hence, a moderate association between USP25 and epilepsy was noted, indicating that USP25 is potentially a predisposing gene for epilepsy. Our results from Usp25 null mice and the patient-derived variants indicated that USP25 would play an epileptogenic role via loss-of-function or gain-of-function effects. The truncated variant p.Gln889Ter would have a profoundly different effect on epilepsy. Together, our results underscore the significance of USP25 heterozygous variants in epilepsy, thereby highlighting the critical role of USP25 in the brain.

Retinoic acid, an essential component of the roof plate organizer, promotes the spatiotemporal segregation of dorsal neural fates.

Dorsal neural tube-derived retinoic acid promotes the end of neural crest production and transition into a definitive roof plate. Here, we analyze how this impacts the segregation of central and peripheral lineages, a process essential for tissue patterning and function. Localized in ovo inhibition in quail embryos of retinoic acid activity followed by single-cell transcriptomics unraveled a comprehensive list of differentially expressed genes relevant to these processes. Importantly, progenitors co-expressed neural crest, roof plate and dI1 interneuron markers, indicating a failure in proper lineage segregation. Furthermore, separation between roof plate and dI1 interneurons is mediated by Notch activity downstream of retinoic acid, highlighting their crucial role in establishing the roof plate-dI1 boundary. Within the peripheral branch, where absence of retinoic acid resulted in neural crest production and emigration extending into the roof plate stage, sensory progenitors failed to separate from melanocytes, leading to formation of a common glia-melanocyte cell with aberrant migratory patterns. In summary, the implementation of single-cell RNA sequencing facilitated the discovery and characterization of a molecular mechanism responsible for the segregation of dorsal neural fates during development.

Paving the way to a neural fate – RNA signatures in naive and trans-differentiating mesenchymal stem cells

Mesenchymal Stem Cells (MSCs) derived from the embryonic mesoderm persist as a viable source of multipotent cells in adults and have a crucial role in tissue repair. One of the most promising aspects of MSCs is their ability to trans-differentiate into cell types outside of the mesodermal lineage, such as neurons. This characteristic positions MSCs as potential therapeutic tools for neurological disorders. However, the definition of a clear MSC signature is an ongoing topic of debate. Likewise, there is still a significant knowledge gap about functional alterations of MSCs during their transition to a neural fate. In this study, our focus is on the dynamic expression of RNA in MSCs as they undergo trans-differentiation compared to undifferentiated MSCs. To track and correlate changes in cellular signaling, we conducted high-throughput RNA expression profiling during the early time-course of human MSC neurogenic trans-differentiation. The expression of synapse maturation markers, including NLGN2 and NPTX1, increased during the first 24 h. The expression of neuron differentiation markers, such as GAP43 strongly increased during 48 h of trans-differentiation. Neural stem cell marker NES and neuron differentiation marker, including TUBB3 and ENO1, were highly expressed in mesenchymal stem cells and remained so during trans-differentiation. Pathways analyses revealed early changes in MSCs signaling that can be linked to the acquisition of neuronal features. Furthermore, we identified microRNAs (miRNAs) as potential drivers of the cellular trans-differentiation process. We also determined potential risk factors related to the neural trans-differentiation process. These factors include the persistence of stemness features and the expression of factors involved in neurofunctional abnormalities and tumorigenic processes. In conclusion, our findings contribute valuable insights into the intricate landscape of MSCs during neural trans-differentiation. These insights can pave the way for the development of safer treatments of neurological disorders.

Mouse cortical organoids reveal key functions of p73 isoforms: TAp73 governs the establishment of the archetypical ventricular-like zones while DNp73 is central in the regulation of neural cell fate.

Neurogenesis is tightly regulated in space and time, ensuring the correct development and organization of the central nervous system. Critical regulators of brain development and morphogenesis in mice include two members of the p53 family: p53 and p73. However, dissecting the in vivo functions of these factors and their various isoforms in brain development is challenging due to their pleiotropic effects. Understanding their role, particularly in neurogenesis and brain morphogenesis, requires innovative experimental approaches. To address these challenges, we developed an efficient and highly reproducible protocol to generate mouse brain organoids from pluripotent stem cells. These organoids contain neural progenitors and neurons that self-organize into rosette-like structures resembling the ventricular zone of the embryonic forebrain. Using this model, we generated organoids from p73-deficient mouse cells to investigate the roles of p73 and its isoforms (TA and DNp73) during brain development. Organoids derived from p73-deficient cells exhibited increased neuronal apoptosis and reduced neural progenitor proliferation, linked to compensatory activation of p53. This closely mirrors previous in vivo observations, confirming that p73 plays a pivotal role in brain development. Further dissection of p73 isoforms function revealed a dual role of p73 in regulating brain morphogenesis, whereby TAp73 controls transcriptional programs essential for the establishment of the neurogenic niche structure, while DNp73 is responsible for the precise and timely regulation of neural cell fate. These findings highlight the distinct roles of p73 isoforms in maintaining the balance of neural progenitor cell biology, providing a new understanding of how p73 regulates brain morphogenesis.

Individual variation in the emergence of anterior-to-posterior neural fates from human pluripotent stem cells

Variability between human pluripotent stem cell (hPSC) lines remains a challenge and opportunity in biomedicine. In this study, hPSC lines from multiple donors were differentiated toward neuroectoderm and mesendoderm lineages. We revealed dynamic transcriptomic patterns that delineate the emergence of these lineages, which were conserved across lines, along with individual line-specific transcriptional signatures that were invariant throughout differentiation. These transcriptomic signatures predicted an antagonism between SOX21-driven forebrain fates and retinoic acid-induced hindbrain fates. Replicate lines and paired adult tissue demonstrated the stability of these line-specific transcriptomic traits. We show that this transcriptomic variation in lineage bias had both genetic and epigenetic origins, aligned with the anterior-to-posterior structure of early mammalian development, and was present across a large collection of hPSC lines. These findings contribute to developing systematic analyses of PSCs to define the origin and consequences of variation in the early events orchestrating individual human development.

Metabolic states influence chicken retinal pigment epithelium cell fate decisions.

During tissue regeneration, proliferation, dedifferentiation and reprogramming are necessary to restore lost structures. However, it is not fully understood how metabolism intersects with these processes. Chicken embryos can regenerate their retina through retinal pigment epithelium (RPE) reprogramming when treated with fibroblast factor 2 (FGF2). Using transcriptome profiling, we uncovered extensive regulation of gene sets pertaining to proliferation, neurogenesis and glycolysis throughout RPE-to-neural retina reprogramming. By manipulating cell media composition, we determined that glucose, glutamine or pyruvate are individually sufficient to support RPE reprogramming, identifying glycolysis as a requisite. Conversely, the activation of pyruvate dehydrogenase by inhibition of pyruvate dehydrogenase kinases, induces epithelial-to-mesenchymal transition, while simultaneously blocking the activation of neural retina fate. We also identified that epithelial-to-mesenchymal transition fate is partially driven by an oxidative environment. Our findings provide evidence that metabolism controls RPE cell fate decisions and provide insights into the metabolic state of RPE cells, which are prone to fate changes in regeneration and pathologies, such as proliferative vitreoretinopathy.

A single-cell transcriptomic map of the developing Atoh1 lineage identifies neural fate decisions and neuronal diversity in the hindbrain

Proneural transcription factors establish molecular cascades to orchestrate neuronal diversity. One such transcription factor, Atonal homolog 1 (Atoh1), gives rise to cerebellar excitatory neurons and over 30 distinct nuclei in the brainstem critical for hearing, breathing, and balance. Although Atoh1 lineage neurons have been qualitatively described, the transcriptional programs that drive their fate decisions and the full extent of their diversity remain unknown. Here, we analyzed single-cell RNA sequencing and ATOH1 DNA binding in Atoh1 lineage neurons of the developing mouse hindbrain. This high-resolution dataset identified markers for specific brainstem nuclei and demonstrated that transcriptionally heterogeneous progenitors require ATOH1 for proper migration. Moreover, we identified a sizable population of proliferating unipolar brush cell progenitors in the mouse Atoh1 lineage, previously described in humans as the origin of one medulloblastoma subtype. Collectively, our data provide insights into the developing mouse hindbrain and markers for functional assessment of understudied neuronal populations.

Chemical Coaxing of Mesenchymal Stromal Cells by Drug Repositioning for Nestin Induction.

Mesenchymal stromal cells (MSCs) display heterogeneity in origin and functional role in tissue homeostasis. Subsets of MSCs derived from the neural crest express nestin and serve as niches in bone marrow, but the possibility of coaxing MSCs into nestin-expresing cells for enhanced supportive activity is unclear. In this study, as an approach to the chemical coaxing of MSC functions, we screened libraries of clinically approved chemicals to identify compounds capable of inducing nestin expression in MSCs. Out of 2000 clinical compounds, we chose vorinostat as a candidate to coax the MSCs into neural crest-like fates. When treated with vorinostat, MSCs exhibited a significant increase in the expression of genes involved in the pluripotency and epithelial-mesenchymal transition (EMT), as well as nestin and CD146, the markers for pericytes. In addition, these nestin-induced MSCs exhibited enhanced differentiation towards neuronal cells with the upregulation of neurogenic markers, including SRY-box transcription factor 2 (Sox2), SRY-box transcription factor 10 (Sox10) and microtubule associated protein 2 (Map2) in addition to nestin. Moreover, the coaxed MSCs exhibited enhanced supporting activity for hematopoietic progenitors without supporting leukemia cells. These results demonstrate the feasibility of the drug repositioning of MSCs to induce neural crest-like properties through the chemical coaxing of cell fates.

Evolutionary origin of the chordate nervous system revealed by amphioxus developmental trajectories.

The common ancestor of all vertebrates had a highly sophisticated nervous system, but questions remain about the evolution of vertebrate neural cell types. The amphioxus, a chordate that diverged before the origin of vertebrates, can inform vertebrate evolution. Here we develop and analyse a single-cell RNA-sequencing dataset from seven amphioxus embryo stages to understand chordate cell type evolution and to study vertebrate neural cell type origins. We identified many new amphioxus cell types, including homologues to the vertebrate hypothalamus and neurohypophysis, rooting the evolutionary origin of these structures. On the basis of ancestor-descendant reconstruction of cell trajectories of the amphioxus and other species, we inferred expression dynamics of transcription factor genes throughout embryogenesis and identified three ancient developmental routes forming chordate neurons. We characterized cell specification at the mechanistic level and generated mutant lines to examine the function of five key transcription factors involved in neural specification. Our results show three developmental origins for the vertebrate nervous system: an anterior FoxQ2-dependent mechanism that is deeply conserved in invertebrates, a less-conserved route leading to more posterior neurons in the vertebrate spinal cord and a mechanism for specifying neuromesoderm progenitors that is restricted to chordates. The evolution of neuromesoderm progenitors may have led to a dramatic shift in posterior neural and mesodermal cell fate decisions and the body elongation process in a stem chordate.

Specification and survival of post-metamorphic branchiomeric neurons in a non-vertebrate chordate.

Tunicates are the sister group to the vertebrates, yet most species have a life cycle split between swimming larva and sedentary adult phases. During metamorphosis, larval neurons are replaced by adult-specific ones. The regulatory mechanisms underlying this replacement remain largely unknown. Using tissue-specific CRISPR/Cas9-mediated mutagenesis in the tunicate Ciona, we show that orthologs of conserved hindbrain and branchiomeric neuron regulatory factors Pax2/5/8 and Phox2 are required to specify the 'neck', a cellular compartment set aside in the larva to give rise to cranial motor neuron-like neurons post-metamorphosis. Using bulk and single-cell RNA-sequencing analyses, we characterize the transcriptome of the neck downstream of Pax2/5/8. We present evidence that neck-derived adult ciliomotor neurons begin to differentiate in the larva and persist through metamorphosis, contrary to the assumption that the adult nervous system is formed after settlement and the death of larval neurons during metamorphosis. Finally, we show that FGF signaling during the larval phase alters the patterning of the neck and its derivatives. Suppression of FGF converts neck cells into larval neurons that fail to survive metamorphosis, whereas prolonged FGF signaling promotes an adult neural stem cell-like fate.

SoxB1 transcription factors are essential for initiating and maintaining neural plate border gene expression.

SoxB1 transcription factors (Sox2/3) are well known for their role in early neural fate specification in the embryo, but little is known about functional roles for SoxB1 factors in non-neural ectodermal cell types, such as the neural plate border (NPB). Using Xenopus laevis, we set out to determine whether SoxB1 transcription factors have a regulatory function in NPB formation. Here, we show that SoxB1 factors are necessary for NPB formation, and that prolonged SoxB1 factor activity blocks the transition from a NPB to a neural crest state. Using ChIP-seq, we demonstrate that Sox3 is enriched upstream of NPB genes in early NPB cells and in blastula stem cells. Depletion of SoxB1 factors in blastula stem cells results in downregulation of NPB genes. Finally, we identify Pou5f3 factors as potential Sox3 partners in regulating the formation of the NPB and show that their combined activity is needed for normal NPB gene expression. Together, these data identify a role for SoxB1 factors in the establishment and maintenance of the NPB, in part through partnership with Pou5f3 factors.

Cell-type-specific loops linked to RNA polymerase II elongation in human neural differentiation

DNA is folded into higher-order structures that shape and are shaped by genome function. The role of long-range loops in the establishment of new gene expression patterns during cell fate transitions remains poorly understood. Here, we investigate the link between cell-specific loops and RNA polymerase II (RNA Pol II) during neural lineage commitment. We find thousands of loops decommissioned or gained de novo upon differentiation of human induced pluripotent stem cells (hiPSCs) to neural progenitor cells (NPCs) and post-mitotic neurons. During hiPSC-to-NPC and NPC-to-neuron transitions, genes changing from RNA Pol II initiation to elongation are >4-fold more likely to anchor cell-specific loops than repressed genes. Elongated genes exhibit significant mRNA upregulation when connected in cell-specific promoter-enhancer loops but not invariant promoter-enhancer loops or promoter-promoter loops or when unlooped. Genes transitioning from repression to RNA Pol II initiation exhibit a slight mRNA increase independent of loop status. Our data link cell-specific loops and robust RNA Pol II-mediated elongation during neural cell fate transitions.

Substrate stress relaxation regulates neural stem cell fate commitment

Adult neural stem cells (NSCs) reside in the dentate gyrus of the hippocampus, and their capacity to generate neurons and glia plays a role in learning and memory. In addition, neurodegenerative diseases are known to be caused by a loss of neurons and glial cells, resulting in a need to better understand stem cell fate commitment processes. We previously showed that NSC fate commitment toward a neuronal or glial lineage is strongly influenced by extracellular matrix stiffness, a property of elastic materials. However, tissues in vivo are not purely elastic and have varying degrees of viscous character. Relatively little is known about how the viscoelastic properties of the substrate impact NSC fate commitment. Here, we introduce a polyacrylamide-based cell culture platform that incorporates mismatched DNA oligonucleotide-based cross-links as well as covalent cross-links. This platform allows for tunable viscous stress relaxation properties via variation in the number of mismatched base pairs. We find that NSCs exhibit increased astrocytic differentiation as the degree of stress relaxation is increased. Furthermore, culturing NSCs on increasingly stress-relaxing substrates impacts cytoskeletal dynamics by decreasing intracellular actin flow rates and stimulating cyclic activation of the mechanosensitive protein RhoA. Additionally, inhibition of motor-clutch model components such as myosin II and focal adhesion kinase partially or completely reverts cells to lineage distributions observed on elastic substrates. Collectively, our results introduce a unique system for controlling matrix stress relaxation properties and offer insight into how NSCs integrate viscoelastic cues to direct fate commitment.