Hippo signalling in mammalian cortical development

The cerebral cortex in humans is composed of billions of morphologically and functionally distinct neurons. Development of the neocortex requires an orchestrated succession of a series of processes, the appropriate generation, migration, and positioning of neurons, the acquisition of layer-specific transcriptional hallmarks, and the establishment of precise axonal projections. We have primarily focussed on elucidating the transcriptomic landscape of murine embryonic neural stem cells (NSCs), basal progenitors (BPs) and newborn neurons (NBNs) at the population level. I have focussed on one underexplored signalling pathway in the brain- the Hippo signalling pathway. Hippo signalling effectors are expressed dynamically during the course of development in NSCs and BPs at mRNA level. Hippo transcription factors (TFs), Tead1 and Tead3 show higher expression during gliogenesis while Tead2 is expressed at relatively higher levels during early phases of neural expansion. Known to be redundant in other biological systems, I explored different effects of three Tead TFs in NSCs using gain and loss of function. I observe reciprocal effects on neuronal migration and fate with Tead1, Tead3 and Tead2. We identified ApoE, Cyr61 and Dab2 as potential direct targets of Tead TFs in NSCs. ApoE gain of function partially recapitulates the gain of function of Tead2, reducing cell migration to the cortical plate (CP) and Dab2 gain of function recapitulates the gain of function of Tead1, an increased migration to CP. ApoE and Dab2 are involved in Reelin signalling and hence we provide the first link between Hippo and Reelin signalling pathways controlling cortical development

Temporal and sequential transcriptional dynamics define lineage shifts in corticogenesis

The cerebral cortex contains billions of neurons, and their disorganization or misspecification leads to neurodevelopmental disorders. Understanding how the plethora of projection neuron subtypes are generated by cortical neural stem cells (NSCs) is a major challenge. Here, we focused on elucidating the transcriptional landscape of murine embryonic NSCs, basal progenitors (BPs), and newborn neurons (NBNs) throughout cortical development. We uncover dynamic shifts in transcriptional space over time and heterogeneity within each progenitor population. We identified signature hallmarks of NSC, BP, and NBN clusters and predict active transcriptional nodes and networks that contribute to neural fate specification. We find that the expression of receptors, ligands, and downstream pathway components is highly dynamic over time and throughout the lineage implying differential responsiveness to signals. Thus, we provide an expansive compendium of gene expression during cortical development that will be an invaluable resource for studying neural developmental processes and neurodevelopmental disorders

Untangling Cortical Complexity During Development

The cerebral cortex is composed of billions of morphologically and functionally distinct neurons. These neurons are produced and organized in a regimental fashion during development. The ability of neurons to encode and elicit complex cognitive and motor functions depends on their precise molecular processes, identity, and connectivity established during development. Elucidating the cellular and molecular mechanisms that regulate development of the neocortex has been a challenge for many years. The cerebral cortical neuronal subtypes are classified based on morphology, function, intrinsic synaptic properties, location, connectivity, and marker gene expression. Development of the neocortex requires an orchestration of a series of processes including the appropriate determination, migration and positioning of the neurons, acquisition of layer-specific transcriptional hallmarks, and formation of precise axonal projections and networks. Historically, fate mapping, genome-wide analysis, and transcriptome profiling have provided many opportunities for the characterization of neuronal subtypes. During the course of this review, we will address the regimental organization of the cerebral cortex, dissect the cellular subtypes that contribute to cortical complexity, and outline their molecular hallmarks to understand cellular diversity in the cerebral cortex with a focus on the excitatory neurons

Ultra-multiplexed analysis of single-cell dynamics reveals logic rules in differentiation

Dynamical control of cellular microenvironments is highly desirable to study complex processes such as stem cell differentiation and immune signaling. We present an ultra-multiplexed microfluidic system for high-throughput single-cell analysis in precisely defined dynamic signaling environments. Our system delivers combinatorial and time-varying signals to 1500 independently programmable culture chambers in week-long live-cell experiments by performing nearly 106 pipetting steps, where single cells, two-dimensional (2D) populations, or 3D neurospheres are chemically stimulated andtracked. Using our system and statistical analysis, we investigatedthe signaling landscape of neural stem cell differentiation and discovered "cellular logic rules" that revealed the critical role of signal timing and sequence in cell fate decisions. We find synergistic and antagonistic signal interactions and show that differentiation pathways are highly redundant. Our system allows dissection of hidden aspects of cellular dynamics and enables accelerated biological discovery

Tead transcription factors differentially regulate cortical development

Neural stem cells (NSCs) generate neurons of the cerebral cortex with distinct morphologies and functions. How specific neuron production, differentiation and migration are orchestrated is unclear. Hippo signaling regulates gene expression through Tead transcription factors (TFs). We show that Hippo transcriptional coactivators Yap1/Taz and the Teads have distinct functions during cortical development. Yap1/Taz promote NSC maintenance and Satb2+ neuron production at the expense of Tbr1+ neuron generation. However, Teads have moderate effects on NSC maintenance and do not affect Satb2+ neuron differentiation. Conversely, whereas Tead2 blocks Tbr1+ neuron formation, Tead1 and Tead3 promote this early fate. In addition, we found that Hippo effectors regulate neuronal migration to the cortical plate (CP) in a reciprocal fashion, that ApoE, Dab2 and Cyr61 are Tead targets, and these contribute to neuronal fate determination and migration. Our results indicate that multifaceted Hippo signaling is pivotal in different aspects of cortical development.ISSN:2045-232

Tropism of SARS-CoV-2 for Developing Human Cortical Astrocytes

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) readily infects a variety of cell types impacting the function of vital organ systems, with particularly severe impact on respiratory function. It proves fatal for one percent of those infected. Neurological symptoms, which range in severity, accompany a significant proportion of COVID-19 cases, indicating a potential vulnerability of neural cell types. To assess whether human cortical cells can be directly infected by SARS-CoV-2, we utilized primary human cortical tissue and stem cell-derived cortical organoids. We find significant and predominant infection in cortical astrocytes in both primary and organoid cultures, with minimal infection of other cortical populations. Infected astrocytes had a corresponding increase in reactivity characteristics, growth factor signaling, and cellular stress. Although human cortical cells, including astrocytes, have minimal ACE2 expression, we find high levels of alternative coronavirus receptors in infected astrocytes, including DPP4 and CD147. Inhibition of DPP4 reduced infection and decreased expression of the cell stress marker, ARCN1. We find tropism of SARS-CoV-2 for human astrocytes mediated by DPP4, resulting in reactive gliosis-type injury

LIF signaling regulates outer radial glial to interneuron fate during human cortical development.

Radial glial (RG) development is essential for cerebral cortex growth and organization. In humans, the outer radial glia (oRG) subtype is expanded and gives rise to diverse neurons and glia. However, the mechanisms regulating oRG differentiation are unclear. oRG cells express leukemia-inhibitory factor (LIF) receptors during neurogenesis, and consistent with a role in stem cell self-renewal, LIF perturbation impacts oRG proliferation in cortical tissue and organoids. Surprisingly, LIF treatment also increases the production of inhibitory interneurons (INs) in cortical cultures. Comparative transcriptomic analysis identifies that the enhanced IN population resembles INs produced in the caudal ganglionic eminence. To evaluate whether INs could arise from oRGs, we isolated primary oRG cells and cultured them with LIF. We observed the production of INs from oRG cells and an increase in IN abundance following LIF treatment. Our observations suggest that LIF signaling regulates the capacity of oRG cells to generate INs

Temporal and sequential transcriptional dynamics define lineage shifts in corticogenesis

The cerebral cortex contains billions of neurons, and their disorganization or misspecification leads to neurodevelopmental disorders. Understanding how the plethora of projection neuron subtypes are generated by cortical neural stem cells (NSCs) is a major challenge. Here, we focused on elucidating the transcriptional landscape of murine embryonic NSCs, basal progenitors (BPs), and newborn neurons (NBNs) throughout cortical development. We uncover dynamic shifts in transcriptional space over time and heterogeneity within each progenitor population. We identified signature hallmarks of NSC, BP, and NBN clusters and predict active transcriptional nodes and networks that contribute to neural fate specification. We find that the expression of receptors, ligands, and downstream pathway components is highly dynamic over time and throughout the lineage implying differential responsiveness to signals. Thus, we provide an expansive compendium of gene expression during cortical development that will be an invaluable resource for studying neural developmental processes and neurodevelopmental disorders.ISSN:0261-4189ISSN:1460-207