Inhibitory Smads differentially regulate cell fate specification and axon dynamics in the dorsal spinal cord

During neural development, progenitor cells generate different types of neurons in specific time windows. Despite the characterisation of many of the transcription factor networks involved in these differentiation events, the mechanism behind their temporal regulation is poorly understood. To address this question, we studied the temporal differentiation of the simple lateral floor plate (LFP) domain in the zebrafish spinal cord. LFP progenitors sequentially generate early-born Kolmer-Agduhr″ (KA″) interneurons and late-born V3 interneurons. Analysis using a Notch signalling reporter demonstrates that these cell populations have distinct Notch signalling profiles. Not only do V3 cells receive higher total levels of Notch response, but they collect this response over a longer duration compared to V3 cells. To test whether the duration of Notch signalling determines the temporal cell fate specification, we combined a transgene that constitutively activates Notch signalling in the ventral spinal cord with a heat shock inducible Notch signalling terminator to switch off Notch response at any given time. Sustained Notch signalling results in expanded LFP progenitors while KA″ and V3 interneurons fail to specify. Early termination of Notch signalling leads to exclusively KA″ cell fate, despite the high level of Notch signalling, whereas late attenuation of Notch signalling drives only V3 cell fate. This suggests that the duration of Notch signalling is instructive in cell fate specification. Interestingly, knockdown experiments reveal a role for the Notch ligand Jag2b in maintaining LFP progenitors and limiting their differentiation into KA″ and V3 cells. Our results indicate that Notch signalling is required for neural progenitor maintenance while a specific attenuation timetable defines the fate of the postmitotic progeny.

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Regeneration of dorsal spinal cord neurons after injury via in situ NeuroD1-mediated astrocyte-to-neuron conversion

10.1101/818823 ◽

2019 ◽

Cited By ~ 3

Author(s):

Brendan Puls ◽

Yan Ding ◽

Fengyu Zhang ◽

Mengjie Pan ◽

Zhuofan Lei ◽

...

Keyword(s):

Spinal Cord ◽

Dorsal Horn ◽

Cell Fate ◽

High Efficiency ◽

Reactive Astrocytes ◽

Dorsal Spinal Cord ◽

Spinal Cord Neurons ◽

Cord Injury ◽

Dorsal Spinal

AbstractSpinal cord injury (SCI) often leads to impaired motor and sensory functions, partially because the injury-induced neuronal loss cannot be easily replenished through endogenous mechanisms. In vivo neuronal reprogramming has emerged as a novel technology to regenerate neurons from endogenous glial cells by forced expression of neurogenic transcription factors. We have previously demonstrated successful astrocyte-to-neuron conversion in mouse brains with injury or Alzheimer’s disease by overexpressing a single neural transcription factor NeuroD1 via retroviruses. Here we demonstrate regeneration of dorsal spinal cord neurons from reactive astrocytes after SCI via adeno-associated virus (AAV), a more clinically relevant gene delivery system. We find that NeuroD1 converts reactive astrocytes into neurons in the dorsal horn of stab-injured spinal cord with high efficiency (∼95%). Interestingly, NeuroD1-converted neurons in the dorsal horn mostly acquire glutamatergic neuronal subtype, expressing spinal cord-specific markers such as Tlx3 but not brain-specific markers such as Tbr1, suggesting that the astrocytic lineage and local microenvironment affect the cell fate of conversion. Electrophysiological recordings show that the NeuroD1-converted neurons can functionally mature and integrate into local spinal cord circuitry by displaying repetitive action potentials and spontaneous synaptic responses. We further show that NeuroD1-mediated neuronal conversion can occur in the contusive SCI model, allowing future studies of evaluating this reprogramming technology for functional recovery after SCI. In conclusion, this study may suggest a paradigm shift for spinal cord repair using in vivo astrocyte-to-neuron conversion technology to generate functional neurons in the grey matter.

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Tlx3 Exerts Direct Control in Specifying Excitatory Over Inhibitory Neurons in the Dorsal Spinal Cord

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.642697 ◽

2021 ◽

Vol 9 ◽

Author(s):

Filipe A. Monteiro ◽

Rafael M. Miranda ◽

Marta C. Samina ◽

Ana F. Dias ◽

Alexandre A. S. F. Raposo ◽

...

Keyword(s):

Spinal Cord ◽

Cell Fate ◽

Homeobox Gene ◽

Spinal Cord Dorsal Horn ◽

Inhibitory Neurons ◽

Dorsal Spinal Cord ◽

Somatosensory Information ◽

Spinal Cord Dorsal ◽

Tightly Coupled ◽

Dorsal Spinal

The spinal cord dorsal horn is a major station for integration and relay of somatosensory information and comprises both excitatory and inhibitory neuronal populations. The homeobox gene Tlx3 acts as a selector gene to control the development of late-born excitatory (dILB) neurons by specifying glutamatergic transmitter fate in dorsal spinal cord. However, since Tlx3 direct transcriptional targets remain largely unknown, it remains to be uncovered how Tlx3 functions to promote excitatory cell fate. Here we combined a genomics approach based on chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) and expression profiling, with validation experiments in Tlx3 null embryos, to characterize the transcriptional program of Tlx3 in mouse embryonic dorsal spinal cord. We found most dILB neuron specific genes previously identified to be directly activated by Tlx3. Surprisingly, we found Tlx3 also directly represses many genes associated with the alternative inhibitory dILA neuronal fate. In both cases, direct targets include transcription factors and terminal differentiation genes, showing that Tlx3 directly controls cell identity at distinct levels. Our findings provide a molecular frame for the master regulatory role of Tlx3 in developing glutamatergic dILB neurons. In addition, they suggest a novel function for Tlx3 as direct repressor of GABAergic dILA identity, pointing to how generation of the two alternative cell fates being tightly coupled.

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