Subtype Diversification and Synaptic Specificity of Stem Cell-Derived Spinal Interneurons

Abstract

SUMMARY Neuronal diversification is a fundamental step in the construction of functional neural circuits, but how neurons generated from single progenitor domains acquire diverse subtype identities remains poorly understood. Here we developed an embryonic stem cell (ESC)-based system to model subtype diversification of V1 interneurons, a class of spinal neurons comprising four clades collectively containing dozens of molecularly distinct neuronal subtypes. We demonstrate that V1 subtype diversity can be modified by extrinsic signals. Inhibition of Notch and activation of retinoid signaling results in a switch to MafA clade identity and enriches differentiation of Renshaw cells, a specialized MafA subtype that mediates recurrent inhibition of spinal motor neurons. We show that Renshaw cells are intrinsically programmed to migrate to species-specific laminae upon transplantation and to form subtype-specific synapses with motor neurons. Our results demonstrate that stem cell-derived neuronal subtypes can be used to investigate mechanisms underlying neuronal subtype specification and circuit assembly.

Establishing neuronal diversity in the spinal cord: a time and a place

“…For example, the RA-synthesizing enzyme Aldh1a2 is downregulated in somites around E10 (Niederreither et al, 1997), which temporally coincides with the point at which neurons switch from expressing Onecut family members to expressing Pou2f2 and Zfhx2-4. Consistent with this idea, RA has been shown to promote Renshaw cell identity over other V1 fates and LMC identity in MNs (Hoang et al, 2018;Sockanathan and Jessell, 1998), and both Renshaw neurons and LMC neurons are neuronal subtypes that specifically depend on Onecut TFs for their generation (Roy et al, 2012;Stam et al, 2012). Conversely, later neuronal identities may be favoured by signalling pathways that are involved in progenitor maintenance and promote gliogenesis (outlined above).…”

“…Thus, Notch-dependent cell-cell interactions sequentially subdivide the V2 lineage. Similarly, Notch signalling has been implicated in regulating the fate of excitatory and inhibitory neurons in the dorsal spinal cord at later developmental stages (Mizuguchi et al, 2006), the columnar identity of MNs (Tan et al, 2016), and V1 interneuron subtype formation (Hoang et al, 2018).…”

“…Stem cell-based in vitro differentiation and reprogramming assays have started to reveal the binding sites of TFs, how they affect chromatin dynamics, and how they specify cell fates (Aydin et al, 2019;Kutejova et al, 2016;Lai et al, 2011;Lee et al, 2008;Mazzoni et al, 2013b;Metzis et al, 2018;Nishi et al, 2015;Rhee et al, 2016;Velasco et al, 2017). Furthermore, protocols for the in vitro differentiation of neurons from most spinal cord domains have been established (Butts et al, 2017;Gouti et al, 2014;Gupta et al, 2018;Hoang et al, 2018;Sternfeld et al, 2017;Wichterle et al, 2002;Xu and Sakiyama-Elbert, 2015). These now provide the means to further elucidate how morphogen signalling dynamics and the binding patterns of morphogen effectors and patterning TFs specify distinct progenitor and neuronal cell identities.…”

A Stem Cell-Based Screening Platform Identifies Compounds that Desensitize Motor Neurons to Endoplasmic Reticulum Stress

“…For this analysis, we generated a new ESC line that expresses tdTomato in dorsal spinal inhibitory interneurons derived from Ptf1a-expressing progenitors. 45 Following differentiation of this cell line under conditions that promote the specification of dorsal interneuron identity, tdTomato-expressing interneurons were co-cultured with GFP-expressing stem cell-derived motor neurons of spinal identity ( Figures 1H and 1I). Quantification of RFP-versus GFP-positive neurons revealed that CPA treatment reduced dorsal spinal interneuron survival by only $17%, compared to a $70% decrease in the survival of co-cultured motor neurons.…”

Single cell transcriptomics reveals spatial and temporal dynamics of gene expression in the developing mouse spinal cord

“…Extrinsic signals may contribute to the temporal stratification of neuronal subtypes. Recent in vitro work suggested that retinoic acid promotes Renshaw cell identity in V1 neurons (Hoang et al, 2018), and the timepoint of their generation correlates with expression of the RA-synthesizing enzyme Aldh1a2 in the adjacent somites (Niederreither et al, 1997). Thus, besides promoting neurogenesis (Novitch et al, 2003), somite-derived RA may be a determinant for early Onecut-positive neuronal subtypes.…”