Spinal Motor Neuron Differentiation Regulatory Dynamics Uncovered

The inner workings of a gene network that regulates the development of spinal motor neurons in the growing chicken and mouse embryo has been discovered by researchers from University of California, Los Angeles.

The research also answers a long-standing question about why motor neurons, the nerve cells of the spinal cord that control muscle movement, form much faster than other types of neurons.

The findings have important implications for facilitating the production of motor neurons from stem cells in the lab. Stem cell-derived motor neurons could be used to repair the diseased or damaged spinal cord and to study neurodegenerative diseases such as amyotrophic lateral sclerosis (also known as Lou Gehrig’s disease) and spinal muscular atrophy.

Unprecedented Detailed View

Co-senior author Bennett Novitch, member of the Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research at UCLA, said:

“This study provides an unprecedented detailed view of how embryos produce the different cell types found in the mature spinal cord. The new experimental techniques we used, combined with collaborative efforts of biologists and computer scientists, are allowing us to gain new insight into the exquisite accuracy of embryonic development.”

During embryonic development of the spinal cord, different types of nerve cells are formed from precursor cells, called neural progenitors.

Reconstruction of transcriptional changes during Motor Neuron differentiation.

Reconstruction of transcriptional changes during Motor Neuron differentiation.
(A) Identification of NP cell states using hierarchical clustering of gene expression profiles of the individual cells.
(B) Cell state graph constructed from minimum spanning trees, color coded for the cell populations identified in A. Stars indicate start and end cells for the reconstruction of transcriptional changes along pseudotime. Shading of edges between cells indicates how often the edge was used in the reconstruction of gene expression along pseudotime.
(C) Cell state graph color coded for expression levels of Irx3, Olig2, Ngn2, Lhx3, Isl1, and Chat.
(D) Inferred changes in gene expression over pseudotime from 9,000 shortest paths connecting start and end cells (stars in B). Each shortest path was resampled to a length of 41 pseudo–time points to enable statistical measurements of gene expression. Cell IDs are color coded according to cell states in (A).
Quantification of the global rate of change in gene expression identifies three metastable states (light gray) separated by transition states, during which the rate of change in gene expression is increased (dark gray). Transition phases are defined as intervals along the pseudo-temporal timeline at which the second derivative of the global gene variation is negative, while metastable states are characterized by a positive second derivative.
(E) Gene expression profiles along pseudo-time for NP TFs (Irx3, Pax6, Nkx6.1, and Olig2), genes associated with the transition to MNs (Ngn2, Lhx3, and Neurod4) and MN markers (Isl1/2, Tubb3, and Chat).
(F) Levels of gene expression for Hes1/5, Olig2, and Ngn2 over pseudotime.
Note that Olig2 expression appears biphasic, with up-regulation of Olig2 concommitant to Ngn2 induction and repression of Hes1/5 in the transition phase from NP to MN. Chat, choline acetyltransferase; KSP, K shortest paths; MN, motor neuron; NP, neural progenitor; pMN, MN progenitor; p3, V3 interneuron progenitor; Tubb3, neuronal class III beta-tubulin.
Credit: PLOS Biology/UCLA Broad Stem Cell Research Center

It has been known for a long time that different types of neurons form at differing speeds during development, with motor neurons forming faster than the other types of nerve cells that populate the spinal cord. However, researchers didn’t know why or how motor neurons develop this way.

Single Cell Transcriptomics

The research team used the latest molecular techniques to assess how the activity of genes change as neurons form. This was achieved using an approach called single cell transcriptional profiling, which allows the activity of all genes in individual cells to be measured simultaneously.

This gave the team snapshots of global gene activity in about 200 cells that were in the process of becoming motor neurons. To analyze these data, the team developed custom computer software to reconstruct how gene activity changes as motor neurons form.

The analysis suggested that a protein produced by a gene called Olig2, which is only expressed in the neural progenitors that create motor neurons, promotes motor neuron formation by interfering with the activity of several members of another gene family — the Hes genes. The Hes genes are known to prevent the development of neurons.

Proposed model of the Olig2-controlled gene regulatory network

(A) Proposed model of the Olig2-controlled gene regulatory network.
Olig2 not only acts as central organizer for dorsal-ventral patterning in the spinal cord but also controls the rate of MN differentiation through direct repression of Hes TFs. This leads to a higher levels of Ngn2 expression and, consequently, a higher rate of neuronal differentiation in the pMN domain, compared to adjacent progenitor domains.
(B) Olig2 is a core component of the Shh-controlled gene regulatory network that patterns the ventral spinal cord.
(C) Olig2-mediated down-regulation of the Notch effectors Hes1/5 relieves repression of Ngn2 in the pMN domain.
(D) Consolidated activities of Ngn2 and Olig2 cause differentiation of NPs to MNs.
Olig2 promotes differentiation of MNs through repression of alternative IN cell fates. bHLH, basic helix-loop-helix; IN, interneuron; MN, motor neuron; NP, neural progenitor; pMN, MN progenitor; p2, V2 interneuron progenitor; p3, V3 interneuron progenitor; RA, retinoic acid; Shh, sonic hedgehog; TF, transcription factor; V2, V2 interneuron; V3, V3 interneuron.
Credit: PLOS Biology/UCLA Broad Stem Cell Research Center

The researchers confirmed Olig2’s role in promoting motor neuron formation by increasing or blocking the function of Olig2 in the spinal cords of developing mouse and chicken embryos, as well as during motor neuron formation in mouse embryonic stem cells.

“During embryonic development, the nervous system is constructed following a precise blueprint, with key parts forming at precise times and places, and appropriate numbers. Our research sheds light into how this process is orchestrated specifically for motor neuron development,”

said Novitch.

Funding for the work came from Wellcome Trust, EMBO Long-term fellowship, Cancer Research UK, Whitehall Foundation, March of Dimes Foundation, NINDS, UK Medical Research Council, Human Frontiers Science Program, and People Programme (Marie Curie Actions) of the European Union’s Seventh Framework Programme.

Sagner A, Gaber ZB, Delile J, Kong JH, Rousso DL, Pearson CA, et al. (2018)
Olig2 and Hes regulatory dynamics during motor neuron differentiation revealed by single cell transcriptomics
PLoS Biol 16(2): e2003127. https://doi.org/10.1371/journal.pbio.2003127

Top Image: left: Olig2 expression (blue) results in reduced Hes gene expression (red) and increased motor neuron formation (green). Right: in the absence of Olig2, Hes expression increases and motor neuron formation decreases. Credit: PLOS Biology/UCLA Broad Stem Cell Research Center