Mouse embryonic stem cells can differentiate via multiple paths to the same state
- Harvard Medical School, United States
- Boston Children's Hospital, United States
Figures
Figure 1
Summary of core research questions.
(a) Direct programming could in principle skip progenitor states in one of two ways. It may either transition from early states directly into later states through a ‘short-circuit’ of the natural …
Figure 2 with 3 supplements
Dissection of DP and SP motor neuron differentiation strategies using InDrops single cell RNA sequencing.
(a) Summary of the direct programming (DP) and standard protocol (SP) differentiation strategies. (b–c) tSNE visualization of single cell RNA sequencing data from each differentiation strategy. …
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Figure 2—source data 1
Summary of criteria used to annotate DP cell states.
Table indicates the expected expression level of key marker genes for each cell state. Hi = high expression, Lo = low expression, ‘-’=no expression. If an expression level is not specified for a given state-gene pair, then that gene was not a component of the criteria used to annotate state. Real expression values are shown in Figure 2D, and Figure 2—figure supplement 1.
- https://doi.org/10.7554/eLife.26945.007
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Figure 2—source data 2
Summary of criteria used to annotate SP cell states.
Table indicates the expected expression level of key marker genes for each cell state. Hi = high expression, Lo = low expression, ‘-’=no expression. If an expression level is not specified for a given state-gene pair, then that gene was not a component of the criteria used to annotate state. Real expression values are shown in Figure 2E, and Figure 2—figure supplement 2.
- https://doi.org/10.7554/eLife.26945.008
Figure 2—figure supplement 1
tSNE visualization of marker genes for DP subpopulations.
This figure relates to main text Figure 2. It shows the raw expression of sample marker genes that were used to identify each subpopulation. Two marker genes are shown per state.
Figure 2—figure supplement 2
tSNE visualization of marker genes for SP subpopulations.
This figure relates to main text Figure 2. It shows the raw expression of sample marker genes that were used to identify each subpopulation. Two marker genes are shown per state.
Figure 2—figure supplement 3
qPCR validation that MN gene expression in DP follows the expected dynamics and does not involve Olig2 induction.
(a) Measurement of the bulk population gene expression over time helps to confirm that the trajectory inferred from single-cell analysis matches the true dynamical events of our system. These …
Figure 3 with 2 supplements
DP and SP induce distinct differentiation paths to the motor neuron state.
(a) Visualization of differentiation paths for both protocols using SPRING reveals two paths to the same state. DP and SP overlap during early neural commitment, but then bifurcate and converge to …
Figure 3—figure supplement 1
Consolidation of DP vs SP end-state comparisons with additional SP EMN and LMN cells does not distort joint topology.
In our initial InDrops map of the SP, we found a low yield of the most mature EMN and LMN states in the SP (Figure 2C). To ensure that low representation of the SP end states did not interfere with …
Figure 3—figure supplement 2
Joint visualization of DP and SP differentiation paths by tSNE.
Like SPRING, tSNE shows similarity between early and late states, but it is ambiguous with respect to the continuous global topology of either path. Between DP and SP, tSNE confirms that ESC, NP, …
Figure 4 with 1 supplement
During DP cells transition through an abnormal intermediate state with a forebrain gene expression defect.
(a) The DP (red, EMN from Figure 2) and SP (black, PVNP + MNP + EMN from Figure 2) differentiation paths diverge into distinct intermediate states following early neural commitment. These distinct …
Figure 4—figure supplement 1
Cell-cycle-associated gene expression decreases as cells enter the DP and SP intermediate states and then progressively mature.
This figure relates to main text Figure 4B. (a) Cell cycle gene expression score overlayed on SPRING trajectory shown in main text. The score is the summed expression of a panel of 21 cell-cycle …
Figure 5 with 1 supplement
Both DP and SP differentiation trajectories approach the transcriptional state of primary MNs (pMNs), but DP does so with higher precision.
(a) tSNE visualization of 874 single cell transciptomes from FACS purified Mnx1+ MNs from embryos reveals heterogeneity within this population. To make comparisons between DP and SP with pMNs we …
Figure 5—figure supplement 1
Heterogeneity within the Mnx1+ E13.5 primary motor neuron population.
This figure shows the raw expression of a set of marker genes that were used to identify the subpopulations indicated in main text Figure 5A. Two markers are shown per subpopulation, with their …
Figure 6 with 2 supplements
Validation that DP cells become functional MNs despite their abnormal differentiation trajectory.
(a) Immunostaining of MN markers in DP MNs confirming expression and correct subcellular localization of Tubb3, Map2, VACht, Isl1, and Hb9. DP MNs also: b) can fire single or multiple action …
Figure 6—video 1
Contractions induced by DP MNs in C2C12 myotubes.
https://doi.org/10.7554/eLife.26945.017
Figure 6—video 2
Contractions induced by DP MNs in ES-MyoD myotubes.
https://doi.org/10.7554/eLife.26945.018Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.26945.019
