Figure 1. Dynamic Changes in Enhancer Organization and Gene Expression during Motor Neuron Differentiation.

(A) Overview of mouse ESC-directed differentiation to postmitotic hypaxial motor neuron (MN). Differentiating cells become nascent postmitotic MNs on day 5 and maturing MNs on day 6. PE, primitive ectoderm; pMN, MN progenitor.

(B) Expression of Isl1, Lhx3, and Hb9 in embryoid bodies. Lhx3 is transiently expressed in nascent MNs on day 5 and rapidly downregulated in maturing MNs on day 6 (Figure S1). Scale bars represent 50 μm.

(C) H3K27ac intensity relative to active enhancer midpoints (30,648 rows, Table S1) during MN differentiation. Active enhancers were grouped and sorted by stage-specific H3K27ac intensity, demonstrating that the majority of enhancers are transient during MN differentiation.

(D) Expression levels of 19,326 annotated RefSeq genes grouped by fold changes in expression between individual MN differentiation stages. Left: FPKM (fragments per kilobase of exon per million fragments mapped) values were median normalized and log₂ transformed. Middle: Log₂ RNA fold change, ordered as shown in the left panel. Right: representative GO terms and examples in each group of genes.

(E) Percentage of enhancers and genes that dynamically change more than 2-fold between indicated time points. Enhancers remain dynamic between day 5 and day 6 MNs, while gene expression becomes more stable.

See also Figure S1.