Polycomb repressive complex 1 activities determine the columnar organization of motor neurons

Abstract

Polycomb repressive complexes (PRCs) establish and maintain gene repression through chromatin modifications, but their specific roles in cell fate determination events are poorly understood. Here we show an essential role for the PRC1 component Bmi1 in motor neuron (MN) subtype differentiation through dose-dependent effects on Hox gene expression. While Bmi1 is dispensable for generating MNs as a class, it has an essential role in specifying and determining the position of Hox-dependent MN columnar and pool subtypes. These actions are mediated through limiting anterior Hox expression boundaries, functions deployed in post-mitotic MNs, temporally downstream from morphogen gradients. Within the HoxC gene cluster, we found a progressive depletion of PRC-associated marks from rostral to caudal levels of the spinal cord, corresponding to major demarcations of MN subtypes. Selective ablation of Bmi1 elicits a derepression of more posterior Hox genes, leading to a switch in MN fates. Unexpectedly, Hox patterns and MN fates appear to be sensitive to absolute PRC1 activity levels; while reducing Bmi1 switches forelimb lateral motor column (LMC) MNs to a thoracic preganglionic (PGC) identity, elevating Bmi1 expression at thoracic levels converts PGC to LMC MNs. These results suggest that graded PRC1 activities are essential in determining MN topographic organization.

Figure 1.

Chromatin marks and expression of Hox genes along the rostrocaudal axis. (A) Schematic of Hox protein expression and Hox-dependent MN columnar subtypes. Rostral is to the left. LMC MNs innervate the limbs, and PGC MNs project to sympathetic chain ganglia. For simplicity, Hox-independent motor columns are not shown. (B) H3K27me3 status of Hoxc6, Hoxc9, and Hoxc10 promoter regions at cervical, brachial, thoracic, and lumbar levels. Fold enrichment is shown over IgG controls for each level. (C) Distribution of Bmi1 binding at columnar Hox loci shows distribution similar to that of H3K27me3. The absence of Bmi1 and H3K27me3 binding to lumbar chromatin indicates that antibodies specifically recognize marks at other levels. (D) Distribution of H3K4me3 marks. (E) Levels of Hoxc6, Hoxc9, and Hoxc10 mRNA at each spinal level.

Figure 2.

Bmi1 depletion does not affect generation of MNs. (A) Expression of Bmi1 is diminished upon electroporation of Bmi1 dsRNA. The electroporated half of the cord is indicated by a bolt. A nuclear LacZ construct was coelectroporated to grossly mark the electroporated region. (B) Expression of H3K27me3 and Eed is not affected by Bmi1 dsRNA. (C) Expression of post-mitotic MN markers Hb9 and Isl1/2 is also not affected. (D) Expression of Olig2, Pax6, and Nkx6.1 in MN progenitors is unaffected. (E) Time line of Bmi1 depletion upon electroporation of dsRNA at HH stage 13. Embryos were fixed and analyzed for Bmi1 expression at different time points following electroporation. Expression of Isl1/2 marks post-mitotic MNs.

Figure 3.

Effect of Bmi1 loss on Hox patterns in spinal MNs. (A) Summary of the effects of Bmi1 depletion at brachial levels. (B–D) Hoxc9 extends to brachial levels after Bmi1 depletion. (E–G) Hoxc6 is repressed in cells that express Hoxc9. (H) Serial sections showing extension of Hoxc9 and loss of Hoxc6. There is no change in Hoxc10 in MNs, although some Hoxc10⁺ cells are observed in dorsal regions. Results shown are typical of electroporations where >80% of cells have lost Bmi1. (I) Quantification of Hoxc9⁺ cells along the rostrocaudal axis of the electroporated and nonelectroporated halves of chick spinal cord HH stages 25–28. Results show averaged cell counts from two embryos. Error bars show standard error. (J) Effects of Bmi1 loss at thoracic/lumbar levels. (K–N) Hoxc10 and Hoxd10 are derepressed. (O,P) Ectopic Hoxd10 cells do not express Hoxc9. (Q) Serial section showing extension of Hox10 proteins and loss of Hoxc9. (R) Quantification of Hoxc10⁺ cells along the rostrocaudal axis of the electroporated and nonelectroporated halves of the spinal cord. Results show averaged cell counts from four embryos.

Figure 4.

Bmi1 depletion converts forelimb LMC MNs to a PGC fate. (A–D) Expression of the LMC markers FoxP1 and Raldh2 is diminished after depletion of Bmi1 at brachial levels. (E,F) Expression of the PGC marker pSmad is detected at brachial levels and coexpresses Hoxc9. Not all ectopic Hoxc9 cells label with pSmad, as Hoxc9 is derepressed in non-MN cells. (G,H) Expression of pan-MN markers Hb9 and Lhx3 is not affected at brachial levels. (I) Summary showing changes in MN molecular identity following Bmi1 depletion. (J,K) Vibratome sections of chick embryos electroporated with a Hb9∷GFP construct to label motor axons (green). Sympathetic chain ganglia are indicated by yellow arrows and are stained by Isl1/2. (L,M) MNs coelectroporated with Bmi1 dsRNA and Hb9∷GFP express ectopic Hoxc9, and their axons are redirected to the sympathetic chain ganglia at brachial levels.

Figure 5.

The developmental timing of Bmi1-mediated control of Hox expression. (A) Elevation of FGF signaling induces Hoxc9 in brachial progenitors. HH stage 14 chick embryos were electroporated and analyzed after 24 h. A GFP expression construct was coelectroporated to grossly mark the electroporated region. Bmi1 expression is unchanged by FGF8 overexpression. (B) Effects of Bmi1 depletion on brachial progenitors. Hoxc9 immunostaining and in situ hybridization show that brachial Hoxc9 expression does not change in the absence of Bmi1. (C,D) Hoxc9 is grossly unaffected at thoracic levels after FGF elevation or Bmi1 depletion. (E,F) Rescue of Bmi1 expression at brachial levels of the spinal cord. Embryos were coelectroporated with Bmi1 dsRNA and Hb9∷Bmi1-ires-GFP. (G,H) Expression of Hoxc9 is shifted rostrally into the brachial level on the electroporated side of the cord; however, Hoxc9 is excluded from cells rescued for Bmi1. (I,J) Expression of Hoxc6 is reduced on the electroporated side of the cord but is restored in rescued cells. (K,L) pSmad is excluded from cells expressing GFP. (M,N) Expression of FoxP1 is restored in cells expressing GFP.

Figure 6.

Elevation of Bmi1 expression at thoracic levels extinguished Hoxc9. (A) Schematic of the effects of Bmi1 elevation at thoracic levels of the spinal cord. (B,C) Bmi1 expression is elevated after electroporation of a pCIG∷Bmi1 expression construct. (D) Bmi1 elevation does not affect MN generation, marked by Isl1/2. (E–G) At brachial levels, Hoxc6 is unaffected, while Hoxc8 expression is diminished. No ectopic Hoxc9 is observed. (H–J) At thoracic levels, Hoxc9 expression is ablated, and ectopic Hoxc6 is detected. (K) Serial sections showing that Hoxc6 expression is not affected at brachial levels but is extended caudally. (L–V) Effects of post-mitotic overexpression of Bmi1 using an Hb9∷Bmi1-ires-EGFP expression construct. (M–O) Bmi1 expression is increased on the electroporated half of the embryo but not within the progenitor zone. Isl1/2 expression is unchanged. (P–R) Effects of Bmi1 elevation on brachial Hox expression. (S–U) Hoxc9 expression in GFP⁺ cells is reduced, and ectopic Hoxc6 is observed. (V) Extension of Hoxc6 to thoracic levels.

Figure 7.

Effects of Bmi1 elevation on MN columnar identity. (A–D) Effects of Bmi1 elevation on MN columnar identity at thoracic levels. LMC markers Raldh2 and (high) FoxP1 are ectopically expressed, while pSmad expression is extinguished. (E,F) Vibratome sections of chick embryos electroporated with Hb9∷GFP construct to label motor axons (green). Sympathetic chain ganglia are indicated by yellow arrows. (G,H) MNs coelectroporated with pCIG∷Bmi1 have reduced Hoxc9 expression, and their axons are directed away from the sympathetic chain ganglia. (I–L) Effects of Hb9∷Bmi1-ires-EGFP electroporation at thoracic levels. FoxP1 expression is elevated, while pSmad expression is reduced. (M–P) Vibratome sections of chick embryos electroporated with Hb9-GFP construct to label motor axons (green). Sympathetic chain ganglia are indicated by yellow arrows. MNs coelectroporated with Hb9∷Bmi1-ires-EGFP have reduced Hoxc9 expression, and their axons are redirected away from the sympathetic chain ganglia.

Figure 8.

PRC1 reprogramming of early Hox patterning. (A) Effects of elevating FGF8 at brachial levels. Hoxc9 is expressed ectopically at brachial levels on the electroporated side of the cord, while expression of Hoxc6 is ablated. Expression of FoxP1 is also ablated. (B) Effects of pCIG∷Bmi1 and CMV∷FGF8 coelectroporation. Bmi1 expression levels are increased in the electroporated cells. Hoxc9 is expressed ectopically at brachial levels, except in cells expressing Bmi1/GFP. Expression of Hoxc6 is reduced at brachial levels, but cells expressing Bmi1/GFP are rescued for Hoxc6. Likewise, expression of FoxP1 is reduced at brachial levels but rescued in cells expressing Bmi1.

Figure 9.

Model for PRCs in controlling Hox expression and MN columnar topography. (A) Summary of the effects of Bmi1 manipulations on Hox expression and columnar position at the brachial/thoracic boundary. Bmi1 depletion leads to ectopic Hoxc9 expression at thoracic levels and converts LMC MNs to a PGC fate. Loss of Hoxc6 is due to ectopic Hoxc9, as combined Hoxc9 and Bmi1 depletion rescues the phenotype. Elevation of Bmi1 at thoracic levels represses Hoxc9 and converts PGC MNs to an LMC fate. (B) Control of Hox gene expression in MNs by signaling gradients, PRCs, and cross-repression. Hox transcription is initiated in neural progenitors through the actions of positional gradients. Anterior boundaries are reinforced through the action of PRC1. Posterior boundaries are controlled through Hox cross-repressive interactions.