Chx10 Consolidates V2a Interneuron Identity through Two Distinct Gene Repression Modes

Abstract

During development, two cell types born from closely related progenitor pools often express identical transcriptional regulators despite their completely distinct characteristics. This phenomenon implies the need for a mechanism that operates to segregate the identities of the two cell types throughout differentiation after initial fate commitment. To understand this mechanism, we investigated the fate specification of spinal V2a interneurons, which share important developmental genes with motor neurons (MNs). We demonstrate that the paired homeodomain factor Chx10 functions as a critical determinant for V2a fate and is required to consolidate V2a identity in postmitotic neurons. Chx10 actively promotes V2a fate, downstream of the LIM-homeodomain factor Lhx3, while concomitantly suppressing the MN developmental program by preventing the MN-specific transcription complex from binding and activating MN genes. This dual activity enables Chx10 to effectively separate the V2a and MN pathways. Our study uncovers a widely applicable gene regulatory principle for segregating related cell fates.

Graded Arrays of Spinal and Supraspinal V2a Interneuron Subtypes Underlie Forelimb and Hindlimb Motor Control

“…The generation of type I and type II V2a interneurons is analogous to motor neuron diversification in which Lhx3 is initially present in all embryonic spinal motor neurons, but subsequently it is downregulated in all subtypes except the medial motor column (Sharma et al, 1998). The overlap in Lhx3 and Chx10 ( Figure S4D) combined with previous observations that ectopic expression of Lhx3 is sufficient to induce Chx10 V2a interneuron differentiation in the dorsal spinal cord (Clovis et al, 2016;Tanabe et al, 1998;Thaler et al, 2002) suggested Lhx3 might be central to the diversification process that generates type I and type II neurons. We generated transgenic mouse lines that express these Lin11-Isl1-Mec3 (LIM) homeodomain factors in a Cre-dependent fashion (tgCAG:lsl:Lhx3 and tgCAG:lsl:Lhx4).…”

“…Tissue preparation, immunohistochemistry, and imaging Antibodies used were: Guinea pig anti-Chx10 (1:4000) (Thaler et al, 1999), Neurotrace-Alexa647 (life technologies, 1:100), Guinea pig anti-Vglut2 (Millipore, 1:3000), Rabbit anti-GFP (Lifetechnologies, 1:1,000), Goat anti-GFP (Millipore, 1:1,000), Rabbit anti-RFP (MBL, 1:1000), Rabbit anti-NeuN (Millipore, 1:1000), Rabbit anti-Lhx3 (1:5000) (Sharma et al, 1998), Rabbit anti-Lhx4 (1:5000) (Sharma et al, 1998), Guinea pig anti-Lhx3 (1:5,000) (Sharma et al, 1998), Guinea pig anti-Lhx4 (1:20,000) (Sharma et al, 1998), Guinea pig anti-Sox14 (Gift from the Lee lab, 1:2,000) (Clovis et al, 2016), Guinea pig anti-Shox2 (1:20,000, antibody raised against peptide sequence: PELKDRKDDAKGMEDEG), Rabbit anti-Shox2 (1:20,000, antibody raised against peptide sequence: PELKDRKDDAKGMEDEG), Goat anti-Bhlhb5 (Santa Cruz, 1:200), Rabbit anti-Nfib (Novus, 1:1,000), Sheep anti-Zfhx3 (Novus, 1:1,000), Rabbit anti-NeuroD2 (abcam, 1:1,000), Goat anti-Sp8 (Santa Cruz, 1:500).…”

Rnf220 cooperates with Zc4h2 to specify spinal progenitor domains

“…6B). At E12.5, Rnf220 mutant embryos showed a dramatic reduction in the number of spinal V2 INs, as assessed by expression of the marker proteins Chx10, Gata3, Sox14 and Lhx3 (Al-Mosawie et al, 2007;Clovis et al, 2016;Del Barrio et al, 2007;Hargrave et al, 2000;Li et al, 2005;Muroyama et al, 2005) (Fig. 7A,D).…”

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

“…3E). These targets include those encoding further TFs, such as Mnx1 in MNs and Vsx2 (also known as Chx10) in V2a neurons, that subsequently consolidate the respective neuronal identities (Arber et al, 1999;Clovis et al, 2016;Thaler et al, 1999). Neurog2 has been demonstrated to interact with LIM-HD complexes in a phosphorylation-dependent manner (Ma et al, 2008), but the sites bound by these LIM protein complexes appear to be distinct from those bound by pro-neural bHLH TFs.…”