Abstract
Sonic hedgehog (Shh) signaling patterns the vertebrate spinal cord by activating a group of transcriptional repressors in distinct neural progenitors of somatic motor neuron and interneuron subtypes. To identify the action of this network, we performed a genome-wide analysis of the regulatory actions of three key ventral determinants in mammalian neural tube patterning: Nkx2.2, Nkx6.1 and Olig2. Previous studies have demonstrated that each factor acts predominantly as a transcriptional repressor, at least in part, to inhibit alternative progenitor fate choices. Here, we reveal broad and direct repression of multiple alternative fates as a general mechanism of repressor action. Additionally, the repressor network targets multiple Shh signaling components providing negative feedback to ongoing Shh signaling. Analysis of chromatin organization around Nkx2.2-, Nkx6.1- and Olig2-bound regions, together with co-analysis of engagement of the transcriptional activator Sox2, indicate that repressors bind to, and probably modulate the action of, neural enhancers. Together, the data suggest a model for neural progenitor specification downstream of Shh signaling, in which Nkx2.2 and Olig2 direct repression of alternative neural progenitor fate determinants, an action augmented by the overlapping activity of Nkx6.1 in each cell type. Integration of repressor and activator inputs, notably activator inputs mediated by Sox2, is probably a key mechanism in achieving cell type-specific transcriptional outcomes in mammalian neural progenitor fate specification.
Highlights
Sonic hedgehog (Shh) signaling is crucial for the specification of ventral neural progenitor types that give rise to molecularly and functionally distinct classes of ventral neurons in the developing vertebrate central nervous system (Dessaud et al, 2008)
Nkx6.1 and Olig2 as direct transcriptional targets of the ventral neural patterning activity of Shh (Lei et al, 2006; Oosterveen et al, 2012; Peterson et al, 2012; Wang et al, 2011). Each of these factors has been shown to function as a transcriptional repressor in neural patterning: Olig2 is required for the specification of somatic motor neuron progenitors, Nkx2.2 for the specification of V3 interneuron progenitors, whereas Nkx6.1 expression overlaps V2 and V3 interneurons and somatic motor neuron progenitors and is essential for the normal specification of both populations (Briscoe et al, 2000, 1999; Lu et al, 2002; Mizuguchi et al, 2001; Muhr et al, 2001; Novitch et al, 2001; Sander et al, 2000; Vallstedt et al, 2001; Zhou and Anderson, 2002; Zhou et al, 2001) (Fig. 1A)
To examine the direct regulatory actions of the Shh-initiated transcriptional network (Lei et al, 2006; Oosterveen et al, 2012; Peterson et al, 2012), we performed Chromatin immunoprecipitation (ChIP)-seq for Nkx2.2, Nkx6.1 and Olig2 on neural progenitors derived in vitro from mouse embryonic stem cells; a model system that recapitulates in vivo patterning processes (Peterson et al, 2012; Wichterle et al, 2002)
Summary
Sonic hedgehog (Shh) signaling is crucial for the specification of ventral neural progenitor types that give rise to molecularly and functionally distinct classes of ventral neurons in the developing vertebrate central nervous system (Dessaud et al, 2008). Nkx6.1 and Olig as direct transcriptional targets of the ventral neural patterning activity of Shh (Lei et al, 2006; Oosterveen et al, 2012; Peterson et al, 2012; Wang et al, 2011) Each of these factors has been shown to function as a transcriptional repressor in neural patterning: Olig is required for the specification of somatic motor neuron progenitors, Nkx2.2 for the specification of V3 interneuron progenitors, whereas Nkx6.1 expression overlaps V2 and V3 interneurons and somatic motor neuron progenitors and is essential for the normal specification of both populations (Briscoe et al, 2000, 1999; Lu et al, 2002; Mizuguchi et al, 2001; Muhr et al, 2001; Novitch et al, 2001; Sander et al, 2000; Vallstedt et al, 2001; Zhou and Anderson, 2002; Zhou et al, 2001) (Fig. 1A). Our data highlight a previously unappreciated breadth of direct fate exclusion, modulation of ongoing upstream Shh signaling input through multiple signaling nodes (Lek et al, 2010) and Sox input into available enhancers, with a resulting cell type-specific output directing a specific neural progenitor type (Oosterveen et al, 2012; Peterson et al, 2012)
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