Mechanistic Differences in the Transcriptional Interpretation of Local and Long-Range Shh Morphogen Signaling

Abstract

Morphogens orchestrate tissue patterning in a concentration-dependent fashion during vertebrate embryogenesis, yet little is known of how positional information provided by such signals is translated into discrete transcriptional outputs. Here we have identified and characterized cis-regulatory modules (CRMs) of genes operating downstream of graded Shh signaling and bifunctional Gli proteins in neural patterning. Unexpectedly, we find that Gli activators have a noninstructive role in long-range patterning and cooperate with SoxB1 proteins to facilitate a largely concentration-independent mode of gene activation. Instead, the opposing Gli-repressor gradient is interpreted at transcriptional levels, and, together with CRM-specific repressive input of homeodomain proteins, comprises a repressive network that translates graded Shh signaling into regional gene expression patterns. Moreover, local and long-range interpretation of Shh signaling differs with respect to CRM context sensitivity and Gli-activator dependence, and we propose that these differences provide insight into how morphogen function may have mechanistically evolved from an initially binary inductive event.

A direct fate exclusion mechanism by Sonic hedgehog-regulated transcriptional repressors

“…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 Sox2 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).…”

“…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 ChIP-seq for Nkx2.2, Nkx6.1 and Olig2 on neural progenitors derived in vitro from mouse embryonic stem cells (mESCs); a model system that recapitulates in vivo patterning processes (Peterson et al, 2012;Wichterle et al, 2002) (supplementary material Table S1). The binding events were reproducibly detected in biological replicates (supplementary material Fig.…”

“…Detailed analyses showed that a number of known neural fate determinants as well as components of the Hedgehog pathway were co-targeted ( Fig. 1E,F (Lei et al, 2006;Oosterveen et al, 2012;Peterson et al, 2012;Wang et al, 2011), intersectional analysis showed a limited overlap with Gli1-and Gli3-bound regions overall ( Fig. 1G; supplementary material Fig.…”

“…Exceptions were putative cis-regulatory regions around ventral neural progenitor subtype specifiers, including Nkx2.2, Olig2 and Nkx6.1 (supplementary material Fig. S2) (Lei et al, 2006;Oosterveen et al, 2012;Peterson et al, 2012;Wang et al, 2011). At the target gene-level, Gli factors showed extensive overlap, particularly with genes targeted by all three repressors ( Fig.…”

“…Several lines of evidence, including the direct binding of Gli transcription factors to their cis-regulatory modules, have identified Nkx2.2, 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, , 1999Lu 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.…”