How cell diversity and pattern are generated during tissue development is a long-standing question. Graded signals, often referred to as morphogens, have been suggested to provide the positional information that organizes gene expression and cellular differentiation in many tissues (Grimm et al., 2010; Ibañes and Izpisúa Belmonte, 2008; Lander, 2007
). The textbook explanation for their activity is that an extracellular concentration gradient of the morphogen establishes distinct levels of signaling in responding cells and thereby regulates target genes in a concentration-dependent manner (Wolpert et al., 1998
). In this view, the pattern of cellular differentiation is a direct and causal readout of a concentration gradient. However, recent studies challenge this idea. First, it is unclear whether a gradient can be sufficiently reliable and precise to pattern a tissue (Bollenbach et al., 2008; Gregor et al., 2007; Kerszberg and Wolpert, 2007; Manu et al., 2009a
). Second, evidence from several systems indicates that tissue patterning can take place in the absence of a stable gradient of a morphogen (Harfe et al., 2004; Nahmad and Stathopoulos, 2009; Ochoa-Espinosa et al., 2009
). Finally, in addition to the levels of signal, the duration of signaling can contribute to patterning (Ahn and Joyner, 2004; Dessaud et al., 2007; Harfe et al., 2004; Pagès and Kerridge, 2000
One tissue where these issues are particularly relevant is the vertebrate central nervous system. Here, Sonic Hedgehog (Shh) protein, emanating from the ventrally located notochord and floor plate, forms a gradient (Chamberlain et al., 2008
) that is responsible for subdividing the ventral neuroepithelium into five neural progenitor domains, each of which generates distinct neuronal subtypes (Jessell, 2000
) (A). In vitro, increasing concentrations of Shh ligand or increasing levels of Gli activity, the intracellular transcriptional effectors of Shh signaling, induce successively more ventral neural fates (Dessaud et al., 2007; Ericson et al., 1997; Stamataki et al., 2005
). In addition, however, neuronal subtype identity depends on the duration of Shh signaling. Accordingly, more ventral neural progenitor identities require longer durations of Shh signaling (Dessaud et al., 2007; 2010
). In vitro studies suggest that cells respond to ongoing exposure to Shh through an adaptation mechanism in which cells become desensitized to Shh (Dessaud et al., 2007
). An important question arising from these studies is how progenitors transform dynamic changes in Shh signaling into spatial patterns of gene expression.
Comparison of Spatial and Temporal Dynamics of Intracellular Shh Signaling and Ptch1 Protein
The transcriptional network acting downstream of Shh signaling might offer an answer to this question. Roles in the refinement and elaboration of patterning have been identified for the transcriptional circuits engaged in other tissues patterned by morphogens (Davidson, 2010; Davidson and Levine, 2008; Jaeger and Reinitz, 2006; Manu et al., 2009a; Xu et al., 2005
). Within the neural tube, three transcription factors, Pax6, Olig2, and Nkx2.2, which identify three spatially distinct ventral progenitor domains, are controlled by Shh signaling (Briscoe et al., 1999; Ericson et al., 1997; Novitch et al., 2001
) (A). Importantly, the final position of the boundaries of the p3 and pMN progenitor domains is regulated, at least in part, by cross repression between these factors (Briscoe et al., 1999, 2000; Ericson et al., 1997; Novitch et al., 2001
). Moreover, these transcription factors have been suggested to modulate the level of Shh signaling in responding cells as the neural tube is patterned (Lek et al., 2010
). Together, these studies identify an important role for the transcriptional circuit in refining the pattern of gene expression in the neural tube, but it leaves unresolved the question of how different levels and durations of Shh signaling control appropriate gene expression in responding cells. Furthermore, the in vivo temporal-spatial profile of Shh signaling and how this produces stable gene expression patterns are unclear.
Here, we use an in vivo reporter of Gli activity to determine the dynamics of Shh signaling in the neural tube, and we provide in silico and in vivo evidence that the regulatory logic of Pax6, Olig2, and Nkx2.2 transcriptional circuit is responsible for interpretation of the Shh signaling gradient. Strikingly, the design of the transcriptional circuit explains both the temporal and graded response to Shh signaling. In addition, it appears to render cells insensitive to transient increases in Shh signaling and produces hysteresis, providing cells with a memory of the signal. Together, these data indicate that the morphogen response of neural cells to Shh is an emergent property of a transcriptional circuit and suggest general principles that are likely to be relevant for morphogen interpretation in many developing tissues.