A homeodomain feedback circuit underlies step-function interpretation of a Shh morphogen gradient during ventral neural patterning
Madelen Lek
0
Jos M. Dias
Ulrika Marklund
Christopher W. Uhde
Sanja Kurdija
Qiubo Lei
LoriSussel
John L. Rubenstein
Michael P. Matise
Hans-Henning Arnold
Thomas M. Jessell
JohanEricson
0
Present address: UBE
,
Nobels vag15A
,
Karolinska Institutet
,
S-17177 Stockholm
,
Sweden
SUMMARY The deployment of morphogen gradients is a core strategy to establish cell diversity in developing tissues, but little is known about how small differences in the concentration of extracellular signals are translated into robust patterning output in responding cells. We have examined the activity of homeodomain proteins, which are presumed to operate downstream of graded Shh signaling in neural patterning, and describe a feedback circuit between the Shh pathway and homeodomain transcription factors that establishes non-graded regulation of Shh signaling activity. Nkx2 proteins intrinsically strengthen Shh responses in a feed-forward amplification and are required for ventral floor plate and p3 progenitor fates. Conversely, Pax6 has an opposing function to antagonize Shh signaling, which provides intrinsic resistance to Shh responses and is important to constrain the inductive capacity of the Shh gradient over time. Our data further suggest that patterning of floor plate cells and p3 progenitors is gated by a temporal switch in neuronal potential, rather than by different Shh concentrations. These data establish that dynamic, non-graded changes in responding cells are essential for Shh morphogen interpretation, and provide a rationale to explain mechanistically the phenomenon of cellular memory of morphogen exposure.
INTRODUCTION
A general strategy to establish cellular diversity in developing
organisms is the deployment of morphogen gradients that signal at
long range from localized sources to impart positional information
to surrounding cells (Lander, 2007). A well-studied example in
vertebrate development is the secreted protein Sonic hedgehog
(Shh), which is responsible for conveying such information to cells
of the ventral neural tube (Jessell, 2000) and to other tissues such
as the limb bud (Bastida and Ros, 2008). In the neural tube, this
activity is manifested in the patterned generation of distinct
neuronal subtypes at stereotypic positions in response to graded
Shh signaling by ventral midline cells. A key role for Shh in this
process is to regulate expression of patterning transcription factors,
but the mechanisms by which information is translated from the
extracellular Shh gradient into a precise and robust transcriptional
output remain poorly understood.
Models of graded Shh signaling in the neural tube (Roelink et
al., 1995; Ericson et al., 1997; Dessaud et al., 2007) posit that a
long-range extrinsic spatiotemporal gradient of ligand emanates
first from the notochord and then from the floor plate (FP). The
Shh signal is received by neural progenitors and transduced by the
Patched/Smoothened (Ptc1/Smo)-receptor complex, ultimately
regulating activity of the executors of the Shh pathway, zinc-finger
domain-containing transcription factors of the Gli family. In the
absence of Shh, Gli2 and Gli3 are processed to transcriptional
repressors (GliR), whereas both are stabilized in their activator
forms (GliA) upon exposure to Shh ligand (reviewed by Dessaud
et al., 2008). Gli proteins regulate the patterned expression of
homeodomain (HD) and basic helix-loop-helix (bHLH)
transcription factors in neural progenitors, and the cross-repressive
interactions between these proteins are important for the
establishment and maintenance of ventral progenitor domains
expressing distinctive molecular profiles (Briscoe et al., 2000;
Muhr et al., 2001). These transcription factors provide positional
value to cells and hence determine the identity of neuronal subtypes
generated from distinct progenitor populations (Dessaud et al.,
2008).
Evidence for graded activity of Shh originates primarily from ex
vivo experiments, in which incremental two- to threefold increases
in the ambient Shh concentration (Roelink et al., 1995; Ericson et
al., 1997) or in the duration of exposure to Shh (Dessaud et al.,
2007) result in induction of cell types that are characteristic of
progressively more ventral regions of the neural tube. Shh signaling
acts at long range in vivo (Briscoe et al., 2001) and a graded
distribution of the Shh ligand has been demonstrated (Chamberlain
et al., 2008). Together with genetic analyses of Gli genes (Ding et
al., 1998; Matise et al., 1998; Persson et al., 2002) and
gain-offunction studies in chick (Stamataki et al., 2005), these data are
consistent with a model in which the extracellular Shh gradient is
translated into an intracellular Gli activator-to-repressor gradient
along the ventral-to-dorsal axis of the neural tube (Dessaud et al.,
2008). Consistent with this view, ventral subtype identities are lost
in mice that lack Shh, as well as in Smo mutants that are unable to
transduce the Shh signal (Chiang et al., 1 (...truncated)