Limb anterior-posterior polarity integrates activator and repressor functions of GLI2 as well as GLI3

Developmental Biology Program, Sloan-Kettering Institute, New York, NY 10065, USA.
Developmental Biology (Impact Factor: 3.55). 07/2012; 370(1):110-24. DOI: 10.1016/j.ydbio.2012.07.017
Source: PubMed


Anterior-posterior (AP) limb patterning is directed by sonic hedgehog (SHH) signaling from the posteriorly located zone of polarizing activity (ZPA). GLI3 and GLI2 are the transcriptional mediators generally utilized in SHH signaling, and each can function as an activator (A) and repressor (R). Although GLI3R has been suggested to be the primary effector of SHH signaling during limb AP patterning, a role for GLI3A or GLI2 has not been fully ruled out, nor has it been determined whether Gli3 plays distinct roles in limb development at different stages. By conditionally removing Gli3 in the limb at multiple different time points, we uncovered four Gli3-mediated functions in limb development that occur at distinct but partially over-lapping time windows: AP patterning of the proximal limb, AP patterning of the distal limb, regulation of digit number and bone differentiation. Furthermore, by removing Gli2 in Gli3 temporal conditional knock-outs, we uncovered an essential role for Gli2 in providing the remaining posterior limb patterning seen in Gli3 single mutants. To test whether GLIAs or GLIRs regulate different aspects of AP limb patterning and/or digit number, we utilized a knock-in allele in which GLI1, which functions solely as an activator, is expressed in place of the bifunctional GLI2 protein. Interestingly, we found that GLIAs contribute to AP patterning specifically in the posterior limb, whereas GLIRs predominantly regulate anterior patterning and digit number. Since GLI3 is a more effective repressor, our results explain why GLI3 is required only for anterior limb patterning and why GLI2 can compensate for GLI3A in posterior limb patterning. Taken together, our data suggest that establishment of a complete range of AP positional identities in the limb requires integration of the spatial distribution, timing, and dosage of GLI2 and GLI3 activators and repressors.

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    • "This point is illustrated by the observation that the Gli2 activator plays a dominant role in neural tube development (Ding et al. 1998; Matise et al. 1998; Bai et al. 2004), while the Gli3R is a key determinant of limb patterning (Bowers et al. 2012; Cao et al. 2013), and a different group of Hh targets is activated accordingly. Moreover, complex interactions between various Gli proteins exist in both neural tube (Liu et al. 2012) and limb patterning (Bowers et al. 2012), and pinpointing the contribution of a given Gli protein is nontrivial. The basic framework of Hh signaling is established through the identification and characterization of various Hh pathway components, many of which were initially identified by genetic screens in Drosophila. "
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    ABSTRACT: Control of Gli function by Suppressor of Fused (Sufu), a major negative regulator, is a key step in mammalian Hedgehog (Hh) signaling, but how this is achieved in the nucleus is unknown. We found that Hh signaling results in reduced Sufu protein levels and Sufu dissociation from Gli proteins in the nucleus, highlighting critical functions of Sufu in the nucleus. Through a proteomic approach, we identified several Sufu-interacting proteins, including p66β (a member of the NuRD [nucleosome remodeling and histone deacetylase] repressor complex) and Mycbp (a Myc-binding protein). p66β negatively and Mycbp positively regulate Hh signaling in cell-based assays and zebrafish. They function downstream from the membrane receptors, Patched and Smoothened, and the primary cilium. Sufu, p66β, Mycbp, and Gli are also detected on the promoters of Hh targets in a dynamic manner. Our results support a new model of Hh signaling in the nucleus. Sufu recruits p66β to block Gli-mediated Hh target gene expression. Meanwhile, Mycbp forms a complex with Gli and Sufu without Hh stimulation but remains inactive. Hh pathway activation leads to dissociation of Sufu/p66β from Gli, enabling Mycbp to promote Gli protein activity and Hh target gene expression. These studies provide novel insight into how Sufu controls Hh signaling in the nucleus. © 2014 Lin et al.; Published by Cold Spring Harbor Laboratory Press.
    Full-text · Article · Nov 2014 · Genes & Development
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    • "Gli2 functions primarily as a transcriptional activator upon Hh stimulation and can initiate transcription of Gli1, a constitutive transcriptional activator that indicates high levels of pathway activity [8] [9] [10] [11]. However, in some contexts, Gli3 may act as a weak activator, and Gli2 may act as a weak repressor [12] [13]. The relative levels of the Gli transcription factors, and the balance between repression and activation of Hh pathway target genes, are a major mechanism by which cells in the developing neural tube, one of the bestcharacterized Shh-responsive tissues, translate a gradient of Hh ligand into a pattern of distinct neuronal fates [6] [14] [15] [16] [17] [18] [19] [20] [21] [22]. "
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    ABSTRACT: Sonic hedgehog (Shh) is a pleiotropic factor in the developing central nervous system (CNS), driving proliferation, specification, and axonal targeting in multiple sites within the forebrain, hindbrain, and spinal cord. Studies in embryonic CNS have shown how gradients of this morphogen are translated by neuroepithelial precursors to determine the types of neurons and glial cells they produce 0005 and 0010. Shh also has a well-characterized role as a mitogen for specific progenitor cell types in neural development 0015 and 0020. As we begin to appreciate that Shh continues to act in the adult brain, a central question is what functional role this ligand plays when major morphogenetic and proliferative processes are no longer in operation. A second fundamental question is whether similar signaling mechanisms operate in embryonic and adult CNS. In the two major germinal zones of the adult brain, Shh signaling modulates the self-renewal and specification of astrocyte-like primary progenitors, frequently referred to as neural stem cells (NSCs). It also may regulate the response of the mature brain to injury, as Shh signaling has been variously proposed to enhance or inhibit the development of a reactive astrocyte phenotype. The identity of cells producing the Shh ligand, and the conditions that trigger its release, are also areas of growing interest; both germinal zones in the adult brain contain Shh-responsive cells but do not autonomously produce this ligand. Here, we review recent findings revealing the function of this fascinating pathway in the postnatal and adult brain, and highlight ongoing areas of investigation into its actions long past the time when it shapes the developing brain.
    Full-text · Article · Sep 2014 · Seminars in Cell and Developmental Biology
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    • "In the presence of Hh signaling, full-length, activated Gli proteins translocate into the nucleus where they activate transcription (Bai et al., 2004; Bowers et al., 2012; Dai et al., 1999; Matise et al., 1998; Sasaki et al., 1999; Wang et al., 2000, 2007). In the absence of Hh signaling, Gli2 and Gli3 are processed by proteolytic cleavage into truncated proteins that act as transcriptional repressors (Litingtung et al., 2002; Pan et al., 2006; Wang et al., 2000; Wen et al., 2010). "
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    ABSTRACT: The transcriptional response to the Hedgehog (Hh) pathway is mediated by Gli proteins, which function as context-dependent transcriptional activators or repressors. However, the mechanism by which Gli proteins regulate their target genes is poorly understood. Here, we have performed the first genetic characterization of a Gli-dependent cis-regulatory module (CRM), focusing on its regulation of Grem1 in the mouse limb bud. The CRM, termed GRE1 (Gli responsive element 1), can act as both an enhancer and a silencer. The enhancer activity requires sustained Hh signaling. As a Gli-dependent silencer, GRE1 prevents ectopic transcription of Grem1 driven through additional CRMs. In doing so, GRE1 works with additional GREs to robustly regulate Grem1. We suggest that multiple Gli CRMs may be a general mechanism for mediating a robust transcriptional response to the Hh pathway.
    Full-text · Article · Apr 2014 · Development
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