Hedgehog and stem cells
Transcription Factors

Author: Monica Pradotto
Date: 28/06/2010



In screening for mutations that affect the segmental pattern of Drosophila larvae, Nusslein-Volhard and Wieschaus discovered a group of mutants that affected patterning within the segments but at the same time left the number of segments unaltered. One of these so-called segment polarity mutants caused denticles to occur not only on the anterior, but also on the posterior half of the segments, covering the back of the larvae with a continuous lane of bristles.
This mutant was therefore named after the spiny little mammal Hedgehog. Drosophila has a single hh gene, mammals have three paralogous genes, called Sonic Hedgehog (Shh), Indian Hedgehog (Ihh), and Desert Hedgehog (Dhh).
The Hh proteins act as morphogens controlling multiple different developmental processes.
All mammalian Hh proteins are thought to have similar physiological effects; the differences in their roles in development result from diverse pattern of expression.
Dhh expression is largely restricted to gonads, including sertoli cells of testis and granulosa cells of ovaries.
Consistent with its expression in a very narrow tissue range, Dhh-deficient mice do not show notable phenotypes is most tissues and are viable. However, males are infertile due to complete absence of mature sperm.
Ihh is specifically expressed in a limited number of tissues, including primitive endoderm, gut, and prehypertrophic chondrocytes in the growth plates of bones. Approximately 50% of Ihh−/− embryos die during early embryogenesis due to poor development of yolk-sac vasculature. Surviving embryos display cortical bone defects as well as aberrant chondrocyte development in the long bones.
Shh is the most broadly expressed mammalian Hh signaling molecule. During early vertebrate embryogenesis, Shh expressed in midline tissues such as the node, notochord, and floor plate controls patterning of the left– right and dorso-ventral axes of the embryo. Shh expressed in the zone of polarizing activity (ZPA) of the limb bud is also critically involved in patterning of the distal elements of the limbs. Later in development, during organogenesis, Shh is expressed in and affects development of most epithelial

Hedgehog: an unusual signal transducer

Hedgehog: functions and mechanisms

Hh signal transduction

Of the mammalian Hh genes, only the mechanisms controlling Shh expression have been studied in detail.
After translation, Hh undergoes multiple processing steps that are required for generation and release of the active ligand from the producing cell. The mechanisms involved in Hh processing and secretion are evolutionary conserved.
Hh proteins are synthesized as precursor proteins (about 400-460 amino acids long) and comprise several different motifs and domains: a signal peptide for protein export, a secreted amino-terminal HhN (Hedge) domain that acts as a signaling molecule, and an autocatalytic carboxy-terminal HhC (Hog) domain that contains a Hint module. HhC binds cholesterol in the sterol recognition region (SRR). The catalytic activity of the Hint module cleaves Hh into two parts and adds the cholesterol moiety to the carboxyl terminus of HhN. The structure of HhC is globular, composed of β strands, and starts with a cysteine residue critical for auto processing.
The structure of the HhN domain is a relatively globular domain with two antiparallel α helices and several β strands wrapping one face of the two helixes. In addition to the cholesterol modification, the HhN domain is also modified at its amino terminus by palmitate through the action of a transmembrane acyltransferase, named Skinny hedgehog in Drosophila, and hedgehog acyltransferase (HHAT) in mammals. Because of these lipid modifications, the modified HhN domain (M-HhN) can form multimeric complexes and can interact with lipoproteins.
Although Hh is tightly associated with the plasma membrane, it is able to act directly over a long range. In both Drosophila and vertebrates, the secretion of Hh from the producing cell requires the activity of the 12-span transmembrane protein, Dispatched (Disp). Loss of Disp leads to accumulation of Hh in the producing cells and failure of long-range signaling.

Figure 1. Intramolecular autoprocessing of Hh.

Once released into the extracellular environment, M-HhN interacts with a number of different proteins: the heparan-sulfate proteoglycan Dally-like (Dlp), and the proteins Ihog and growtharrest-specific 1 (Gas1) are positive regulators of Hh signaling, whereas Hh-interacting protein (Hip) acts as a negative regulator by sequestering M-HhN.
The lipid modification of HhN as well as the extracellular protein interactions influence its extracellular movement and ensure correct shorthand long-range signaling.
In the absence of Hh ligand, Ptc catalytically inhibits the activity of the seven-transmembrane-span receptor-like protein Smo. Binding of Hh to Ptc results in loss of Ptc activity, and consequent activation of Smo. Smo then transduces the Hh signal to the cytoplasm.
Smo is negatively regulated by pro-vitamin D3, and is positively, but indirectly, regulated by oxysterols (oxygenated derivatives of cholesterol). 7-Dehydrocholesterol reductase, which converts pro-vitamin D3 into cholesterol, is also a regulator of Hh signaling. Smo activity can be modulated by many synthetic small molecules and natural products, including the steroidal alkaloids cyclopamine.

Intracellular Hh signaling

In the absence of Hh, Ptc keeps Drosophila Smo in an unphosphorylated state. Unphosphorylated Smo is cleared from the cell surface via endocytosis and is degraded in lysosomes.
After Hh stimulation, Smo is hyperphosphorylated and its endocytosis and degradation are blocked. dSmo C terminus binds directly to the kinesin-like protein Cos2, which acts as a scaffolding protein, bringing together multiple cytoplasmic components of the pathway. These include the full-length transcriptional activator form of Ci, CiA (155 kDa), and multiple serine–threonine kinases, including a kinase that specifically acts on the Hh pathaway, Fused (Fu) and the multifunction kinases PKA, GSK3, CKI, and CKI.
In the absence of Hh, CiA is hyperphosphorylated by the combined action of PKA, which acts as a priming kinase, and GSK3 and the casein kinases, which further phosphorylate the primed substrate.
The hyperphosphorylation promotes recognition of CiA by the ubiquitin E3 ligase Slimb (-TrCP in vertebrates), leading to the generation of a truncated transcriptional repressor form of Ci, CiR (75 kDa).
In addition to promoting CiR formation, Cos2 regulates Ci by tethering it to the cytoplasm and preventing its nuclear translocation.
In the presence of Hh, Smo accumulates and the binding of Cos2 to Smo prevents conversion of CiA to CiR. However, this mechanism alone is not sufficient to fully activate the pathway, as some CiA is still retained in the cytoplasm by another protein, Supressor of Fused [Su(Fu)]. Genetic evidence from Drosophila indicates that full activation of the pathway in response to Hh requires the Fu protein kinase, which blocks the negative influence of Su(Fu) on Ci.
Upon entering the nucleus, CiA binds to specific sequences in promoter and enhancer regions and controls the transcription of the Hh target gene(s).
Despite the conservation of the Hh signaling pathway and many of its roles in development between invertebrate and vertebrate specie, the mechanisms by which Smo regulates the Ci/GLI transcription factors appears to be distinct between Drosophila and mammals. The C-terminal domains of vertebrate Smo proteins are significantly shorter than those of invertebrates and lack the main phosphorylation. In addition, the two mammalian orthologs of Cos2, Kif27, and Kif7 have none of the unique sequence characteristics of Cos2 that differentiate Cos2 from the kinesin family of motor proteins. Based on sequence, Kif7 and Kif27 appear to be functional molecular motors, whereas Cos2 has apparently lost its ability to bind ATP and function as a motor protein.
Drosophila Smo activation is coupled to the hyperphosphorylation of 26 serine/threonine residues located within the C-terminal cytoplasmic tail by PKA and CKI.
None of these PKA or CKI phosphorylation sites are conserved in vertebrate Smo. Cos2–Fu complex, which is centrally important in Drosophila, plays little role in mammalian Hh signaling. Instead, it appears that mammalian Hh signaling critically depends on Su(Fu) and on several components involved in formation of the primary cilia.
Primary cilium is an organelle that protrudes from the surface of most vertebrate cells. It has been reported that activated mammalian Smo accumulates to primary cilia in response to Shh treatment; in the absence of Shh, this accumulation is prevented by Ptc.
Other components involved in Hh signaling, including Su(Fu) and unprocessed GLI proteins, have also been localized to the primary cilium.
In contrast to the differences in signaling between Smo and GLI, the activities of the GLI proteins themselves are regulated similarly to Ci,. The activator and repressor functions of Ci are divided in mammals to three GLI proteins, GLI1–3.
GLI1 and GLI2 are responsible for most activator functions and have similar activities at protein level. Whereas loss of GLI2 is embryonic lethal, GLI1 is dispensable for normal development.
GLI1 expression is induced by Hh ligands, and its function appears to be primarily to provide positive feedback and to prolong cellular responses to Hh. GLI3, in turn, functions primarily as a repressor and its loss or mutation leads to limb malformations in mice and humans.
GLI activity appears to be regulated by Hh in a way that is very similar to that observed in Drosophila.
In theabsence of Hh, GLI3 is phosphorylated, recognized by -TrCP, and proteolytically processed to a truncated repressor form. Addition of Shh leads to inhibition of processing and accumulation of full-length forms of both GLI2 and GLI3.

Figure.2 A simplified Hh signaling pathway, constructed from combined Drosophila and mammalian data.

The Hedgehog protein family

Role of Hh signaling

The developmental processes that the Drosophila and vertebrate Hh signaling pathways regulate appear remarkably conserved. At the cellular level, the effects of Hh range from growth and self-renewal to cell survival, differentiation, and/or migration. During embryogenesis, the Hh cascade is used repeatedly and in different tissues to induce a large number of developmental processes. The ability of a single morphogen to affect almost every part of the vertebrate body plan is made possible by the fact that cellular responses to Hh depend on the type of responding cell, the dose of Hh received, and the time the cell is exposed to Hh. At the molecular level, the diverse cellular responses are effected by induction of different sets of target genes. Among the genes regulated tissue specifically by Hh signaling are those encoding other secreted signaling proteins, including bone morphogenetic protein 4 (BMP4), fibroblast growth factor 4 (FGF4), and vascular endothelial growth factor (VEGF)-A, genes involved in cell growth and division, and many transcription factors that are essential for animal development, including members of the Myod/Myf, Pax, Nkx, Dbx, and Irx families.
The effect of Hh dose on target tissue responses is best characterized in the specification of cell identities in the ventral neural tube. During neural tube development, Shh protein diffuses from the notochord and floor plate, creating a concentration gradient across the ventral neural tube. Different doses of Shh within this gradient specify five neuronal subtypes at precise positions along the floor plate–roof plate axis.
Initially, Shh induces Class II homeodomain (e.g., Nkx2.2, Nkx6.1) and represses Class I homeodomain (Pax6, Pax7, Irx3, and Dbx1/2) transcription factors. Cross-repressive interactions between these factors then act to sharpen the expression boundaries and to subsequently direct cells to differentiate into specific lineages.
Much less is known about the roles played by Hh in pupal development and in maintaining homeostasis of tissues during adult life. The best-characterized role for Hh signaling in adults is in the reproductive system, and Hh proteins are expressed and required for maturation of the germ cells in multiple species.
In Drosophila ovary, Hh acts as a somatic stem cell factor, directly controlling the proliferation and maintenance of ovarian somatic stem cells.

Hedgehog signal transduction: recent findings

Hh signaling in stem cell maintenance

Stem cells either self-renew or differentiate depending upon various micro-environmental cues, including soluble growth factors, extracellular matrix and mechanical forces. Critical role in cell function and in context of stem cells is the concept of the “stem cell niche”. Such a niche is a cellular environment that can provide critical stem cell maintenance signals to support and maintain the undifferentiated phenotype of stem cell maintenance include those involving the Wnt, Notch and Hedgehog pathways, all of which play a role in early embryonic development and in the control of cell differentiation and proliferation.
Transient Hh pathway activity promote stem cell self-renewal in normal tissues, whereas continuous activation is associated with the initiation and growth of many types of human cancer. This pathway thus provide a potential link between the normal self-renewal of stem cells and the aberrantly regulated proliferation of cancer stem cells.
In the mammalian CNS, Shh is essential for the establishment of the ventral pattern along the entire neuraxis, including the telencephalon. In addition, Shh has been demonstrated to play a mitogenic role in the expansion of granule cell precursors during CNS development. Several lines of evidence support a role for Shh regulating progenitor cell proliferation in the CNS.
Shh null animals have a marked decrease in the size of the brain. Conversely, Shh gain-of-function studies have demonstrated both in vitro and in vivo that ectopic exposure of the telencephalon and other areas of the neural tube to Shh results in hypertrophy.
Shh emanating from Purkinje cells mediates the proliferation of granule precursors in the external granule cell layer. Further, human disorders resulting in an increase in Shh signaling, as a result of Ptch1 heterozygosity or activation of Shh signaling in target cells, results in the formation of cerebellar medulloblastomas.

In vivo analysis of quiescent adult neural stem cells responding to Sonic hedgehog

Shh is also a potent mitogen for neuronal progenitor cells of the adult hippocampous. Rat hippocampal progenitors proliferated when cultured with Shh and clonal populations retained their multipotency. Moreover cyclopamine, a potent inhibitor of Smo, reduce in a significant way the proliferation of neuronal progenitors.

Sonic hedgehog controls stem cell behavior in the postnatal and adult brain

SVZ (subventricular zone) cells express Shh and Gli1 and that blockage of hedgehog (Hh) signaling in adult and perinatal mice results in diminished expression of Gli1 and deficits in SVZ cell proliferation in vivo.

Sonic hedgehog is required for progenitor cell maintenance in telencephalic stem cell niches

The development of hematopoietic assays for human stem cells permits the use of primary human cells as a model system to study human stem cell biology. Using these in vitro and in vivo assays to detect candidate human stem cells of hematopoietic tissue, combinations of cytokines have been examined for their ability to maintain stem cells in ex vivo cultures. Results from these studies show that human cytokines induce the proliferation of primitive blood cells and progenitor cell expansion, however, this proliferation occurs at the expense of stem cell differentiation and loss of pluripotent regenerative capacity.
Therefore, factors capable of inducing proliferation of primitive stem cells without differentiation have yet to be identified in the human.
There are evidence for a functional role of Hh proteins in the development of human blood cells. Hh and its putative receptors, Ptc and Smo, along with downstream transcription factors Gli-1, Gli-2 and Gli-3 are expressed in primitive human blood cells and stromal cells of the hematopoietic microenvironment.
However, the downstream transcriptional regulators of the Hh pathway, Gli-1, Gli-2 and Gli-3, were not expressed in lineage restricted hematopoietic cells, which suggested that Hh signaling is not essential to hematopoietic cell maturation.
Blocking of endogenously produced Hh or addition of exogenous soluble Hh can control the proliferation of uncommitted human hematopoietic cells. This shows that Hh signals are critical to the proliferative regulation of hematopoietic progenitor cells. These effects of Shh regulation were dominant to the regulatory mechanisms that control proliferation and differentiation, which are orchestrated by hematopoietic cytokines.
Shh signals regulate the proliferation and differentiation of primitive human blood cells by modulating the morphogenic function of Bone morphogenetic protein 4. BMP-4 is a member of the transforming growth factor β (TGF-β) superfamily and is expressed in ventral mesoderm where induces hematopoietic commitment.
Proliferation of human stem or progenitor cells is controlled by cytokines, which promote differentiation and subsequent loss of stem cell function. The response of primitive human blood cells to cytokines induces the transcriptional up-regulation of BMP-4 and Noggin, a specific inhibitor of BMP-4. Cytokine responsiveness is dependent on expression of soluble or cell-surface Hh proteins that may function in an autocrine or paracrine fashion, which results in the differentiation of primitive cells. Blocking endogenous Hh protein inhibits cytokine-induced proliferation and up-regulation of BMP-4 , which results in decreased differentiation through maintenance of functional stem or progenitor cells. However, enhanced Hh signals, which are induced by the addition of exogenous Shh, cause a mitogenic response in combination with hematopoietic cytokines that results in a greater expansion of stem or progenitor cells. This is achieved by Shh via BMP-4 morphogenic signaling.

Figure 3. model for the function of Hh in the proliferation and differentiation of primitive human hematopoietic cells.

Sonic hedgehog induces the proliferation of primitive human hematopoietic cells via BMP regulation Hh and Wnt pathways control cells proliferation

Hh and Wnt pathways control cells proliferation

There are evidences of an interaction between canonical Wnt activity and the Shh pathway in the regulation of cell cycle progression of neural cells.
The activity of Shh is epistatic to Wnt. By controlling the expression of the Tcf3/4 transcription factors, Shh activity is required for canonical Wnt/β-catenin transcriptional activity. This provides a genetic mechanism through which Shh and Wnt activities are integrated in the control of Ccnd1 expression and of progression through the G1 phase of the cell cycle. Ccnd1 is a
direct target gene of the Wnt canonical pathway and active TCF
consensus binding sites are present in the Ccnd1 promoter, but in the absence of Shh signalling, Wnt was
unable to activate Ccnd1 expression as a consequence of reduced TCF expression in the absence of Shh.
Wnt activity is restricted to the G1 phase of the cell cycle, whereas Shh plays a role in the regulation of the late cell cycle. Expression of late cyclins depends on Shh/Gli but not on Wnt/β-catenin activities. This analysis confirmed that the Shh pathway controls cell cycle progression of neural cells through the regulation of G1 and G2 cyclins.

Figure 4. Summary of Shh and Wnt activities in the regulation of the G1 and G2 progression of neuroepithelial cells.

Hedgehog activation is required upstream of Wnt signalling to control neural progenitor proliferation

Shh and response to hypoxia

One of the most important features of the stem cell microenvironment is the presence of hypoxia. Approximately 1% to 1.5% of the genome is transcriptionally regulated by hypoxia, and many of these genes are known to be regulated by hypoxia-inducible factor-1 (HIF-1). Studies with normal bone marrow stem cells in vitro indicated that very early progenitor cells were protected from commitment and remained in a quiescent state under hypoxic conditions (1% O2). Low oxygen tension (3%-5% O2) has also been reported to inhibit the differentiation and enhance the stemness of populations of mesenchymal stem cells. In addition hypoxia promotes survival and proliferation of certain populations of mammalian neuronal stem cells or neuronal progenitors cells.
Brain injury invokes a recapitulation of established developmental programs and the Sonic hedgehog (Shh) pathway has been implicated. Ischemia/ hypoxia to brain and cultured cells upregulated the expression of Shh, particularly in neural progenitor cells (NPCs), and modulated their proliferative response to ischemia/hypoxia. Shh gene expression and protein levels increase in neural progenitors after hypoxia. hypoxia and exogenous Shh had an additive effect on proliferation. Cyclopamine reduced the proliferation stimulus of hypoxia or Shh and their combined treatment.

Sonic Hedgehog Regulates Ischemia/Hypoxia-Induced Neural Progenitor proliferation

Generation of dopaminergic neurons from human fetal mesencephalic progenitors after co-culture with striatal-conditioned media and exposure to lowered oxygen

Hh Signaling and cancer

If cancer stem cells arise from tissue stem cells, and if Hh pathway activity is critical for the renewal of at least some of these stem cell types, then continuous Hh activity may promote cancer growth by continuously recapitulating their roles in promoting normal stem cell renewal. But stem cell renewal must be tightly regulated (otherwise tumours might arise), raising the critical question of how and under what circumstances normal regulation can be circumvented in cancer. A connetion is strongly suggested by the know association between chronic tissue injury and cancer.
Acute injury is accompanied by the expansion of stem cell pools and by the transient activation of the Hh and also Wnt signaling pathways. Under conditions of chronic injury, pathway activation and presumed expansion of stem cell pools would persist so long as repeated injury prevents full regeneration. This state of continuous pathway and progenitor-cell activation resembles the continuous pathway activity and cell division seen in cancer.
These observations suggest that cancer growth may represent the continuous operation of an unregulated state of tissue repair and that continuous Hh/Wnt pathway activities in carcinogenesis may represent a deviation from the return to quiescence that normally follows regeneration. The simplest model for the emergence of this state is that genetic or epigenetic events prevent the return to quiescence of an activated stem or progenitor cell on completion of regeneration, thus initiating a tumour by trapping the cell in an
activated state of continuous renewal. Consistent with this model, the Bmi-1 gene required for HSC renewal is also required for the propagation of leukaemias in transfer experiments. The expression of Bmi-1 and nestin, which are both associated with stem cell renewal, is dependent on Hh pathway activity in Hh dependent tumours.
Moreover, aberrant activation of Hh signaling can cause basal cell carcinoma (BCC, the most common type of skin cancer), medulloblastoma (a childhood cancer with an invariably poor prognosis), and rhabdomyosarcoma. These tumor types occur at an increased rate in patients or mice with germline mutations in Ptc, and sporadic cases are often associated with mutations in the Hh pathway components Ptc, Smo, or Su(Fu), or more rarely, the amplification of GLI1.
Aberrantly activated Shh signaling has also been suggested to play a role in other cancers, such as glioma, breast, esophageal, gastric, pancreatic, prostate, and small-cell lung carcinoma.
With the exception of rare GLI1 amplifications found in gliomas, the mutational basis of Hh pathway activation in these types of cancer has not been ascertained.
Multiple lines of evidence suggest that Hh acts to promote cancer by directly regulating cellular growth and/ or survival.

Tissue repair and stem cell renewal in carcinogenesis

Hedgehog Signaling in Development and Cancer

Sonic Hedgehog signaling impairs ionizing radiation – induced checkpoint activation and

induces genomic instability

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