**6. Hedgehog pathway**

128 Neuroimaging for Clinicians – Combining Research and Practice

flow; ciliary motility loss leads to reduced fluid flow in brain ventricles, resulting in

Motility is the main feature of the unique 9+0 primary cilium at the embryonic node, which is essential for correct embryonic development. Impairment of these cilia result in embryos showing randomized left-right asymmetry and randomized turning and heart looping [17

Polarization of cell division: Recent advances have demonstrated that ciliary proteins are involved in the regulation of the cell cycle. Centrioles play a dual role in the cell. They form the centrosomes that can interconvert with basal bodies upon ciliation. At the same time, they also give rise to the poles of the mitotic spindle. Centrioles may function as a signaling platform, through proteins that promote the transition from one phase to another in mitosis

The Centrosome is an organelle located in the center of eukaryotic cells, and acts as an organizing center rather than a microtubule [19]. The centrosome is known as the cytoskeleton's microtubule organizing center of the eukaryotic cell animal, that radiates in a star way or ASTER during mitosis. Experiments show that centrosome absence prevents the

The centrosome consists of: a) the *Diplosome,* two cylindrical centrioles arranged perpendicularly, located near the nucleus (9 groups of 3 microtubules in cylindrical provision, diameter of 200 nm and 400 nm long). The centrioles of the centrosome are distinguished by a Mother centriole (mature) and a Daughter one; the *Mother* centriole is associated with proteins forming appendices: distal (related to cilia and flagella; a centriole at the base of each cilium or flagellum) and subdistal, involved in microtubule nucleation. b) the *pericentriolar material*, the dense part of the cytosol (amorphous-looking material) and c)

The centrosome´s main function is to form and organize the microtubules that comprise the achromatic spindle in division of the cell nucleus. The paternal centrosome plays the motor role in meiosis, while the maternal centrosome disappears. Only the mature mother centriole, likely due to the existence of subdistal appendages, transforms into the peculiar structure at the distal end, the basal body, which gives rise to the cilia and flagella of eukaryotic cells. If certain factors are absent (the ODF2 in mice), the cells are unable to

The centrioles are surrounded by an electron-dense matrix called the pericentriolar material (PCM), composed of sets of proteins that modulate or assist in centrosome involving processes. To this day, genome sequencing and comparison has detected and described about 300 proteins that most likely work with the centrosome, although few of them are

Mutations in intraflagellar transport (IFT) genes have clearly demonstrated a correlation between primary cilia and cell cycle control. The basal body-centrosome complex also plays a crucial role in coordinating IFT and the formation of cilia. The centrosome is surrounded by pericentriolar material (PCM), which serves as a nucleation site for microtubules. In

hydrocephalus [14, 15, 16]

[18].

**4. Cell cycle: Centriole union link** 

 Apc2, anaphase promoting complex protein 2 *Platelet-derived growth factor receptors (Pdgfr)*

the *aster fibers* (microtubules organized in rays).

organize cilia or flagella [20].

well known.

cytogenesis process; the cycle does not progress beyond G1.

The process and transfer of information signals to the cell are mediated by specialized proteins [3]:


Hedgehog proteins (Hh) are a group of signaling proteins generating extracellular ligands that play an important role in regulating cell differentiation. In humans, there are three known Hedgehog proteins: Sonic Hh (Shh), Indian Hh and Desert Hh. When these ligands bind to various specific receptors, they trigger a signaling cascade resulting in activation of transcription factors of the Gli family that regulate the transcription of effector genes. The Hh receptors in vertebrates are the integral membrane "Patched" proteins Ptc1 and Ptc2 .

Ciliopathies: Primary Cilia and Signaling Pathways in Mammalian Development 131

Formation of finger-type depends on morphogen levels to which precursor cells were subjected during development. When another source of Shh is introduced at previous levels, either naturally or through experimental means, two gradients are generated in the outline of the limb which, depending on their geometry, may overlap with each other in the central area of the limb. This configuration leads to mirror duplications of the digits (d4-d3-d2-d3-

In the developing mammalian ventral spinal cord, five different neuronal cell types, comprising the motoneuron (MN) and four different interneurons (V0-V3), are generated from their respective neural progenitors (pMN, pV0-pV3) under the influence of signals from the notochord [43]. Shh, one of the Hedgehog (Hh) family of proteins, is strongly expressed in the notochord and later in the floor plate, and is able to mimic the ability of the notochord for inducing different ventral spinal cord cell types. In response to Shh signaling, neural progenitors activate or suppress the expression of several homeobox and basic helixloop-helix (bHLH) transcription factors. The combinatorial expression of these transcription

Although Shh has been shown to be sufficient for the induction of distinct ventral spinal cord neurons, recent studies have suggested an alternative derepression mechanism whereby many neuronal cell types could be generated in the absence of Shh signaling. For example, although most of the ventral cell types are absent from Shh or Smo mutants because of the ectopic production of Gli3 repressor, most of the ventral neurons are generated in Shh; Gli3 and Smo;Gli3 double mutants [45,46]. Similarly, in embryos that lack all Gli transcription factors and cannot respond to Hh signaling, many of the ventral neurons are also present [47, 48]. These results suggest that perhaps the primary role of Hh signaling is to repress excess Gli3 repressor in the ventral spinal cord so that progenitors can respond to other signals. An additional role for Shh signaling is to organize the formation of distinct progenitor domains, as different cell types intermix in the absence of Shh signaling

The mouse mutation, hennin (hnn), causes coupled defects in cilia structure and Sonic hedgehog (Shh) signaling. Gli3 repressor activity is normal in hnn embryos, but Gli activators are constitutively active at low levels. The hnn phenotypes are whole e10.5 wildtype (A) and hnn (B and C) embryos, which show exencephaly and spina bifida. As a result, IFT mutants display a loss of Hh signaling phenotype in the neural tube, where Gli activators play the major role in pattern formation, and a gain of Hh signaling phenotype in the limb, where Gli3 repressor plays the major role. Because both anterograde and retrograde IFT are essential for positive and negative responses to Hh, and because cilia are present on Hh responsive cells, it is likely that cilia act as organelles that are required for all

During development of the central nervous system, *Shh secreted* in the prechordal plate, notochord and floor plate, disseminates and establishes a concentration gradient across the dorsal ventral axis of the neural tube, with maximum levels at the ventral side, forming an appropriate dorsoventral pattern of cell differentiation. Finally, mediated by the three Gli transcription factors´ combined activity, Shh, in the neural tube, not only controls the specification but also regulates cell proliferation and survival through cell cycle regulation

**6.1.2 Hh and central nervous system development** 

factors defines the fate of the neural progenitor cells [44].

activity of the mouse Hh pathway [50].

d4) [41].

[49].

The 1st step in the Hh signaling process is the physical union with Ptc, activating the pathway and producing internalization by endocytosis of the Hh-Ptc complex and its subsequent lysosomal degradation. This eliminates the extracellular morphogen and sets the concentration gradient.

 The transduction system involves Smoothened (Smo), a transmembrane protein. The Hh to Ptc union begins its internalization and releases Smo that, from intracellular vesicles, shifts and accumulates on the cell surface where it is translocated to the cilia, initiating the signaling cascade that culminates in the modulation of the transcription factors Ci / Gli (Gli protein 1, 2 and 3) [33, 34]

#### **6.1 Embryonic development**

The Hedgehog (Hh) pathway determines growth patterns and embryonic differentiation in a large number of organs. Approximately half of all mutations affect the initial establishment of the body plan, and several of these produce phenotypes that have not been described previously. A large fraction of the genes identified affect cell migration, cellular organization, and cell structure. Findings indicate that phenotype-based genetic screens provide a direct and unbiased method for identifying essential regulators of mammalian development [35]. The mutant cilia are short, with a specific defect in the structure of the ciliary axoneme [36].


#### **6.1.1 Hh and limb development**

Cilia are present in the forebrain neurectoderm and in ectodermal and mesenchymal cells of the limb, but are aberrant or absent from these tissues in IFT mutants [33, 41, 42]. The zone of polarizing activity (ZPA), a small cluster of mesodermal cells located at the rear edge of the developing limb, is responsible for the anteroposterior pattern of the limb by secreting Shh, the morphogen controlling the number and identity of the fingers plus antero-posterior specification. The normal expression of Shh in chicken wing ZPA is accompanied by its diffusion gradient model scheme along the anteroposterior axis. Normal skeletal pattern of the chicken wing consists of 3 digits known as 2 (d2), 3 (d3) and 4 (d4).

Formation of finger-type depends on morphogen levels to which precursor cells were subjected during development. When another source of Shh is introduced at previous levels, either naturally or through experimental means, two gradients are generated in the outline of the limb which, depending on their geometry, may overlap with each other in the central area of the limb. This configuration leads to mirror duplications of the digits (d4-d3-d2-d3 d4) [41].

#### **6.1.2 Hh and central nervous system development**

130 Neuroimaging for Clinicians – Combining Research and Practice

The 1st step in the Hh signaling process is the physical union with Ptc, activating the pathway and producing internalization by endocytosis of the Hh-Ptc complex and its subsequent lysosomal degradation. This eliminates the extracellular morphogen and sets the

 The transduction system involves Smoothened (Smo), a transmembrane protein. The Hh to Ptc union begins its internalization and releases Smo that, from intracellular vesicles, shifts and accumulates on the cell surface where it is translocated to the cilia, initiating the signaling cascade that culminates in the modulation of the transcription

The Hedgehog (Hh) pathway determines growth patterns and embryonic differentiation in a large number of organs. Approximately half of all mutations affect the initial establishment of the body plan, and several of these produce phenotypes that have not been described previously. A large fraction of the genes identified affect cell migration, cellular organization, and cell structure. Findings indicate that phenotype-based genetic screens provide a direct and unbiased method for identifying essential regulators of mammalian development [35]. The mutant cilia are short, with a specific defect in the structure of the

 The Hh signaling pathway is essential in embryogenesis due to its involvement in the development of multiple organs where it controls such important aspects as specification of different kinds of cells, cell proliferation and cell survival (apoptosis

 The ligand Shh (Sonic hh) is responsible for the functions of the best characterized development signaling centers such as the notochord and the zone of polarizing activity

 The morphogen concentration provides cells with *positional value* commensurate with gradient position, determining cell destiny, generating an appropriate pattern or morphology, and foreshadowing the final organ or tissue pattern. Hh proteins act as morphogens in the development of the central nervous system, limbs, heart, blood vessels, gonads, intestine and kidney. In vertebrates, cilia also function in the Shhdependent patterning of the developing neural tube and limb [38]. Mutant mice with defects in ciliogenesis resulting from mutations in IFT protein-encoding genes, such as polaris, wimple (Ift172), Ngd5 (Ift52) and the gene encoding the retrograde motor Dnchc2, have neural tube defects and preaxial polydactyly phenotypes similar to

Cilia are present in the forebrain neurectoderm and in ectodermal and mesenchymal cells of the limb, but are aberrant or absent from these tissues in IFT mutants [33, 41, 42]. The zone of polarizing activity (ZPA), a small cluster of mesodermal cells located at the rear edge of the developing limb, is responsible for the anteroposterior pattern of the limb by secreting Shh, the morphogen controlling the number and identity of the fingers plus antero-posterior specification. The normal expression of Shh in chicken wing ZPA is accompanied by its diffusion gradient model scheme along the anteroposterior axis. Normal skeletal pattern of

mutants with defects in Shh pathway proteins [33, 39 a 42].

the chicken wing consists of 3 digits known as 2 (d2), 3 (d3) and 4 (d4).

concentration gradient.

**6.1 Embryonic development** 

ciliary axoneme [36].

control).

(ZPA) of the tip [37].

**6.1.1 Hh and limb development** 

factors Ci / Gli (Gli protein 1, 2 and 3) [33, 34]

In the developing mammalian ventral spinal cord, five different neuronal cell types, comprising the motoneuron (MN) and four different interneurons (V0-V3), are generated from their respective neural progenitors (pMN, pV0-pV3) under the influence of signals from the notochord [43]. Shh, one of the Hedgehog (Hh) family of proteins, is strongly expressed in the notochord and later in the floor plate, and is able to mimic the ability of the notochord for inducing different ventral spinal cord cell types. In response to Shh signaling, neural progenitors activate or suppress the expression of several homeobox and basic helixloop-helix (bHLH) transcription factors. The combinatorial expression of these transcription factors defines the fate of the neural progenitor cells [44].

Although Shh has been shown to be sufficient for the induction of distinct ventral spinal cord neurons, recent studies have suggested an alternative derepression mechanism whereby many neuronal cell types could be generated in the absence of Shh signaling. For example, although most of the ventral cell types are absent from Shh or Smo mutants because of the ectopic production of Gli3 repressor, most of the ventral neurons are generated in Shh; Gli3 and Smo;Gli3 double mutants [45,46]. Similarly, in embryos that lack all Gli transcription factors and cannot respond to Hh signaling, many of the ventral neurons are also present [47, 48]. These results suggest that perhaps the primary role of Hh signaling is to repress excess Gli3 repressor in the ventral spinal cord so that progenitors can respond to other signals. An additional role for Shh signaling is to organize the formation of distinct progenitor domains, as different cell types intermix in the absence of Shh signaling [49].

The mouse mutation, hennin (hnn), causes coupled defects in cilia structure and Sonic hedgehog (Shh) signaling. Gli3 repressor activity is normal in hnn embryos, but Gli activators are constitutively active at low levels. The hnn phenotypes are whole e10.5 wildtype (A) and hnn (B and C) embryos, which show exencephaly and spina bifida. As a result, IFT mutants display a loss of Hh signaling phenotype in the neural tube, where Gli activators play the major role in pattern formation, and a gain of Hh signaling phenotype in the limb, where Gli3 repressor plays the major role. Because both anterograde and retrograde IFT are essential for positive and negative responses to Hh, and because cilia are present on Hh responsive cells, it is likely that cilia act as organelles that are required for all activity of the mouse Hh pathway [50].

During development of the central nervous system, *Shh secreted* in the prechordal plate, notochord and floor plate, disseminates and establishes a concentration gradient across the dorsal ventral axis of the neural tube, with maximum levels at the ventral side, forming an appropriate dorsoventral pattern of cell differentiation. Finally, mediated by the three Gli transcription factors´ combined activity, Shh, in the neural tube, not only controls the specification but also regulates cell proliferation and survival through cell cycle regulation

Ciliopathies: Primary Cilia and Signaling Pathways in Mammalian Development 133

Recently, many vertebrate-specific components have been identified that act between the GOS and Gli. These include intraflagellar transport proteins which link vertebrate Hh signaling to cilia. Because abnormal Hh signaling can cause birth defects and cancer, these vertebrate-specific components may play a role in human health. Hh signaling has been involved in development of several human cancers including small cell lung carcinoma, medulloblastoma, basal cell carcinomas, digestive tract (pancreas) tumors, brain, prostate,

PTC has been considered a tumor suppressor gene: Ptc inhibits the signaling pathway, so

Many oncogenic factors may converge on Gli activity to promote tumor progression, thus pointing towards the Hh signaling cascade as a phenomenon to be considered in potential treatments for many different types of cancer. For example, Gorlin syndrome or nevoid basal cell carcinoma syndrome is caused by a disorder of chromosome 9 (q22.3 and q31) and 1 (p32), leading to mutations in the PTCH tumor suppressor gene, a human homologue of *Drosophila melanogaster patched* gene. The PTCH gene encodes the signal's Sonic Hedgehog (Shh) transmembrane protein receptor, a regulatory molecule in embryogenesis and

There are 3 known pathways, activated after docking of Wnt to its "Frizzle" family receptors

It acts through β-catenina/Arm and specifies different cell types, controlling proliferation and apoptosis depending on the development context. It is activated in virtually all tumors. Induces stabilization and accumulation in the cytoplasm of ß-catenin and its subsequent translocation to the cell nucleus where it affects transcription of target genes [2, 3, 70, 71]. a. When the Wnt signal is not activated, ß-catenin binds to the degradation complex consisting of APC axin and the serine/threonine kinases CK1 and GSK3. The degradation complex´s main role is to phosphorylate ß-catenin, guiding it to its degradation through addition of multiple ubiquitins and its enzymatic process in the proteasome. There are several negative regulators operating at the ligand receptor-like level, such as Cer1, DKK, and sFRP, whose function is to modulate Wnt-induced

b. Wnt contact with its receptors leads to stabilization of ß-catenin and its accumulation in the cytoplasm. ß-catenin displaces Groucho transcriptional repression from the complex formed by LEF/TCF, leading to activation of target genes such as C-MYC and

c. *Canonical Wnt signaling is involved in the* generation of different cell types in the ventral spinal cord: The identity of distinct cell types in the ventral neural tube is generally believed to be specified by sonic hedgehog (Shh) in a concentration-dependent manner. However, recent studies have questioned whether Shh is the sole signaling molecule determining the fate of ventral neuronal cells. There is evidence showing that canonical Wnt signaling is involved in the generation of different cell types in the ventral spinal

CCND1, which are involved in cell cycle progression and proliferation.

**6.3 Oncology and carcinogenicity** 

the loss of activity results in activation of the Shh pathway.

skin, etc. [62 a 67].

carcinogenesis [68].

**7. Wnt Signaling pathways** 

**7.1 Canonical wnt pathway** 

positive signals.

(FZD) (*See Gerdes JM y Katsanis N. 2008* review[69]):

and expression of antiapoptotic genes. Moreover, the neocortex, cerebellum and tectum growth also depend on the mitogenic activity of Shh [51 a 55]. Recently, it has been attributed a role in axonal growth [56], and in maintaining stem cell niches in the adult.

#### **6.2 Stem cell proliferation and differentiation: neurogenesis**

In adults, the Hh signal is involved in maintaining stem cell and tissue homeostasis. In neurogenesis, the brain's mature CM receives signals to divide and differentiate into neurons. The primary cilium plays a key role in the CM receiving orders to divide by the uptake of cell growth factors through the route called "Sonic hedgehog".

#### Degenerative defects:

Neural stem cells (NSCs) are present in the mammalian brain from embryo to adult. It has been shown that these cells are a significant source of new neurons, and promise to be the origin of a new central nervous system restorative therapy: Hedgehog (Hh) signaling is involved in a wide range of important biological activities. Within the vertebrate central nervous system, Sonic Hedgehog (Shh) can act as a morphogen or mitogen that regulates the patterning, proliferation, and survival of neural stem cells (NSCs). However, its role in embryonic stem cell (ESC) neurogenesis has not been explored in detail. Hh signaling is required for ESC neurogenesis; in order to elucidate the underlying mechanism, Cai C. et al [57] utilized the Sox1-GFP ESC line, which has a green fluorescent protein (GFP) reporter under the control of the Sox1 gene promoter, providing an easy means of detecting NSCs in live cell culture. That ESC differentiation in adherent culture follows the ESC--> primitive ectoderm --> neurectoderm transitions observed in vivo. Selective death of the Sox1-GFPnegative cells contributes to the enrichment of Sox1-GFP-positive NSCs. Interestingly, Shh is expressed exclusively by the NSCs themselves and elicits distinct downstream gene expression in Sox1-GFP-positive and -negative cells. Suppression of Hh signaling by antagonist treatment leads to different responses from these two populations as well: increased apoptosis in Sox1- GFP-positive NSCs and decreased proliferation in Sox1-GFP-negative primitive ectoderm cells. Hedgehog agonist treatment, in contrast, inhibits apoptosis and promotes proliferation of Sox1-GFP-positive NSCs. These results suggest that Hh acts as a mitogen and survival factor during early ESC neurogenesis, and evidence is presented to support a novel autocrine mechanism for Hh-mediated effects on NSC survival and proliferation.

An intravenous Hh agonist at doses that upregulate spinal cord Gli1 transcription also increases the population of neural precursor cells after spinal cord injury in adult rats. These data support previous findings based on injections of Shh protein directly into the spinal cord [58].

A common feature of embryonic and adult NSCs is that they have a primary cilium emerging from a mother centriole, and through its receptors Sonic Hedgehog (Shh) is involved in cell specification and neurogenesis with neural progenitors´ expansion in brain development [59]. Shh is active in some groups of cells in mature organs which seem to be involved in maintaining stem cell numbers.

In mammalian telencephalon, two postnatal neurogenesis areas are known: the hippocampus dentate gyrus and the telencephalic ventricles´ subventricular zone. Quiescent cells express low levels of Gli1, a marker of responding Shh cells, but the Shh/Gli pathway is activated to regulate the generation of new neurons [60]. The Shh signaling cascade is also involved in the maintenance of other types of adult stem cells such as hematopoietic cells [61].
