Use of Xenopus laevis Neural Crest Cells to study the role of PDGF-B in gliomas tumor progression

University of Pisa

Research Doctorate in Biology

Cycle XXVIII

(2012-2016)

Use of Xenopus laevis Neural Crest

Cells to study the role of PDGF-B in

gliomas tumor progression

Supervisor: prof. Michela Ori

Candidate: Kety Giannetti

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TABLE OF CONTENTS

ABSTRACT 5

1.INTRODUCTION 7

1. Platelet Derived Growth Factor (PDGF) 8

1.1 PDGF family members 8

1.2 PDGF roles during embryogenesis 12

1.2.1 PDGF roles in Central Nervous System (CNS) development 16

1.3 PDGF and cancer 18

1.3.1 PDGF-B in gliomagenesis 19

2. Neural Crest Cells (NCC) 23

2.1 Cell adhesion properties of NCC 26

2.2 Wnt/PCP pathway roles in NCC 31

2.3 Chemotaxis signals and NCC migration 33

2.4 Cell sorting and NCC streams segregation 37

3. Xenopus laevis as a model system 38

2. MATHERIALS AND METHODS 41

1. Embryos handling 42

2. Embryos microinjection and manipulation 42

2.1 Capped mRNAs 43

2.1.1 Constructs used for micro-injection:

linearization and capped mRNA transcription 44

2.2 Morpholino antisense oligonucleotide 44

3. RNA Extraction, cDNA Synthesis, PCR and qPCR 45

3.1 Primers used for PCR or for qPCR gene expression analysis 46

4. Whole-mount In situ hybridization (WISH) 47

4.1 Synthesis of antisense RNA probes 47

4.1.1 Constructs used for gene expression analysis:

linearization and RNA transcription to generate antisense RNA probes 48

5. Neural Crest Cells explants 48

6. in vivo chemotaxis assay 49

7. Western blot 50

3. RESULTS 51

1. pdgf-b expression during Xenopus laevis embryonic development 52 2. PDGF-B over-expression affects Xenopus laevis Neural Crest Cells behavior 57

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2.1 PDGF-B changes NCC cell-adhesion properties 61

2.1.1 R-CAD over-expression partially resemble PDGF-B effect on NCC 63 2.2 PDGF-B alters the chemoattraction signals that rule NCC migration 65 2.3 NCC cell sorting signals are affected by PDGF-B over-expression 69

2.4 PDGF-B possibly cross-talk with the wnt/PCP pathway 71

3. Down-regulation of pdgf-b in Xenopus embryos affects NCC migration 76

4. DISCUSSION 80

1. PDGF-B over-expression affects Xenopus laevis NCC molecular asset 81 2. pdgf-b expression pattern and function during Xenopus embryonic development 86

5. CONCLUSIONS 89

6. REFERENCES 92

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Gliomas are the most common primary tumours of the central nervous system; one of their major features is the ability of individual cells to infiltrate the brain parenchyma, which renders these tumours also the most aggressive. Evidences gained from studies on animal models have firmly established a causal connection between aberrant PDGF-B signalling and the formation of some gliomas. The aim of my project is to unravel the mechanisms underlying PDGF-B driven gliomagenesis and tumour progression, especially focusing on alteration of motility and migration. For this purpose, I used the animal model Xenopus laevis and in particular I focused on Neural Crest Cells (NCC), considering the many similarities shared by this population with cancer cells.

I demonstrated that over-expression of human PDGF-B in Xenopus embryos, obtained by mRNA microinjection, can alter NCC behaviour in terms of migration and streams segregation, validating the system. I then analysed, in PDGF-B injected embryos, the expression of some of the key players known to regulate NCC development such as: Cadherins, Chemokines and their receptors, Neuropilins and Semaphorins and Wnt/PCP pathway members. The results obtained highlighted a significant down-regulation of

neuropilin-2 and of wnt11, a Wnt/PCP pathway member, suggesting for the first time a

link between their expression and the PDGF signalling. The discovery of these new putative targets of the PDGF-B signalling pathway opens new possible lines of investigations aimed to identify alternative therapeutic approaches for glioma treatment. Interestingly, I also evidenced that the gene expression pattern of Xenopus pdgf-b during embryogenesis relies in domains close to NCC, suggesting a possible physiological role for it in NCC development. In this regard, I deeper characterised the spatial and temporal expression profile of Pdgf-b during Xenopus embryogenesis and I performed loss of function experiments that showed how NCC migration is affected after pdgf-b down-regulation. Furthermore, with an in vivo chemotaxis assay, I observed that PDGF-BB can act as a chemoattractant for NCC in vivo, suggesting a putative chemotactic role for it and strengthening the hypothesis that this growth factor might play a physiological role in NCC migration.

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1. Platelet Derived Growth Factor (PDGF)

1.1 PDGF family members

PlateletDerived Growth Factor (PDGF) family comprises four ligands (PDGFA, B, C, and -D), that form disulfide-linked homodimers or the heterodimer AB, and two tyrosine kinase receptors (PDGFR-α and -β) (Fig. 1.1) (Demoulin and Essaghir, 2014). The active ligand-receptor complex consists of two receptor chains associated with one dimeric ligand; while PDGFR-α binds to all PDGF isoforms except for PDGF-DD, PDGFR-β binds only to PDGF-BB and –DD; also an heterodimeric αβ receptor has been reported that binds to PDGF-AB, -BB and possibly -CC and –DD (Demoulin and Essaghir, 2014).

Figure 1.1: Platelet Derived Growth Factor (PDGF) family members and their interactions. The four ligands

A, B, C and D (in dark blue, light blue, violet and dark brown respectively) and the two tyrosine kinase receptors α and β (light brown and dark orange respectively) are shown; ligands need to dimerize in order to be active, after binding to the ligand, receptors dimerize. Arrows indicate interactions, dashed arrows indicate hypothetical interactions.

Adapted from (Giannetti et al., 2016).

All PDGF ligands, schematic represented in Fig. 1.2, contain in their C-term portion “PDGF/VEGF domains” essential for the dimerization, as they bear 8 conserved Cysteine

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residues which form intra- and inter- molecular disulfide bonds, but the rest of the molecule can be very different, indeed this group is further classified into 2 subfamilies: one comprising PDGF –A and –B and the other comprising -C and –D (Hoch and Soriano, 2003). The most relevant difference is the exclusive presence of a CUB domain in the N-term portion of PDGF –C and –D, which must be cleaved extracellularly in order for the dimer to be active; on the other hand, PDGF –A and –B do not contain this domain and their N-term pro-domain is cleaved intracellularly, so the dimer is already active when it is secreted from the cell.

Figure 1.2: PDGF ligands structures. Different domains of PDGF ligands are represented as indicated in the

legend at the bottom: PDGF-domain (dashed rectangle), retention motif (light blue), CUB domain (light pink). Al PDGF ligands have a cleavage site, indicated as a red line.

Adapted from (Hoch and Soriano, 2003).

Although PDGF –A and –B dimers are released as active forms, their biological activity can be affected by interfering with their spatial distribution; this modulation is possible thanks to the short retention motif at the very C-term of these ligands, which can be cleaved either by alternative splicing (only for PDGF-A) or by alternative processing (only for PDGF-B) or by Matrix- Metallo-Proteases (MMP) from the Extra-Cellular Matrix (ECM) (Fig. 1.3) (Andrae et al., 2008). The absence or presence of the retention motif affects their affinity to ECM components and subsequently their distribution along it.

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Figure 1.3: Processing of PDGF dimers. Different forms of active dimers and their processing. Intracellular

space is divided from extracellular space by a yellow line, ligands are represented as curved shapes with the following colors: PDGF –A (light blue), -B (purple), -C or -D (dark blue). Retention motif of either PDGF –A or –B is shown in green, while CUB domain of PDGF-C and -D is shown in light brown.

From (Andrae et al., 2008).

PDGF receptors α and β, differently from their ligands, do not have significant structural differences, indeed they are both composed by 5 immunoglobulin-like domains at the N-term portion, displayed at the extracellular side, and by 2 split Tyrosine kinase domains at the C-term, displayed intracellularly; after the ligand is bound to the extra-cellular portion, a conformational change triggers the trans-phosphorylation of Tyr residues within the cytoplasmic portion. After the phosphorylation event, the two receptors are then subjected to the docking of different adaptor proteins, which in turns is responsible for the divergences in the signalling pathways triggered downstream of each of them (Demoulin and Essaghir, 2014). A representative scheme illustrating the

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intracellular modulators and the signalling pathways activated by PDGFs, that are mainly: PI3 kinase, RAC/JNK, PKC & calcium and MAP kinase, are shown in Fig. 1.4.

Figure 1.4: PDGF Signalling pathways and receptors docking proteins. Different domains of the proteins

are represented with specific shapes/colors enlisted on the left. Ligand-receptor couple is on the center-top, a light brown horizontal line defines the cellular boundary; after binding, phosphorylation occurs at specific sites (marked with red circles and line) which are recognized by selective docking proteins. The main signalling pathways activated are: PI3 kinase, RAC/JNK, PKC &calcium, MAP kinase.

Adapted from (Demoulin and Essaghir, 2014).

From their first identification in the 70s, the members of the PDGF family have been extensively studied in embryogenesis, development, adult homeostasis and disease (Andrae et al., 2008, Heldin, 2013, Heldin, 2014, Hoch and Soriano, 2003). For the purposes of this thesis, exclusively the roles of PDGF in embryonic development and in cancer progression will be articulated in the following paragraphs.

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1.2 PDGF roles during embryogenesis

Most of the studies aimed to elucidate the roles of PDGF signalling during embryonic development were performed in transgenic mice models and limited functional studies were also performed in Xenopus; the results of these studies are nicely reviewed in (Andrae et al., 2008, Hoch and Soriano, 2003). More recently, functional studies have been extended to the zebrafish model by several groups (Eberhart et al., 2008, McCarthy et al., 2016, Wiens et al., 2010), supporting and implementing the information already available, while for Xenopus only a few new information were provided.

Regarding the very early phases of embryonic development, it was shown in Xenopus that

pdgf-a and pdgfr-α are essential for gastrulation (Ataliotis et al., 1995), during which pdgf-a controls mesoderm cells orientation and migration (Damm and Winklbauer, 2011),

while no appearance of a similar role in mouse and zebrafish was observed. Anyhow, an essential role during the formation of Neural Crest Cells (NCC) derived structures was observed for pdgfr-α both in mouse and in zebrafish, in fact mice pdgfr-α null or with NCC-conditional loss of Pdgfr-α (Fig. 1.5) and zebrafish hypomorphic pdgfr-α mutants (Fig. 1.6) exhibit cleft face and palate (Eberhart et al., 2008, Tallquist and Soriano, 2003).

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Figure 1.5: External appearance of NCC conditional Pdgfr-α mice mutants. (A-B) Frontal and ventral view of

a neonate NCC conditional Pdgfr-α mutant mouse, the genotype is indicated on the top. (C) pdgfr-α null embryo Embryonic Stage 14.5 (E14.5) and (D) NCC conditional pdgfr-α mutant mouse embryo E13.5. Arrowheads indicate blebs.

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Figure 1.6: pdgfr-α down-regulation leads to defects of palate formation in zebrafish embryos. (A-C)

zebrafish embryos 48 hours post fertilization (hpf), live imaging of (A) an Un-Injected Control embryo (UIC), an Mirn140 injected embryo and (C) an hypomorphic pdgfr-α mutant embryo; Mirn140 is a MicroRNA that targets pdgfr-α mRNA and blocks its translation, arrowheads point to cranial hemorrhaging. (D-F) zebrafish embryos 6 days post fertilization (dpf), same order as for A-C; arrowheads point to hypoplastic upper lip.

(G-L) alcian staining/alizarin staining of 6dpf zebrafish embryos, (G) UIC embryo, (H) Mirn140 injected embryo

with mild phenotype, (I) hypomorphic pdgfr-α mutant embryo with mild phenotype, (J) Mirn140 mismatched injected embryo, (K) Mirn140 injected embryo with severe phenotype, (L) hypomorphic pdgfr-α mutant embryo with severe phenotype; ep stands for ethmoid plate, tr stands for trabeculae. Mirn140 mismatched is a microRNA with mutations that block the binding to its target.

Adapted from (Eberhart et al., 2008).

The phenotypes described for either pdgfr-α null mice or NCC-conditional pdgfr-α knock out mice were not observed for Pdgf-a (Bostrom et al., 1996) or Pdgf-b (Leveen et al., 1994) or Pdgfr-β (Soriano, 1994) or Pdgf-d (Gladh et al., 2016) null mice, while for Pdgf-c null mice (Ding et al., 2004) similar palate formation defects were observed, suggesting a possible role for this ligand in palate structures formation and, consequently, in NCC development. In comparison with Pdgf-c null mice, it was noted that combined Pdgf-a and Pdgf-c ko mutants phenotypes correlate better with Pdgfr-α ko phenotypes, considering this and that Pdgf-a and Pdgf-b double mutants do not result in any of the phenotypes associated with Pdgfr-α mutations, it was hypothesized that Pdgf-a and Pdgf-c are the principal ligands for Pdgfr-α in mice during NCC development (Ding et al., 2004);

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nevertheless it must be noted that Pdgf-a null mice did not show cleft palate or face and that the effects of combined loss of Pdgf-c and Pdgf-b are yet not known, therefore this conclusion might be not correct. The same authors also pointed out that loss of function of Pdgfr-β leads to completely different phenotypes, suggesting that this receptor is not involved in NCC development, however, more recently, a genetically interaction between the two receptors was found during NCC development in mice and zebrafish (McCarthy et

al., 2016); anyway the characterization of Pdgfr-β expression pattern in mouse at these

stages is still lacking.

Although it is clear that NCC development is affected when pdgfr-α is down-regulated or ablated, in both mouse and zebrafish embryos it is not established yet if this is due to a migration defect rather than a differentiation aberration. Nevertheless, PDGFs are known mesenchymal cells chemoattractant (Andrae et al., 2008) and in the zebrafish embryo, pdgf-aa was shown to be capable of attracting NCC in vivo (Eberhart et al., 2008), so it is reasonable to assume that PDGF signalling might be directly involved in NCC migration, with PDGF ligands being chemoattractant for them.

Little is known about pdgf-b and pdgfr-β roles during early stages of embryonic development, in fact abrogation of both of them shows that although they are not crucial for mouse gastrulation, vascular development is drastically affected (Fig. 1.7), with mice dying perinatally for extensive hemorrhages (Hellstrom et al., 1999, Leveen et al., 1994, Soriano, 1994); this phenomenon was addressed to Pdgf-b being able to act as a chemoattractant for mural cells, which express Pdgfr-β and are recruited from endothelial cells to the neo-forming vessels (Hellstrom et al., 1999).

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Figure 1.7: Pdgf-b knock out mice embryos. (A) mutant dead embryo E18.5, (B-C) live mutant embryo E19,

arrows indicate subcutaneous purpura, (D) live normal embryo E19. Adapted from (Leveen et al., 1994).

Unfortunately, there are no available data about pdgf-b and pdgfr-β abrogation in

Xenopus, while for zebrafish a recent study demonstrated that pdgfr-β is essential for

intersegmental vessel formation (Wiens et al., 2010).

All the vertebrates animal models analyzed, express pdgfr-α in NCC, however up to date it is still not fully understood the precise role of PDGF signalling in NCC development and which ligands are involved, therefore further work as well as new insights from other animal models could be useful.

Despite gastrulation and NCC development, PDGFs have many functions also during organogenesis, but this thesis will not debate on that except for a special attention on the role of PDGFs during Central Nervous System (CNS) development, which is treated in the following paragraph.

1.2.1 PDGF roles in Central Nervous System (CNS) development

The role of PGDFs during Central Nervous System (CNS) development regards glial cells development with the specific involvement of Pdgfr-α and Pdgf-a and was investigated only in the mouse. Around E12, Oligodendrocyte Precursors (OPs) arise as bilateral rows of cells in the periventricular zone of the murine neuroepithelium; among them a

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subpopulation of Pdgfr-α expressing cells, which are bipotential glial cells progenitors giving rise to oligodendrocytes and type-2 astrocytes (O2-A), proliferate and migrate to fill the neural tube/spinal cord (Calver et al., 1998). At this stage, Pdgf-a is expressed by neurons and astrocytes throughout the spinal cord while Pdgf-c is expressed in the floor plate and ventral horn regions (Fig. 1.8) (Aase et al., 2002, Calver et al., 1998, Hoch and Soriano, 2003). The importance of Pdgf-a in O2-A development is evident since in pdgf-a null mice mutant pups show a reduction in the number of O2-A cells present at birth, followed by a parallel reduction in the number of oligodendrocytes and the amount of myelin that is deposited postnatally (Fruttiger et al., 1999). On the other hand, it was recently showed that there is no correlation between O2-A cells and Pdgf-c expression, suggesting that this growth factor does not play a role in oligodendrocytes development (Andrae et al., 2016).

Figure 1.8: Oligodendrocyte Precursors development and PDGF signalling in the mouse embryo. Scheme

of Pdgf-a and Pdgf-c expression and the spatial distribution of O2-A cells. Adapted from (Hoch and Soriano, 2003).

The other members of PDGF family are currently not known to be involved in the development of CNS, however Pdgf-b and Pdgfr-β are known to be expressed by post-natal neurons and are thought to be involved in neuroprotection mechanisms since the ablation of Pdgfr-β leads to exacerbated brain damages following certain brain injuries (Enge et al., 2003, Ishii et al., 2006). Nevertheless, since the over-expression of Pdgf-B in immature neural progenitors leads to the generation of the same kind of tumors, an

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instructive role such as inducing oligodendroglial fate was suggested (Calzolari and Malatesta, 2010), it remains to be determined whether this function is played during CNS development or not.

1.3 PDGF and cancer

The importance of PDGFs in cancer biology was first brought up in 1983, when the v-sis gene of the transforming Simian Sarcoma Virus (SSV) was identified as a retroviral copy of the PDGF-B gene (Doolittle et al., 1983, Waterfield et al., 1983); during the last four decades many efforts have been made about unraveling the roles beard by PDGF family members in cancer biology, however the situation is highly complicated and much is yet to be understood. Alterations of PDGF pathway have been observed in several cancer types such as breast cancer, melanoma, colorectal cancer, hepatocellular cancer, ovarian serous carcinoma, prostate cancer, lung adenocarcinoma, lung squamous cell cancer, renal cancer, acute myeloid leukemia, glioblastoma multiforme and low grade glioma (Farooqi and Siddik, 2015); the highest frequencies of alteration in PDGF genes have been observed in studies about melanoma (up to 30%), lung cancer (up to 20%) and glioblastoma (up to 20%) (Farooqi and Siddik, 2015). PDGF members expression can be also a prognostic biomarker, for instance positive immunostaining of PDGFs are adversative prognostic factors in patients with advanced breast cancer (Seymour and Bezwoda, 1994) and the expression of PDGFRα in ovarian cancer is associated with poor prognosis (Henriksen et al., 1993).

A very interesting aspect of PDGF relevance in cancer biology is the fact that the over-expression of a structurally and functionally normal Pdgf-b encoding gene can cause high-grade brain tumors, identified as an homogeneous class of oligodendrogliomas (Calzolari and Malatesta, 2010); given the widely accepted concept that malignant tumors are caused by multiple genetic events and display a diversity of phenotypic aberrations (Westermark, 2014), this apparently opposing phenomenon is of high interest for cancer

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biology researchers. It is also notable that despite the existence of several PDGF receptor kinase activity inhibitors, their capacity of treating human cancers have been proved on several types with the notable exception of brain tumors (Demoulin and Essaghir, 2014). In this scenario, it is easy to understand the importance of underlying the mechanisms involved in PDGF-B driven tumorigenesis and tumor progression.

1.3.1 PDGF-B in gliomagenesis

Despite the fact the PDGF signalling is altered in a wide range of human tumors (Farooqi and Siddik, 2015), a special attention have been paid for gliomas thanks to the observation made by Uhrbom and colleagues in 1998 (Uhrbom et al., 1998) about Pdgf-b over-expression being capable of transforming in vivo mouse proliferating neural cells, with the final result of obtaining high grade gliomas. Gliomas are the most aggressive tumors originating in the CNS, they are currently divided on the base of histopathology and cell type-specific features in several groups including: Astrocytic tumors, oligodendroglial tumors, oligoastrocytic tumors and ependymal tumors; another classification is made according to the malignancy of tumors: a grade is assigned from I (low malignancy) to IV (high malignancy), and the highest grade of malignancy was observed only for astrocytic and oligodendroglial tumors (Calzolari and Malatesta, 2010). The latter two kinds of gliomas are considered to be almost incurable for their poor accessibility to surgical and pharmacological treatment as well as for their highly infiltrative nature, which renders their complete surgical removal almost impossible (Calzolari and Malatesta, 2010). Since the prognosis is very poor in the vast majority of these kinds of gliomas cases, the necessity of finding new therapeutic targets and strategies is highly relevant. Glioma mouse models can be obtained simply by over-expressing Pdgf-b in mouse neural cells, which is usually achieved by in vivo transduction of mouse neural cells using replication incompetent retroviral vectors bearing Pdgf-b gene (Fig. 1.9); depending on the time point at when transduction is performed, either on

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neonates (Uhrbom et al., 1998) or on adults (Assanah et al., 2006) or on embryos (Appolloni et al., 2009), different results were obtained. Indeed, only when transduction is performed on embryos, high-grade pure oligondendrogliomas are obtained (Appolloni et

al., 2009), while for neonates and adult injections, although high grade gliomas are

obtained, contamination of other cell types were observed (Assanah et al., 2006, Uhrbom

et al., 1998).

Figure 1.9: Murine models of Pdgf-b induced glioma. The scheme shows the time point at when

transduction is performed (A- neonate, B- Adult and C- embryo) and the different outcomes. Adapted from (Calzolari and Malatesta, 2010).

This difference is highly relevant since it suggests a putative instructive role for Pdgf-b in PDGF responsive neural progenitors, in fact, as mentioned in the former paragraph, Pdgfr-α positive embryonic neural progenitors can give rise to many cell types, but the

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administration of Pdfg-b implies the selective transformation of these cells in oligodendrogliomas (Appolloni et al., 2009).

The work of this thesis is based on a collaboration with prof. Malatesta at University of Genoa, his group is currently involved in the study of the Pdgf-b driven mouse glioma model mentioned above and described in (Appolloni et al., 2009), therefore a special attention will be given to that model in this dissertation. The transduction of murine immature neural progenitor cells, as shown in fig 1.9C, is achieved by in utero injection of retroviral vectors carrying the Pdgf-b gene in E14 embryos, specifically within the lateral telencephalic ventricles. The retroviral vector used is a replication incompetent vector based on a Moloney Murine Leukemia Virus (MoMLV)- derived retroviral vector and containing an IRES sequence between the mouse Pdgf-b gene and the GFP sequence; the advantage brought by the usage of such a vector is that since transduced cells will simultaneously express Pdgf-b and GFP, it is possible to trace transduced cells thanks to GFP and to later on distinguish tumor cells from recruited cells. Transduced cells display tumorigenic features as they proliferate over confluence and infiltrate the brain parenchyma, bypassing cell–cell contact inhibition of both proliferation (CIP) and migration (CIM). The molecular analyses made on mice brains of new born puppies till 6 months of life, performed both via immunostaining (Fig. 1.10) and via microarray analyses on cultured cells derived from 3 different tumors, highlighted that the population of transformed cells did not show expression of neuronal (NeuN) or astroglial (GFAP) markers, while they showed expression of oligodendroglial markers (NG2, Olig2, Pdgfr-α) along with proliferative (Ki67) and stem cells (Nestin) markers, suggesting that these cells belong to a very coherent group of tumors and show unambiguous traits of oligodendroglial identity.

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Figure 1.10: Immunostaining on sections from Pdgf-b induced mice brain tumors. Each frame indicates a

different staining for the antigen indicated in red (bottom left): (A) neuronal marker NeuN, (B) astroglial marker Glial Fibrillary Acidic Protein (GFAP), (C) proliferative marker Ki67, (D) stem cell marker Nestin, (E-H) oligodendroglial markers (E) Neural/Glial antigen 2 (Ng2), (F) Oligodendrocyte transcription factor (Olig2), (G) Pdgfr-α and (H) mature oligodendrocyte marker Adenomatous Polyposis Coli (APC). All sections where stained with anti-GFP antibody (in green) to reveal transduced cells and with DAPI (in blue) to highlight nuclei. Adapted from (Appolloni et al., 2009).

Another important observation made by prof. Malatesta group is that GFP negative cells are present within the tumor mass, but when tumor GFP positive and GFP negative cells were separated and re-injected in adult mice brain, only GFP positive cells were capable of developing oligodendroglioma. Moreover they also showed that the tumorigenic potential of these cells increases with time: only late-onset tumor derived cells were capable to induce a secondary tumor in an adult mouse brain when re-injected, while cells derived from early-onset tumors were not capable of inducing secondary tumors (Calzolari et al., 2008). This indicates a phenomenon called oncogene addiction: the oncogenic activity of these cells is strictly related to the expression of the oncogene (Pdgf-b), and the tumorigenic potential is instructed with time by the oncogene in an autocrine way (Calzolari et al., 2008).

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2. Neural Crest Cells (NCC)

The Neural Crest (NC) is an embryonic highly migratory multipotent cell population induced within the Neural Plate Border (NPB), at the interface between the neuroepithelium and the prospective epidermis of a developing embryo (Mayor and Theveneau, 2013); while NC cells (NCC) can be distinguished morphologically from their neural tube sister cells only as they exit the neural tube, the molecular events of NC patterning start earlier during gastrulation (Pegoraro and Monsoro-Burq, 2013). NC induction is the result of a precise combination of BMP, Wnt, FGF, Retinoic Acid and Notch signals produced by the ectoderm, the neuroepithelium and the underlying mesoderm (Mayor and Theveneau, 2013), in a time window ranging from gastrulation through neurulation as illustrated in Fig 1.11, that shows Xenopus NCC development. NCC induction begins when a broad progenitor domain named the “neural border” is specified between neural and non-neural ectoderm during gastrulation (step 1, fig 1.11), then NC-specific markers are activated in a subset of neural border cells which in turn induce NC specifiers that are responsible for the further development of NCC, including Epithelial-to-Mesenchymal Transition (EMT) inducers and initiation of migration signals (step 2 and 3, fig 1.11); as organogenesis proceeds, NCC extensively migrate along defined routes, proliferate, and populate their target tissues and organs (step 4 fig. 1.11), where they stop and differentiate (step 5, Figure 1.11) (Pegoraro and Monsoro-Burq, 2013).

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Figure 1.11: Main steps of Neural Crest Cells (NCC) development in Xenopus laevis embryos. The steps of

NC formation are temporally sorted during gastrulation, neurulation, and organogenesis. For each step, a characteristic marker expression pattern is shown from pictures of Whole Mount In Situ Hybridization (WISH) on Xenopus embryos (from left to right): pax3 anterior view of a stage 14 embryo), snail2 (dorsal-anterior view of a stage 18 embryo), sox10 (dorsal-(dorsal-anterior view of a stage 22 embryo), melanocytes (lateral view of a tadpole embryo, stage 31-33).

Adapted from (Pegoraro and Monsoro-Burq, 2013).

After migration, NCC can differentiate into an astonishing plethora of derivatives depending on the domain of origin (fig. 1.12); there are four main functional (but overlapping) domains (Pegoraro and Monsoro-Burq, 2013):

 Cranial (cephalic) NCC, which arise from the diencephalon to the third somite, produce the craniofacial mesenchyme that differentiates into the cartilage, bone, cranial neurons, glia and connective tissues of the face; these cells also enter the pharyngeal arches and pouches to give rise to thymic cells, odontoblasts of the tooth primordia and the bones of middle ear and jaw (Mayor and Theveneau, 2013, Shyamala et al., 2015).

 Trunk NCC, which arise caudal to the fourth somite, either become the pigment-synthesizing melanocytes or migrate ventro-laterally to each sclerotome and

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form the dorsal root ganglia containing the sensory neurons (Mayor and Theveneau, 2013, Shyamala et al., 2015).

 Enteric NCC, which delaminate from the neural tube facing somites 1 to 7, generate the parasympathetic (enteric) ganglia of the gut (Mayor and Theveneau, 2013, Shyamala et al., 2015).

 Cardiac NCC, a subset of cranial NCC, produce the entire musculo-connective tissue wall of the large arteries as they arise from the heart, as well as contribute to the septum that separates the pulmonary circulation from the aorta (Shyamala

et al., 2015).

Figure 1.12: NCC domains spatial localization in the developing embryo. This scheme is obtained

combining result coming from 4 different animal models illustrated on top left: Xenopus laevis, chicken, mouse and Zebrafish. Color legend is indicated on the bottom left, to be noted that dashed lines corresponding to the different domains are drawn on the top of the schematic embryo. Adapted from (Mayor and Theveneau, 2013).

Despite the differences on differentiating cell types among the domains, no matter which domain they belong, the event that triggers NCC delamination from the territory of induction and migration is the EMT. This event permits the passage between an epithelial cell type, where cells establish close contact with their neighbors and a tightly regulated

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apico-basal polarity, to a mesenchymal cell type, were cells are loosely organized in the three dimensional space and therefore have a higher motility; such a conversion involves profound phenotypic changes including loss of cell-cell adhesion, loss of cell polarity and acquisition of infiltrative and invasive properties (Thiery et al., 2009). EMT is also known to be a crucial event in acquisition of malignancy during tumor progression and was shown to occur in metastatic primary epithelial tumors including breast, colon, ovarian, endometrial and pancreatic cancer or in non-epithelial tumors such as melanoma and sarcoma; in fact malignant cells mimic many of the behavioral, molecular and morphologic aspects of NCC development, aiming to achieve survival, growth and invasion goals, which are shared characteristic of both cell types (Maguire et al., 2015). In this scenario is then comprehensible why NCC can represent a good model for cancer cells and how it is relevant to shed light on mechanisms involved in NCC migration in order to contribute on understanding tumor progression events.

The molecular modulators of NCC development are various and interconnected, they can be divided into 4 groups depending on the functions and on the belonging signalling in: cell adhesion modulators, Wnt/PCP pathway members, chemotaxis signals and cell sorting signals; the role of molecules from the same group can differ depending on the NCC domain examined, therefore the following paragraphs will discuss individually the functions of each one of them focusing on cranial NCC, those selected to be examined for this project.

2.1 Cell adhesion properties of NCC

Cell adhesion is a process mediated by molecules of the cell surface by which cells interact and attach to a surface, substrate or another cell; it occurs from the action of transmembrane glycoproteins, called cell adhesion molecules (CAMs) which are classified into five principal classes: cadherins, immunoglobulins, selectins, mucins and integrins (Lodish H, 2000). The integrins mediate cell-matrix interactions whereas the other types

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of CAMs participate in cell-cell adhesion; cell-cell adhesion involving cadherins and selectins depends on Calcium (Ca2+) ions, whereas interactions involving integrin and

Ig-superfamily CAMs do not (Lodish H, 2000). Cadherins, a family of Ca2+_{-dependent CAMs,} are the major molecules of cell-cell adhesion and play crucial roles in NCC development, particularly during EMT, migration and aggregation of cranial ganglia; data coming from several animal models indicate that, among them, cadherin-2 (cdh-2 or N-cad), cadherin-6 (cdh-6) and cadherin-11 (cdh-11) play major roles at different time points of cranial NCC development (McKeown et al., 2013).

 cdh-2 (n-cad) function in NCC has been studied mostly in Xenopus; it was shown that both its down- and up- regulation leads to NCC migration inhibition (Fig. 1.13G-J) (Theveneau et al., 2010), a phenomenon that was explained by the postulation of a model in which n-cad mediated cell-cell adhesion causes contact-dependent cell polarity (Theveneau et al., 2010), which in turned is involved in contact inhibition of locomotion (CIL), an event that was previously proved to be involved in guidance of NCC migration (Carmona-Fontaine et al., 2008). More recently this hypothesis was supported by the founding that an e- to n-cadherin switch controls CIL acquisition upon EMT, in other words: e-cad usually suppresses CIL while n-cad, which is not expressed in premigratory NCC while it is then expressed by migrating NCC (Fig. 1.13A-F), promotes CIL, thus upon EMT promotion of the e- to n- cadherin switch, initiation of NCC migration is triggered (Fig. 1.13L) (Scarpa et al., 2015).

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Figure 1.13: n-cad expression and modulation in Xenopus NCC. (A-F) n-cad expression in Xenopus embryos

at (A-C) a premigratory stage and (D-F) a migratory stage analyzed via (A,D) WISH and (B,C,E,F) immunostaining on sections (green signal); dashed lines delimitate cranial NCC territory, b stands for branchial streams, h stands for hyoid stream, NT stands for neural tube, NC stands for neural crest. (G-J) effects of n-cad gene expression modulation on NCC, analyzed via WISH monitoring NCC marker twist expression: (G-H) injection of Morpholino for knock-down of n-cad, first frame is control side while second frame is injected side, (I-J) injection of mRNA for over-expression of N-cad, first frame is control side while second frame is injected side; red asterisks indicate the eye. (L) Schematic representation of the proposed e- to n- cadherin switch involved in CIL.

Adapted from (A-J) (Theveneau et al., 2010) and (L) (Scarpa et al., 2015).

A recent work, though, suggests that e-cadherin is constantly expressed during cranial NCC migration thanks to qPCR and immunostaining on endogenous protein analyses (Huang et al., 2016), contradicting the postulated cadherin switch by (Scarpa et al., 2015). This recent finding re-opens the debate on how cadherins expression and function is modulated in migrating Xenopus NCC.

 Cadherin-6 (cdh-6) or cadherin-6B, in chicken embryos is expressed in the dorsal neural tube prior to neural crest migration, but is then repressed by the

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transcription factor snail2, expressed by pre-migratory and early migrating cranial neural crest cells; functional studies revealed that knock-down of cdh-6B leads to premature NCC migration (Fig. 1.14), whereas cdh-6B overexpression inhibits migration (Coles et al., 2007).

Figure 1.14: Knock-down of cdh-6 results in increased NCC migration in chick embryo. (A-F)

electroporation of control morpholino (MO) compared to (H-M) electroporation of cdh-6B MO. (A-M) WISH detecing (A,B,H,I) sox10 or (C,D,J,K) snail2 or (E,L) foxD3 or (F,M) rhoB; picture of (A,D,H,K) whole embryo or

(B,C,E,F,I,J,L,M) transverse sections; in all panels, the electroporated side is on the right, as indicated by the

inset or overlaid lissamine (red) fluorescence staining showing cells that have received the MO, arrows identify an increased migration when compared to the contralateral control side.

Adapted from (Coles et al., 2007).

Further support to these observations was provided by the very recent demonstration that cdh-6 proteolysis in chicken embryos not only promotes delamination, but also provides transcriptional regulatory feedback into the NC gene regulatory network leading to the up-regulation of pro-EMT genes including β-catenin, CyclinD1, and most notably snail2 (Schiffmacher et al., 2016). On the other hand, in Zebrafish, cdh-6 over-expression was observed to promote apical detachment of hindbrain pre-migratory NCCs, an important early step in EMT, thus promoting NCC migration (Clay and Halloran, 2014). Integrating this information with data from other animal models might help in clarifying the role of cdh-6 in

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NCC development and to understand if this role is conserved among certain species or not.

 Cadherin-11 (cdh-11) function in NCC was studied in Xenopus; similarly to the results obtained for n-cad, both cdh-11 down-regulation and over-expression lead to inhibition of NCC migration, although for inhibition the effect is more visible for the hyoid and branchial streams (Kashef et al., 2009), while all streams are equally affected when over-expression is performed (Borchers et al., 2001). This is apparently not in agreement with the observations that reduction of cdh-11 adhesive functions leads to augmented invasive properties of NCC by affection of CIL sensitivity (Becker et al., 2013) and that cdh-11 cleavage by ADAM13 promotes NCC migration (Abbruzzese et al., 2016). Actually, one must consider the fact that NCC migration is a precise orchestrated event and the improvement of invasive properties does not necessarily correlate whit an improved migration, moreover cadherins-mediated signalling is affected by their proteolytic processing, therefore down-regulation and cleavage are very likely to give different results.

Although the studies mentioned above lead to no straight conclusions on what is the role of a specific cadherin in NCC development across species, also considering the poor available set of information concerning the topic, their relevance in NCC is undoubtable. In this project an additional cadherin was considered named cadherin-4 (cdh-4 or r-cad); although there are currently no evidence of a role for this cadherin in NCC development and considering that it is not expressed in Xenopus at the stages when NCC form and migrate, the observation made from prof. Malatesta group about an N- to R- cadherin switch underlying malignancy in the Pdgf-b driven mouse glioma model (Appolloni et al., 2014) prompted us to investigate a putative modulation of r-cad expression by PDGF-B. The results of this investigation will be shown in the “Results” chapter.

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2.2 Wnt/PCP pathway roles in NCC

“Wingless-related integration site” proteins (Wnts) are evolutionary conserved secreted Cys-rich proteins that couple to various receptors and thereby activate different downstream pathways; on the basis of early studies, these pathways have been classified as either canonical (β‑catenin-dependent) or noncanonical (β‑catenin-independent) signalling pathways (Niehrs, 2012). However, this classification is not strict, in fact within canonical and noncanonical Wnt signalling, various sub-branches are used in different cellular contexts (Niehrs, 2012). The Wnt family is highly various, in humans there are at least 19 genes encoding for different WNTs (Kikuchi et al., 2011) and the downstream signalling variety is further enhanced by the fact that not only there are more than 15 among receptors and co-receptors, but also the activity of a specific Wnt depends both on the cellular context and on the receptor, and thus Wnts cannot be rigorously subdivided according to the pathway they induce (Niehrs, 2012). However, a useful approximation that Wnt1, Wnt3a and Wnt8 are more commonly encountered in canonical Wnt signalling, while Wnt5a and Wnt11 are predominantly involved in noncanonical Wnt signalling can be done; furthermore, Frizzled (Fz) proteins act as common receptors for both the canonical and noncanonical pathways, and the use of LRP5 and LRP6 or ROR1 and ROR2 determines canonical and noncanonical pathways, respectively (Niehrs, 2012). The key cytoplasmic partner transducing all major Wnt subpathways is the scaffold protein Dishevelled (Dsh), representing an almost universal molecule downstream of Wnt receptors (Niehrs, 2012).

The best-characterized noncanonical pathway is the Wnt/PCP pathway: it involves Fzs to activate a cascade for modulation of the small GTPases Rac1 and RhoA and JUN‑N‑terminal kinase (JNK) (Kikuchi et al., 2011), leading in turn to changes in the cytoskeleton and cell polarity (Niehrs, 2012). The Wnt/PCP pathway is prominently involved in regulating cell polarity in morphogenetic processes, for instance, in vertebrates it regulates cell movement during gastrulation and neural tube closure

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(Niehrs, 2012). Wnt/PCP and canonical Wnt signalling are well known to antagonize each other, and inhibiting one will typically upregulate the other (Niehrs, 2012).

In all vertebrate models examined, canonical Wnt pathway was proved to be essential for NCC specification (Wu et al., 2003), while for Wnt/PCP pathway the situation is less clear. The role of Wnt/PCP pathway in NCC has been studied in Xenopus by several groups from the first observation that injection of dsh mutated forms, which inhibit specifically the Wnt/PCP pathway, leads to affection of NCC migration but has no effects on specification (De Calisto et al., 2005); these findings were supported by the observation that wnt11 inhibition, a noncanonical wnt, gives the same NCC phenotypic alterations (Matthews et

al., 2008). Wnt/PCP pathway in NCC migration was also studied at the cellular level

proving that it interferes with Contact Inhibition of Locomotion (CIL) (Carmona-Fontaine

et al., 2008). CIL is a mechanism that controls polarized localization of RhoGTPases

depending on cell contact and it was proposed as an important factor in controlling NCC directional migration by (Carmona-Fontaine et al., 2008). In order for a cell to move directionally, it needs to be polarized; active protrusions, such as lamellipodia, filopodia, blebs and invadopodia, are usually formed at the front of the cell, whereas a retraction area is formed at the back (Mayor and Theveneau, 2014). On the basis of in vitro studies, it was observed that this front–back polarity is controlled by the localized polymerization of actin, which in turn is usually regulated by the Rho smallGTPases and membrane phospholipids (Ridley, 2011). Upon collision between two neural crest cells, different PCP components become localized at the cell–cell contact site, leading to a localized inhibition of Rac and activation of Rho, which in turn results in establishment of a new cell-polarity (Carmona-Fontaine et al., 2008, Mayor and Theveneau, 2014). The PCP components involved in CIL have been proposed to be wnt11 and fz7 (Carmona-Fontaine et al., 2008) and the fact that the actin binding protein calponin 2 (cnn2), a downstream effector of Rho GTPases, impairs NCC migration when inhibited and act downstream of wnt11 (Ulmer et al., 2013), support this idea (Figure 1.15).

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Figure 1.15: Schematic representation of Wnt/PCP pathway involvement in CIL of migrating NCC. (A)

Collision between two neural crest cells. RhoA (in purple) and Rac1 (in red) activities are initially polarized, upon cell–cell interaction, polarization is lost and at the region of contact RhoA is activated comporting establishment of a new cell-polarity. (B) Wnt/PCP signalling cascade in NCC involves activation of RhoA/ROCK signalling. Rho/ROCK inhibits Rac1 and calponin 2 (cnn2) and promotes actomyosin contractility favoring both the collapse of cell protrusions and retraction of the cell body away from the contact.

Adapted from (Mayor and Theveneau, 2014).

Even though a more crucial role in NCC migration was hypothesized for Wnt/PCP pathway, results are controversial and more recently, involvement of Wnt/PCP pathway in NCC specification was also observed in Xenopus (Ossipova and Sokol, 2011). Very recent data coming from Xenopus suggest that Canonical Wnt activity needs to be tightly controlled to enable NC migration (Maj et al., 2016), an observation in line with the known antagonism between the two pathways. However the available information is not sufficient to clearly define the border between canonical and noncanonical Wnt associated functions. Nonetheless it is more than clear that Wnt/PCP components are major players of NCC migration regulation.

2.3 Chemotaxis signals and NCC migration

Chemotaxis is a fundamental process by which cells migrate directionally when they are exposed to external chemical gradients; it is exhibited by a wide variety of cell types and involves distinct strategies that depend on the environmental conditions (Bagorda and

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Parent, 2008). Eukaryotic chemotaxis is thought to be a key component in a multitude of biological processes and sensing the gradient results in directed cell motion in the form of crawling (Rappel and Loomis, 2009). The first event that occurs during chemotaxis is the binding of a chemoattractant molecule to its specific receptor, which triggers major cytoskeleton rearrangements by promoting F-actin polymerization at the front and actomyosin assembly at the back of cells; to migrate directionally in a chemo-attractant gradient, these events must occur in a coordinated fashion (Bagorda and Parent, 2008). These cytoskeleton rearrangements, mediated by members of the Rho family small G proteins, ultimately promote leading-edge formation at the cell front and retraction at the cell back (Bagorda and Parent, 2008).

In the cephalic region of an embryo, environmental signals force delaminating NCC to assemble into three main subpopulations called streams, which migrate into well-defined pathways (Minoux and Rijli, 2010); the control of migration, despite this segregation, is also played by positive guidance cues that attract NCC along their paths (Mayor and Theveneau, 2013). Among the molecules which are already known to act as chemoattractant for other cell types, from studies performed in Xenopus, the chemochine cxcl-12, also known as stromal cell-derived factor-1 (sdf-1), was observed to have a chemotactic function for NCC. sdf-1, originally identified as a secreted stromal factor, is a chemokine produced by many cell types; it is involved in chemotaxis and migration of leukocytes and it was shown to participate in many developmental processes such as hematopoiesis, angiogenesis, neurogenesis and heart morphogenesis (Nagasawa, 2014). During Xenopus embryonic development, sdf-1 is expressed in the ectoderm next to NCC during pre-migratory stages, then in between migratory streams and ventrally facing NCC at migratory stages (Theveneau et al., 2010); its receptor cxcr-4, at the same stages, is expressed by NCC (Moepps et al., 2000). These expression patterns match with the observation that down-regulation of either sdf-1 or cxcr-4 impairs NCC migration (Fig. 1.16), and also with the discovery that NCC are collectively attracted toward the chemokine sdf-1 thorough cxcr-4, with sdf-1 being involved in stabilization of cell

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protrusions promoted by cell-cell contact rather than initial polarization of cells (Theveneau et al., 2010); the model proposed is shown in Fig. 1.16.

Figure 1.16: sdf-1/cxcr-4 role in Xenopus NCC migration. (J,K,N-Q) WISH on injected embryos revealing

complement 3 (C3) expression, a NCC marker: (J,K) sdf-1 knock-down performed with Morpholino injection,

(N-Q) cxcr-4 knock down performed by injecting either (N-O) a dominant negative form or (P-Q) Morpholino;

frame on the top of each couple is control side, frame on th bottom is injected side, red asterisks indicate the eye. (R) schematic representation of sdf-1 role as guidance cue for NCC migration: stabilization of cell protrusions (big black arrow) cooperates with other events such as CIL (the mechanism regulating RhoA/Rac1 polarity and activity, shown in shades from light blue to red). (S) Schematic representation of NCC collective migration: while the group moves along the path established by surrounding repulsive signals (purple lateral stripes), cell-cell connection loosen and finally cells start to migrate singularly, big green arrow represent chemotactic attractive signal from sdf-1.

Adapted from (Theveneau et al., 2010).

Another interesting chemoattractant for NCC uncovered in Xenopus is complement component 3 (c3), acting through the c3a-receptor (c3ar) (Carmona-Fontaine et al., 2011); c3 is a central component of the complement pathway, it is cleaved to produce C3a, a small anaphylatoxin peptide with known chemotactic properties in the immune system (Ricklin et al., 2010). Its role in NCC, despite its function as a chemoattractant, is not directly related to the guidance though an established path but rather on another aspect involved in regulation of cephalic NCC migration: mutual cell attraction. In fact, the migration of NCC is thought to have an initial phase where NCC migrate as a cohesive group, then only later on they detach from each other and start to migrate individually (Theveneau et al., 2010). After the observation that modulation of c3 and c3ar affects NCC migration, in line with the expression pattern of these molecules that both localize

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in migrating NCC (McLin et al., 2008), cellular analyses demonstrated that they are responsible for mutual attraction of NCC, proposing the following model: while CIL leads to cell dispersion, on the other hand co attraction (CoA) keeps the cells together, therefore a permanent cycle between CIL and CoA is required for collective migration (Fig. 1.17) (Carmona-Fontaine et al., 2011).

Figure 1.17: c3 and c3ar inhibition affects NCC migration in Xenopus. (M-N) pictures of (M) a stage 15

embryo treated with a bead soaked with an antibody against C3a and (N) stage 25 embryos, from left to right: a control embryo treated with an unsoaked bead and two embryos treated with a bead soaked with an antibody against C3a, showing a mild effect or a strong effect. (P-Q) pictures of (P) a stage 15 embryo treated with a bead soaked with morpholino against c3aR and (Q) stage 25 embryos, from left to right: a control embryo and two embryos treated with a bead soaked with morpholino against c3aR showing a mild effect or a strong effect. All the photographed embryos underwent WISH revealing either snail2 or twist, black arrow or asterisk indicate the place where the bead was placed, black and white arrowheads indicate NCC migratory streams. (H) scheme explaining the relationship between contact inhibition of locomotion (CIL) and mutual cell attraction (CoA).

Adapted from (Carmona-Fontaine et al., 2011).

The information about the chemotactic cues which guide NCC are not abundant also as a consequence of the difficulties derived from such an investigation, however new possible players can be suggested by evaluating their expression patterns along with information available about their roles in other cell types,. In this regard PDGF was already suggested to play such a role; even though in the mouse PDGFs have been proposed as differentiation instructors rather than migration regulators (Hoch and Soriano, 2003), it is

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possible that different systems utilize the same signal for different purposes. Further investigation could be helpful in shedding light on the matter.

2.4 Cell sorting and NCC streams segregation

Cranial NCCs follow stereotypical migratory pathways that are conserved among vertebrate species; maintaining the spatial segregation of such streams has an important impact on craniofacial pattern (Minoux and Rijli, 2010) and is regulated by cell sorting signals, which mediate either cell repulsion or attraction. In this regard a major role is played by repulsive signals transmitted by three couples of receptor/ligand: Neuropilins/Semaphorins, Eph receptors/ephrins and Robo/Slit (Maguire et al., 2015). Since this thesis will focus only on Neuropilins, only the Neuropilins/Semaphorins couple will be discussed.

Neuropilins (Nrp-1 and Nrp-2) are transmembrane receptors binding to the guidance molecules class 3 semaphorin (Sema3) and VEGF; Nrp-1 preferentially interact with Sema3A while Nrp-2 prefers Sema3F (Lumb et al., 2014). In the mouse embryo, neuropilins are expressed in a non-overlapping pattern recently characterized: Nrp-1 is expressed by cranial NCC migrating out of rhombomere 4, Nrp-2 is expressed by cranial NCC migrating out of rhombomere 2 (Lumb et al., 2014). The importance of Neuropilins in cranial NCC streams segregation was suggested from the fact that Nrp-2 or Sema3F knock out mice show abnormal cranial neural crest migration, with bridges of migrating cells crossing between the neural crest streams that enter branchial arches 1 and 2, and trigeminal ganglia formation failure (Gammill et al., 2007). Similar affections of cranial NCC migration and derivatives formation were also observed for Nrp-1/Sema3A down-regulation in the mouse embryo (Schwarz et al., 2008). About Xenopus, there are no information on the function of neuropilins in NCC development, however the expression patterns of nrp-1/sema3A and nrp-2/sema3F were published by (Koestner et al., 2008),

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showing that they are complementary and compatible with a possible role in NCC segmentation (Fig. 1.17).

Figure 1.17: schematic representation of neuropilins and semaphorins expression patterns in Xenopus tailbud embryos. (A) semaphorin3A (in light blue) and neuropilin-1 (in red), (B) semaphorin3F (in light blue)

and neuropilin-2 (in red); mh stands for midbrain-hindbrain boundary, sc stands for spinal cord, s stands for somite, r3 stands for rhombomere 3 and r5 stands for rhombomere 5.

Adapted from (Koestner et al., 2008).

Since this thesis will debate on a study of Xenopus NCC behavior in respond to PDGF-B over-expressiong, after having illustrated the available information regarding NCC development key players, in the next paragraph the characteristics of Xenopus as an animal model will be finally considered.

3. Xenopus laevis as a model system

Choosing a suitable animal model system to unveil new molecular mechanisms underlying PDGF-B over-expression effects was one of the most important step for achieving meaningful results. Xenopus is a genus of aquatic frogs, native to southern Africa, that are tolerant to starvation, diseases and other insults that make them very easy to keep in captivity (Ori et al., 2013). Xenopus as a tetrapod shares a long

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evolutionary history with mammals: they share similarities at the genome level, in organ development, anatomy and physiology and therefore represents an excellent model to predict human biology (Ori et al., 2013). Thus, since the 1950s Xenopus laevis has become a very widely used research animal model for a variety of biological and biomedical researches with unique versatility (Hardwick and Philpott, 2015). In particular, Xenopus is one of the most popular system used in the field of developmental biology since it offers experimental advantages over other widely used model systems such as mice; these advantages come from the large number and large size of the eggs, which can be easily fertilized in vitro producing huge numbers of synchronized embryos with fast external development: the embryo can be directly observed from the very first cell division and all stages of development have been classified and described in details (Nieuwkoop, 1967). Moreover the embryos can be easily manipulated, injected, grafted or labeled and in particular, researchers can easily manipulate gene expression simply by directly micro-injecting into the eggs either Morpholino-modified antisense oligonucleotides (for knock down) or mRNA (for gain of function). Since the first cleavage already establishes the left-right axis of the embryo, by injecting only one blastomere the modulation of gene expression will be performed in only one side of the embryo, which can be distinguished from the non-injected (N.I.) one thanks to the use of tracers such as LacZ or GFP, so in the same embryo there will be also a control N.I. side that allows more reliable evaluation of the phenotypes obtained (Fig. 1.18). In addition, the availability of a well-defined fate map of each blastomere allows addressing the microinjection in specific regions of the embryo so that lethal effects can be avoided and the study of defects that appear at later stages of development is therefore possible (Ori et al., 2013).

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Figure 1.18: Tracers used for micro-injection in Xenopus embryos: LacZ and GFP. From left to right: a stage

2 (2 cells) embryo is injected in only one blastomere (needle with red solution on the top right), the same embryo is reared to stage 18 then fixed and stained to reveal β-galactosidase activity (enzyme product of the gene LacZ), another possibility is to use a live tracer such as the Green Fluorescent Protein (GFP): before fixation, green fluorescence is observed in correspondence of the injected side of a stage 18 embryo.

Adapted from (Ori et al., 2013).

The bright side of using a classical developmental biology model in the field of cancer biology research derives from the parallels between early development and tumorigenesis: signalling pathways that are critical during embryonic development can be instrumental in tumor progression and high cell proliferation rates are seen mostly only in developmental and cancer cells; indeed cancer can perhaps be viewed as an inappropriate re-activation of normal embryonic pathways or as disorder of cellular-differentiation (Hardwick and Philpott, 2015). With these perspectives, my project, in collaboration with prof. Malatesta at University of Genoa, was developed with the aim of using an alternative and advantageous model that could bring valuable insights to boost up the research around PDGF-B induced gliomagenesis; the final goal of the entire research is then to provide new putative targets for glioma treatment, a still poorly successful branch of medicine.

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1. Embryos handling

In order to obtain Xenopus laevis embryos, each adult female used was injected with 800-1000 units of Human Chorionic Gonadotropin (HCG, Pregnyl, 5000 U.I., Organon) 12-14 hours prior to eggs collection. Ovulated eggs were fertilized with testis homogenate and let to develop in 0.1X Marc’s Modified Ringer’s Solution (MMR 1X: 0.1 M NaCl, 2 mM KCl, 1 mM MgCl2, 5 mM HEPES pH 7.5). Jelly coats were removed by using a dithiothreitol (DTT) containing dejelling solution (3.2mM DTT in in Tris pH 8.8, 0.2 M).

2. Embryos microinjection and manipulation

For knock-down experiments, a morpholino antisense oligonucleotide was used (Mo, Gene Tools, LLC) while for over-expression experiments, in vitro transcribed capped mRNAs were injected; in both cases, in vitro transcribed capped mRNAs of lineage tracers were co-injected.

Morpholinos or capped mRNAs were injected into one blastomere of two- four- or eight cells stage embryos. Microinjections were performed using a “Femtoject" apparatus (Eppendorf) on embryos kept in 0.1X MMR and 3% Ficoll-400 (GE Healthcare) and cultured overnight (ON) at 14° C in the same solution. Embryos were subsequently transferred in 0,1X MMR and incubated at 14°C until the desired developmental stage. Embryos were staged according to Nieuwkoop and Faber (1967).

When n-β-gal mRNA (LacZ) was used as a lineage tracer in microinjection experiments, β -gal staining was detected by fixing embryos in 1X MEMFA (MEMFA salts 10X: MOPS pH 7.4 100 mM, EGTA 2 mM, MgSO₄ 1 mM, Formaldehyde 3.7%) for 30 min at Room Temperature (RT), washing in 1X PBS pH 7.3 and then incubating inβ-gal staining solution (Potassium Ferricyanide K₃Fe(CN)₆ 5mM, Potassium Ferrocyanide K₄Fe(CN)₆ 5mM,

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MgCl₂ 2mM and Red-gal substrate 1 mg/ml in 1X PBS) for 20- 30 min at 37°C. Embryos were then fixed again in 1X MEMFA for 1 hour at RT, washed twice in 1X PBS and then dehydrated with pure ethanol to be processed by whole mount in situ hybridization. When animal cap explants were analyzed, embryos were reared in 0.1X MMR till stage 8, then transferred in 0.7X MMR with Gentamycin where the animal cap region was excited using forceps after removal of the vitelline membrane (fig. 2.1). Explants were then kept at RT in 0.7X MMR till wound healed then kept at 14°C in 0.1X MMR till sibling embryos reached the desired stage.

Figure 2.1: schematic representation of animal cap explant. On the left, a stage 2 embryo is injected within

both blastomere with GFP (green circles) as a live tracers. On the right, the same embryo at stage 8 is used for animal cap explant, the green area represent the region to be cutted out with the forceps (scissors in this drawing).

2.1 Capped mRNAs

Capped mRNAs were synthesized in vitro from the template cDNAs using the SP6 mMESSAGE mMACHINE Kit (Ambion). After DNase digestion to remove template cDNA, transcripts were purified with 0.1 volume of Ammonium Acetate to avoid embryos gastrulation defects, extracted with 1 volume of phenol/chloroform, Precipitate with 2 volume of 100% ethanol, resuspended in water at the 500 ng/μl concentration and stored at -20°C. Concentration and length of transcripts were estimated by agarose gel electrophoresis.

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2.1.1 Constructs used for micro-injection: linearization and capped mRNA transcription

- n-β-gal or LacZ (NotI/Sp6), containing the nuclear-β-galactosidase reporter inserted into the pCS2+ vector (Chitnis et al,. 1995).

- pCMT-EGFP (NotI/Sp6), containing the Enhanced Green Fluorescent Protein reporter gene inserted into the pCS2+ vector (a gift from D. Gilmour).

- PDGFB (Acc65I/Sp6), inserted into the pCS2+ vector. - RCAD (NotI/Sp6), inserted into the pCS2+ vector.

- pRN3P-dshGFP (SfiI/T3), fusion protein of disheveled (dsh) with GFP at the C-term. - pXT7- fz8 (XbaI/T7) containing frizzled 8.

- PDGFR-β (KpnI/Sp6) inserted into the pCS2+ vector. - pXT7- ΔNfz8 (XbaI/T7).

- pSP64T- wnt5a (XbaI/Sp6)

2.2 Morpholino antisense oligonucleotide

For knockdown experiments, a splice-blocking Morpholino was purchased from

Gene

Tools, LLC

gene as illustrated in the following scheme (Fig. 2.2); spliceMO int1EX2 sequence: 5’- GATCTCCCTGCCAAAAACACACAGA-3’

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Figure 2.2: annealing of splice-blocking Morpholino used fo pdgf-b down-regulation. Gene sequence of

pdgf-b corresponding to intron1-exon 2 (highlighted by grey rectangle)-intron2. Morpholino targeting sequence is highlighted by red rectangle.

3. RNA Extraction, cDNA Synthesis, PCR and qPCR

Total RNA was extracted from 10 embryos and isolated using either TRIZOL (Invitrogen) or “Nucleospin® RNA” (Macherey-Nagel) according to manufacturer’s instructions. cDNA was synthesized using either “iScript™ cDNA Synthesis Kit” (BIO-RAD) according to manufacturer’s instructions. cDNAs were then used for PCR or quantitative Real-Time PCR (qPCR).

For PCR, DreamTaq Green PCR Master Mix (ThermoFisher) was used according to manifacturer’s instruction, 5μl of cDNA out of 1:5 diluted 20μl total amount was used for each reaction. All PCRs were conducted at 95°C for 4 min and then 35 cycles for: 95°C for 1 min, 55°C for 1:30 min and 72°C for 1 min with and additional final step at 72°C for 4 min.

For qPCR, to quantify gene expression levels, equal amounts of cDNA (32 ng) were mixed with either “GoTaq® qPCR Master Mix” (Promega) or” iQ™ SYBR® Green Supermix” (Bio-Rad) 1X, primers 0,25μM. odc or gapdh were amplified as an internal control. All qPCRs were conducted at 50°C for 5 min, and then 45 cycles for: 95°C for 15s, 60°C for 20 s and 72°C for 40 s. The specificity of the reaction was verified by melt curve analysis. The threshold crossing value was noted for each transcript and normalized to the internal