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INTRODUCTION
1. Serotonin
1.1. Discovery of serotonin
Serotonin history begins in the early 1930’s with Vittorio Erspamer working in the Institute of Comparative Anatomy and Physiology, University of Pavia in Italy. Dr Erspamer was interested in the smooth muscle constricting or contracting properties of various amine substances found in pharmacologically active molecules extracted from animals. One of these substances, which was capable of causing smooth muscle contraction, especially in the uterus of rats, was found in the enterochromaffin cells of the gut. He named this substance enteramine(Erspamer, 1937). In the late 1940’s Irvine Page, who was interested in the etiology and treatment of hypertension at the Cleveland Clinic, found the presence of endogenous constricting factors in blood serum during his studies. Dr. Page had the foresight to recruit a highly skilled organic chemist, Maurice Rapport, and an equally skilled and distinguished
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biochemist, Arda Green. Page, Rapport, and Green succeeded in isolating and characterizing the serum substance and named it serotonin(Rapport, 1948). In May 1949, the structure of serotonin was determined to be 5- hydroxytryptamine(Rapport, 1949). Around 1952 it was realized that enteramine and serotonin were the same substance, and in the same year Dr. Betty Mack Twarog found serotonin in mammalian brain (Twarog BM, 1953) at the Page Lab and this brought serotonin into the field of neuroscience(Azmitia, 1999).
Nowadays the role of serotonin is recognized in the adult organism and its function during embryonic development are being gradually revealed.
1.2. Biosynthesis and metabolism of
serotonin
Serotonin (5-hydroxytryptamine, 5-HT) is a monoamine synthesized from L-tryptophan in two steps (Fig. 1.1): 1. Hydroxylation of L- tryptophan in 5-hydroxytryptophan (5-HTP) is catalyzed by tryptophan hydroxylase (Tph), which is the rate limiting enzyme of the synthesis. This enzyme requires for its activity the presence of tetrahydrobiopterine (BH4), oxygen (O2), NADPH2 and iron (Fe2+).
2. Decarboxylation of 5-HTP in 5-HT is catalyzed by L-aromatic amino acid decarboxylase (Addc) with pyridoxal-phosphate as coenzyme.
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Serotonin is converted into inactive molecules by oxydative deamination of the lateral amino chain by monoamine oxidase (Mao), leading to indol-acetaldehyde which is then oxidized into 5-hydroxy-indol-acetic acid (5-HIAA) found in urines.
Fig.1.1 5-HT Synthesis Pathway
The enzyme tryptophan hydroxylase (Tph) belongs to a superfamily of aromatic amino acid hydroxylases, with phenylalanine and tyrosine hydroxylases. Tph activity is encoded by two genes (Tph1 and Tph2) that are expressed in different organs and cells and modulated post-transcriptionally by phosphorylation(Ehret et al., 1991; Hasegawa and Ichiyama, 1987; Makita et al., 1990; Walther et al., 2003; Zhang et al., 2004).
Tph1, the non-neuronal Tph isoform, is expressed in the
pineal gland and in the enterochromaffin cells of the gut. In the pineal gland, 5-HT represents an intermediate product for melatonin synthesis having the serotonin N-acetyltransferase (AANAT) as the rate-limiting enzyme.
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Melatonin has been primarily implicated in the circadian rhythmicity of physiological functions. In the periphery about the 95% of the 5-HT is synthesized by the enterochromaffin cells in the gastrointestinal tract(Gershon, 2004), where it initiates responses as diverse as nausea, intestinal secretion, and
peristaltis and has been implicated in gastroenteric diseases, such as the irritable bowel syndrome(Kim and Camilleri, 2000). 5-HT produced in the gastrointestinal tract is released in circulating blood where it is uptaken and stored by platelets. Here 5-HT participates to blood coagulation, homeostasis and in T cell-mediated immune responses. In contrast, Tph2 is the neuronal isoform as it is expressed exclusively in the neurons of the raphe nuclei and the myenteric plexus.
1.3 Serotonin and Development
It is now appreciated that 5-HT plays a crucial role during embryogenesis by influencing neural, craniofacial, cardiac and limb development(Levitt et al., 1997; Moiseiwitsch and Lauder, 1997; Persico et al., 2003; Vitalis et al., 2002).
5-HT is present in oocytes and early embryos of all vertebrates and invertebrates studied, from sea urchins to mammals, even before the appearance of neural structures(Buznikov et al., 2005; Levin et al., 2006). D uring mammalian embryogenesis 5-HT is supplied by the maternal blood(Cote et al., 2007; Yavarone et al., 1993)
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and in Xenopus embryos 5-HT is present as a maternal pool in the eggs(Fukumoto et al., 2005).5-HT is synthesized by embryonic stem cells(Walther and Bader, 1999) and is likely to play a role in egg maturation(Buznikov et al., 1993), mitogen-induced vascular remodelling(Fanburg and Lee, 1997), control of cleavage and cell division rates, patterning of cytoskeletal structures(Lee et al., 1991), blastomere adhesion, gastrulation movements(Colas et al., 1999) (Hamalainen and Kohonen, 1989; Colas et al., 1999), control of gap-junctional communications(Moore and Burt, 1995; Rorig and Sutor, 1996), establishment of left-right asymmetry(Levin et al., 2006) and specification of neural identity and connectivity in the developing nervous system(Fukumoto et al., 2005; Whitaker-Azmitia, 2001). The most straightforward way to understand the function of the transient expression of genes that control 5-HT levels during development has been to investigate the effects of invalidation of these genes.
The Tph1 null mice exhibit larger heart size than the wild-type mice and suffer a progressive cardiovascular dysfunction leading to heart failure(Cote et al., 2003).
Tph1 gene disruption leads to a dramatic loss of 5-HT
from the periphery. Thus Tph1 KO mice provide a convenient tool to address the developmental role of maternal 5-HT. In a recently report Tph1 null females were mated with wild-type, Tph1 heterozygous or null males and the phenotype of pups obtained has been
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analysed. Côté and colleagues have demonstrated that the dramatic abnormalities in the development of the brain and other tissue of the littermates is linked to the genotype of the mothers and that 5-HT of maternal origin is require for normal embryonic development(Cote et al., 2007). Recently three knock out mouse lines for Tph2 have been generated by three different research groups(Gutknecht et al., 2008; Migliarini et al., 2013; Savelieva et al., 2008). Savelieva and colleagues (2008) generated both the Tph2 KO mouse line and the Tph1/2 double KO mouse line. Surprisingly, none of these mutations resulted in an overt phenotype. Tph2KO and double KO mice were smaller in body length and weight compared to WT controls. Tph2KO and double KO mice were generally normal in many of the behavioural tests employed in this study. However, differences in behaviour were evident in tests sensitive to serotonergic drugs like SSRIs and some compounds acting at 5-HT receptors. Pasqualetti’s group also demonstrated that mice lacking Tph2 revealed impairment of growth rate compared to heterozygous and wt littermates(Migliarini et al., 2013).
It has been also generated a knockin mouse line expressing a mutant form of the Tph2, equivalent to a rare human variant (R441H) identified in few individuals with unipolar major depression. Expression of mutant Tph2 in mice results in markedly reduced (~80%) brain 5-HT production and leads to behavioral abnormalities in tests assessing 5-HT mediated emotional states (Beaulieu
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et al., 2008). The invalidation of the gene that encodes
the main enzyme responsible for 5-HT degradation, the monoamine oxidase A (MaoA), causes a nine fold increase in 5-HT concentration in the brain during the 1st postnatal week which results in the disruption of the clustering and segregation of thalamocortical and retinal fibres that normally occurs during this time (Upton et al., 1999; Rebsam et al., 2002). Other
developmental alterations in these mice concern the control of locomotor and respiratory rhythms in neonates (Bou-Flores et al., 2000).
In the knock out mouse for serotonin transporter (Sert), 5-HT continue to be released but cannot be removed from the synaptic cleft, leading to a decreased rate of synaptic 5-HT clearance and to up to six-fold increase in basal level of forebrain extracellular 5-HT (Mathews et
al., 2004). This causes abnormal development of the
thalamocortical and retinal axons similar to those observed in MaoA KO mice, indicating that an excessive build-up of 5-HT in the extracellular space is responsible for developmental changes (Upton et al., 1999; Persico et
al., 2001). Moreover Sert-/-
mice show increase anxiety-like behaviour and reduction in aggressive behavior (Holmes et al., 2002; Holmes et
al., 2003). Thus they have been extensively employed as
animal models complementary to direct studies of genetically complex brain disorders and traits in human, with important findings in biochemical, pharmacological, anatomical and behavioural areas (Bengel et al., 1999).
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Other neurons could benefit from this increased stimulation of 5-HT receptors, as indicated by the reduction in the amount of developmental cell death in the telencephalon of Sert KO mice (Persico et al., 2003). Pet-1 KO mice which have 80% depletion of 5-HT in the central nervous system, the general structure of the brain seems to be normal. However these mice have marked behavioural changes, such as heightened anxiety and aggressive behavior(Hendricks et al., 2003). In the mouse, 5-HT derived from the maternal-embryonic circulation (Yavarone et al., 1993), acts as a morphogenetic signals to regulate various aspects of craniofacial development (Lauder et al., 1988; 1993). During the development of the branchial arches, sites of 5-HT reuptake and degradation are transiently expressed by ephitelia (Shuey et al., 1993). This sites appear to protect the underlying mesenchyme from exposure to inappropriate levels of 5-HT, as indicated by patterns of cell death and malformations caused by exposure of cultured embryos to selective 5-HT reuptake inhibitors (SSRIs), like fluoxetine (Prozac) and sertraline (Zoloft) (Shuey et al., 1992). As shown in a variety of in vitro models 5-HT acts as a dose-dependent growth regulatory signal for craniofacial cells, regulating neural crest cells migration (Moiseiwitsch and Lauder 1995), mesenchymal cells proliferation (Bhasin et al., 2004b; Buznikov et al., 2001; Nebigil et al., 2000), tooth germ development (Moiseiwitsch and Lauder, 1996), and expression of growth factors and extracellular matrix
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molecules, including those of the cartilage matrix (Lambert and Lauder, 1999; Lambert et al., 2001; Moiseiwitsch and Lauder, 1997; et al., 1998). Serotonin carries out these diverse developmental functions by activating different receptor subtypes, including 5-HT1, 5-HT2 and 5-HT3 receptors (Moiseiwitsch and Lauder 1995; Tecott et al., 1995; Lauder et al., 2000; Bhasin et
al., 2004a,b). In Xenopus the serotonin 2B receptor
signaling has been shown to modulate the shape and functionality of distinct elements of the jaw, including the jaw joint (Reisoli et al., 2010).
1.4 Serotonin Receptors
Serotonin, like other neurotransmitters such as acetylcholine, glutamate, and γ-aminobutyric acid (GABA), acts via two categories of receptors: ionotropic and metabotropic receptors. Ionotropic receptors are named also channel receptors and they have a low affinity for their neurotransmitter ligand but a rapid activation constant (a few milliseconds).
In contrast, metabotropic receptors which are G-proteins-coupled receptors act throught a second messengers production and exhibit both high affinity for their neurotransmitter and slow activation constant (in seconds or longer). The genes encoding 5-HT receptors are fifteen. They have been cloned in the mammalian brain(Hoyer and Martin, 1997). Only one family receptor, the 5-HT3 are ionotropic receptors. The other families are metabotropic receptors with seven
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transmembrane domains. They can be categorized into four groups according to their main second messenger system (Raymond et al., 2001):
1. 5-HT1 receptors, coupled to Gαi/Gαo proteins; 2. 5-HT2 receptors, coupled to Gαq proteins; 3. 5-HT4, 5-HT6, and 5-HT7 receptors, coupled to
Gs proteins;
4. 5-HT5 receptors for which the coupling is still uncertain.
I focused my study about the second category, the 5-HT2 receptors (5-HT2Rs). This is composed by three members of the 5-HT2 receptor family, termed 5-HT2A, 5-HT2B, and 5-HT2C (Hoyer et al., 1994). The 5-HT2 receptors couple consistently to the PLC-β second messenger pathway(Peroutka, 1995) but they can also couple to other second messenger pathways in a cell-specific manner.
The 5-HT2A receptor is widely distributed in the brain (cortex, caudate nucleus, olfactory tubercle, nucleus accumbens, and hippocampus)(Pazos et al., 1985), skeletal(GuilletDeniau et al., 1997; Hajduch et al., 1999) and smooth muscles(Ellwood and Curtis, 1997; Kuemmerle et al., 1995), kidney(Garnovskaya et al., 1995), and platelets(Cook et al., 1994). In the brain, 5-HT2A receptor is certainly the site of action of hallucinogens, such as LSD(Aghajanian and Marek, 1999), and of some atypical anti-psychotics, such as
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clozapine and risperidone(Adell et al., 2005). Alterations in 5-HT2AR function may be a contributing factor to the cognitive impairment in mental disorders such as schizophrenia and depression(Kroeze and Roth, 1998). 5-HT2BR will be discussed in detail in the following paragraph.
5-HT2CR expression has been detected in choroid plexus, cortex, nucleus accumbens, amygdala, hippocampus, caudate nucleus and substantia nigra(Abramowski et al., 1995). 5-HT2CR activation inhibits dopaminergic neurons(Gobert et al., 2000; Ji et al., 2006). Atypical anti-psychotics and particularly those with inverse agonism, release this inhibition, providing a way to activate dopaminergic systems(Berg et al., 2005; De Deurwaerdere et al., 2004; Gobert et al., 2000). This activation may be relevant for the treatment of depression and the negative symptoms of schizophrenia. Serotonin 5-HT2C receptor null-mutant mice displayed both an epilepsy and obesity phenotype. These findings implicate 5-HT2CR in the regulation of neuronal network excitability. It has also been observed that body weight and adipose tissue deposition are elevated in adult mutant mice compared to their wild type littermates. Paired-feeding studies suggest that the obesity syndrome is a result of increased food intake. In addition, mutants display reduced sensitivity to the appetite suppressant actions of non-specific serotonergic agonists. These studies establish a role for 5-HT2CR in the serotonergic
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regulation of body weight and food intake(Tecott et al., 1995).
1.5 The 5-HT2B Receptor
The expression of 5-HT2B receptor in the adult rat appeared restricted to the stomach fundus(Foguet et al., 1992; Kursar et al., 1992), was subsequently found in the brain, more specifically in the cerebellum, cortex, hippocampus and amygdala(Duxon et al., 1997). Similar locations express 5-HT2B receptor in the mouse brain(Choi and Maroteaux, 1996). The 5-HT2B receptors are also found in the adult gut and cardiovascular system(Loric et al., 1992), including the stomach, intestine and pulmonary smooth muscle, as well as in pulmonary and vascular endothelial cells and cardiomyocytes in mouse(Choi and Maroteaux, 1996), human(Bonhaus et al., 1995; Choi et al., 1994; Schmuck et al., 1994) and rat(Baxter et al., 1995; Foguet et al., 1992; Kursar et al., 1992; Ullmer et al., 1995). Choi and colleagues observed that the 5-HT2B receptor is expressed in embryos with a peak of expression at 8.5 days post-fecondation in mice and by whole mount in situ hybridization and immunohistochemistry they detected 5-HT2B expression in neural crest cells, heart myocardium, somites and neural tube(Choi et al., 1997; Lauder et al., 2000).
Our laboratory also demonstrated that 5-HT2B receptor is expressed in the proliferative region of the retina and the neural tube during Xenopus laevis development(De
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Lucchini et al., 2003). These observations strongly suggested that 5-HT2B receptors are actively involved in a transient action of 5-HT during embryonic morphogenesis.
As already said, the 5-HT2 family acts through metabotropic receptor and the main transduction pathway for this family is the activation of phospholipase C second messenger via the alpha subunit of the Gq GTP binding protein. This messenger activates inositoltrisphosphate and diacylglycerol production leading to Ca2+ release and protein kinase C activation(Loric et al., 1995). In addition, 5-HT2B receptor stimulation has the ability to activate the arachidonic acid pathway via phospholipase A2(Tournois et al., 1998) and the nitric oxide synthesis pathway via a PDZ type I C-terminal motif(Manivet et al., 2000). Nebigil and colleagues (2000) demonstrated in vitro that 5-HT2B receptors activity is mitogenic. In this mitogenic signaling, c-Src is the crucial molecule that links 5-HT2B receptor and the cell-cycle regulators. C-Src alone controls cyclin E induction and transactivates PDGF-receptor tyrosine kinase activity to induce cyclinD1 expression through p42mapk/p44mapk (ERK2/ERK1) mitogen-activated protein kinases (MAPK) pathway(Nebigil et al., 2000). All these pathways are resumed in Fig.1.2.
Moreover Nebigil and colleagues (2003) showed that 5-HT2B receptor is also cytoprotective for cardiomyocytes by activating both phosphatidylinositol-3 kinase/Akt and
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ERK1/2 kinases pathway and preventing caspase activity(Nebigil et al., 2003; Nebigil and Maroteaux, 2001).
Fig.1.2 Signal transduction pathways of the HT2BR. The
5-HT2BR couples with phospholipase C (PLC), to generate inositoltrisphosphate (IP3), which then triggers both intracellular calcium release and diacylglycerol (DAG) accumulation, leading to stimulation of protein kinase C (PKC). The 5-HT2BR activates the phospholipase A2 (PLA2)/arachidonic acid (AA). Stimulation of the 5-HT2BR triggers intracellular cGMP production through activation of the nitric oxide synthase (NOS) pathway. Activation of the 5-HT2BR stimulates the ras-mitogen-activated protein kinase (ERK/MAPK) cascade. It appears that c-Src provides a link between PDGFR/MAPK and cell cycle regulators in Gq-coupled 5-HT2BR mitogenic signaling. (From Nebigil and Maroteaux, 2001)
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1.6 The role of 5-HT2B receptor during
development
The first evidence of a role of 5-HT2B receptor during embryonic development has been provided by culture of embryos exposed to the 5-HT2 family receptor high affinity antagonists: ritanserin. These embryos presented strong growth retardation, abnormal flexure of the cephalic region, underdeveloped forebrain and hindbrain, small pharyngeal arches and severe morphological defects in the heart. In particular it has been suggested that ritanserin treatment interfered with neural crest cells (NCCs) migration and in the heart, it was responsible for premature sarcomeric organization of the subepicardial layer and for the absence of the trabecular cell layer in the ventricular myocardium(Choi et al., 1997; Nebigil and Maroteaux, 2001).
Consistently with these studies, the KO mouse line for 5-HT2B receptor presented severe heart abnormalities, including trabecular defects accounting for mid-gestation lethality (Fig.1.3)(Nebigil et al., 2000).
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Fig.1.3 Heart abnormalities in 5-HT2B KO mouse line causes
mid-getation lethality (from Nebigil et al., 2000)
On the contrary, 5-HT2B over-expression in the heart leads to cardiac hypertrophy associated with abnormal mitocondrial proliferation and enzymatic activity, indicating an essential role for 5-HT in the hypertrophic signaling(Nebigil et al., 2003). Collet and colleagues (2008) demonstrated that 5-HT2B receptor play also a major role in bone formation. They revealed that 5-HT2B KO mice which survived until adulthood presented osteopenia and reduced bone formation in aging and that osteoblast recruitment and proliferation was reduced in HT2B-depleted primary cultures(Collet et al., 2008). 5-HT and retinoids play important roles in embryogenesis, and recent evidences suggest that these signaling pathways may act together to regulate development of
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embryonic tissues(Choi et al., 1997). Moreover similar teratogenics effects obtained by exposing cultured mouse embryos to an excess of retinoic acid (RA) and ritanserin has suggested a possible functional relationship between 5-HT2B receptor and RA signaling during normal embryogenesis(Choi et al., 1997; Lauder et al., 2000).
1.6.1 Relationship between RA and 5-HT2B
during development
Bhasin and colleagues (2004) established that even if 5-HT2B promoter contains several potential retinoids response elements, RA does not regulate 5-HT2B transcription but they revealed that 5-HT2B receptor and RA promote and inhibit respectively chondrogenic differentiation in hindlimb micromass cultures by acting as opposing signals on different MAPK signal transduction pathways(Bhasin et al., 2004a). These authors also demonstrated that RA and 5-HT via 5-HT2B receptor, may act as opposing signals to regulate proliferation in mouse frontonasal mass, further supporting their hypothesis that RA and 5-HT2B signals may act as opposing signals for common cellular mechanisms(Bhasin et al., 2004b).
1.6.2 The role of 5-HT2B during Xenopus
development
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Fig 1.4 Expression pattern of 5-HT2B (A) in the branchial arches,
(B) at stage 37 in the periocular mesenchyme,visualized by in situ hybridization on section, (C) in the retina in embryos at stage 45.
During Xenopus laevis development the 5-HT2B is expressed in the periventricular zone of the neural tube of late tailbud embryos(De Lucchini et al., 2003). Moreover, X5-HT2B mRNA expression was detected at stage 37, in scattered cells of the dorsal neural tube, in the diencephalon, in the NCCs migrated into the branchial arches (Fig.1.4 A) and in the periocular mesenchyme (Fig.1.4 B) and in the retina(Fig.1.4 C). Our laboratory by means of a loss of function study demonstrated that 5-HT via the 5-HT2B receptor, is involved in Xenopus laevis retinal histogenesis by controlling cell proliferation and cell survival(De Lucchini et al., 2005). Furthermore it has been unveiled that 5-HT2B signaling pathway regulates the mandibular arch morphogenesis. 5-HT2B signaling is, in fact, required for craniofacial morphogenesis in Xenopus (Reisoli 2010) and it modulates postmigratory skeletogenic cranial NCC behavior in an-autonomous
A
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manner through the phospholipase C beta3. This signaling is required to define and sustain the BapX1 expression necessary for jaw joint formation (Fig.1.5)(Reisoli et al., 2010).
Fig.1.5 Dorsal view of a 5-HT2B over expressing embryos after the Alcian staining The skeletal phenotype is characterized by the
presence of an ectopic cartilage and by the reduction of the palatoquadrate and subocular cartilages (Reisoli et al.2010).
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2. The importance of Cranial Neural
Crest Cell
Cranial neural crest contribution in building the vertebrate head is so crucial that the acquisition of the NCC by protochordate ancestors is considered to be a turning point in the evolution of vertebrates(Le Douarin et al., 2004).
During development cranial neural crest cells (NCCs) form over a time period that spans from gastrulation to late organogenesis in response to both intrinsic and extrinsic influences. The presuntive cranial neural crest is first induced by a set of signaling events that occurs at the border between the future neural and non-neural ectoderm. As neurulation progresses, these precursors come to lie within the elevating neural folds and dorsal aspect of the neural tube, from which they subsequently emigrate. During this period, cranial NCCs lose cell-cell adhesion and undergo cytoskeletal rearrangements and morphological changes, that allow them to delaminate and emigrate from the neuroepithelium. They concomitantly acquire migratory ability, with acquisition of cell-surface receptors, metalloproteases and adhesion molecules that allow them to respond properly to cell-cell interactions and environmental cues that influence their pathways of migration. Once NCCs arrive at their final destinations, they often self-aggregate during initiation of terminal differentiation. This complex succession of events, from NCCs induction, to specification to overt differentiation, is guided by a carefully orchestrated gene
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regulatory network. This network is comprised of multiple regulatory modules that guide acquisition of specific properties such as multipotency and migratory capacity in NCCs. It involves signaling pathways and transcription factors that are responsible for the induction of NCCs (i.e. Wnt, bone morphogenetic proteins (BMPs), fibroblast growth factors (FGFs)), the specification of the neural plate border (i.e. Msx1, Msx2; Pax3 and Zic1) and the specification of bona fide NCCs.
Previous work in several species, has implicated a set of transcriptional regulators in conferring neural crest identity. This set of genes, termed neural crest specifier genes, include Sox9, Sox10,FoxD3, Snail2/Slug, c-Myc, and AP2(Meulemans and Bronner-Fraser, 2005). These transcription factors are expressed in premigratory and/or early migrating neural crest cells and are thought to be involved in induction and survival of the premigratory population, their epithelial to mesenchymal transition from the neural tube, migratory ability, and cell lineage decisions, by regulating a number of downstream targets(Adams et al., 2008; Sauka-Spengler and Bronner-Fraser, 2006; Sauka-Spengler and Bronner-Bronner-Fraser, 2008). In all vertebrates, cranial NCCs migrate dorso-laterally from the neural tube (Fig.1.6) in a conserved and characteristic pattern of three distinct streams termed mandibular, hyoid and branchial, into the 1st, 2nd and 3rd/4th pharyngeal arch, respectively, where they
differentiate into different derivatives such as
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and bones of head skeleton(Knecht and Bronner-Fraser, 2002; Le Douarin et al., 2007; Sadaghiani and Thiebaud, 1987).
Fig.1.6 Schematic representation of Xenopus laevis migration of cranial NCCs I, mandibular crest stream; II, hyoid crest stream;
III-IV, branchial crest streams. Frontal view on neurula stage and lateral view on tailbud stage. Modified by Sadaghiani and Thiebaud (1987).
Sometimes the neural crest-derived mesenchyme is known as ‘‘mesectoderm’’ to reflect its ectodermal origin(Hall, 1971). The mesectoderm also provides head connective-tissue cells and the vascular smooth-muscle cells associated with the vessels derived from the aortic arches and with the vessels irrigating the forebrain and face(Etchevers et al., 2001). Although NCCs populations are specified early in development, NCCs that reach target sites are pluripotent and further restrictions occur only late in development depending on their location within the embryo. There is a direct correlation between the time of migration of NCCs from the neural tube and their developmental potential. For instance, in the
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Xenopus laevis head, the early migratory cells populate
the pharyngeal arches and will generate ectomesenchymal derivatives: cartilage, and connective tissue of the face, while the later migrating cells stay closer to the developing central nervous system and generate neurons and glia of the cranial ganglia(Hensey and Gautier, 1998).
3. Eye Development
Vertebrate eye development is a complex process that involves multiple inductive interactions between the forebrain neuroectoderm (which gives rise to retina and the optic nerve), the surface ectoderm (which gives rise to the lens, epithelia of the cornea, conjunctiva and eyelids) and the neural crest cells-derived periocular mesenchyme (POM) which gives rise to the choroids, sclera, stroma of the cornea and iris anterior chamber of vitreous body(Cvekl and Tamm, 2004; Johnston et al., 1979; Trainor and Tam, 1995). The vertebrate eye develops from the neuroepithelium of the ventral diencephalon which evaginates laterally to form the optic vesicles. Subsequent invagination of the distal portion of the optic vesicle leads to the formation of a 2-layered optic cup. The distal layer differentiates into the multilayered neural retina, the proximal layer develops into the retinal pigmented epithelium, consisting of a single layer of nonneural pigment cells. The presuntive neural retina of the optic vesicle is in close contact with
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the overlying surface ectoderm which invaginates to form the lens. The presuntive retinal pigmented epithelium is surrounded by extraocular mesenchyme originating from mesodermal cells and NCCs migrating from the diencephalic and mesencephalic brain regions (Fig.1.7)(Fuhrmann, 2010; Fuhrmann et al., 2000).
Fig1.7 Summary of vertebrate early eye develelopment A)
Factors from surrounding tissues (extraocular mesenchyme, optic stalk and surface ectoderm) regulate patterning of the neural retina and RPE in the vertebrate optic vesicle, which then expresses the specific transcription factors Vsx2 and Mitf, respectively. B) Invagination of the distal optic vesicle (presumptive retina) and the overlying lens placode results in formation of the optic cup and lens vesicle (Modified by Fuhrmann 2010).
The vertebrate mature neural retina is made up of approximately 50–60 different neuronal cell types that can be grouped into seven major classes: rods, cones, horizontal cells, bipolar cells, amacrine cells, Müller glia, and ganglion cells. These cells are organized in a stratified structure of three nuclear layers, (ganglion cell,
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inner nuclear, and outer nuclear layers), which are separated by two synaptic (inner and outer plexiform) layers. Adiacent to the pigmented epithelium, the outer nuclear layer (ONL) is made by the photoreceptor cell bodies; the outer plexiform layer (OPL) separates the ONL and the inner nuclear layer (INL), composed in turn by bipolar, amacrine and horizontal cell bodies; the inner plexiform layer (IPL) precedes the last layer in the retina, the ganglion cell layer (GCL), facing the vitreous body. Within such a complex laminar structure, bipolar cells establish communication between photoreceptors and ganglion cells, whereas horizontal neurons provide horizonal information transport and integration. The highly intricate retina structure is achieved through a complex system of events, comprising cell proliferation and differentiation, migration and apoptosis. Cell differentiation in the retina follows a conserved timing in vertebrates: the first cells to differentiate are the ganglion cells, followed in order by cones, amacrine, horizontal, rods, bipolar, and then Müller glia cells. Lineage studies have demonstrated that retinal progenitor cells are multipotent and that a single retinal progenitor cell can give rise to any one of the seven cell classes(Turner and Cepko, 1987; Turner et al., 1990): the differentiation of a specific cell type instead of another thus depends on the cellular environment in which cells become localized and not by their precursors(Cepko et al., 1996; Harris, 1997). In fish and amphibians, the retina grows throughout life by adding new cells of all types from the ciliary marginal
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zone (CMZ), a region at the peripheral edge of the retina(Johns, 1977; Reh, 1992; Straznicky K., 1971; Wetts et al., 1989). The CMZ has the exceptional advantage of being spatially ordered with respect to cellular development and differentiation, with the youngest and least determined stem cells closest to the periphery, the proliferative retinoblasts in the middle, and the cells that have stopped dividing at the central edge(Dorsky et al., 1995; Wetts et al., 1989). In mammals, a homologous structure exists, called pigmented ciliary margin (PCM): this region contains quiescent stem cells that constitute a reservoir for the regeneration of all different cell subtypes in the adult retina(Tropepe et al., 2000).
Classical experiments suggest that signals from surrounding tissues are essential during early eye development. Holtfreter (1939) observed in explants cultures derived from amphibian anterior neural plate that eye development arrests at the optic vesicle stage in the absence of contact with the epidermis and the neural crest-derived mesenchyme (Fuhrmann et al., 2000). The primary function of periocular mesenchyme is to provide multiple mature cell lineages that are necessary for the development of normal anterior segments (including the corneal endothelium, anterior chamber and iris stroma). A second essential function is that cells originating from the surface epithelium need to interact with periocular mesenchyme for proper eyelid development(Lievre and Ledouarin, 1975). Finally, the periocular mesenchyme
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provides essential signals for patterning of ocular ectoderm primordial, including specification of retinal pigmented epithelium and the differentiation of the optic stalk from neural ectoderm(Evans and Gage, 2005; Fuhrmann et al., 2000; Matt et al., 2008).
3.1 Retinoic Acid guides correct eye
morphogenetic movements
Retinoic Acid (RA) is an important developmental signaling molecule(Duester, 2000). Some studies suggest a connection between impaired RA function and development eye defects in humans and mice(Lohnes et al., 1994; Seeliger et al., 1999). Moreover RA has been defined as a signaling molecule needed for pattering of other regions of the central nervous system including the hindbrain and the spinal cord(Maden, 2002). Now it is clear that RA controls eye development, but its mechanism is different from that of other eye signaling molecules that control patterning or cell fate determination, such as fibroblast growth factor, which is released by the surface ectoderm to stimulate neural retina differentiation(Russell, 2003), or activin, which is released by periocular mesenchyme to stimulate retinal pigment epithelium differentiation(Fuhrmann et al., 2000). Genetic studies in mice suggest that RA controls eye development by acting within the neural crest-derived periocular mesenchyme(Matt et al., 2005). The mechanism of RA action during eye development
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remains unclear largely because of an incomplete understanding of the complex spatiotemporal properties of RA synthesis in this organ and of the target tissues upon which RA acts. Molotkov et al (2002) have been demonstrated that RA is involved in eye neural development, through its ability to stimulate the morphogenetic movements needed for retinal invagination.
During optic cup formation the sources of RA are Raldh3 (RA synthetized enzymes 3) expressed in the future Retinal Pigmented Epithelium (RPE) and Raldh2 expressed in mesenchyme on the temporal side of the optic vesicle; the RA target tissue is the neural retina. The outcome of this early phase of RA signaling is the stimulation of morphogenetic movements leading to ventral invagination of the optic vesicle to form a complete optic cup. Although Raldh2 provides sufficient RA to initiate ventral invagination of the neural retina, it alone is an incomplete source of RA, as its expression ends too early to provide the RA needed for proper distribution of neural retinal cells as the optic cup forms. However, Raldh3 expression continues and it alone can provide RA that is sufficient for both the initiation of ventral invagination and the proper distribution of neural retinal cells leading to formation of a normal vitreous body cavity. RA control of neural retina morphology does not involve the regulation of cell proliferation or apoptosis. During anterior eye formation the sources of RA are Raldh1 expressed in the dorsal neural retina and
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Raldh3 expressed in the ventral neural retina, and the RA
target tissue is the periocular mesenchyme (POM). The outcome of this late phase of RA signaling is the limitation of POM morphogenetic movements that provide tissue contributing to the corneal mesenchyme and eyelids(Cvel and Tamm, 2004). Different studies illustrate the usefulness of RALDH null mice in revealing the spatiotemporal role of RA signaling in tissues where more than one RALDH contributes to RA
Raldh3 alone can supply all of the RA needed for eye
morphogenetic movements in the mouse(Molotkov et al., 2002).
4.Xenopus laevis as a model system
Choosing a suitable animal model system to unveil new roles of the serotonergic signaling during development is 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. Thus Xenopus has become a very widely used model organism for a variety of biological and biomedical researches. 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,
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accessibility and large size of the eggs, which can be easily fertilized in vitro producing huge numbers of synchronized embryos that can be observed developing from the first cell division to a tadpole in about 36 hours
(Fig.1.8) The embryos can be easily manipulated,
injected, grafted or labeled. In particular, researchers can easily manipulate gene functions in large numbers of embryos by using morpholino-modified antisense oligos and by mRNA mis-expression. This can be performed by injecting morpholinos or in vitro transcribed mRNAs into one cell of the two-cell stage embryos. This procedure provides an useful internal control, as the first cleavage results in gene down or up-regulation in only one side of the embryo. In addition, the availability of a well-defined fate map of each blastomere allows to address the microinjection in specific regions of the embryo so that a morpholino-mediated conditional knock-down approach can be easily obtained. This approach can overcome lethal effects and allows to study defects that appear in later stages of development. Homotipic transplantation assay system has also been settled up in Xenopus embryos allowing to detect cell contribution to specific structures as well as the cell autonomous or non autonomous contribution of specific gene activity in developmental processes (Borchers et al., 2000; Reisoli et al., 2010). Xenopus is also successfully used for chemical genetics and drug screen studies (Wheeler and Brändli, 2009). In particular, chemical genetics provides a complementary approach to loss- or gain-of function
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mutations in the study of complex biological processes. Public databases harbor the nucleotide sequences of almost 2 million expressed sequences tag (EST) from both X. laevis and X. tropicalis (the diploid sister species of X. laevis, whose genome is nearly sequenced) and microarrays are available for both species, thus an impressive collection of genomic and transcriptomic tools is rapidly becoming available for this model system.
Xenopus as a tetrapod shares a long evolutionary history
with mammals: they share similarities at the level of the genomes, in organ development, anatomy and physiology and therefore represents an excellent model to predict human biology (Raciti et al., 2008). The closer a model organism is in evolutionary terms to the target organism, i.e., humans, the more reliably results may be translated from studies in the model organism to humans.
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