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Keywords:

  • Orthopedia;
  • Otp;
  • hypothalamus;
  • development;
  • neurohormones;
  • hypothalamic nuclei;
  • dopamine, DA

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. TRACING THE EVOLUTION OF Otp
  5. REGULATION OF Otp DURING HYPOTHALAMIC DEVELOPMENT
  6. Otp ROLES IN HYPOTHALAMIC NUCLEI DEVELOPMENT
  7. PERSPECTIVES
  8. Acknowledgements
  9. REFERENCES

The wealth of expression and functional data presented in this overview discloses the homeogene Orthopedia (Otp) as critical for the development of the hypothalamic neuroendocrine system of vertebrates. Specifically, the results depict the up-to-date portrait of the regulation and functions of Otp. The development of neuroendocrine nuclei relies on Otp from fish to mammals, as demonstrated for several peptide and hormone releasing neurons. Additionally, the activity of Otp is essential for the induction of the dopaminergic phenotype in the hypothalamus of vertebrates. Recent insights into the pathways required for Otp regulation have revealed the implication of the main extracellular signals acting during hypothalamic development. Alterations in these pathways are involved in several neuronal disorders, and the resultant downstream misregulation of Otp might impair the development of the hypothalamus, and be therefore responsible for the neuroendocrine dysfunctions that typify these diseases. Developmental Dynamics 237:2295–2303, 2008. © 2008 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. TRACING THE EVOLUTION OF Otp
  5. REGULATION OF Otp DURING HYPOTHALAMIC DEVELOPMENT
  6. Otp ROLES IN HYPOTHALAMIC NUCLEI DEVELOPMENT
  7. PERSPECTIVES
  8. Acknowledgements
  9. REFERENCES

In vertebrates, the development of the central nervous system (CNS) is an extremely complicated process that starts during gastrulation, when the organizer induces the competent ectoderm to become neural tissue. The newly formed neural tube is regionalized along the anteroposterior (AP) and dorsoventral (DV) axes into increasingly complex areas with different functions, so that several specialized cell types differentiate at precise positions in subsequent waves of neurogenesis. Finally, specific connections between neurons in the CNS and peripheral nervous system (PNS) are established.

AP regionalization comprises three major areas defined by morphological boundaries: the forebrain, that will give rise to the telencephalon and diencephalon, the midbrain, and the hindbrain. The hypothalamus (from Greek “under the thalamus”), according to the prosomeric morphological model (Puelles and Rubenstein,2003) we adopt in this review, lies at the rostral end of the neural tube, anterior to the rostralmost part of the caudal diencephalon (thalamus). Acting as an interface between endocrine and autonomic system, the hypothalamus has been claimed to be involved in a great number of physiological functions, such as sexual differentiation, reproductive behavior, birth, blood pressure, circadian rhythmicity, energy balance, homeostasis, as well as various developmental disorders including mental retardation, sudden infant death syndrome (SIDS), and Kallman, Tourette, and Prader-Willi syndromes, among others (Sandyk et al.,1987; Leckman et al.,1994; Swaab,1995; Guillemin,2004).

Although the physiological and clinical functions of the hypothalamus have been extensively addressed, the knowledge of the developmental mechanisms connecting the prosencephalic signaling pathways, the expression of specific transcription factors and the specification of diencephalic neuronal individuality remain largely unexplored.

The homeogene Orthopedia (Otp) may allow a meaningful understanding of the mechanisms that lead to the proper development of the hypothalamus, being crucial for the establishment of several neuronal phenotypes in the rostral diencephalon of vertebrates. From an evolutionary point of view, the identification of Otp orthologs throughout Metazoa supports a conservative and fundamental role in neural patterning and differentiation processes (Del Giacco et al.,2006).

In vertebrates, Otp is expressed in alternating and highly conserved hypothalamic domains (Simeone et al.,1994; Del Giacco et al.,2006; Bardet et al.,2008), where it operates in the proper differentiation of several neurohormone-secreting nuclei (Acampora et al.,1999,2000; Wang and Lufkin,2000; Michaud,2001; Eaton and Glasgow,2007; Ryu et al.,2007; Blechman et al.,2007; Eaton et al.,2008). Moreover, it has been recently demonstrated that Otp is involved in the development of diencephalic dopaminergic (DA) neurons in zebrafish and mouse (Del Giacco et al.,2006; Blechman et al.,2007; Ryu et al.,2007; Pistocchi et al.,2008).

Although the signaling cascades controlling Otp remain largely uncovered, a recent study proved that, in zebrafish, the expression of an Otp ortholog, otp1, is regulated by the main signaling pathways acting during hypothalamic development (i.e., Hedgehog, fibroblast growth factor, and Nodal; Del Giacco et al.,2006).

In view of the crucial properties of the hypothalamus, we provide a description of the progress in research on Otp, an essential factor in the development of the neurosecretory system.

TRACING THE EVOLUTION OF Otp

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. TRACING THE EVOLUTION OF Otp
  5. REGULATION OF Otp DURING HYPOTHALAMIC DEVELOPMENT
  6. Otp ROLES IN HYPOTHALAMIC NUCLEI DEVELOPMENT
  7. PERSPECTIVES
  8. Acknowledgements
  9. REFERENCES

Otp was first described in Drosophila and mouse in the context of controlling regionalization of the hypothalamus and spinal cord, and it was named after the homeodomain sequence homology with Antennapedia and orthodenticle genes of Drosophila (Simeone et al.,1994). To date, Otp orthologs have been identified in almost all multicellular organisms, in support of the notion that the ancestor of this transcription factor likely arose early in the evolution of stem metazoans (Del Giacco et al.,2006). The less complex animal known to carry an Otp gene is the radiate cnidarian Hydra magnipapillata. In addition, Otp has been identified in all major branches of bilaterally symmetrical animals, including (lophotrochozoan and ecdysozoan) protostomes and deuterostomes. Within protostomes, expression of Otp was documented in the platyhelminth Dugesia japonica, the mollusc Patella vulgata, the annelid Platynereis dumerilii, and the arthropod D. melanogaster (Simeone et al.,1994; Umesono et al.,1997; Nederbragt et al.,2002; Tessmar-Raible et al.,2007).

The brain of the planarian D. japonica, an acoelomate flatworm, consists of a cephalic ganglion and ventral nerve cords. In both adult and regenerating heads, Otp is expressed in the branch structures of the planarian brain, but not in the medullary cords (Umesono et al.,1997,1999). In the mollusc P. vulgata, the mRNA encoded by Otp is clearly associated with the anlage of the apical tuft, the brain of the trochophore larva where both photoreceptor and serotonergic cells will initiate (Nederbragt et al.,2002). Otp transcripts in Platynereis embryos are observed near the vasotocinergic and RFamidergic neurons of the apical plexus, positioned in the medial forebrain region (Tessmar-Raible et al.,2007). In Drosophila, Otp is expressed in neuroblasts with a metameric pattern, and peculiarly in the ectodermal cells of the gut (Simeone et al.,1994).

Echinoderms stands alone among phyla because Otp activity is not coupled with neural patterning as determinant of neurosecretory cell types, replaced by a completely new function in skeletogenesis, where Otp appears to control short-range cell signaling factors. In detail, Otp transcription is activated by the ARID-class transcription factor deadringer within a symmetric domain of the oral ectoderm at mid-late gastrula stage, close to active sites of spiculogenesis (Di Bernardo et al.,1999; Amore et al.,2003; Cavalieri et al.,2003,2007; Zhou et al.,2003). In hemichordate embryos, Otp expression occurs in mid-level neural domains labeling the apical and basal layers of the prosome ectoderm (Lowe et al.,2003). The ascidian tunicate larva preserves the primitive chordate body plan from which vertebrate arose during evolution. During Ciona intestinalis development, Otp expression starts in the rostral edge of the neural tube at mid-tailbud stage. Later, Otp expression occurs in three domains of the larval brain that anteroposteriorly correspond to the stomodeum primordium, which contributes to the formation of the adult neural complex, the sensory vesicle, and the visceral ganglion, the integrative center of motor activity control (Manni et al.,2005; Joly et al.,2007; te Welscher and Sordino, unpublished data).

It has been recently suggested that the neurosecretory brain of the early Bilateria was also performing sensory functions (Tessmar-Raible et al.,2007). In this view, phylogenetic studies of structure, function, and transcriptional activity let us propose that the twofold recruitment of Otp in terminal differentiation of the neuroendocrine and sensory processes occurred very early in the evolution of brain patterning and formation. Besides its essential function in terminal differentiation of neuronal cell types, compelling evidence of divergence is provided by insects and echinoderms, where Otp functions switched fully or partially to the formation of non-neural ectoderm.

REGULATION OF Otp DURING HYPOTHALAMIC DEVELOPMENT

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. TRACING THE EVOLUTION OF Otp
  5. REGULATION OF Otp DURING HYPOTHALAMIC DEVELOPMENT
  6. Otp ROLES IN HYPOTHALAMIC NUCLEI DEVELOPMENT
  7. PERSPECTIVES
  8. Acknowledgements
  9. REFERENCES

Compared with other organisms, the zebrafish genome contains two Otp orthologs, otp1 (otpb) and otp2 (otpa) (Del Giacco et al.,2006), likely arose from the ancient rayfinned fish-specific event of genome duplication (Amores et al.,1998; Wittbrodt et al.,1998; Force et al.,1999; Postlethwait et al.,2000). otp1 transcript is already detectable by reverse transcriptase-polymerase chain reaction (RT-PCR) at the oocyte stage, demonstrating its maternal origin, and remains visible throughout the following stages of development analyzed. Although the more sensitive RT-PCR technique detects otp1 mRNA at all stages of embryonic development, the first evidence of expression by whole-mount in situ hybridization (WISH) is visible at the three somites (s) stage in the hindbrain (rhombomere 5) and in the rostralmost part of the forebrain (Del Giacco et al.,2006). Similar analysis of otp2 expression discloses a marked divergence in the temporal profile of expression in comparison to otp1. Indeed, otp2 is not maternally expressed, and its zygotic transcription is initially detected by RT-PCR at 8 s, and at 14 s by WISH (Del Giacco and Pistocchi, unpublished data; Ryu et al.,2007). Later on, otp1 and otp2 are expressed in almost identical domains in all rhombomeric and diencephalic districts, similarly to Otp patterns in mouse (Simeone et al.,1994). In the forebrain, by 24 hr postfertilization (hpf) otp1 and otp2 label the hypothalamic preoptic area (PO; anterior alar plate), and the posterior tuberculum (PT; posterior basal plate of prosomere 3, according to Rink and Wullimann,2002), a thalamic derivative (Del Giacco et al.,2006; Ryu et al.,2007). These territories are within or in close proximity to organizing centers expressing signaling factors involved in hypothalamic development, such as Nodal, Sonic Hedgehog (SHH), and Fibroblast Growth Factor 8 (FGF8; Reifers et al.,1998; Schauerte et al.,1998; Sekimizu et al.,2004; Wolff et al.,2004; Fig. 1). Intriguingly, these are the same pathways that regulate otp1 expression during formation of neural stem cells in the zebrafish forebrain (Del Giacco et al.,2006; Fig. 2).

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Figure 1. Expression domains of otp1 and otp1-regulators in the fish forebrain. Schematic lateral view of a 24 hours postfertilization (hpf) zebrafish forebrain with anterior to the left and dorsal up. otp1 (in purple) label the preoptic area (PO), and the territory comprising the posterior tuberculum (PT), and the hypothalamic area rostral to PT. These territories are within, or in close proximity to, the expression domains of shh (light blue), fgf8 (yellow), fez1/tof (dashed), and prox1 (blue). cf, cephalic flexure; hyp, hypothalamus; pit, pituitary; os, optic stalks; or, optic recess; t, telencephalon.

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Figure 2. Genetic interactions controlling the development of diencephalic isotocin (IST), vasotocin (AVT), somatostatin (SS), corticotrophin-releasing hormone (CRH), and dopaminergic (DA) nuclei in zebrafish. otp1 expression in the PO is positively regulated by shh and negatively by fgf8. The effect of shh on otp1 expression is diminished by Dzip1, a shh negative regulator, while Nodal activates otp1 expression through shh. In the PT area, otp1 expression is independent from shh and fgf8 pathways but it is activated by Nodal. otp1 expression in the PT is also positively regulated by prox1, which controls neuronal differentiation of otp1-positive cells. fezl and med12 activate the expression of otp1 in PO and PT, and the posttranscriptional modulation of Otp1 protein is exerted by PAC1 and its ligand PACAP in both areas. No data concerning the signaling pathways regulating otp2 expression are currently available. otp1 and otp2 are critical for the development of the DA neurons in the PT region and are required for the development of the IST, AVT, SS, and CRH neurons in the PO.

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shh acts as a positive regulator of otp1 in the PO, but not in the PT, as shown by the analysis of two mutants of the shh regulatory module, sonic you (syu) and iguana (igu), characterized by functionally null alleles of, respectively, shh (Schauerte et al.,1998) and dzip1, a repressor of Hedgehog signaling gradients (Sekimizu et al.,2004; Wolff et al.,2004). In syu mutants, the otp1 pattern in the PO is drastically reduced in size, while loss of Dzip1 functions in igu embryos induces a phenotype featuring supernumerary PO otp1-positive cells. The ability of the Shh pathway to regulate otp1 in a positive manner has been confirmed by means of shh overexpression experiments resulting in otp1 ectopic activation in the optic vesicle and in few cells of the thalamus. Remarkably, these data let us hypothesize that alterated interactions between SHH and OTP in humans might be at the origin of the neuroendocrinopathies that characterize many cases of holoprosencephaly (HPE), a congenital disorder of neural induction (Sarnat and Flores-Sarnat,2001). In some cases, the disease is characterized by the disruption of the SHH pathway, suggesting that the failure of hypothalamic development is consequential to the secondary down-regulation of OTP caused by the absence of SHH signaling.

Tissues that express fgf8 are also observed near the otp1 cluster in the PO, where some neuronal progenitors coexpress the two genes (Del Giacco et al.,2006). Investigating a potential implication of fgf8 in otp1 regulation, injection of synthetic fgf8 mRNA resulted in the transcriptional repression of otp1, in accord with the observation that the PO cluster was expanded in embryos of the fgf8 null mutant acerebellar (ace). These results are indicative of a negative role for fgf8 in the control of PO-specific otp1 transcription. As for shh, otp1 expression is not altered in the PT region after fgf8 misregulation (Del Giacco et al.,2006).

The requirement of the Nodal signaling for the proper expression of otp1 has been proved by the profound modifications of the diencephalic otp1-positive domains in cyclops (cyc) embryos. cyc embryos carry mutations in the nodal-related 2 gene (ndr2) that alter the development of ventral CNS structures, with most severe phenotypes featuring cyclopia and lack of floor plate and hypothalamus (Hatta et al.,1991,1994; Rebagliati et al.,1998; Sampath et al.,1998). Analysis of otp1 expression in cyc embryos with milder phenotypes (reduction, rather than absence, of the hypothalamic basal plate) showed a significant decrease of the PO otp1 cluster size determined, at least in part, by the repression of shh transcription (Krauss et al.,1993; Del Giacco et al.,2006). In the same embryos, no otp1 expression occurred in the PT. Absence of the otp1 signal in this area is not determined by shh deficiency as for the PO cluster, because otp1 transcription in the posterior basal plate of the hypothalamus is independent from Shh signaling (see above), but is rather caused by the actual positive regulation exerted by the Nodal signaling on otp1 expression. Indeed, ndr2 overexpression determines the expansion of the posterior tubercular otp1 domain, in agreement with the opposite phenotype displayed by cyc embryos. Interestingly, there are some indications that also the Nodal signaling is involved in HPE. Indeed, the HPE-candidate homeogene TGIF, a SMAD2 corepressor, directs transcriptional silencing of target genes along the Nodal-related signaling pathway. A TGIF mutant with potential “gain-of-repression” activity that may lead to HPE has been identified (Wotton et al.,1999; Kuang et al.,2006), suggesting that, as for SHH, the neuroendocrine aspects of the disorder due to the defective development of the hypothalamus might be mediated by the secondary down-regulation of OTP, determined in this case by the excessive repression of the Nodal signaling.

The mediator complex subunit med12 (mot/med12) is an important component of neuronal subtype differentiation. med12 regulates commitment and differentiation of progenitor cells by controlling the expression of neuronal determination genes (Wang et al.,2006). Moreover, it is implicated in otp1 expression in zebrafish hypothalamus and PT (Del Giacco et al.,2006). Indeed, the number of otp1-positive neurons in both areas is reduced in mutant embryos carrying a deletion in the med12 gene (motionless, mot), with a more severe phenotype in the PT.

otp1 expression in hypothalamic neurons is also affected by prox1, the vertebrate homolog of the Drosophila gene prospero where it regulates the transition from proliferation to differentiation (Dyer,2003; Choski et al.,2006; Shimoda et al.,2006). prox1 is widely expressed in several districts of the developing CNS, including some otp1-positive neurons located in the posterior hypothalamus adjacent to the PT. Prox1 down-regulation causes a significant decrease in otp1 expression in discrete areas of the hypothalamus by impeding neuronal differentiation of otp1-positive cells and, by consequences, determining their misspecification (Pistocchi et al.,2008).

Recently, novel genetic pathways controlling otp1 transcription and modulating Otp1 protein level have been revealed (Blechman et al.,2007). The zinc-finger-containing protein Fezl (Levkowitz et al.,2003) regulates the expression of otp1 in PO and PT, as shown by the diminished expression of otp1 in the hypomorph fezl mutant too fewm808 (tofm808). In addition, a posttranscriptional modulation of otp1 is exerted by the G-protein-coupled receptor PAC1 and its ligand, the pituitary adenylate cyclase activating polypeptide (PACAP), as shown using both in vivo and in vitro approaches (Blechman et al.,2007).

At this juncture, no data concerning the signaling pathways regulating otp2 expression are available (Fig. 2).

Otp ROLES IN HYPOTHALAMIC NUCLEI DEVELOPMENT

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. TRACING THE EVOLUTION OF Otp
  5. REGULATION OF Otp DURING HYPOTHALAMIC DEVELOPMENT
  6. Otp ROLES IN HYPOTHALAMIC NUCLEI DEVELOPMENT
  7. PERSPECTIVES
  8. Acknowledgements
  9. REFERENCES

Otp Requirement in Neuroendocrine System Differentiation

In mouse, Otp forebrain-specific activity is detected in structures such as the supraoptic/paraventricular (spv), the anterior hypothalamic (AH) and the retrochiasmatic (rch) regions (Simeone et al.,1994). These areas give rise to anterior paraventricular (aPV), paraventricular (PVN), supraoptic (SON), and arcuate (ARN) nuclei (Puelles and Rubenstein,1993,2003; Rubenstein et al.,1994; Alvarez-Bolado et al.,1995). In particular, Otp is involved in developmental pathways leading to terminal neuroblast differentiation, such as proliferation, survival, migration, and differentiation of postmitotic cells. For these reasons, Otp null mice fail to develop properly aPV, PVN, SON, and ARN nuclei and therefore they die soon after birth (Acampora et al.,1999; Wang and Lufkin,2000; Fig. 3). Otp and Sim1, a bHLH-PAS transcription factor also expressed by PVN, SON and aPV neurons (Michaud et al.,1998), influence the development of the same groups of cells in the neurosecretory system (Fig. 4). The two genes operate along parallel pathways, as suggested by their respective null mice, whereas the knockout of Otp does not result in the alteration of Sim1 expression pattern or vice versa (Acampora et al.,1999; Wang and Lufkin,2000). Together, they are required in the PVN for the differentiation of the neurons secreting thyrotropin-releasing hormone (TRH), in the aPV for the differentiation of the neurons that secrete somatostatin (SS), and in the PVN and SON nuclei for the maintenance of Brn2 expression, a POU domain transcription factor necessary for the development of oxytocin (OT), arginine vasopressin (AVP), and corticotrophin-releasing hormone (CRH) producing neurons. Otp is also essential for the development of SS-secreting neurons in the ARN (Michaud et al.,1998,2000; Acampora et al.,1999; Wang and Lufkin,2000). New insights into the development of the vertebrate hypothalamus disclose the evolutionarily conserved role of Otp in the differentiation of the neurohormone-secreting cells in fish and mammals. Specifically, otp1 is required for the development of the zebrafish isotocin-neurophysin (IST or, alternatively, ITNP), and arginine vasotocin-neurophysin (AVT or, alternatively, VSNP or VTN) producing neurons (Tessmar-Raible et al.,2007; Eaton and Glasgow,2007; Eaton et al.,2008; Fig. 2).

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Figure 3. Mammalian hypothalamic nuclei that require Otp for their development. The hypothalamus (lateral view) consists of several nuclei containing different types of secretory neurons. The illustration portraits those neuronal populations, defined by the molecules that they synthesize, that necessitate Otp to develop. Paraventricular nucleus (PVN), producing oxytocin (OT), arginine vasopressin (AVP), corticotrophin-releasing hormone (CRH), and thyrotropin-releasing hormone (TRH); anterior paraventricular nucleus (aPV), producing somatostatin (SS); supraoptic nucleus (SON), producing oxytocin (OT), and arginine vasopressin (AVP); arcuate nucleus (ARN), producing somatostatin (SS); A11 nucleus, producing dopamine (DA). A, anterior; P, posterior.

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Figure 4. Transcription factors necessary for hypothalamic nuclei development in mouse. Otp and Sim1 function along parallel pathways in the paraventricular nucleus (PVN) and anterior paraventricular nucleus (aPV), and they are both required in the PVN and supraoptic nucleus (SON) nuclei for the maintenance of Brn2 expression. Otp alone is also essential for the development of somatostatin (SS) -secreting neurons in the arcuate nucleus (ARN) and dopaminergic (DA) neurons in the A11 group.

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IST is the teleost homolog of mammalian OT, a neurohormone and brain neurotransmitter that is involved in modulating several physiological functions and behavioral traits (Gainer and Wray,1992; Insel et al.,1997; Young et al,.1999; Ferguson et al.,2002), as well as various developmental psychiatric disorders (Leckman et al.,1994; Swaab,1995; Purba et al.,1996; Insel et al.,1999; Hollander et al.,2003). ist and otp1 are coexpressed at the level of the parvocellular and magnocellular neurons of the PO, and morpholino-mediated knockdown of otp1 results in a dramatic reduction of ist-expressing cells in this area of the hypothalamus. As for Otp and Sim1 in mice, the two zebrafish genes operate in parallel genetic pathways during IST-secreting cell development, as demonstrated by otp1 and sim1 knockdown experiments (Eaton and Glasgow,2007).

Differentiation of AVT neurons in the PO area is also strictly dependent on otp1 (Eaton et al.,2008). AVT is the homolog of the mammalian AVP, a nonapeptide neurohormone structurally similar to OT, which regulates a broad array of vertebrate physiological and behavioral responses, such as water body retention and blood pressure (Goodson and Bass,2001; Balment et al.,2006). avt is expressed in the parvocellular, magnocellular, and gigantocellular neurons of the PO (Venkatesh and Brenner,1995; Acher et al.,1997) and in an additional cluster of cells also belonging to the rostral hypothalamus (Eaton et al.,2008). All these avt-expressing neurons are positive for otp1 expression. Both otp1 and sim1 knockdown abolish avt expression in the PO area. On the contrary, otp1 inactivation does not interfere with the expression of avt in the second hypothalamic cluster of cells that, despite high level of avt mRNA, do not produce AVT. Interestingly, according to the authors, this group of avt-cells could represent the analogue of the mammalian hypothalamic suprachiasmatic nucleus (SCN), a modulator of circadian rhythms (Eaton et al.,2008).

Additional data substantiating the conserved role of Otp as a determinant of hypothalamic neuroendocrine nuclei are available in the work of Ryu and colleagues (2007), describing a reduction in the number of diencephalic corticotropin releasing hormone (CRH) and somatostatin (SST) producing neurons in m866 mutant embryos, in which the otp2 gene is disrupted.

As depicted in Figure 2, no data concerning Otp1 and Otp2 roles in the development of, respectively, CRH-/SST-, and IST-/AVT-secreting cells are currently available.

These studies demonstrate the evolutionary conserved role of Otp in the differentiation of the neuroendocrine system of vertebrates.

Otp Is Necessary to Induce the Dopaminergic Phenotype in the Diencephalon of Vertebrates

The catecholaminergic neurons of the vertebrate CNS participate in a wide variety of tasks, including motor coordination, mood regulation, and cognitive function. Neurotransmitter catecholamines (CA), namely Dopamine (DA), Adrenaline (AD), and Noradrenaline (NA), are neuroactive molecules that exert strong influence on vertebrate behavior (Swanson and Sawchenko,1983) and serve a variety of central and peripheral functions (Mason,1984). In zebrafish, the exhaustive description of the CA neurotransmitter pathway makes this organism a favorite model for the comprehension of morphogenesis in the neurosecretory system of vertebrates. Among the populations of CA neurons described in the zebrafish brain during development, distinct groups of dopaminergic (DA) neurons in the hypothalamus and the PT (Ma,1994a,b,1997,2003; Guo et al.,1999; Holzshuh et al.,2001; Rink and Wullimann,2001; Rink and Wullimann,2002; Ma and Lopez,2003) are found in the diencephalic otp1 (Del Giacco et al.,2006) and otp2 (Ryu et al.,2007) expression domains. Indeed, otp1 and otp2 signals overlap with the expression of tyrosine hydroxylase (TH), the rate-limiting enzyme in the synthesis of CAs, in almost all the posterior tubercular and hypothalamic DA neurons (Del Giacco et al.,2006; Ryu et al.,2007; Blechman et al.,2007). Otp1 and Otp2 are critical for the development of these cells, as demonstrated by loss-of-function experiments, such that the ablation of each of these proteins determines the development of a lower number of DA neurons (Del Giacco et al.,2006; Ryu et al.,2007) (Fig. 2). Despite available data are slightly controversial regarding the role of otp1 in posterior tubercular and hypothalamic DA neurons development (Ryu and colleagues did not observe a decrease in the number of DA neurons in these areas after otp1 knockdown in wild-type embryos; Ryu et al.,2007), two independent studies converge on the dramatic effect of Otp1 repression on the development of diencephalic DA neurons (Del Giacco et al.,2006; Blechman et al.,2007).

m866 mutant embryos display the same phenotype obtained after otp2 down-regulation. Remarkably, all the affected DA neurons are otp1- otp2-positive, while all the nearby otp1- otp2-negative DA neurons are unaffected, suggesting a cell-autonomous control of the otp-dependent DA phenotype. The coordinate requirement of otp1 and otp2 for diencephalic DA neurons development has been analyzed by performing otp1 translational inhibition in m866 embryos, resulting in a striking enhancement of the mutant phenotype. In the same study, the authors demonstrated that otp2 can determine some aspects of the DA phenotypes by inducing ectopic TH expression (Ryu et al.,2007). From an evolutionary point of view, it is remarkable how the two zebrafish Otp orthologs synergize to perform the functions of the single-copy mammalian Otp. In this regard, it is noteworthy that, unlike Otp−/− mice, the otp2m866 mutants are viable (Acampora et al.,1999; Wang and Lufkin,2000; Ryu et al.,2007). Such difference might be explained not exclusively with the hypothesis of partial redundancy between otp1 and otp2, but also considering the option that otp1 could be more relevant than otp2 in diencephalic neurodifferentiation, as suggested by the significant difference in the timing of expression of the two genes (i.e., otp1 is maternally expressed and characterized by an earlier onset of zygotic transcription in comparison to otp2; Del Giacco et al.,2006; Ryu et al.,2007; Del Giacco and Pistocchi, unpublished data). The availability of a fish carrying a mutation in the otp1 gene will provide the answer to this issue.

Further evidence emphasizes the importance of otp1 for the development of the vertebrate diencephalic DA system. For instance, mot mutants have been originally identified because of a severe developmental deficit involving the monoaminergic neurons and, in particular, the DA subtype (Guo et al.,1999). As mentioned above, med12 is a positive regulator of otp1, as indicated by the partial loss of otp1 expression in the diencephalon of mot mutant embryos (Del Giacco et al.,2006). Interestingly, in addition to mental retardation syndromes and schizophrenia, human polymorphisms in med12 are also associated with positive syndrome psychosis, that are thought to be an outcome of alterations in DA neurotransmission (Philibert and Madan,2007), suggesting that other genes involved in med12-dependent DA neurodifferentiation, such as Otp, might be implicated in human diseases and behavioral disorders.

The essential role of otp1 in DA neuron development is confirmed by the analysis of tofm808 mutant embryos, lacking Fezl activity and characterized by the impairment of DA hypothalamic neurons development, where otp1 expression in the PT area is completely lost (Blechman et al.,2007).

In the past few months, novel insight into the involvement of otp1 in the differentiation of DA neurons has emerged, pointing to the synergistic effect exerted by otp1 and prox1 in DA neuronal development. Indeed, not only prox1 down-regulation determines a decrease in the number of posterior tubercular and hypothalamic DA neurons, but it is also capable of inducing the TH phenotype in concert with otp1. otp1/prox1 mRNA coinjection is responsible for a higher number of DA neurons in the posterior hypothalamic/PT area and induces ectopic TH-positive cells in non-neuroectodermal regions. Interestingly, otp1 mRNA appearance in the hypothalamus precedes the onset of prox1 expression in this area, suggesting that prox1 is not involved in the activation of otp1 transcription. Rather, prox1 activity might be vital for controlling the transition of the otp1-positive cells toward their final DA fate (Pistocchi et al.,2008).

It is worth pointing out that, similarly to fish, where the role of otp1 and otp2 in diencephalic DA neurodifferentiation has been extensively demonstrated (Del Giacco et al.,2006; Ryu et al.,2007; Blechman et al.,2007; Pistocchi et al.,2008), Otp in mouse is required for the development of diencephalic TH-positive neurons (Ryu et al.,2007; Figs. 3, 4). In particular, only the diencephalic neurons of the A11 group, expressing both Otp and TH, were lost in Otp−/− embryos, while the TH-positive Otp-negative neurons of the mesencephalic A8-A10 and diencephalic A12-A15 groups resulted unaffected, suggesting a minor role of murine Otp in the formation of diencephalic DA neurons. Hence, the wider control exerted by Otp in fish DA neurons has been restricted to the sole mammalian A11 group during evolution. Of interest, dysfunction of this specific group of DA neurons has been associated to the Restless Legs Syndrome (RLS), a medical condition characterized by abnormal limb sensations (urge to move), felt mostly at rest, that can severely disrupt sleep (Clemens et al.,2006). A fascinating possibility would be that Otp-induced developmental defects of the DA system might determine the hypofunctioning of the A11 neurons and, by consequence, the onset of this pathological condition.

PERSPECTIVES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. TRACING THE EVOLUTION OF Otp
  5. REGULATION OF Otp DURING HYPOTHALAMIC DEVELOPMENT
  6. Otp ROLES IN HYPOTHALAMIC NUCLEI DEVELOPMENT
  7. PERSPECTIVES
  8. Acknowledgements
  9. REFERENCES

Impairment in the development of the hypothalamus leads to several behavioral disorders and pathologies that dramatically impact on human life. In this review we have focused on the Orthopedia gene and its crucial role in hypothalamic development, as a key reference point to advance into the clarification of normal and altered functioning of the neuroendocrine brain. In this scenario, it is worth pointing out that master genes involved in CNS development, and previously associated with human disorders (i.e., HPE, positive syndrome psychosis, RLS), influence Otp neuronal specification-activity in the hypothalamic region. Even subtle alterations in Otp expression and/or functions might affect the number or type of specific neuronal groups, thus determining pathological conditions with various level of severity. Further investigating the molecular mechanisms that impinge upon Otp activity will improve the elucidation of the developmental foundations of relevant behavioral traits as well as complex clinical disorders of neuronal origin, potentially providing novel diagnostic and therapeutic approaches.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. TRACING THE EVOLUTION OF Otp
  5. REGULATION OF Otp DURING HYPOTHALAMIC DEVELOPMENT
  6. Otp ROLES IN HYPOTHALAMIC NUCLEI DEVELOPMENT
  7. PERSPECTIVES
  8. Acknowledgements
  9. REFERENCES

The authors thank the two unknown reviewers for their careful reading and helpful comments. L.D.G. thanks C. Cattaneo for her critical comments and priceless support.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. TRACING THE EVOLUTION OF Otp
  5. REGULATION OF Otp DURING HYPOTHALAMIC DEVELOPMENT
  6. Otp ROLES IN HYPOTHALAMIC NUCLEI DEVELOPMENT
  7. PERSPECTIVES
  8. Acknowledgements
  9. REFERENCES