Development of the zebrafish hypothalamus - Wiley Online Library

Ann. N.Y. Acad. Sci. ISSN 0077-8923

A N N A L S O F T H E N E W Y O R K A C A D E M Y O F SC I E N C E S Issue: Trends in Neuroendocrinology

Development of the zebrafish hypothalamus Yossy Machluf, Amos Gutnick, and Gil Levkowitz Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel Address for correspondence: Gil Levkowitz, Department of Molecular Cell Biology, The Weizmann Institute of Science, P.O. Box 26, Rehovot 76100, Israel. [email protected]

Hypothalamic neurons regulate fundamental body functions including sleep, blood pressure, temperature, hunger and metabolism, thirst and satiety, stress, and social behavior. This is achieved by means of the secretion of various hypothalamic neuropeptides and neurotransmitters that affect endocrine, metabolic, and behavioral activities. Developmental impairments of hypothalamic neuronal circuits are associated with neurological disorders that disrupt both physiological and psychological homeostasis. Hypothalamic cell specification and morphogenesis can be uniquely studied in zebrafish, a vertebrate organism readily amenable to genetic manipulations. As embryos are optically transparent and develop externally, they provide a powerful tool for in vivo analyses of neurons and their circuits. Here, we discuss the current knowledge regarding the neuroanatomy of the zebrafish hypothalamus and recent studies identifying critical determinants of hypothalamic differentiation. Taken together, these reports demonstrate that the molecular pathways underlying development of the hypothalamus are largely conserved between zebrafish and mammals. We conclude that the zebrafish has proved itself a valuable vertebrate model for understanding the patterning, specification, morphogenesis, and subsequent function of the hypothalamus. Keywords: Danio rerio; Fezf2; Otp; Sim1; hypothalamic disorders; peptidergic neurons

Introduction The hypothalamus integrates multiple sensory stimuli, processes them, and subsequently triggers the systemic responses needed to regain and/or maintain homeostasis. This is achieved by two primary modes of action that operate in parallel within the hypothalamus: direct innervations with synaptic neurotransmission; and secretion of neuromodulators into surrounding tissue or vasculature. The hypothalamus consists of multiple nuclei, each composed of distinct neuronal cell types that form connections with many parts of the nervous system to control a variety of physiological and behavioral functions.1 Abnormalities in the development of the hypothalamus have been associated with adverse neurological conditions, such as depression, chronic stress, autism, and obesity.2,3 If we are to understand the genetic causes of such neurodevelopmental disorders, we must gain a better understanding of hypothalamic development. Deciphering the

biochemical mechanisms underlying neural patterning, specification, and connectivity of hypothalamic circuits will no doubt contribute to our understanding of the selective vulnerability of hypothalamic neurons in neurological disorders that are associated with defects in energy balance, as well as neuroendocrine and psychiatric disorders. Although the physiological and clinical aspects of the hypothalamic system have been intensely studied, relatively little is known about the developmental cues that regulate the embryonic development of the hypothalamus. This is mainly due to a lack of early markers that are specifically expressed in hypothalamic progenitors, the difficulties in studying the migration and specification of neural progenitors in the mammalian brain, and limited knowledge of signals that control progenitor specification. Furthermore, few studies have directly addressed the link between the development of hypothalamic neurons and their function. These questions are now being addressed by employing zebrafish as a genetic model for hypothalamic development in

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vertebrates. The zebrafish, Danio rerio, is a tropical teleost that has emerged in recent years as a valuable and prominent model system for the study of genetic, cellular, physiological, and behavioral aspects of neural development.4–8 Zebrafish provide attractive features as a model organism: relatively low housing costs, rapid development of optically transparent embryos, short generation time, robust reproduction with large progeny size, and amenability to genetic manipulation and to application of pharmacological tools. This review will highlight recent discoveries in the field of hypothalamic development. These studies indicate that, despite the large evolutionary distance between fish and mammals, the overall organization, basic structures, and functional capacities of major hypothalamic components are highly conserved between zebrafish and mammalian brains. Comparative studies between zebrafish and rodents have unearthed both conserved and unique genetic programs governing the specification of and differentiation of hypothalamic neurons. Taken together, these studies reveal a hierarchy of signals that dictate different hypothalamic cell fates in vertebrates and may help to clarify how coordinated development of multiple hypothalamic cell types is achieved. Neuroanatomy of the vertebrate hypothalamus Acting as an interface between the autonomic and endocrine systems, the hypothalamus regulates a

broad spectrum of physiological and behavioral processes, including homeostasis, metabolism, energy balance, circadian rhythms, sexual differentiation, and reproductive behavior in all vertebrates.

Mammals The mammalian hypothalamus is located at the medio-basal region of the brain, ventral to the thalamus and dorsal to the pituitary gland. Its structure has historically been divided into distinct but intricately interconnected nuclei, agglomerations of cell bodies that are visually distinct in histological sections.9,10 During the recent years, efforts have been invested to identify the molecular markers that label major hypothalamic nuclei11 and genetic pathways underlying the development of hypothalamic neurons (reviewed in Refs. 12–14). The neuroendocrine hypothalamus comprises two main types of neurosecretory cells that differ morphologically, form different types of connections to the pituitary, and serve different functions. Magnocellular neurons located in the paraventricular (PVN) and supraoptic (SON) nuclei of the anterior hypothalamus project their axons directly to the posterior lobe of the pituitary (a.k.a. the neurohypophysis), where they release oxytocin (OXT) and arginine–vasopressin (AVP) into the general blood circulation in response to homeostatic cues, such as osmotic, cardiovascular, and reproductive signals.15 In contrast, parvocellular neurons of the PVN and anterior periventricular nuclei control the anterior pituitary (a.k.a. the adenohypophysis)

Figure 1. Neuronal cell types in the embryonic zebrafish hypothalamus. (A) Schematic representation of stereotypical positions of several types of hypothalamic neurons in a 2-day-old zebrafish embryo. (B) Oxytocinergic and dopaminergic neurons in the zebrafish hypothalamus: 2-day-old zebrafish embryos underwent in situ hybridization with antisense RNA probes directed against oxtl to identify oxytocinergic neurons, and then were immunostained with an anti-tyrosine hydroxylase-1 (TH1) antibody to detect dopaminergic (DA) neurons. Both panels show lateral views of the embryo, anterior to the left. DA, dopamine; Oxtl, oxytocin-like; Hcrt, hypocretin; Sst, somatostatin; NPO, neurosecretory preoptic area; PTv, ventral posterior tuberculum; Tel, telencephalon. Scale bar: 50 ␮m. (In color in Annals online.)

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through an indirect mechanism: they project to the median eminence, where they release hypophysiotropic hormones that reach the adenohypophysis via the hypophyseal–portal–vascular system. Among these hormones are corticotropinreleasing hormone (CRH), thyrotropin-releasing hormone (TRH), somatostatin (SST), and dopamine (DA). SST and DA are also produced by neurons in the neighboring periventricular nuclei (PeVN) and arcuate nuclei, respectively.1

Teleosts Although the anatomical organization of the fish hypothalamus does not closely resemble its mammalian counterparts, brains of teleosts contain equivalents to most if not all of the mammalian hypothalamic cell types. Teleostian hypothalamic neurons are all located in stereotypical clusters within the ventral diencephalon (Fig. 1). Numerous studies in zebrafish have mapped the expression of orthologs of many known hypothalamic peptides, including OXT,16 AVP,17 CRH,18 TRH,19 SST,20 hypocretin (Hcrt),21,22 and growth hormonereleasing hormone.23 One major difference between the teleostian and mammalian hypothalamus lies in their mode of connectivity to the adenohypophysis: fish lack an equivalent to the portal blood vessel system that is used in mammals to deliver hormones into the adenohypophysis. Instead, the teleost adenohypophysis is directly innervated by hypothalamic neurons (Fig. 2; Ref. 24). However, the hypothalamic neuronal populations that control the pituitary in fish have been conclusively shown to be functionally analogous to their mammalian counterparts. For example, the physiological activities of the fish neurohypophyseal hormones OXT-like and AVP-like (a.k.a. isotocin and arginine–vasotocin) in controlling water balance and reproduction (egg laying) are clearly conserved (reviewed in Ref. 24), and the teleost hormones CRH and Hcrt (a.k.a. orexin) were found to control stress response and arousal, as in mammals.25–27 Zebrafish as a model for hypothalamic neuronal specification As discussed earlier, the hypothalamus is a complex anatomical structure containing multiple nuclei, each composed of several neuronal cell types that form connections with many parts of the nervous system. This complexity bears the question: how

Figure 2. Magnocellular and parvocellular neurons of the zebrafish hypothalamus. (A–C) Three coronal slices through the diencephalon of a 14-day-old zebrafish embryo, which was subjected to triple immunofluorescence staining for tyrosine hydroxylase-1 (TH1) to detect dopaminergic (DA) neurons, oxytocin-like (Oxtl), and the homeodomain protein Orthopedia (Otp). Oxytocinergic neurons in the NPO and dopaminergic neurons in the PTv send axonal projections to directly innervate the neuro- and adeno-hypophyseal parts of the pituitary, respectively. Scale bar: 20 ␮m. (In color in Annals online.)

are so many types of neurons generated alongside one another simultaneously and with such coordination? Specifically, we lack understanding of the precise cellular processes and lineage relationships that underlie hypothalamic neuronal patterning and specification. In contrast to mammals, zebrafish embryos develop externally and are transparent, and highly amenable to genetic manipulation, making them an ideal vertebrate model for in vivo studies of neural patterning and neuronal specification. As such, zebrafish models have been used extensively in recent years to study the roles played by key signaling pathways in controlling the development of hypothalamic neurons. These studies have already revealed a large degree of cross-species conservation in developing hypothalamic circuits and have shed new light on the activity of key transcriptional determinants of hypothalamic differentiation.20,24,28–35 OXT and DA neurons represent the two major neuroendocrine cell types The mechanism by which coordinated generation of hypothalamic neurons is achieved can be best

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studied by focusing on selected hypothalamic neuronal types. In this respect, zebrafish dopaminergic (DA) and OXT-like (Oxtl) neurons are representative of mammalian parvocellular and magnocellular hypothalamic cell types affecting the adenohypophysis and neurohypophysis, respectively.20 Impairment of these cell types is associated with defects in energy balance, as well as neuroendocrine and psychiatric disorders.2 For example, patients with Prader-Willi syndrome have a deficit in OXT neurons that is accompanied by hyperphagia and severe obesity.36,37 Degeneration and dysfunction of diencephalic DA neurons are implicated in a variety of human diseases including sleep disorders, restless leg syndrome, and hyperprolactinemia.38,39 In zebrafish, DA and Oxtl neurons differentiate in close proximity to one other at approximately the same time and each of these neuronal cell clusters reaches a fixed, stereotypical cell number (Fig. 1; Ref. 40). Within the context of adult physiology, concurrent activation of OXT and DA receptors results in interneuronal communication between DA and OXT neurons, which acts to modulate social behavior, pair bonding, sexual behavior and arousal, anxiety, and stress responses, as well as lactation.15 Thus, generation and maintenance of DA and OXT neurons must be tightly coordinated for the brain to function properly and is essential for the organism’s overall fitness. Zebrafish Oxtl neurons develop in the neurosecretory preoptic nucleus (NPO), which is analogous to the mammalian SON. Zebrafish hypothalamic DA cells develop in several clusters located at the basal plate posterior tuberculum (PTv), adjacent to Oxtl neurons (Fig. 1). Detectable oxtl mRNA is found in zebrafish embryos at around 36 h postfertilization (hpf), and a gradual increase in the number of oxtl-expressing neurons is observed over the course of development. Oxtl cells and their projections, including axons projecting into the neurohypophysis, can be readily detected using antibodies directed against the mammalian OXT peptide (Fig. 2). Development of the DA neurons of the zebrafish diencephalon is somewhat more complicated. DA cell bodies and their projections41–48 can be identified in the embryo and adult by visualizing expression of the biosynthetic enzyme tyrosine hydroxylase (TH), as well as the DA transporter.49,50 The first DA neurons and processes

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become detectable at around 18 hpf in the prospective PTv. Over the course of the following 3.5 days, other major DA groups and tracts become visible throughout the ventral diencephalon. The zebrafish genome contains two nonallelic paralogs of the th gene, th1 and th2. The former has higher sequence similarity to other vertebrate th genes.51 Analysis of their expression patterns indicates that th1 is the predominant paralog in the brain during embryonic and early larval stages.52–54 Careful analyses of development and connectivity of the DA system in teleosts and mammals have revealed many conserved features as well as some distinct differences, the most striking of which is the complete absence of DA neurons from the zebrafish midbrain.41,42,46,48,55–57 This major difference has been attributed to a caudal shift of DA cells during evolution from fish to mammals.58 It has been suggested that the DA neurons in the mammalian midbrain (groups A8–A10) are a more recent acquisition of the avian and mammalian brain.29,30 Recent reports prove that the zebrafish DA neurons that reside in the PTv express known hypothalamic markers that functionally correlate these neurons to the mammalian hypothalamic DA system.20,32,34,35,59–61 Most likely, they are homologous to the mammalian A12 (tuberoinfundibular) and A14 (periventricular hypohyseal/tubero hypohyseal) neurons.29,30 It should be noted that some DA neurons of the PTv that project into the subpallium likely correspond to the mammalian ascending nigrostriatal DA system, suggesting that selected diencephalic DA neurons in zebrafish are functionally homologous to mammalian mesodiencephalic DA neurons.43–45 The roles of extrinsic factors in hypothalamic patterning Progenitor cells acquire their neuronal identity through a hierarchical sequence of events involving lineage commitment, choice of neuromodulator, migration, axonal growth, and survival. This course of events is mediated by both extrinsic and intrinsic signals, which act bidirectionally and in concert to specify neural cell fate.62–64 We briefly review recent studies concerning the involvement of several extrinsic signals in hypothalamic patterning and differentiation.

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SHH Experiments in the mouse, chick, and zebrafish indicate that Sonic hedgehog (SHH) is required for induction and early patterning of the hypothalamus.65–69 Gain of function of SHH in zebrafish was found to induce ectopic expression of hypothalamic markers.70,71 However, the floor plate derived SHH signal may not be required for overall neuronal specification, as hypothalamic DA neurons remain unaffected in the zebrafish mutants sonic you (syu) and smoothened (smu), which harbor null mutations in shh and its coreceptor, respectively.72 Nodal and BMP In zebrafish and mice, the prechordal plate mesendoderm is a source for secreted factors that induce hypothalamic fates. Axial secretion of Nodal and BMP, both members of the transforming growth factor beta (TGF-␤) superfamily, is necessary for hypothalamic induction and regionalization.68,69,71,73 Studies in the zebrafish and chick have provided evidence that Nodal may affect the secretion of Wnt, BMP, and SHH from the prechordal mesendoderm, which may affect the development of specific hypothalamic precursors, such as DA neurons.67,68,72 Wnt Wnt signaling serves multiple functions in the zebrafish forebrain. It acts as a posteriorizing signal driving the fates of the posterior diencephalon and mesencephalon74 and is important for acquisition of hypothalamic versus floor plate identity.75 At later developmental stages, the Lef1 transcription factor, a critical component of canonical Wnt signaling, drives neurogenesis in the posterior-ventral hypothalamus.76 Notably, Wnt signaling restricts the initial pool of DA precursors and thus determines the size of DA cell population but not of neighboring hypothalamic populations.40 Taken together, analyses of various zebrafish mutants have highlighted a requirement of Nodal/ TGF-␤ and Wnt signaling pathways for hypothalamic neuronal specification, as well as general involvement of SHH in hypothalamic patterning. It remains to be determined whether other extrinsic factors, such as fibroblast growth factors (FGFs) or retinoic acid, are involved in hypothalamic patterning and specification.

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Transcriptional networks coordinating hypothalamic specification A key step toward understanding the development of the zebrafish hypothalamus is to identify the intrinsic transcriptional code underlying hypothalamic differentiation. Insights into the transcriptional programs driving the specification of individual hypothalamic neurons have been obtained by analyzing phenotypes and gene expression patterns in mutant embryos and by targeted gene knockdown of candidate regulators of hypothalamic specification. These studies have uncovered networks of regulatory genes that control different stages of hypothalamic development, as well as individual cell fate decisions. Most of the mutations found to affect hypothalamic neuronal specification have been mapped to genes involved in transcriptional regulation: either transcription factors or known cofactors of transcriptional machinery (Table 1). Many of these intrinsic regulatory pathways are required for development of more than one hypothalamic cell type, suggesting a common origin for many hypothalamic neurons. Specific roles of some of these transcription regulators during hypothalamic development are discussed below. Zinc finger protein Fezf2 and the zebrafish hypothalamus The gene encoding a Kruppel-type, zinc fingercontaining transcriptional regulator, called forebrain embryonic zinc finger 2 (fezf2) is disrupted in the too few (tof m808 ) mutant zebrafish.77–79 This mutant displays a dramatic reduction the number of hypothalamic DA, serotonergic (5HT), and oxytocinergic (Oxtl) neurons.79–81 Although the deficit of DA and 5HT neurons in tof m808 mutants is maintained throughout adulthood, the development of Oxtl neurons in the NPO is merely delayed.20 Complete inactivation of Fezf2 leads to more severe developmental defects, suggesting that tof m808 allele is, in fact, a hypomorphic allele that retains residual transcriptional activity.20,82 Experiments in rodents showed that Fezf2 is mainly expressed in subcerebral projection neurons in the cortex, and is required for their specification, development, and axonal projection to spinal cord and brainstem.83–89 Fezf2 is the most abundant transcription factor found in magnocellular cells of the SON, and changes in blood osmolarity led to

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Table 1. Mutations affecting zebrafish hypothalamic neurons

Mutant name

Alleles

Phenotype DA

Oxtl

Avpl

Crh

Trh

Sst

Hcrt

Affected gene

Function

Reference

TM, elongation factor TM, mediator complex TM, mediator complex TF TF TF Atpase, Chaperone

119

foggy (fog)

m806



Spt5

motionless (mot),

m807,



Med12

crsp34m855

m885



Med27

too few (tfu/tof ) otpam866 arnt2m1055 nsf st53

m808 m866 m1055 st53

   –

≤  –

 

 



–  

–



Fezf2/fezl Otpa Arnt2 NSF

120 121 80 32 62 122

–, not affected; , reduced or absent; ≤, delayed development; empty, not determined. TF, transcription factor; TM, transcriptional machinery.

marked alterations in the levels of fezf2 transcript in these neurons.90,91 As Fezf2 is expressed in the developing mouse hypothalamus,11,84,87 it has yet to be determined whether fezf2 is required for the development of hypothalamic neurons in mammals. Apparent differences in the sensitivity of developing Oxtl and DA cells to fezf2 activity seem to correlate with fezf2 expression within hypothalamic progenitors: fezf2 colocalizes with Oxtl neurons in the NPO but does not colocalize with DA neurons in the neighboring PTv.20 However, our understanding of fezf2 activity is further complicated by mosaic genetic analyses of tof m808 mutants that have shown that fezf2 actually regulates hypothalamic DA neurogenesis in a non-cell-autonomous manner.79 It is possible that cell-autonomous versus non-cellautonomous regulation by fezf2 may prove to be the molecular basis for the observed differential coordination of DA and Oxtl neuronal development. Relationships between hypothalamic patterning, Fezf2, and cell number Fezf2 is one of the earliest markers delineating the prospective forebrain, and its expression is tightly controlled by the canonical Wnt signaling pathway.77,92–94 The expression of fezf2 in the prospective diencephalon is enhanced following overexpression of Wnt antagonists, indicating that fezf2 expression is repressed by canonical Wnt signaling.40,92 The

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Wnt8b/Fz8a ligand–receptor interaction downregulates the transcription of fezf2, which is required for DA development. Unlike mouse embryos, in which quantification of cell number is difficult, the small population size in the zebrafish hypothalamus enables accurate analysis of cell number. Russek-Blum et al. took advantage of this trait to quantify the number of individual neuronal cell types.40 This study revealed that each hypothalamic cluster consists of an almost invariant cell number. This finding raises the question of which developmental cues determine and ensure the nearly fixed number of hypothalamic neurons of each cell type and how. Russek-Blum et al.40 demonstrated that canonical Wnt signaling controls DA cell number by acting upstream of the Fezf2 protein. Surprisingly, this study showed that the number of DA cells has already been determined by the end of gastrulation, at the initial stages of primary neurogenesis. Thus, the interplay between the activity of fezf2 and canonical Wnt signaling controls DA population size during early forebrain development. Regulation of hypothalamic differentiation by Fezf2 targets Given the importance of fezf2 for zebrafish hypothalamic development, identifying its downstream targets may provide vital insights into the molecular

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Figure 3. LIM homeobox 5 (lhx5) is required for hypothalamic cell fates. (A) Lhx5 expressed in dopaminergic neurons in the PTv. One-day old embryos were subjected to whole-mount in situ hybridization with probes directed against lhx5 followed by immunostaining with an anti-tyrosine hydroxylase-1 (TH1) antibody to detect dopaminergic (DA) neurons (lateral view, anterior to the left). (B) Targeted knockdown of lhx5 was achieved by injecting 1-cell stage embryos with antisense morpholino oligonucliotides, which targets the translation start site of lhx5 mRNA.100 Similar results were obtained when using splice blocking antisense targeting exon3–intron3 junction (data not shown). Injected and control embryos were fixed two days after injection and subjected to wholemount in situ hybridization with antisense RNA probes directed against oxtl, followed by immunostaining with an anti-TH1 antibody to detect DA neurons. Knockdown of lhx5 led to deficits in both DA and Oxtl neurons (ventral view, anterior to the top). Scale bars: (A) 50 ␮m and (B) 22 ␮m. (In color in Annals online.)

events underlying hypothalamic neuronal specification. Fezf2 was found to affect neurogenesis by controlling the expression of the proneural gene neurogenin 1, which is necessary for zebrafish DA development.82 Similarly, dlx2, which controls specification of ventral thalamic (A13) DA progenitors in the mouse, is regulated by Fezf2.77,93,95 These two Fezf2 targets may mediate early diencephalic commitment of hypothalamic neurons. The homeodomain-containing protein Orthopedia (Otp) is expressed in both DA and Oxtl cells and is a downstream effector of Fezf2.20 Otp is a critical cell-autonomous determinant that controls the fates, migration, and terminal differentiation of mammalian hypothalamic neuroendocrine cells.96,97 A forward genetic screen in zebrafish revealed a null mutant allele, otpam866 , which displays a DA phenotype resembling the aforementioned fezf2 tof m808 deficiency.98 Quantitative differences in Otp protein levels were found to regulate specific differentiation programs that promote distinct dopaminergic and oxytocinergic identities.20 We recently found that Fezf2 regulates the expression of LIM homeobox 5 (lhx5) protein, as expression of lhx5 was markedly reduced in the ventral diencephalon of fezf2-deficient zebrafish embryos (Y.M. and G.L., unpublished data). A similar observation was recently reported in fezf1−/− fezf2−/− double mutant mice.99 Targeted knockdown of lhx5

led to deficits in both DA and Oxtl neurons (Fig. 3). Given that Lhx5 is known to regulate expression of secreted Frizzled-related Wnt antagonists, it might affect DA development by mitigating canonical Wnt signaling, which normally restricts DA cell number.40,100 Otp and Sim1 are evolutionarily conserved neuroendocrine determinants

Otp As outlined earlier, the homeodomain-containing protein Otp is a critical determinant of hypothalamic differentiation. The zebrafish genome contains two otp paralogs, otpa (a.k.a. otp2) and otpb (a.k.a. otp1), which display functional redundancy during hypothalamic development.32 Forward- and reverse-genetic analyses of otp-deficient zebrafish embryos indicate that the two paralogs are both required for the development of DA neurons in the hypothalamus.20,33 Expression of otpa and otpb in the NPO and PTv is regulated by Fezf2, and is detected in the DA progenitor zone as well as in mature DA neurons.20,32,33 Otpb is also required for the development of the magnocellular neurons that secrete Oxtl and Avpl.17,20,31,35 Targeted inactivation of Otp in mice leads to severe deficits in major hypothalamic neuroendocrine cells, including neurons secreting SST, AVP, OXT, CRH, TRH, and DA.96,97,101 These defects are

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associated with reduced cell proliferation, abnormal cell migration, and failure to terminally differentiate. Studies in zebrafish have demonstrated that Otp expression, which is highly conserved in tetrapods,102 is tightly regulated by the developmental growth factors SHH, FGF8, and Nodal.33,60 Although all three factors regulate otp expression in the NPO, only Nodal regulates its expression in the PTv.33 Activity of Prox1, the vertebrate homolog of the Drosophila gene prospero, was also found to be crucial for differentiation of Otpb-positive neuronal precursors.103 Finally, the G protein-coupled receptor PAC1 (a.k.a. adcyap1r1) regulates Otp levels and the development of zebrafish DA and Oxtl neurons by controlling the rate of Otp protein synthesis.20

Sim1 Genetic analyses in mice and zebrafish have unearthed a parallel pathway driving development of the neurosecretory system. The two bHLH-PAS transcription factors, Aryl-hydrocarbon receptor nuclear translocator 2 (Arnt2) and single-minded 1 (Sim1), heterodimerize to specify neurons of the PVN and SON. Consistent with this mode of action, inactivation of either Arnt2 or Sim1 results in elimination of all neurons of PVN, SON, and PeVN, as well as hormone secretion.104–107 Among these neurons, only OXT- and AVP-producing cells are sensitive to Sim1 dosage, whose hemizygosity causes early onset of obesity and hyperphagia in mammals.108,109 Notably, in mammals, Sim1 acts mainly at later stages to control differentiation of postmitotic neurons, although Otp is also required at early stages to regulate proliferation and specification of neuronal precursor cells.96,104 Loss- and gain-of-function studies of sim1 and arnt2 in zebrafish have highlighted several evolutionarily conserved, as well as species-specific features. Sim1 and Otp act in parallel pathways to specify hypothalamic neurons.35,61 Similar to the phenotype observed in otp-mutants, a selective and strong reduction in the number of hypothalamic sim1-positive DA cells is evident in arnt2m1055 zebrafish mutants as well as in sim1 morphants.59,61 Sim1 and arnt2 are required for the formation of the neurosecretory hypothalamus, as expression of mRNA encoding crh, trh, sst, oxtl, and avpl hormones was reduced or lost in the NPO of arnt2m1055

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mutants.17,34,61 In contrast, overexpression of Otp or Sim1 displayed a synergistic effect and lead to supernumerary DA and Crh cells within defined diencephalic competent fields.61 Lineage tracing of defined neural progenitors within the zebrafish neural plate has allowed the creation a fate map of the zebrafish prospective diencephalon including the hypothalamus.68,73,110 A subsequent study of the transcriptional code that instructs these early progenitors to acquire hypothalamic identity revealed that hypothalamic specification requires the sequential activity of pan-neural bHLH protein Olig2, which regulates Sim1 expression in specific hypothalamic progenitors.59 These findings indicate that Olig2, which is expressed in multiple neuronal and glial progenitors, invokes a subtype specification signal that is mediated by Sim1. The regulatory interaction between Olig2 and Sim1 links early hypothalamic neurogenesis with specific differentiation programs. Hypothalamic development through evolutionary lenses It is known that a common ancestor of all teleosts experienced a whole genome duplication event early in evolution, about 350 million years ago.111–114 Evolutionary theory predicts that the redundancy caused by gene-duplication events is often resolved by subsequent mutation and degeneration of one of the duplicated genes, rapidly rendering it nonfunctional. Less commonly, both duplicates are preserved when one acquires a beneficial function whereas the other retains its original function (termed “neofunctionalization”). Preservation of both duplicate genes can also come about when functions and/or expression domains of the gene are partitioned between the duplicates due to degenerative mutations in both (termed “subfunctionalization”).115,116 Understanding hypothalamic development in the zebrafish allows us to identify such functional complementation between duplicates and piece together the evolutionary processes that separated them from one another. This understanding can, in turn, provide us with critical information regarding the role these genes play in both teleost and mammalian development. Analyses of th1 and th2 expression domains reveal only partial spatio-temporal overlap, while function seems to be retained by both proteins, consistent with partitioning of expression patterns.51–53

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Although otpa and otpb share a similar expression patterns, both are coordinately and synergistically required for the development of different hypothalamic DA subclasses,20,32 indicating partitioning of ancestral function within the genetic program. Although sim1a and sim1b are nearly identically expressed in the embryonic diencephalon, only sim1b expression is regulated by arnt2, and it seems that sim1a, but not sim1b, plays a role during zebrafish DA development.61 In fact, the function of sim1b has yet to be determined. Regardless of differential control mechanisms of their transcription, this may argue for nonfunctionalization of sim1b.61 Finally, zebrafish pacap1b, which was shown to regulate otp protein levels and development of DA and Oxtl neurons,20 differs from its paralog pacap1a in both expression pattern and putative functions.117 These examples demonstrate that at least some duplicated teleostian genes acquired changes to their function and/or expression pattern—changes that likely represent partitioning (subfunctionalization) or novel (neofunctionalization) characteristics. As additional examples of these evolutionary processes are uncovered, we may gain new insights into the molecular spatial and temporal requirements of hypothalamic development. Concluding remarks Vertebrate developmental genetics is now flourishing, with the zebrafish playing a key role due to advanced molecular, cellular, and behavioral tools. New data from mutant screens, reverse genetics, transgenesis, and expression pattern analyses have all made valuable contributions to our understanding of genes and their role in embryonic development. Application of these tools has lead to identification of novel genes that are essential for specification and differentiation of the numerous neuronal cell types that make up the vertebrate hypothalamus, and has done much to piece together the intricate networks of interaction between them. A summary of the regulatory gene network driving the development of the zebrafish hypothalamus is presented in Figure 4. It has become evident that all vertebrates share common genetic programs that drive hypothalamic development. Regulatory modules governing DA neuronal development in zebrafish, such as Otp and Sim1/Arnt2, are evolutionarily conserved, suggesting a shared ancestral origin of the mesodiencephalic DA system. Com-

Hypothalamic neuronal specification

Figure 4. Regulatory gene network in the developing hypothalamus. Summary of signaling cascades involved in differentiation of zebrafish hypothalamic neurons showing positive and negative regulatory interactions between a variety of extrinsic and intrinsic factors (see text). Numbers indicate references supporting each interaction; the asterisk refers to evidence presented in this report. Broken arrows signify putative regulatory interactions that have yet to be demonstrated. Arnt2, Arylhydrocarbon receptor nuclear translocator 2; Fezf2, Forebrain embryonic zinc finger 2; Fz8a, Frizzled 8a; Irx, Iroquois homeobox protein; Lhx5, LIM homeobox protein 5; Ngn1, neurogenin 1; Olig2, oligodendrocyte transcription factor 2; Otp, Orthopedia; sFRPs, soluble Frizzled-related proteins; Sim1, singleminded 1.

parative analyses of zebrafish with other vertebrates may provide new insights into the evolutionary processes that helped shape the complex genetics of brain development in all vertebrates. Finally, recent insights regarding the transcriptional code of hypothalamic development may soon prove invaluable to our understanding of hypothalamic function. Several ongoing studies have already begun applying new genetic tools and imaging capabilities to the relatively simple zebrafish hypothalamus to shed much-needed light on the molecular biology of hypothalamic function. Acknowledgments We thank Dr. Jan Kaslin for performing the immunofluorescence staining shown in Figure 2 and

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Ziv Arieli for his help with the figure graphics. The research in the Levkowitz lab is supported by the German-Israeli Foundation (grant number 183/2007), the Israel Science Foundation (grant number 928/08), and the Harriet & Marcel Dekker Foundation. G.L. is an incumbent of the Tauro Career Development Chair in Biomedical Research. Conflicts of interest

18.

19.

20.

The authors declare no conflicts of interest. 21.

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