Sim1, Arnt2 and Otp function along parallel pathways, as they are all required for Sim2 expression in the PVN for the differentiation of the neurons secreting TRH, and in the aPV for the differentiation of the neurons that secrete SST. In the PVN and SON nuclei, these TFs are required for the maintenance of Brn2 expression, a POU domain TF necessary for the development of OXT, AVP, and CRH producing neurons (Schonemann et al., 1995; Michaud et al., 1998, 2000; Acampora et al., 1999; Wang and Lufkin, 2000; Keith et al., 2001; Goshu et al., 2004).

### Steroidogenic Factor 1 (SF-1)

The VMN of the hypothalamus is involved in the regulation of many homeostatic functions, such as the maintenance of energy balance, sexual behavior, anxiety and circadian rhythms (McClellan et al., 2006; Cheung et al., 2013). However, the development of the VMN is less characterized in comparison to the highly studied periventricular zone of the hypothalamus. The finding that SF-1 null mice lack a recognizable hypothalamic VMN, now allows the investigation of neuroanatomical and functional development of this non-peptidergic hypothalamic nucleus. SF-1 is an orphan nuclear receptor (also known as adrenal 4-binding protein; Ad4BP or Nr5a1). It was originally identified as a transcriptional regulator of cytochrome P450 steroid hydroxylases enzymes that are involved in the biosynthesis of steroid hormones (Omura and Morohashi, 1995; Parker and Schimmer, 1995). It is highly conserved both in structure and function throughout evolution (Luo et al., 1994; Achermann et al., 1999; Takase et al., 2000; Allen and Spradling, 2008). SF-1 is expressed in steroidogenic cells in the gonads, adrenal cortex and spleen as well as in the VMN and the anterior pituitary. At E11.0 SF-1 is expressed by diencephalic cells which will form the VMN (Ikeda et al., 2001; Tran et al., 2003). It is expressed as early as E9.0 during embryonic development of the mouse by cells which at later stages, will form the gonads in both sexes.  Notably, SF-1 is important for endocrine cell-fate specification (Lee et al., 2011). Mice lacking SF-1 expression die shortly after birth of adrenocortical insufficiency (Luo et al., 1994; Parker et al., 2002). These knock-out mice do not develop an adrenal gland or gonads. Similar defects in adrenal and gonadal development are also apparent in humans who carry mutations in the sf-1 gene (Luo et al., 1994; Achermann et al., 1999). Disruption of SF-1 also results in structural and neuronal connectivity alterations in the VMN (Shinoda et al., 1995; Tran et al., 2003; Zhao et al., 2008; Cheung et al., 2013). Yet it seems that in the absence of functional SF-1 the initial migration and proliferation of the neuronal precursors remains unaffected, whereas it is important for terminal differentiation of these hypothalamic neurons (Tran et al., 2003). Interestingly, in vitro analysis of various promoters reveals potential SF-1 binding sites in the fezf1, A2bp1, Nkx2-2, Slitrk1 and Slitrk5 genes that are involved in neuronal differentiation and patterning (Kurrasch et al., 2007).

## Receptors and Ligands Regulating Hypothalamic Development

The migration of a specific neuronal type into the hypothalamus, settlement of specific neuronal populations in distinct hypothalamic nuclei as well as their proper connectivity with a variety of target sites further requires extrinsic signals such as growth factors, neuropeptides and their receptors. These act concomitantly with the aforementioned intrinsic TFs to regulate the above processes. Moreover, several recent findings indicate that some neuropeptides, are involved in the development of neural circuits in which they function in the mature hypothalamus. This suggests that at least some neuropeptides act as ''developmental autoregulators''. This section demonstrates the importance of these extrinsic factors for the patterning and assembly of neurocircuits in the developing hypothalamus.

### Extrinsic Developmental Factors

Sonic hedgehog (SHH) which is probably the most characterized morphogen was shown to be crucial for the growth and axial patterning of the hypothalamus (Mathieu et al., 2002; Szabó et al., 2009; Alvarez-Bolado et al., 2012). Moreover, SHH was shown to directly regulate the expression of its cognate receptor Patched 1 (PTCH1) through which it probably signals to promote anterior-dorsal hypothalamic fate and to antagonize Nodal activity in the development of the posteriorventral hypothalamus (Concordet et al., 1996; Koudijs et al., 2008; Szabó et al., 2009). SHH cooperates with Nodal in the maintenance of the anterior-dorsal hypothalamus (Mathieu et al., 2002) and with bone morphogenetic proteins (BMPs) to drive hypothalamic dopaminergic neuronal specification (Ohyama et al., 2005). Genetic disruption of BMP-receptor1a from olig1 cell lineage led to a decreased number of dopaminergic and proopiomelanocortin (POMC) neurons and increased neuropeptide Y (NPY) neurons in the Arc leading to hypophagic phenotype (Peng et al., 2012). Although this genetic manipulation led to increased expression of the orexigenic AgRP, there was a profound impairment in their fiber numbers (Peng et al., 2012). As both Nodal and BMP are members of the transforming growth factorβ (TGF-β) superfamily, this data suggests interaction between SHH and TGF-β signaling pathways during hypothalamic development.

Wnt and its receptor Frizzled are also important regulators of hypothalamic differentiation and Wnt signaling components such as wnt8b, Frizzled8a and Lef1 were shown to regulate the patterning, neurogenesis and differentiation of posterior hypothalamic cells of zebrafish (Kim et al., 2002; Lee et al., 2006; Russek-Blum et al., 2008). In addition, other members of the Wnt cascade were identified in the Arc of the mouse (Benzler et al., 2013). As the expression of SHH, Nodal, BMP and Wnt is maintained in the mature brain it would be interesting to see whether these molecules are released in a synaptic manner, or maintain their activity via a diffusion mechanism in the adult hypothalamus.

### Peptides and Their Receptors

PAC1 (A.K.A ADCYAP1R1) which is the most specific (i.e., high-affinity) receptor for the pleiotropic neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) was shown to regulate the development of zebrafish dopaminergic and OXT neurons by controlling the rate of Otp protein synthesis (Blechman et al., 2007). The anorexigenic peptide Leptin is known to regulate metabolic related homeostatic functions in the hypothalamus through its cognate Leptin receptor (LepR; reviewed by Münzberg and Morrison, 2015). Interestingly, leptin was shown to directly regulate developmental neurite formation of Arc neurons (Bouret et al., 2004). It regulates the neural projections of both orexigenic NPY and anorexigenic POMC neurons (Bouret et al., 2012). This seemingly contradictory effect in which leptin regulates the plasticity of two neuronal populations with opposing effects on energy balance, could be partly explained by the developmental switch in leptin regulation of NPY neurons during the postnatal weaning maturational period (Baquero et al., 2014).

The roles of PACAP, Leptin and their receptors in regulating adult brain and hypothalamic functions were thoroughly investigated in the last decades (reviewed by Blechman and Levkowitz, 2013; Matsuda et al., 2013; Münzberg and Morrison, 2015). The above examples show that these peptidergic systems, which were previously considered as regulators of mature functions, also play a role in the developing hypothalamus. Thus, at least some neuropeptides regulate not only the function but also the development of the systems they control.

## Adult Functions of Developmental Factors

Several key developmental factors have been found to be expressed in the mature hypothalamus, however, their postdevelopmental roles in homeostatic regulation remain elusive. This section presents the current data regarding the adult function of the developmental-related TFs described in previous sections of this manuscript.

### Otp−

It has been suggested that a common ancestor of all rayfinned fish experienced a whole genome duplication event early in evolution, about 350 million years ago (Amores et al., 1998; Vandepoele et al., 2004; Dehal and Boore, 2005; Brunet et al., 2006). As a result, the zebrafish genome contains two Otp genes, otpa and otpb, which share high sequence and expression-pattern homology and present partial redundancy in function (Ryu et al., 2007; Fernandes et al., 2013). Otp expression is maintained in the mature hypothalamus of mouse and zebrafish (Amir-Zilberstein et al., 2012; Herget et al., 2014; **Figure 3**). This suggests that alongside its crucial role in embryonic development, Otp is also involved in adult hypothalamic function. While Otp-null mice die shortly after birth and conditional allele for the gene currently does not exist, the duplication and partitioning of the gene in zebrafish allows both otpa−/− or otpb−/− fish to survive into adulthood (Ryu et al., 2007; Fernandes et al., 2013). This enables the investigation of Otp's role in post-developmental and adult hypothalamic function. Data acquired from adult Otp-null zebrafish, point to the homeostatic activities Otp regulates in adult brains. Adult Otpa-null zebrafish demonstrated impaired anxiety-like behavior compared with their wild-type siblings in response to ''novel-tank'' diving test. Mutant fish spent more time in the top zone of the tank during the first 2 min of the assay suggesting that Otp is involved in the regulation of novelty related stressors (Amir-Zilberstein et al., 2012; Blechman and Levkowitz, 2013). Otp mutant zebrafish also display deficits in the molecular response of the hypothalamopituitary-adrenal (HPA) axis to stressors. These include the regulation of CRH transcription and cortisol response. Thus, Otp directly regulates CRH gene expression in zebrafish and mouse (Amir-Zilberstein et al., 2012). Moreover, Otp indirectly regulates the alternative splicing of the aforementioned PAC1 receptor during stress adaptation phase that follows various homeostatic challenges (Amir-Zilberstein et al., 2012). Thus in addition to its role in hypothalamic development, Otp may act as a cellular sensor, which mediate between a given homeostatic challenge (''input'') and the following hypothalamic hormonal response (''output''). This assumption is reinforced by the finding that Otp-positive neurons in the zebrafish NPO modulate the visual motor response through the regulation of the melanopsin 4a (opn4a) receptor. This apparent Otp-mediated ''non-visual'' deep brain light-sensing system indicates that Otp neurons serve as an extra-ocular photoreception center in dark photokinesis behavior (Fernandes et al., 2012).

Taken together with its known role in regulating several types of neuropeptidergic neurons in the NPO/PVN, these findings suggest that Otp orchestrates the physiological response to environmental challenges. Given the importance of Otp in hypothalamic function, comprehensive research regarding its expression patterns and stress-induced molecular targets in response to physiological and psychological challenges, is further required.

### Sim1−

Sim1 displays haploinsufficiency unveiling its function in metabolic regulation.  Sim1 heterozygotes display normal energy expenditure, and treatment with the melanocortin receptor

Immunofluorescence staining of Otp (red) and oxytocin (OXT) EGFP reporter (Blechman et al., 2011) (green) in a two year-old zebrafish brain. The image shows tiled maximum intensity projection of a mid-sagital

and OXT in the NPO. CC, crista cerebellaris; CCe, corpus cerebelli; NPO, neurosecretory preoptic area; OB, olfactory bulb; Tel, telencephalon; TeO, tectum opticum. Scale bar, 200 µm.

agonist, MTII, increases energy expenditure in both WT and Sim1 heterozygous mice (Kublaoui et al., 2006). This phenotype is further supported by data from humans with balanced chromosomal translocations or genomic mutations, which interrupts the Sim1 gene. In spite of their normal basal metabolic rate, these subjects usually suffer from early onset obesity, increased food intake, and display evidence of neurobehavioral abnormalities (Holder et al., 2000; Ramachandrappa et al., 2013).

Sim1+/− mice display reduced expression of several hypothalamic neuropeptides such as TRH, CRH, AVP, and SST (Kublaoui et al., 2008). In line with their low OXT levels, Sim1 heterozygotes also show higher sensitivity to the orexigenic effect of the OXT receptor antagonist, OVT (Kublaoui et al., 2008). Furthermore, intracerebroventricular administration of OXT to Sim1+/− rescues the hyperphagic phenotype and reduces the characteristic weight increase of heterozygotes. Postnatal chemical ablation of Sim1 expressing neurons leads to hyperphagic obesity and reduced expression of OXT and TRH. However, while Sim1 heterozygotes or post-developmental knockouts display normal energy expenditure, ablation of Sim1-positive neurons leads to decreased energy expenditure (Xi et al., 2012). Postnatal PVN-specific ablation of Sim1 combined with chow diet leads to a hyperphagic obesity phenotype. However, when fed with high-fat diet, the trend is reversed and these mice display reduced food intake and weight loss. Since PVN specific ablation of Sim1 neurons also leads to increased Sim1 expression in the Amygdala, the key regulating region of this phenotype remains to be determined (Xi et al., 2013).

In view of the above data, the authors suggested a model arguing that Sim1 heterozygous phenotype of obesity and hyperphagia occur due to the Sim1 regulatory effect on OXT which is also severely depleted in Sim1 heterozygotes and melanocortin recptor-4 (Mc4r), both of which are known to function in appetite regulation (Kublaoui et al., 2006, 2008; Tolson et al., 2010). The accumulating data further support the fact that at least some of the phenotypes correlated with lack of Sim1 are caused by the perturbation of its mature brain functioning rather than from developmental impairments.

### SF-1−

SF-1 positive neurons are directly involved in the regulation of body weight in the mature brain. This function is mediated, among others, by their responsiveness to the hormone leptin. Hence, specific deletion of leptin receptor in SF-1 neurons results in defects in the ability to maintain normal energy homeostasis and these mice display increased body weight (Dhillon et al., 2006). In addition, there are some indications that SF-1 may directly regulate the expression of the brain-derived neurotrophic factor (BDNF; Tran et al., 2003, 2006), a growth factor which is involved in energy balance (Xu et al., 2003). Yet, it is not clear whether SF-1 indeed regulates BDNF in vivo, and if so—what the physiological significance of this regulation is (Dhillon et al., 2006).

Alongside its role in the maintenance of energy balance SF-1 may be involved in the modulation of the HPA axis in response to stress. SF-1 heterozygous mice develop normal VMN and pituitary, but have defects in their adrenal and in their stress response. These mice display abnormal circulating levels of ACTH and corticosterone during the day and following stressful challenges (Bland et al., 2000). In addition, mice with CNS-specific SF-1 knock-out display anxiety-like behaviors in response to various environmental challenges (Zhao et al., 2008). Beyond the developmental defects that disrupt the normal stress response, in vitro experiments indicate the involvement of SF-1 in the direct regulation of the CRH receptor-2 gene, and in SF-1 knock-out mice there is a marked reduction in CRH receptor expression in the VMN (Zhao et al., 2008).

## Concluding Remarks

The hypothalamus regulates brain and body functions by controlling the activity of a variety of neuropeptidecontaining cell types. By doing so, it allows vertebrates to orchestrate multiple homeostatic processes in order to adapt to the ever-changing environment, thereby maintaining the organism's survival and reproduction. Therefore, mechanistic understanding of the patterning and differentiation of the hypothalamus may further shed light on the function of the mature hypothalamus.

The increased use of zebrafish as a model organism allows the combination of powerful genetic tools with highresolution imaging techniques to advance our knowledge regarding the molecular pathways governing hypothalamic development. One obvious advantage in utilizing both mammalian and non-mammalian models is the ability to gain wider knowledge on evolutionarily conserved regulatory pathways. However, a prerequisite for practical use of the accumulating scientific knowledge is to understand the neuroanatomical organization of the hypothalamus in the key models.

Although the knowledge regarding nucleus-specific markers and TFs continue to expand, many hypothalamic nuclei remain ''uncharted''. Thus, the identification of new regulators of hypothalamic development and function is of fundamental importance. In this regards, the introduction of innovative genome editing methodologies, such as the transcription activator-like effector nuclease (TALEN) or the clustered regularly interspaced short palindromic repeats (CRISPR) genome editing methods now allow efficient genetic manipulation and analysis of new candidate genes (Doudna and Charpentier, 2014; Wright et al., 2014).

Recent findings have demonstrated that some genetic pathways that are involved in hypothalamic development, also play a role in mature hypothalamic functions (summarized in **Table 1**). Thus, key developmental factors maintain their expression in the mature hypothalamus. As developmental perturbation of these genes might lead to lethality, the use of conditional and inducible knockout models is necessary. For example, Sim1 post-developmental knockouts demonstrate the valuable scientific knowledge that can be achieved when using such models, unveiling the highly important role of

#### TABLE 1 | Key functions demonstrated for transcription factors expressed in both developing and mature hypothalamus.

Sim1 in feeding and metabolic regulation in the mature hypothalamus.

### Acknowledgments

We thank Dr. Natalia Borodovsky for her comments on the prosomeric scheme (**Figure 2**). The research in the Levkowitz lab is supported by the Simons Foundation Autism Research

### References

Initiative (SFARI #240085); Israel Science Foundation (ISF); Legacy Heritage Fund for Biomedical Science Partnership; Israel Ministry of Agriculture; Sparr Foundation (in the frame of the Weizmann Research Council); The Henry Chanoch Krenter Institute for Biomedical Imaging and Genomics; The Maurice and Vivienne Wohl Biology Endowment. G.L. is an incumbent of the Elias Sourasky Professorial Chair.

dopaminergic and neuroendocrine cell specification in larval zebrafish. PLoS One 8:e75002. doi: 10.1371/journal.pone.0075002

the hedgehog signaling pathway. BMC Dev. Biol. 8:15. doi: 10.1186/1471- 213X-8-15

Federation (INF) Masterclass Series: Molecular Neuroendocrinology: ''From Genome to Physiology'', eds D. Murphy and H. Gainer (Chichester: Wiley & Sons, Ltd).

**Conflict of Interest Statement**: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2015 Biran, Tahor, Wircer and Levkowitz. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution and reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

## Ontogenesis of peptidergic neurons within the genoarchitectonic map of the mouse hypothalamus

### **Carmen Díaz 1\*, Nicanor Morales-Delgado2 and Luis Puelles 2**

1 Department of Medical Sciences, School of Medicine and Institute for Research in Neurological Disabilities, University of Castilla-La Mancha, Albacete, Spain 2 Department of Human Anatomy and Psychobiology, University of Murcia, School of Medicine and IMIB (Instituto Murciano de Investigación Biosanitaria), Murcia, Spain

#### **Edited by:**

Gonzalo Alvarez-Bolado, University of Heidelberg, Germany

#### **Reviewed by:**

Stuart Tobet, Colorado State University, USA Gonzalo Alvarez-Bolado, University of Heidelberg, Germany

#### **\*Correspondence:**

Carmen Díaz, Department of Medical Sciences, School of Medicine and Institute for Research in Neurological Disabilities, University of Castilla-La Mancha Almansa Street, 14 Albacete 02006, Spain e-mail<EMAIL_ADDRESS>
During early development, the hypothalamic primordium undergoes anteroposterior and dorsoventral regionalization into diverse progenitor domains, each characterized by a differential gene expression code. The types of neurons produced selectively in each of these distinct progenitor domains are still poorly understood. Recent analysis of the ontogeny of peptidergic neuronal populations expressing Sst, Ghrh, Crh and Trh mRNAs in the mouse hypothalamus showed that these cell types originate from particular dorsoventral domains, characterized by specific combinations of gene markers. Such analysis implies that the differentiation of diverse peptidergic cell populations depends on the molecular environment where they are born. Moreover, a number of these peptidergic neurons were observed to migrate radially and/or tangentially, invading different adult locations, often intermingled with other cell types. This suggests that a developmental approach is absolutely necessary for the understanding of their adult distribution. In this essay, we examine comparatively the ontogenetic hypothalamic topography of twelve additional peptidergic populations documented in the Allen Developmental Mouse Brain Atlas, and discuss shared vs. variant aspects in their apparent origins, migrations and final distribution, in the context of the respective genoarchitectonic backgrounds. This analysis should aid ulterior attempts to explain causally the development of neuronal diversity in the hypothalamus, and contribute to our understanding of its topographic complexity in the adult.

**Keywords: hypothalamus, neuropeptides, genoarchitecture, progenitor areas, migrations**

#### **INTRODUCTION**

The hypothalamus is a complex forebrain structure that regulates vital processes and various visceral and somatic behavior. Numerous hypothalamic peptides are involved in modulating such functions. Advances on the molecular mechanisms associated to the ontogeny of peptidergic neurons are helping us

**Abbreviations:** 3v, Third ventricle; A/B, Alar-basal boundary; ac, Anterior commissure; ABas, Anterobasal nucleus; ABasM, Median acroterminal ABas; ABasW, Wing (terminal) portion of ABas; Agrp, Agouti-related peptide; AH, hypothalamic nucleus; AHy, Adenohypophysis; Arc, Arcuate nucleus; Arcc, Core portion of the Arc; ArcM, Median acroterminal arcuate nucleus; Arcs, Shell portion of the Arc; ArcW, Wing (terminal) portion of Arc; Avp, Arginin-vasopressin; ATB, Acroterminal border; BFB, Basal-floor boundary; Cartpt, Cocaine- and amphetamine-regulated transcript prepropeptide; ch, Optic chiasm; CPa, Central portion of the peduncular paraventricular area; Crh, Corticotrophin-releasing hormone; DBLH, Dorsobasal lateral hypothalamic area; DM, Dorsomedial hypothalamic nucleus; DM-P, Peduncular DM; DMcP, Core portion of the peduncular DM; DMcT, Core portion of the terminal DM; DMsP, Shell of the peduncular DM; DMsT, Shell portion of the terminal DM; DM-T, Terminal DM; DPa, Dorsal portion of the peduncular paraventricular area; DPM, Dorsal premamillary nucleus; EPD, Dorsal entopeduncular nucleus; EPV, Ventral entopeduncular nucleus; fx, Fornix tract; Gal, Galanin; Ghrh, Growth hormone-releasing hormone; HDB, to understand developmental defects that cause metabolic and neuroendocrine disorders (Michaud, 2001; Caqueret et al., 2005).

Hypothalamo-diencephalic boundary; Hcrt, Hypocretin/orexin; IHB, Intrahypothalamic boundary; LH, Lateral hypothalamus; LM, Lateral mamillary nucleus; LPa, Lateral paraventricular nucleus; M, Mamillary region; MCLH, Magnocellular lateral hypothalamic nucleus; ME, Median eminence; mtg, Mamillotegmental tract; NHy, Neurohypophysis; Npy, Neuropeptide Y; opt, Optic tract; os, Optic stalk; Oxt, Oxytocin; Pa, Paraventricular hypothalamic complex; PBas, Posterobasal area and nucleus; Pdyn, Prodynorphin; ped, Peduncle (lateral and medial forebrain bundles); Penk, Preproenkephalin; PHy, Peduncular hypothalamus; PM, Perimamillary area; Pmch, Pro-melanin-concentrating hormone; PMS, Superficial perimamillary nucleus; POA, Preoptic area; Pomc, Pro-opiomelanocortin α; PPa, Peduncular part of Pa; PRM, Periretromamillary area; PRML, Lateral

Furthermore, developmental gene expression patterns allowed a molecular characterization of specific areal subdivisions within the alar and basal regions of the hypothalamus. Such regional analysis produces *genoarchitectonic maps* (Ferran et al., 2009; Puelles and Ferran, 2012), which illuminate the variety of molecular mechanisms controlling the specification of differential neuronal fates at each particular hypothalamic subregion. Each neuroepithelial area of the hypothalamus becomes distinct by its unique gene expression profile. As a progenitor domain, it is capable of controlling differentially over time its proliferative and neurogenetic activity, producing specific neuronal populations, and simultaneously generating signals for axonal and neuronal navigation. Different neuron types may originate sequentially at the same domain, due to temporal changes in the local molecular profile. Some neurons migrate tangentially within the mantle layer, invading other hypothalamic domains before they finish developing their phenotype and acquire functional roles within particular circuits.

Tangential migrations from nearby forebrain areas into the hypothalamus, as well as internal migrations, are not exceptional during hypothalamic development (e.g., Keyser, 1972; Alvarez-Bolado et al., 2000; Skidmore et al., 2008; Zhao et al., 2008). Puelles et al. (2012) postulated several other tangential migrations, including distinct cell streams forming the ventromedial nucleus and the ventral premamillary nucleus. Our recent studies likewise suggested widespread tangential migrations of some peptidergic neurons (Morales-Delgado et al., 2011, 2014). As a result, a frequent feature of conventional hypothalamic nuclei is their content of mixed neuron types, using a variety of neuropeptides and neurotransmitters; this intermixing may be functionally relevant (Rhodes et al., 1981; Sofroniew and Glasmann, 1981; Markakis and Swanson, 1997; Broberger et al., 1998; Elias et al., 1998; Sawchenko, 1998; Broberger, 1999; Simmons and Swanson, 2009; Shimogori et al., 2010; Puelles et al., 2012; Tobet and McClellan, 2013).

Puelles et al. (LP, online reference atlases and ontology of the *Allen Developing Mouse Brain Atlas*, 2009; Puelles et al., 2012, 2013) recently updated the prosomeric model, particularly as

part of PRM; PRMM, Medial part of PRM; PRtLH, Prereticular lateral hypothalamus; PSPa, Peduncular part of SPa; PSPa lim, Liminal subarea of PSPa; PTh, Prethalamus; PThE, Prethalamic eminence; RM, Retromamillary region; RPa, Rostral paraventricular nucleus; Rt, Reticular nucleus of prethalamus; RTu, Retrotuberal area; RTuD, Dorsal retrotuberal domain; RTuD sub, Subliminal subarea of RTuD; RTuI, Intermediate retrotuberal domain; RTuV, Ventral retrotuberal domain; SCH, Suprachiasmatic nucleus; SCHs, Shell of SCH; sm, Stria medullaris; SPa, Subparaventricular area; SPall, Subpallium; Sst, Somatostatin; STh, Subthalamic nucleus; Th, Tyrosine hydroxylase; Thal, Thalamus; THy, Terminal hypothalamus; TPa, Terminal part of Pa; TPaC, Central portion of TPa; TPaD, Dorsal portion of TPa; TPaV, Ventral portion of TPa; Trh, Thyrotrophin-releasing hormone; TSO, Terminal supraoptic nucleus; TSPa, Terminal part of SPa; Tu, Tuberal region; TuD, Dorsal tuberal domain; TuD sub, Subliminal subarea of TuD; TuI, Intermediate tuberal domain; TuSbO, Tuberal suboptic nucleus; TuV, Ventral tuberal domain; VBLH, Ventrobasal lateral hypothalamic area; Vip, Vasoactive intestinal polypeptide; VM, Ventromedial hypothalamic nucleus; VMc, Core portion of the VM; VMs, Shell portion of the VM; VPa, Ventral portion of the peduncular paraventricular area; VPM, Ventral premamillary nucleus; ZI, Prethalamic zona incerta.

regards the hypothalamus. It was defined as a bi-neuromeric rostral forebrain territory, lying in front of the diencephalon proper and ventral to the telencephalon (which can be seen as a part of it; **Figure 1A**). The *Mash1/Dlx/Arx/Isl1*-expressing preoptic area was ascribed to the subpallial telencephalon. The neighboring *dorsal* part of the alar hypothalamus—the paraventricular areasingularly expresses the genes *Otp* and *Sim1*. There is thus a clearcut molecular hypothalamo-telencephalic boundary, which is longitudinal (Shimogori et al., 2010; Puelles et al., 2012). At its *ventral* end, the hypothalamus is represented by the mamillary and retromamillary areas, which include at their median plane the *rostral end* of the forebrain floor plate. The hypothalamus has no rostral neighbor, since it represents the rostralmost part of the neural tube. As a consequence, the right and left lateral walls of the neural tube are here continuous one with another *primarily* (from neural plate stages onwards), creating an unique *median* alar + basal subregion recently named "acroterminal area" (Puelles et al., 2012). Terminal lamina, optic chiasma and tuberomamillary midline specializations (e.g., median eminence and neurohypophysis) develop there. The roof plate is telencephalic (partly choroidal and partly septocommissural), and ends at the anterior commissure.

The **Figures 1A–C** illustrate the hypothalamic progenitor domains defined within the prosomeric model, with correlative major derived nuclei, and basic genoarchitectonic patterns. Additional such data will be provided below, as each domain is considered. There is a rostrocaudal partition of the hypothalamus and attached telencephalon into two transverse (neuromeric) parts (hypothalamic prosomeres 1 and 2, or hp1/hp2; Pombal et al., 2009; Nieuwenhuys, 2009; Puelles et al., 2012). To avoid confusion with older terminologies, the resulting two transverse parts of the hypothalamus were renamed as *terminal* and *peduncular* hypothalamus (THy, PHy), referring to the terminal rostral position of the former and to the association of the latter with the course of peduncular telencephalic fibers (Puelles et al., 2012; for genoarchitectonic analysis of these rostrocaudal hypothalamic divisions, see Ferran et al., under review).

THy and PHy are both divided dorsoventrally into longitudinal alar, basal and floor territories, which display differential molecular profiles and various microzonal subdivisions. These longitudinal zones are continuous *caudally* with diencephalic counterparts, being a result of shared dorsoventral patterning. The alar hypothalamus is divided into the *paraventricular area* (continuous with the prethalamic eminence and the prethalamic reticular nucleus), and the *subparaventricular area* (continuous with the prethalamic zona incerta) (TPa, PPa; TSPa, PSPa; **Figures 1A–C**; Puelles et al., 2012). The narrow TPa contains the eye vesicle, the optic stalk and the chiasma. The SPa contains subpially the optic tract.

The basal hypothalamus largely corresponds to the classical *tuberal* and *mamillary* hypothalamic regions. However, these two regions belong exclusively to the voluminous basal THy. The new terms *retrotuberal* and *retromamillary* regions are needed for the corresponding PHy basal territories; accordingly, the tuberal and retrotuberal domains compose a hypothalamic longitudinal column, and the mamillary and retromamillary domains another (Tu/RTu; M/RM; **Figures 1A–C**; note "retromamillary"

#### **FIGURE 1 | Schematic partial prosomeric model of the forebrain representing the general position, morphologic organization and principal nuclear and genoarchitectonic subdivisions of the**

**hypothalamus**. The rostrocaudal (R-C) and dorsoventral (D-V) spatial directions are indicated. **(A)** Schema of the main hypothalamic progenitor areas distributed across the dorsoventral and rostrocaudal dimensions. The longitudinal alar/basal boundary (A/B), and the intrahypothalamic (IHB) and acroterminal (ATB) boundaries are indicated respectively as thick pink, orange and blue lines. The hypothalamic area is subdivided rostrocaudally into neuromeric peduncular and terminal parts (PHy, THy). Alar territories are shown on the left (yellow) and basal territories on the right (blue). The alar hypothalamus is subdivided into the paraventricular (TPa/PPa) and subparaventricular (TSPa/PSPa) areas (each pair of areas refers to THy and PHy components of a longitudinal zone), plus corresponding acroterminal subregions. The paraventricular area shows a general tripartition into dorsal, central and ventral parts (TPaD, TPaC, TPaV, DPa, CPa, VPa). The subparaventricular area appears subdivided into supraliminal and liminal parts (referring to the alar-basal limit; the liminal domain expresses

Nkx2.2). The basal hypothalamus is also divided dorsoventrally into the large tuberal/retrotuberal (Tu/RTu) area and the primary mamillary/retromamillary (M/RM) area, plus the corresponding acroterminal subregions. The THy/PHy parts of the hypothalamic floor lie underneath (white). Moreover, the Tu/RTu region is subdivided into three dorsoventral parts: TuD/RTuD, TuI/RTuI and TuV/RTuV. The TuD/RTuD contains a dorsal subliminal part (which also expresses Nkx2.2). The primary M/RM area is subdivided into a perimamillary/periretromamillary band (PM/PRM), and the underlying secondary M/RM complex proper. **(B)** Map of the main alar and basal hypothalamic nuclei represented upon the diagram shown in **(C)**. **(C)** Schematic color-coded map of the gene expression patterns distributed across the main dorsoventral hypothalamic subdivisions of E13.5 mouse embryos. Varied combinations of gene markers create a characteristic molecular profile for each territory, as represented in tabular form on the right. Genes expressed in more than one dorsoventral domain are grouped in the white area and genes expressed in an unique domain are shown in the blue area. For abbreviations, see the list.

substitutes for the older "supramamillary" term). Tu/RTu further subdivides dorsoventrally into dorsal, intermediate and ventral longitudinal microzonal domains (TuD/RTuD, TuI/RTuI, TuV/RTuV; **Figures 1A–C**). The TuD/RTuD domain corresponds to the classic, precociously differentiated "hypothalamic cell cord' (Gilbert, 1935; Keyser, 1972). The TuI/RTuI contains the ventromedial, dorsomedial and arcuate nuclei, whereas the narrow TuV/RTuV produces the hypothalamic histaminergic neurons (Puelles et al., 2012). The underlying mamillary/retromamillary basal territory also shows a molecularly distinct dorsal microzonal subdivision, the perimamillary/periretromamillary domain (PM, PRM; **Figures 1A–C**; Bardet et al., 2008; Puelles et al., 2012; Morales-Delgado et al., 2014; Allen Developing Mouse Brain Atlas).

This anteroposterior and dorsoventral map of the hypothalamus provides a scenario where the genoarchitectonically characterized progenitor domains of specific peptidergic neuron groups can be precisely identified (**Figures 1C**, **2**). Moreover, jointly with results from knockout and other transgenic mice, this modern hypothalamic scenario potentiates the analysis of patterning mechanisms implicated in neuronal type specification.

### **MATERIAL**

We examined the development of twelve peptidergic cell populations (*Agrp, Avp, Cartpt, Gal, Hcrt, Npy, Oxt, Penk, Pdyn, Pmch, Pomc, Vip*; **Table 1**), analyzed from *in situ* hybridization images downloaded from the *Allen Developing Mouse Brain Atlas*. 1 These are mostly sagittal sections; while this section plane is appropriate for the analysis of potential dorsoventral and anteroposterior tangential migrations, the visualization of some anatomic landmarks may be compromised. We recurred to careful analysis of all sagittal (eventually also coronal) section planes shown at the *Allen Atlas*, as well as to our extensive experience with multiple planes of sections through the mouse hypothalamus. We correlated the positions of peptidergic cells at the time points available at the *Allen Atlas* (embryonic days E11.5, E13.5, E15.5 and E18.5, and postnatal day P4) with the genoarchitectonically distinct areas (**Figure 1C**) and conventional nuclei (**Figure 1B**), following the model of Puelles et al. (2012). We also recorded aspects of areal heterochrony (**Table 2**), and apparent tangential migrations (**Table 3**; **Figures 3**–**5**). Note that

1http://developingmouse.brain-map.org/

#### **Table 1 | Peptide mRNAs mapped in this work**.

Agouti-related peptide (Agrp) Arginin-vasopressin (Avp) Cocaine- and amphetamine-regulated transcript prepropeptide (Cartpt) Galanin (Gal) Hypocretin/orexin (Hcrt) Neuropeptide Y (Npy) Oxytocin (Oxt) Preproenkephalin (Penk) Prodynorphin (Pdyn) Pro-melanin-concentrating hormone (Pmch) Pro-opiomelanocortin α (Pomc) Vasoactive intestinal polypeptide (Vip)

All transcript patterns correspond to the Allen Developing Brain Mouse Atlas database.

any interpretations of "migration" extracted from our descriptive material are necessarily hypothetic, open to experimental testing, though we hold that these conclusions momentarily represent the most parsimonious interpretations of the data. For overview, we added the similar data on *Sst*, *Ghrh*, *Trh* and *Crh* neurons of Morales-Delgado et al. (2011, 2014) in our present **Figure 2**; **Tables 2**, **3**. Our discussion accordingly contemplates sixteen peptidergic cell types, allowing some general conclusions.

### **RESULTS AND DISCUSSION**

**Figure 2** maps the locations where the different peptide markers listed in **Table 1** first appear expressed, adding the results of Morales-Delgado et al. (2011, 2014) and Morales-Delgado (2012). It reveals that given dorsoventrally disposed longitudinal zones dominate as major sources of neuropeptidic populations. These are the Pa area in the alar hypothalamus and the TuD/RTuD and PM/PRM areas in the basal hypothalamus. It is also apparent that multiple peptidergic cell types emerge within these domains, and, remarkably, some cell types appear independently in two or all three of them. A shared characteristic of the molecular profiles of these three areas is the expression of the transcription factor *Otp* (**Figure 1C**; Morales-Delgado et al., 2011, 2014; Morales-Delgado, 2012; Puelles et al., 2012). This was already shown to be required for the differentiation of a number of peptidergic cell types (Acampora et al., 1999; Wang and Lufkin, 2000). A few other progenitor domains, such as SCH within the acroterminal part of alar TSPa, and DM-P, DM-T, and Arc within the basal hypothalamus, give rise to other peptidergic derivatives, in a more restricted mode. On the other hand, there are hypothalamic progenitor domains that so far do not represent sources of peptidergic neurons. These results are also represented in tabular form within **Figure 2** and in **Table 2**.

#### **ALAR HYPOTHALAMIC SOURCES: THE PARAVENTRICULAR AREA**

This area consists of a large, triangular peduncular paraventricular subarea (PPa) defined by diverse deep populations of the *main paraventricular nucleus* (Pa), and a thin terminal paraventricular subarea (TPa), which develops the deep *rostral paraventricular nucleus* (RPa; the conventional "anterior paraventricular area", or aPV), and the subpial *supraoptic nucleus* (PPa; TPa; RPa; TSO; **Figure 1A**). The main Pa is subdivided into dorsal, central, and ventral parts (DPa, CPa, VPa; **Figure 1B**). Puelles et al. (2012) showed that the PPa/TPa essentially contains glutamatergic cells.

The paraventricular area originates peptidergic cell types expressing *Gal*, *Penk*, *Cartpt*, *Avp* and *Oxt* (present data) as well as *Sst*, *Trh*, *Ghrh*, or *Crh* cells (**Figure 2**; Jing et al., 1998; Morales-Delgado et al., 2011, 2014), with timing differences between the peduncular and terminal sectors. Some peptides may coexist in the same neurons (Swanson, 1987). OXT and GAL, e.g., co-localize in neurons of the adult paraventricular and supraoptic nuclei (Landry et al., 1991; see Rossmanith et al., 1996; Foradori et al., 2006; Furutani et al., 2013, and Bartzen-Sprauer et al., 2014 for other examples). As regards its molecular profile, this domain co-expresses *Otp* and interactive *Sim1*/*Arnt2* genes, within a *Pax6*-positive and *Dlx/Arx/Shh/Nkx2.1*-negative background (**Figure 1C**; Puelles et al., 2012). Mice lacking *Otp* or *Sim1*/*Arnt2* functions lost the differentiation of OXT, AVP, SST, CRH, and TRH neurons at the paraventricular and supraoptic nuclei, as well as at the RPa (Michaud et al., 1998; Acampora et al., 1999; Wang and Lufkin, 2000; Michaud, 2001; Caqueret et al., 2005). Unfortunately, *Gal*, *Penk*, *Cartpt* and *Ghrh* cell types were not studied in these reports. *Ghrh* cells possibly were disregarded because in the adult this phenotype largely seems restricted to the arcuate nucleus. However, *Ghrh* cells separately originate at the basal Arc area, and the alar peduncular Pa, whose derivatives secondarily invade massively the basal plate (**Figure 2**; Morales-Delgado et al., 2014). In *Otp* null mice, *Ghrh* cells were apparently absent in the basal areas that receive tangentially migrated alar *Ghrh* elements, while the intrinsic *Ghrh* cells at the tuberal Arc were not affected.

The topographic and rostrocaudal distribution of *Trh-*, *Ghrh-*, *Cartpt-, Sst-*, *Avp-, Penk, Crh*-, *Oxt-* and *Gal*-expressing cells within the Pa highlights dorsoventral microzonal differences, which are most evident at the main peduncular Pa (DPa, CPa, VPa; TPaD, TPaC, TPaV; **Figure 2**). The earliest differentiated paraventricular *Trh, Ghrh, Cartpt, Sst*, *Avp* and *Penk* cells are circumscribed to the VPa/TPaV subdomains, whereas the earliest *Crh*, *Oxt* and *Gal* cells appear restricted to the CPa/TPaC subdomains (**Figures 3A,B,F,G,I,K,L,P,V; Table 2**; see also Morales-Delgado et al., 2011, 2014). Curiously, none of the analyzed peptidergic populations arises primarily in the DPa/TPaD subdomains, though these are secondarily invaded by some of them. A rostrocaudal peculiarity of the distribution of these paraventricular cells is that the peduncular populations (CPa, VPa sources) largely remain close to the ventricle, forming the *Pa* complex (**Figures 3I,J,L,N,O,U–W,Y**), though there are relatively more superficial elements in the LPa nucleus (radially superficial to VPa) and in the *dorsal entopeduncular nucleus* (EPD; interstitial within the cerebral peduncle) (**Figures 3F,G,S**). In contrast, the terminal paraventricular derivatives (TPaC, TPaV sources) largely eschew the deep RPa (aPV), excepting the *Sst* cells, which are abundant there, and mostly aggregate instead within the subpial *terminal supraoptic nucleus*(TSO; **Figures 3G,H,J',K,M,X**). Others incorporate into a conspicuous cell stream that migrates early on into the tuberal area, forming the subpial *tuberal suboptic nucleus* (TuSbO; this new term corrects the misleading one

Light gray areas are devoid of peptidergic cells, whereas dark gray areas produce two or more types simultaneously. Orange background: Alar domains. Reddish background: Basal domains. Bold letters: peptidergic cells studied in Morales-Delgado et al., 2011, 2014; Morales-Delgado, 2012. All other peptidergic cell types were analyzed from Allen Developing Mouse Brain Atlas datasets.

#### **Table 3 | Alar/basal topography of the different peptidergic cell populations, according to their primary progenitor sources and their perinatal distribution, after tangential migration**.

Bold tags: populations that move from alar into basal domains. Asterisks: populations that move within their primary domain.

"tuberal supraoptic nucleus"; Puelles et al., 2012), and the related deeper *tuber cinereum* (TCi) cell population (**Figures 3H,J,L,M,X**;

see TuSbO and TCi in Puelles et al., 2012; their Figures 8.27, 8.32, 8.33).

*Trh* cells first appear in the VPa at E10.5, and later are found also in the TPaV (E12.5), as well as in the CPa and DPa subnuclei. In the adult rat *Trh* cells were recently described likewise in the tuberal lateral hypothalamus (Horjales-Araujo et al., 2014). *Ghrh* cells also have early E10.5 origins in the VPa and appear in the TPaV at E12.5 (**Table 2;** Morales-Delgado et al., 2014). Some of these *Ghrh* and *Trh* elements then move tangentially ventralwards into basal territories, such as the PBas/ABas, the TuSbO nucleus, and the shells of the VM and DM-P nuclei (Morales-Delgado et al., 2014; their Figure 13). *Cartpt* cells were first identified at VPa at E11.5 (where they persist at P4) and appeared at the TPaV at E13.5 (**Figures 3A,B**; **Table 2**). Some peduncular elements disperse into neighboring domains underneath, such as the alar PSPa and the basal PBas (compare **Figures 3A,C**), while terminal elements seem to migrate into the alar TSPa (**Figures 3B**, **5D**). In newborn mice, *Cartpt*-expressing cells were described as associated to *Lhx6* expression in a population at the ventral part of the PSPa (Shimogori et al., 2010); these might correspond to our migrated *Cartpt* elements.

*Sst* cells emerge within VPa at E12.5 and within TPaV at E13.5, where they largely remain visible, though the deep elements of the terminal RPa possibly disperse partly into TPaC

2http://mouse.brain-map.org

and TPaD (Morales-Delgado et al., 2011; *Allen Developing Mouse Brain Atlas*). Singularly, *Avp* cells were first observed at the superficial LPa/EPD nuclei (peduncular Pa domain) at E13.5; deeper cells appear at the VPa and TPaV at E15.5 (**Figures 3F–I**; **Table 2**). Later, *Avp* cells move from VPa into CPa, where they subsequently predominate (**Figures 3J**, **5C**; *Allen Developing Mouse Brain Atlas*), and from the TPaV (E15.5) into the shell of the suprachiasmatic nucleus (SCH) and the TuSbO; in this case, *Avp* cells nearly disappear from their ventral paraventricular sources postnatally (**Figures 3H,J,J"**, **5C**; **Table 2**). *Penk* cells likewise appear in VPa at E13.5 and TPaV at E18.5 (**Figures 3P,R**); the VPa ones remain at this location thereafter, whereas the TPaV counterparts seem to invade the SCH shell (**Figures 3S,T**, **5E**).

3http://mouse.brain-map.org

migrations are indicated with a question mark. Filled red ovals identify progenitor areas where the relevant peptidergic cells persist and

abbreviations, see the list.

population at its source, as well as at individual recipient areas. For

On the other hand, among the peptidergic populations originated at the central portion of the peduncular Pa, *Crh* cells were first detected there at E13.5 (Morales-Delgado et al., 2014), whereas *Oxt* and *Gal* cells made their earliest appearance at E15.5 (**Figures 3L,V**; **Table 2**).  From E18.5 onwards some peduncular *Oxt* cells appear also in the VPa (and the LPa cell group, which appears radially displaced relative to the VPa), and other cells seem to enter the DPa (compare **Figures 3L,N,O**, **5A**); terminal *Oxt* cells become ventrally displaced into the SCH shell and the TuSbO nucleus (**Figures 3L,M,O**, **5A**), jointly with some terminal *Gal* cells (**Figures 3X**, **5B**). *Oxt* cells also appeared at the TSO at E15.5, while a less dense *Gal* cell population was found there from E18.5 onwards (**Figures 3K,M,X**, **5A,B**; **Table 2**; Jing et al., 1998). The body of terminal paraventricular cells migrating into the TuSbO is observable as a characteristic cellular arch passing dorsoventrally deep to the optic tract, extending from the alar TSO into the basal TuSbO between E11.5 and E13.5. We only illustrate it for *Avp* cells (**Figures 3H**, **5C**). In the adult hypothalamus, *Gal* and *Crh* transcripts in mice (and CRH peptide in rats) are still concentrated mainly at the CPa, with a minority of cells dispersed into VPa and DPa and the cited basal areas; this indicates that migratory dispersion of these elements is limited (*Allen Developing Mouse Brain Atlas*; compare Figure 10 of Swanson, 1987, and Figure 4 of Simmons and Swanson, 2009; note these authors use the old columnar axis ending in the telencephalon; they accordingly describe as "rostral" and "caudal" our prosomeric "dorsal Pa" and "ventral Pa" positions; Puelles et al., 2012).

We mentioned above that the transcriptional functions of *Otp* and *Sim1/Arnt2* are required for the development of at least some of these paraventricular peptidergic neuron types (Michaud et al., 1998; Acampora et al., 1999; Wang and Lufkin, 2000; Michaud, 2001; Goshu et al., 2004). However, the partially overlapping domains of expression of these genes across the peduncular and terminal Pa area do not seem sufficient to explain the above described dorsoventral dissociation of central and ventral paraventricular progenitor subdomains in the paraventricular complex. Some of these differential effects can perhaps be attributed to the *Brain-2* (*Brn2*), and *Sim2* genes, which seem likewise involved in the differential specification of peptidergic cell lineages in this region. See below for data on *Sim2*. The *Brn2* and *Sim1* expression domains selectively overlap within the CPa domain at E12.5 and E13.5, whereas VPa only expresses *Brn2*, and DPa only displays *Sim1* signal (Figure 7 of Michaud et al., 1998; note their columnar rostrocaudal axis again corresponds to our prosomeric dorsoventral axis).

The reported observation that *Cartpt* cells are diminished in number in *Sim1*−/− mice at E12.5 (Caqueret et al., 2006) is difficult to understand, since these cells apparently originate at the VPa, where *Sim1* is not expressed. Moreover, in newborn mice, BRN2 protein co-localizes with *Crh*-, *Avp*- and *Oxt*-expressing cells, which occupy CPa, but not significantly with *Trh* cells, which are produced at the selectively *Brn2*-expressing VPa area (Schonemann et al., 1995; Morales-Delgado et al., 2014). These two apparently inconsistent results may be explained by our earlier (Morales-Delgado et al., 2014) and present data indicating that the *Trh* and *Cartpt* cells born at the VPa normally migrate into the retrotuberal basal plate. The reported absence of *Cartpt* cells in the Pa complex of *Sim1*−/− mice (Caqueret et al., 2006) may be spurious, if these cells were produced normally (no *Sim1* normally at the VPa), and simply migrated away. The same interpretive error perhaps occurred with *Trh* cells with co-localized BRN2 protein, which probably were not searched for where they lie *after migration*. Moreover, in *Brn2-*null mice, no *Crh*, *Avp* and *Oxt* cells were detected in the Pa and TSO nuclei, while the *Sst* and *Trh* mRNA expression in VPa/TPaV was unaffected (Schonemann et al., 1995).

Conversely, *Trh* and SST cells coincide with the alar expression domain of *Sim2*, which is largely restricted to VPa/TPaV, while there is no topographic correlation between the *Sim2* expressing domain and *Crh-*, *Avp-* and *Oxt-*positive cells in neonatal mice (Goshu et al., 2004). As expected, the number of *Trh* and SST cells was reduced in *Sim2* null mice, whereas *Crh*-, *Avp*- and *Oxt*-expressing cells were not affected (Goshu et al., 2004).

In summary, the development of *Crh*, *Oxt* and *Avp* cells within CPa seems to occur under control of *Otp* and *Sim1/Arnt2*, probably associated to maintained *Brn2* expression. In contrast, *Trh* and *Sst* cells, and probably also *Cartpt* cells, emerge in the VPa/TPaV, controlled by overlapping signals of *Otp*, *Brn2* and *Sim2* (in absence of *Sim1*). Unfortunately, no experimental data are available with respect to the possible defects in the distribution of alar *Ghrh*, *Cartpt, Penk* or *Gal* cells in the *Otp*, *Sim1*, *Sim2* and *Brn2* mutants.

Additional spatially restricted signals possibly are also implicated in the fate determination of these and other peptidergic cell lineages in the paraventricular area. For instance, CPa strongly expresses *Dickkopf 3* (*Dkk3*) from E11.5 onwards (see *Allen Developing Mouse Brain Atlas*), making this transcription factor another candidate bearing on the differentiation of CRH, galanin and/or OXT cells.

#### **ALAR HYPOTHALAMIC SOURCES: THE SUBPARAVENTRICULAR AREA**

The subparaventricular domain (TSPa/PSPa; Puelles et al., 2012) is primarily negative for *Otp*/*Sim1*, and positive for *Dlx*, *Arx, Isl1, Lhx6* and *Vax1* (**Figure 1C**; Shimogori et al., 2010; Puelles et al., 2012); the early expression of *Arx* is subsequently downregulated, whereas *Dlx* family signals remain expressed into perinatal stages, associated to the differentiation of gabaergic neurons in the entire domain (Hallonet et al., 1998; Shimogori et al., 2010; Puelles et al., 2012; *Allen Developing Mouse Brain Atlas*). This domain produces the classic anterior hypothalamus (massively developed at the TSPa, compared to the thin PSPa); the derivatives include the periventricular subparaventricular nucleus, the anterior hypothalamic nucleus, and the acroterminal suprachiasmatic nucleus (**Figure 1B**; Puelles et al., 2012). The latter selectively shows *Vip*-, *Avp-* and *Oxt-*expressing cells (**Figure 2**), detected from E18.5 onwards (SCH; **Figures 3D,E,J,J',O**; **Table 2**; *Allen Developing Mouse Brain Atlas*). Jing et al. (1998) and VanDunk et al. (2011) reported *Avp* mRNA at the mouse SCH already at E16.5 and E17.5, respectively. At P4, *Avp* and *Oxt* cells were more abundant than *Vip* cells, the latter being restricted to a superficial SCH locus (SCHs; **Figure 3T**). In the adult, VIP cells largely aggregate at the gabaergic SCH core (Moore and Speh, 1993; Castel and Morris, 2000), while the VP/OXT cells lie within the glutamatergic SCH shell (Silverman and Pickard, 1983; Sofroniew, 1985; VanDunk et al., 2011; Puelles et al., 2012; *Allen Developing Mouse Brain Atlas*).

Due to its acroterminal topography, the SCH primordium is distinct from its nearby subparaventricular neighbor, the anterior hypothalamic nucleus, in that it selectively expresses *Six3*, *Six6, Ror*α and *Lhx1*, a profile that persists in the adult mouse SCH, with *Ror*α in the shell and *Lhx1* in the core; transient selective expression of *Nkx6.2*, *Fzd5* and *Rx* occurs as well in this area (Conte et al., 2005; Shimogori et al., 2010; VanDunk et al., 2011; Puelles et al., 2012). Differential programming of neurogenesis at this rostral SPa locus is therefore to be expected. *Ror*α signal progressively becomes restricted to the SCH shell postnatally, with a distribution overlapping that of *Avp/Oxt* cells. However, neither *Avp* or *Vip* expression was affected in *Ror*α null mice (VanDunk et al., 2011). With respect to *Six3*, this gene first appears widely expressed in the rostral neural plate, down to the prospective isthmo/mesencephalic border (Oliver et al., 1995; Bovolenta et al., 1998; Kobayashi et al., 2001; Sánchez-Arrones et al., 2009; Dutra de Oliveira Melo, 2011; VanDunk et al., 2011). Afterwards, *Six3* is progressively downregulated at its caudal end; from E11.5 onwards, *Six3* appears restricted to the terminal hypothalamic prosomere (hp2; Puelles et al., 2012) (*Allen Developing Mouse Brain Atlas*; Dutra de Oliveira Melo, 2011; VanDunk et al., 2011). Complete *Six3* inactivation stunts the whole rostral prosencephalon (telencephalon and hypothalamus; Carl et al., 2002; Lagutin et al., 2003; Lavado et al., 2008). A recent study using a *Nestin*-Cre transgenic line to limit floxed *Six3* loss to neural progenitors produced unexplained variable results at E15.5–E19.5. Some specimens showed absence of *Ror*α and *Avp* expression specifically at the SCH, while *Avp* expression continued to be present in the neighboring RPa and the supraoptic nucleus (TPa derivatives) (VanDunk et al., 2011).

The recent developmental study of VanDunk et al. (2011) clearly represented a significant advance in our understanding of this specialized area. We argue nevertheless that their columnar descriptions would benefit from a translation into prosomeric terms, in so far as prosomeric theory allows fine dorsoventral and anteroposterior regionalization and description of the hypothalamus, whereas columnar theory does not; that is, there is no precise columnar answer to the question ¿which part of the hypothalamic primordium is occupied by the suprachiasmatic nucleus? In contrast, its position within the prosomeric genoarchitectonic map and the framework of possible signaling mechanisms is clearcut in **Figure 1B**. In spite of the VanDunk et al. (2011) analysis, the cascade of regulators involved in the local differentiation of *Avp* and *Oxt* cell lineages remains unclear (in comparison with the origin of such cells in an *Otp*-expressing molecular background), and so does the mechanism that segregates *Avp/Oxt* cells into the SCH shell (whose glutamatergic profile surprises within the SPa); *Vip* cells unproblematically settle within the SCH gabaergic core. Though VanDunk et al. (2011) concluded there is a single neuroepithelial source for all SCH neurons, experience accrued so far elsewhere in the brain suggests that the same progenitors normally do not produce both glutamatergic and gabaergic cells, leaving apart exceptional observations of neurons displaying both neurotransmitters, whose progenitor mechanisms remain unknown (e.g., Jarvie and Hentges, 2012). Parallel sources for these two components, at the TPa or ABasM, and the TSPa, respectively, should perhaps be considered.

Puelles et al. (2012) conjectured that the vasopressinergic and oxytocinergic cell types (and other glutamatergic cells) of the SCH shell might originate in the suprajacent acroterminal TPa progenitor area, which also expresses *Six3*, and is characterized by *Otp*/*Sim1/Brn2* expression (plausibly a necessary genetic background for these peptidergic phenotypes; note the underlying ABasM area also expresses *Otp* in conjunction with basal markers). According to this hypothesis, prospective *Ror*α, *Avp* and *Oxt* shell components might migrate tangentially at early stages from the acroterminal TPa into the subjacent SCH primordium within TSPa (**Figures 5A,C**). *Ror*α expression is most dense next to the TPa/TSPa boundary in sagittal sections, and the early *Ror*α cells form a marginal stratum covering early *Lhx1* cells, supporting such a nearby source and migration mechanism (Figures 2B, 4 of VanDunk et al., 2011). The variant hypothesis may be contemplated that prospective SCH *Avp/Oxt* cells are produced within the TPa, and a combination of *Six3* with specific SPa markers (*Dlx genes, Arx, Isl1, Vax1*) and/or selective SCH regional markers (*Six6, Ror*α*, Lhx1, Nkx6.2*, *Fzd5*, *Rx)* defines a SCH domain that selectively *attracts* these *Avp/Oxt* cells from the TPa into the incipient SCH shell subregion (question marks in **Figures 5A,C**).

#### **BASAL HYPOTHALAMIC PEPTIDERGIC CELL SOURCES**

As mentioned above, the basal hypothalamus is primarily divided dorsoventrally into the tuberal/retrotuberal (Tu/RTu) and the primary mamillary/retromamillary (M/RM) regions (**Figure 1**). The Tu/RTu territory is subdivided into dorsal, intermediate and ventral subdomains (TuD/RTuD; TuI/RTuI; TuV/RTuV; Puelles et al., 2012). The TuD/RTuD produces glutamatergic cells for the periventricular anterobasal and posterobasal nuclei (ABas, PBas), plus some intermediate populations such as the nucleus of the TCi, the magnocellular lateral hypothalamic nucleus (MCLH), and several ventralwards migrating populations, including the massive ventromedial nucleus (VM; Puelles et al., 2012); ABas can be further subdivided into acroterminal/median—AbasMand wing—AbasW- portions. Leaving apart the migrated ventromedial nucleus, the TuI/RTuI contains intrinsic gabaergic populations belonging to the terminal and peduncular parts of the dorsomedial nucleus *shell domains* (DMsT, DMsP), as well as to the acroterminal arcuate nucleus *shell* (Arcs); these formations all have glutamatergic *core* cell aggregates whose presumptive migratory origins are under study (Puelles et al., 2012). In addition, there exists the glutamatergic ventral premamillary nucleus, which migrates from the retromamillary area into DMsT (VPM; **Figure 1B**; Puelles et al., 2012). The remaining thin TuV/RTuV domain is related to production of the hypothalamic histaminergic neurons (Puelles et al., 2012). On the other hand, the primary M/RM area subdivides into the *Otp/Sim1*-positive perimamillary/periretromamillary band, and the secondary M/RM complex proper, both of which produce exclusively glutamatergic neurons.

#### **BASAL HYPOTHALAMIC SOURCES: THE DORSAL TUBERAL AND RETROTUBERAL AREAS**

The acroterminal part of the anterobasal area (ABasM; TuD), where *Six3* is selectively expressed, develops an early *Otp*-positive cell population, some of whose elements differentiate into diverse peptidergic cell types (e.g., Morales-Delgado et al., 2011). Jointly with the peduncular RTuD, the TuD subdomain is characterized molecularly by the expression of general basal markers such as *Shh* and *Nkx2.1*, and more restricted expression of other markers such as *Vax1*, *Lmo3*, *Enc1*, *Vat1l*, and *Cnr1* (**Figure 1C**; *Allen Developing Mouse Brain Atlas*). A subliminal dorsal section of this domain also expresses *Nkx2.2* (Puelles et al., 2012; their Figure 8.14B).

The first peptidergic cell types detected at the ABasM are *Sst* and *Pomc* cells, first detected at E10.5 and E11.5, respectively (ABasM; **Figure 4A**; **Table 2**; present data; McNay et al., 2006; Morales-Delgado et al., 2011). *Cartpt-*, *Penk*- and *Npy*expressing cells next appear throughout ABas at E13.5 (ABasM and ABasW; TuD; **Figures 3B,P**, **4D**; **Table 2**), whereas *Pmch* cells first appear at E11.5 at the PBas (RTuD; not shown; see *Allen Developing Mouse Brain Atlas*; Croizier et al., 2011), and *Sst*, *Penk, Npy* and *Hcrt* cells next emerge there between E13.5 and E15.5 (**Figures 4D,H**; **Table 2**). The peptidergic cell types mentioned at the ABas differentiate as subtypes within the preexistent abundant population of *Otp/Nkx2.1*-positive postmitotic neurons, whereas, in contrast, the peduncular PBas area contains few *Otp/Nkx2.1* cells (**Figures 1C**, **2**; Morales-Delgado et al., 2014). At subsequent stages, peptidergic cells like those of the ABas appear to invade the subjacent tuberal (acroterminal) arcuate nucleus, as well as the shell of the migrated VM nucleus, which also originates from the TuD area (**Figures 5D–G**; Morales-Delgado et al., 2011; Puelles et al., 2012). As is further discussed below, we conclude that ABas is the source of these topographically tuberal *Sst*, *Cartpt, Penk*, *Pomc*, and *Npy* cells (which also are accompanied by migrated *Otp*-positive cells). In *Otp*-null mice, cells expressing *Sst* mRNA or producing SST peptide were absent in the Arc and in an "adjacent area", probably the VM shell (Acampora et al., 1999; Wang and Lufkin, 2000). It would be interesting to analyze the other peptidergic cell populations originated at ABas/PBas in these mice, in order to check their dependency on *Otp* expression and function.

The ABas domain expresses additionally *Nr5a1* (*SF1*) at E9.5–E11.5 (Figure 7 of Ikeda et al., 2001), coinciding with the appearance of the earliest *Sst* and *Pomc* cells (**Table 2**; Shimogori et al., 2010; Morales-Delgado et al., 2011). SF1- and POMCimmunoreactive neurons overlap at the acroterminal ABasM at E10.5 (Figure 1C of McNay et al., 2006; these authors identified it as "retrochiasmatic nucleus", which is a classic synonym for ABas). Subsequently, at 13.5, *Nr5a1* (*SF1*) labels also the AbasW, as well as a VM subpopulation that moves massively ventralwards into TuI (see *Allen Developing Mouse Brain Atlas*; Puelles et al., 2012). Separately, *Pomc* labels since E12.5 a parallel dorsoventral migration from the acroterminal ABasM into the acroterminal Arc area (**Figures 4B,C**, **5G**; Shimogori et al., 2010; Puelles et al., 2012; *Allen Developing Mouse Brain Atlas*). These migrations thus start selectively at the dorsal tuberal ABasW and ABasM subareas, respectively. A number of *Cartpt*, *Sst*/*Otp* and *Npy* cells accompany both migrations, and thus reach as a common terminus the VM and Arc shell regions (TuI; **Figures 3C**, **4D',E–G**, **5D,F**; Morales-Delgado et al., 2011). In murine *Nr5a1* knockout mutants, the distribution of cells expressing NPY, GAL and estrogen receptor α was altered, but the differentiation of these phenotypes was unaffected (Dellovade et al., 2000). The VM nucleus itself appeared subtly altered in this mutant, with partial fading of its normally sharp boundary, allowing some intermixing of peripheral GAD67 cells, as well as disgregated GFP-labeled SF1-/- neurons and redistribution of other components (e.g., ISL1, BDNF, NKX2.1, NPY immunoreactive cells; Dellovade et al., 2000; Tran et al., 2003; Davis et al., 2004). On the whole, these studies suggest that the final topography of the diverse peptidergic or non-peptidergic neuronal types within and surrounding the VM is altered as a consequence of the dismorphogenesis caused by *Nr5a1* mutation. It may be speculated that other peptidergic cell types originated at the ABas may be also partially relocated in the *Nr5a1*-null phenotype.

In the adult rat, NPY cells are generally ascribed to the Arc, plus transient *Npy* expression at other sites. Our data in the mouse indicate there is in addition early appearance of *Npy* cells within the ABas and PBas areas at E13.5 (**Figures 4D,D'**). Subsequently some of these *Npy* cells migrate into the intermediate tuberal and retrotuberal areas, targeting the Arc and DM nuclei, and the shell of VM (**Figures 4E–G**, **5F**). These migrated elements may correspond with the reported rat dorsomedial, perifornical and lateral hypothalamic *Npy* cells (Singer et al., 2000; Grove et al., 2001).

*Cartpt* cells first appeared at ABas at E13.5, with an apparent later expansion of the source into the peduncular PBas, and subsequent dispersion into terminal and peduncular Tu/RTu regions such as the VM shell and the DM shell (**Figures 3B,C**, **5D**), consistently with Koylu et al. (1997), Broberger (1999), Vrang et al. (1999), and Elias et al. (2001). *Cartpt* cells were first observed at the Arc at P14. We also observed *Cartpt*-positive cells in the mammillary area from P4 to P56, a localization which was not previously reported (M; **Figures 3C**, **5D**; *Allen Developing Mouse Brain Atlas*). This late *Cartpt* cell population may be interpreted either as due to cell dispersion from TuI, with a more dorsal origin (TuD), or as late timing of the expression of the marker.

*Mash1* (*Ascl1*) codes a transcription factor, whose expression generally overlaps and parallels in regional intensity that of *Dlx* genes (SPa ventricular and mantle zones in the alar hypothalamus, and the ventricular zone of the whole basal Tu/RTu region). In the absence of *Mash1* function, a general reduction of cell numbers was observed at the VM and Arc nuclei, affecting the POMC, NPY, GHRH and dopaminergic lineages (McNay et al., 2006). At E10.5 there is already a dramatic reduction in the number of SF1/MASH1 and POMC cells at the ABas, whereas at later stages migrated SF1 (*Nr5a1*) cells in the VM, as well as migrated NPY cells and intrinsic dopaminergic neurons in the Arc are significantly reduced (McNay et al., 2006). As regards the differentiation of GHRH cells at the Arc, see comments below on requirement of *Msh1* and *Gsh1* functions.

The early restricted expression of *Pomc* at the ABasM (acroterminal TuD) without any additional expression at the ABasW and PBas may be related to the local restricted signal of *Six3* (or other genes mentioned above) within the acroterminal area. *Six3* is reportedly necessary for *Shh* activation in the terminal hypothalamus (Geng et al., 2008). General basal *Shh* and *Nkx2.1* expression is necessary for the development of the tuberal hypothalamic regions, though, curiously, the ABas itself is not affected in mice lacking *Nkx2.1* function, whereas the Arc and VM nuclei are absent or abnormal (Kimura et al., 1996). Moreover, the *Neurog3* (*Ngn3*) gene is expressed selectively at ABas as early as E9.5 (described as "Arc/VM region of the hypothalamus" by Pelling et al., 2011). *Mash1* apparently acts upstream of *Ngn3* to regulate neurogenesis in the basal hypothalamus (McNay et al., 2006). Pelling et al. (2011) showed in *Ngn3*-Cre mice that a set of POMC, NPY, TH and SF1 cells originate from *Ngn3* progenitors, presumably at the ABas area. This result suggests that the arcuate TH-positive population of dopaminergic cells may also migrate tangentially down from the ABas domain (an idea never proposed before, but presently corroborated by our finding of ABas *Th*-expressing cells at E11.5 (*Allen Developing Mouse Brain Atlas*). Moreover, loss of *Ngn3* function leads to a significant reduction of POMC cells, combined with an increase of TH and NPY cells (Pelling et al., 2011), implying that this factor promotes the POMC phenotype but represses the NPY and TH fates.

Basal *Pmch* cells were already present superficially at E11.5, restricted to the subliminal dorsal retrotuberal PBas area (not shown; *Allen Developing Mouse Brain Atlas*); we identified this population according to Puelles et al. (2012) as the conventional *magnocellular lateral hypothalamic nucleus* (MCLH; **Figure 4L**); similar cells were included in the lateral hypothalamus by Croizier et al. (2011). Additional PBas *Pmch* cells later disperse between E15.5 and P4 within the *dorsobasal and ventrobasal sectors of the lateral hypothalamus*, deep to the lateral forebrain bundle (see these sectors in Puelles et al., 2012; their Figure 8.32), deep to the migrated subthalamic nucleus. Some of these elements also invade the DM-P shell and the dorsobasal/ventrobasal perifornical nuclei (MCLH, LH, DBLH, VBLH; **Figures 4N–P**, **5H**; see P56: **Figure 4Q**).

The *Pmch* cells found in the adult rat lateral hypothalamus, including the MCLH, were labelled by acute BrdU at E10-E11 (Brischoux et al., 2001; Croizier et al. (2011). The latter authors mapped the earliest MCH neurons in rat embryos, compared their positions with relevant regional molecular markers (emphasizing their origin within a *Nkx2.2/Nkx2.1/Shh*-positive band), and studied the course of their efferent axons. We are essentially in agreement with their primary data, but qualify the interpretation given by these authors about the molecular environment of the early *Pmch* cells. This is partly because we use the more elaborated model of molecularly distinct progenitor domains reported by Puelles et al. (2012), which was not available to these authors. Croizier et al. (2011) convincingly showed in E14 rat embryos that the early *Pmch* cells occupy a restricted posterior sector of the classical longitudinal hypothalamic cell cord, which is formed by precociously differentiating basal neurons (Gilbert, 1935; Keyser, 1972; His, 1892; see also Puelles et al., 1987, 2014). Consistently with this interpretation, this band expresses *Shh* and *Nkx2.1* (typical overall basal plate markers), as well as *Nkx2.2* (an early longitudinal marker expressed *across* the alar-basal boundary, thus labeling a subliminal part of the hypothalamic cell cord, as well as a liminal part of the overlying subparaventricular area in the alar plate; **Figure 1A**; Puelles and Rubenstein, 2003; Puelles et al., 2004, 2012; Figure 8.14B of the 2012 reference). Within our model, the basal subliminal band falls specifically within the TuD/RTuD progenitor area. The restricted *peduncular* position of the early *Pmch* cells, corroborated by our present mouse results, jointly with the molecular environment provided by Croizier et al. (2011), is consistent with a subliminal PBas (RTuD) origin, a site with selective expression of *Lhx9* (**Figures 1**, **5H**; Shimogori et al., 2010).

However, Croizier et al. (2011) assumed that the early basal cell cord is identical with the "intrahypothalamic diagonal" of Shimogori et al. (2010), thinking it correlates with the longitudinal band that expresses *Nkx2.2*. This interpretation seems wrong in two ways. First, the *Nkx2.2* band has alar and basal parts (as contemplated in Puelles et al., 2004, 2012), and the differentiating MCH cells are restricted to the basal component, as was clearly indicated by the overlapping expression of basal markers such as *Shh* and *Nkx2.1* (Croizier et al., 2011; their Figures 3K–P). Second, these authors apparently misinterpreted the description of the molecular profile of the intrahypothalamic diagonal given by Shimogori et al. (2010), insofar as these authors did not identify their diagonal area as expressing *Shh* and *Nkx2.1*. The intrahypothalamic diagonal is the distinct *alar* longitudinal SPa territory that overlies the basal hypothalamic cell cord (TuD/RTuD); it expresses *Dlx*/*Arx*/*Vax1/Isl1* transcripts and some *Lhx* genes, but lacks significant *Shh* or *Nkx2.1* signal (Shimogori et al., 2010; Puelles et al., 2012; **Figure 1C**). A ventral part of this area—the *liminal* SPa subarea- shows *Nkx2.2* signal on top of the cited molecular profile (Puelles et al., 2012; see their Figure 8.14B). The *Pmch* cells clearly lie under it (Croizier et al., 2011; their Figures 3K–P), within the underlying *Nkx2.1/Nkx2.2*-positive subliminal TuD/RTuD basal domain, which corresponds to the true hypothalamic cell cord.

Hypothalamic *Hcrt/orexin* cells also differentiate selectively within the peduncular dorsal retrotuberal region, though later, at E15.5 (**Figures 2**, **4H**). We think that this occurs specifically within the ventral PBas subarea that does not express *Nkx2.2* (present data). Progressively, most *Hcrt/orexin* neurons disperse radially and tangentially into the (retrotuberal) *ventrobasal sector of the lateral hypothalamus*, where *Nkx2.2* is not expressed (VBLH; **Figures 4H,I**, **5I**; compare Figure 8.29 of Puelles et al., 2012), though some remain more dorsally, within their source area PBas. In the adult rat brain, *Hcrt* cells were found just rostral to the zona incerta, within the "lateral and dorsal hypothalamus" and the "perifornical nucleus" (Peyron et al., 1998; their Figures 3, 8 and 14; compare **Figure 1**). This location lies just dorsally to our PRM area, pinpointing the RTuI, or ventrobasal, part of the LH. The singularity of PBas as a cell source, as opposed to the terminal ABas domain, might be due to its distinctive genoarchitectonic properties, such as the lack of *Nr5a1* expression (Puelles et al., 2012; present data) and the coincidence with selective *Lhx9* expression (Shimogori et al., 2010). The latter authors reported that *Lhx9* is co-expressed with *Hcrt* and partially also with *Gal* cells in newborn mice. In conditional *Shh* mutant mice in which *Shh* was selectively abolished in the basal hypothalamus, the entire Tu/RTu region was very reduced, and *Hcrt* cells were not detected (Szabó et al., 2009); on the whole, this supports our conclusion of a restricted *basal Hcrt* source at the ventral part of PBas. In contrast, Zhao et al. (2008) suggested an alar prethalamic origin of at least some *Hcrt* cells, based on the presence of a few LH hypocretin /orexin-expressing cells that coincide with *Foxb1-*derived progeny in newborn mice. This possibility needs additional analysis, since it postulates both a caudorostral (prethalamo-hypothalamic) and dorsoventral (alarbasal) translocation of such cells, which would need to be distinguished from alternative *Foxb1-*derived progeny potentially produced at the basal mamillary domain, some of which move into periretromamillary positions, just under the VBLH (Zhao et al., 2008).

#### **BASAL HYPOTHALAMIC SOURCES: THE INTERMEDIATE TUBERAL AND RETROTUBERAL AREAS**

This section centers on the arcuate and dorsomedial domains. The tuberal arcuate nucleus, an acroterminal basal locus characterized by overlapping *Nkx2.1*/*Dlx/Six3* markers, combined with early downregulation of its initial basal *Shh* expression (Manning et al., 2006), is an important source of some peptidergic neurons, such as *Ghrh* (Morales-Delgado et al., 2014) and *Agrp* cells (present data). We postulated above that the adult local dopaminergic (*Th*), and *Pomc* cell types originate more dorsally at the *Ngn3* expressing ABas domain (ArcM, ArcW; **Figure 5G**; Pelling et al., 2011). It is less clear whether *Npy* cells are included in the same migratory pathway. Such cells do appear first at the ABas area, and later at the Arc. It was established that most adult POMC cells are not gabaergic cells (consistently with the postulated ABas origin), whereas a majority of NPY neurons are gabaergic, which suggests instead a local Arc origin, though they may be complemented with some migrated cells (Horvath et al., 1997; Ovesjö et al., 2001; Hentges et al., 2004; Puelles et al., 2012). Significantly, about one quarter of the mature NPY neurons in the Arc share a common progenitor with POMC cells (Padilla et al., 2010). Accordingly, two subgroups of Arc NPY cells probably exist: 25% is glutamatergic, originated at the *Ngn3*/*Nr5a1/Six3* positive ABas locus, and a majority (75%) gabaergic population is generated within the Arc, in clear correspondence with local *Dlx* and *Gad67* expression (Yee et al., 2009; Puelles et al., 2012; **Figure 5F**).

Dispersed *Ghrh* cells first appear at E13.5 at the wing (nonacroterminal) part of the arcuate nucleus, extending later into the median acroterminal Arc (ArcM) where they form an important *Ghrh* cell population (ArcW, ArcM; **Table 2**; Morales-Delgado et al., 2014; *Allen Developing Mouse Brain Atlas*). On the other hand, *Agrp* cells first develop at the median part of the arcuate nucleus (ArcM) somewhat later, from E15.5 onwards. *Agrp* cells thereafter remain fully restricted to the Arc during postnatal development (**Figures 4J,K**; Nilsson et al., 2005). Some of the genes expressed at the Arc locus, such as *Six3*, *Nkx2.1*, *Mash1*, *Hmx2*, *Hmx3*, *Gsh1*, *Aldh1a2*, and *Isl1* were implicated in the differentiation of GHRH cells. *Nkx2.1* is expressed throughout the mantle of the hypothalamic basal plate at prenatal stages, and conditional *Nkx2.1* mutants are defective in the Arc derivatives (Mastronardi et al., 2006). The NKX2.1 transcription factor acts upstream of *Mash1*, *Hmx2*/*Hmx3* and *Gsh1* (Caqueret et al., 2005). *Mash1* and *Hmx2*/*Hmx3* control of *Gsh1* expression, which is required for the specification of GHRH neurons (Wang et al., 2004; McNay et al., 2006). Unfortunately, these studies did not explore the role of those genes in the differentiation of *Agrp* cells. Interestingly, *Agrp* cells were unaffected in null mutants of *Ngn3* (Arai et al., 2010), a transcript restricted to ABas, consistently with their apparent Arc origin.

Caudal to the Arc, the intermediate Tu/RTu subregion shows the mutually similar DM-T and DM-P areas, which share *Shh*, *Dlx*, *Nkx2.1, Isl1, Cnr1, Peg10* and *Lef1* expression, in absence of *Lhx9/Lhx6* signals (DM-T, DM-P; **Figures 1B,C**). Both DM portions (as well as the Arc) are subdivided into core (glutamatergic) and shell (gabaergic) portions; the core cells were suspected of migrating into these areas from a neighboring origin, probably the TuD/RTuD, whereas the gabaergic shell elements are intrinsic, consistently with local *Dlx* family genes and *Gad67* (Puelles et al., 2012).

Interestingly, the DM-P domain is a major source of *Gal* cells; the latter were first identified there at E15.5 (**Figure 3V**; *Allen Atlas* data). Similar early *Gal* cells were reported to appear in a vaguely defined "precursor hypothalamic region" at E16 in the rat, which we estimate to be peduncular and retrotuberal (RTuI) in topography (Figure 1A of Gundlach et al., 2001). Starting at E18.5, a less important *Gal* population appears likewise at the DM-T area, jointly with a possibly migrated perimamillary subpopulation (not shown; see *Allen Atlas* data). Postnatally these cells concentrate at the DM *shell* domains (Puelles et al., 2012), a pattern that suggests a local origin within TuI/RTuI and a gabaergic nature (DMsP, DMsT, **Figures 3V,W,Y,Z**, **5B**; **Table 2**; Puelles et al., 2012). Additional *Gal* cells appear at the Arc and the ventrobasal lateral hypothalamus postnatally (VBLH; **Figures 3Z**, **5B**; compare Melander et al., 1986). This suggests partial migratory dispersion of DM-P/DM-T *Gal* cells into the PM, Arc and VBLH areas (**Figure 5B**).

#### **BASAL HYPOTHALAMIC SOURCES: THE PERIMAMILLARY AND PERIRETROMAMILLARY AREAS**

Another basal progenitor domain producing peptidergic cell types is the arc-shaped perimamillary/periretromamillary area (PM/PRM), which lies ventral to the linear *Nkx2.1*/*Dlx*/*Arx*/*Lhx6/Wnt8b*-expressing TuV/RTuV domain (tuberomamillary terminal of Shimogori et al., 2010), where histaminergic neurons selectively originate (Puelles et al., 2012; **Figure 1C**). The PRM/PM area represents a domain with precocious neurogenesis, comparable in this regard to the hypothalamic cell cord (TuD/RTuD; see Puelles et al., 2014). It is molecularly distinct from the retarded retromamillary and mamillary areas by its selective expression of *Otp* and *Sim1*, in curious parallelism with the alar paraventricular area (**Figure 1C**; Puelles et al., 2012). *Sst, Penk, Npy* and *Pmch* phenotypes seem to originate from both the peduncular (PRM) and terminal (PM) subdivisions of this domain, in each case with a particular temporal sequence (**Figure 2**; **Table 2**; Morales-Delgado et al., 2011). It is unclear whether the PM/PRM *Gal* cells interpreted above as migrated from the DM-P area might originate instead locally (this point can be decided by examining their gabaergic vs. glutamatergic nature, since PM/PRM produces only the latter type; Puelles et al., 2012). *Sst* and *Npy* cells appeared selectively at the PRM at E13.5 and were detected only at E18.5 and P4, respectively, within the PM; this delay raises the possibility of their tangential translocation from PRM into PM (**Figures 4D–G**, **5F**; **Table 2**; Morales-Delgado et al., 2011; Morales-Delgado, 2012). *Penk* cells were not found at E15.5, but were already well represented at both PRM and PM at E18.5 (**Figure 3R**), which suggests an earlier appearance. At the later stage, as well as postnatally, the *medial PRM nucleus* (PRMM; **Figure 3R**) and the *dorsal premamillary nucleus* (DPM; **Figure 3R**; this is the main PM derivative) show massive *Penk* cell populations, and additional *Penk* cells were found at a previously undescribed rounded perimamillary cell aggregate superficial to the DPM, named here *superficial perimamillary nucleus* (PMS; **Figure 3U**; see also the Allen Developing Mouse Brain Atlas, transversal P56 series [64881286], section 61, for DPM and PMS). The neighboring *ventral premamillary nucleus* (VPM) also contains *Penk-*positive cells; however, it lies in the TuI area, intercalated between the ventromedial nucleus and the PM band (not shown; see the P56 Allen image cited above). Though these tuberal cells might have a different, more dorsal basal origin (see above results on ABas/PBas *Penk* cells), we mention them here because of the possibility that these VPM cells may have migrated either from the underlying PM area or from the more distant PRM area, accompanying the migration of RM cells into VPM (**Figure 5E**; Puelles et al., 2012).

Medial hypothalamic *Pmch* cells were also clearly identified along the characteristic PRM/PM band from E13.5 onwards (**Figures 1**, **2**, **4M–P**; **Table 2**). A neighboring *Pmch* cell group forms a shell around the migrated ventral premamillary nucleus (TuI area) from E15.5 onwards (VPM; **Figures 4N–P**). Remarkably, at postnatal stages few *Pmch* cells remain visible at the PRM/PM locus, suggesting an earlier translocation into the suprajacent TuI/RTuI areas, mainly the DM-P and DM-T nuclei, as well as the mentioned VPM shell neighborhood, where they increasingly become visible (**Figures 4N–P**, **5H**). The PRM/PM *Pmch* cells seem unrelated to the more dorsal and precocious ones described above, which contribute to the MCLH and LH populations (see **Figures 4Q**, **5H**). While Croizier et al. (2011) did not identify this separate set of PM/PRM MCH cells, (Brischoux et al. (2001); their Figure 5E; compare with our **Figure 1**) showed a drawing of a sagittal section, in which rat *Pmch* cells at E18.5 clearly occupy a band that does not agree in shape and position with any part of the basal hypothalamic cell cord (TuD/RTuD), but agrees with our PRM/PM area.

*Otp* and *Sim1*/*2* are potential key genes in the differentiation of the mentioned PRM/PM peptidergic lineages, since they are selectively co-expressed very early at this locus (supplementary figures of Shimogori et al., 2010; Morales-Delgado et al., 2011, 2014; Puelles et al., 2012; *Allen Developing Mouse Brain Atlas*); however, this hypothesis was not tested so far. Other genes expressed at the PRM/PM domain, such as *Shh, Nkx2.1, Pou3f3 (Brn1), Ebf3 and* *Peg10* (**Figure 1C**), might be also involved in the neurogenesis and/or differentiation of the peptidergic cell types originated in this territory.

#### **PRODYNORPHIN PROGENITOR AREAS**

We also studied *Pdyn* cells, which may be a singular case of delayed appearance of the peptidic marker expression in neurons that previously migrated tangentially. *Pdyn* cells first appear at E13.5 within the alar VPa area (peduncular hypothalamus); later they invade the overlying CPa and DPa areas at E15.5 and E18.5, respectively (**Figures 4R–U**, **5J**; **Table 2**). Basal *Pdyn*expressing cells were first observed at the DM-P and migrated (terminal) VM from E13.5 onwards, and at the DM-T at P4 (**Figures 4R',S–U**, **5J**). At perinatal stages, the basal elements became progressively concentrated within the core domains of these nuclei, known to be glutamatergic (e.g., VMc; DMcT in **Figures 4T,U**). Puelles et al., 2012 argued that there might be a relationship of *Pdyn* cells with *Nkx2.2*-expressing immature migrating elements, which all arise from the longitudinal band mentioned above, which overlaps the alar-basal boundary; this band divides into a *liminal* alar sub-band and a *subliminal* basal sub-band, whose derivatives co-express differential alar vs. basal gene markers (**Figures 1A,C**, **2**). Puelles et al. (2012) showed that, from E10.5 onwards, peduncular liminal *Nkx2.2*-positive neurons selectively migrate dorsalwards into the VPa nucleus, from where some of them later move into the LPa, CPa and DPa nuclei. This sequential pattern recalls our present data on early peduncular *Pdyn* cells, though these are first detected at the VPa at E13.5, and not at the *Nkx2.2* band (**Table 2**). If they are identical with the earlier migrating *Nkx2.2*-positive cells, then they apparently start to express *Pdyn* in a delayed manner, after their initial migration into VPa ends. In that case, their proper origin would not be the VPa area, but the underlying liminal PSPa subarea (**Figure 5J**). Co-expression and lineage studies are needed to resolve this issue; *Nkx2.2* continues being expressed in the paraventricular complex even in the adult mouse (Puelles, unpublished observations). It is also relevant to check the potential gabaergic vs. glutamatergic phenotype of these *Pdyn* cells, since local VPa cells are expected to be glutamatergic, whereas PSPa cells are held to be gabaergic (Puelles et al., 2012, note their Figure 8.18 shows gabaergic cells at the VPa and DPa nuclei).

On the other hand, terminal subliminal (basal) *Nkx2.2* positive cells (from the upper TuD area) migrate between E13.5 and E15.5 ventralwards into the VM nucleus, constituting one of its diverse migrated subpopulations (Puelles et al., 2012; their Figure 8.26 and text; **Figure 5J**). In this case, we see *Pdyn* cells emerging at the VM locus at E13.5 (**Figure 4R'**), possibly representing migrated subliminal *Nkx2.2* cells that again express the peptidic marker in a delayed manner, after migration. This interpretation is supported by the fact that all other known populations of the VM migrate from the overlying TuD area. Accordingly, the latter may be suspected to be the true origin of these VM *Pdyn* cells.

The *Pdyn* cells emerging at the DM-P may correlate likewise with peduncular subliminal *Nkx2.2*-positive cells (from the upper RTuD area), since some of them also migrate ventralwards a short distance into the DM-P area at E14.5 (Puelles et al., 2012; their Figure 8.26F). This is where we saw *Pdyn* cells at E15.5, possibly again due to delayed differentiation (**Figure 4S**; **Table 2**). Unfortunately, the Allen Atlas lacks clearcut data on *Nkx2.2* expression at this locus at later embryonic stages (few labelled cells appeared at the DM-P locus at E18.5), or postnatally (no significant signal). Contrarily, *Pdyn* cells become increasingly visible postnatally at the TuI/RTuI area, largely aggregated within the developing core portions of the DM-P and DM-T nuclei, as shown here at E18.5 and P4 (**Figures 4T,U**, **5J**; note that due to its obliquity at 45 degrees relative to the ventricle, the DMcP appears in more lateral sections than the DMcT, which is parallel to the ventricle**)**. We likewise lack evidence that any *Nkx2.2-*expressing cells invade the TuI area where the DMcT *Pdyn* cells emerge postnatally. In defense of the migratory hypothesis, it may be conjectured that migration of such cells into the DM areas might imply downregulation of *Nkx2.2* before the *Pdyn* signal appears. Lineage studies are thus needed to test our conjecture that *Pdyn* cells derive systematically from either alar or basal *Nkx2.2*-espressing progenitors. Note the potential origin of the DM *Pdyn* cells at the overlying TuD/RTuD area would explain their glutamatergic phenotype within the gabaergic DM shell environment (Puelles et al., 2012).

#### **CONCLUSIVE COMMENTS**

**Table 2** summarizes our findings about the earliest topography of the different hypothalamic peptides, separated into terminal and peduncular progenitor areas. We subsumed under the terminal progenitor domains the rostral specialized acroterminal areas (**Figure 1B**; Puelles et al., 2012), which nevertheless show some independent behavior (e.g., SCH, Arc, as indicated in parentheses in **Table 2**).

early *Cartpt* cells appear aligned with the prospective anterior hypothalamic nucleus, whereas the later *Vip, Avp* and *Oxt* cells emerge at the neighboring acroterminal suprachiasmatic nucleus; moreover, the *Avp/Oxt* types have a question mark in **Table 2**, because we suspect these elements may come from the overlying TPa area (see text above). A sequential pattern nevertheless is truly present in other areas; this implies a general fate regulatory mechanism that is heterochronic and position-sensitive (i.e., at a given locus, different cell types are generated over time); moreover, the same cell type may differentiate sooner or later in different areas.

### **ACKNOWLEDGMENTS**

This work was funded by the Local Government of Castilla-La Mancha grant PII1I09-0065-8194 to CD, the Spanish Ministry of Economy and Competitiveness grant BFU2008-04156 and the SENECA Foundation 04548/GERM/06 (no. 10891) to LP. Infrastructure support provided by the University of Castilla-La Mancha and the University of Murcia is also acknowledged.

### **REFERENCES**

Acampora, D., Postiglione, M. P., Avantaggiato, V., Di Bonito, M., Vaccarino, F. M., Michaud, J., et al. (1999). Progressive impairment of developing neuroendocrine cell lineages in the hypothalamus of mice lacking the Orthopedia gene. *Genes Dev.* 13, 2787–2800. doi: 10.1101/gad.13.21.2787

and the lateral hypothalamic area. *J. Comp. Neurol.* 402, 442–459. doi: 10. 1002/(sici)1096-9861(19981228)402:4<442::aid-cne2>3.3.co;2-i

postnatal development in the mouse. *Brain Res. Dev. Brain Res.* 155, 147–154. doi: 10.1016/j.devbrainres.2005.01.009

**Conflict of Interest Statement**: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 30 October 2014; accepted: 12 December 2014; published online: 12 January 2015*.

*Citation: Díaz C, Morales-Delgado N and Puelles L (2015) Ontogenesis of peptidergic neurons within the genoarchitectonic map of the mouse hypothalamus. Front. Neuroanat. 8:162. doi: 10.3389/fnana.2014.00162*

*This article was submitted to the journal Frontiers in Neuroanatomy*.

*Copyright © 2015 Díaz, Morales-Delgado and Puelles. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution and reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms*.

## Ontogenesis of oxytocin pathways in the mammalian brain: late maturation and psychosocial disorders

#### *Valery Grinevich1 \*†, Michel G. Desarménien2†, Bice Chini 3†, Maithé Tauber 4,5† and Françoise Muscatelli 6,7\*†*

#### *Edited by:*

*Gonzalo Alvarez-Bolado, University of Heidelberg, Germany*

#### *Reviewed by:*

*René Hurlemann, University of Bonn, Germany Harold Gainer, National Institutes of Health, USA*

#### *\*Correspondence:*

*Valery Grinevich, Schaller Research Group on Neuropeptides (V078), German Cancer Research Center, CellNetwork Cluster of Excellence, University of Heidelberg, Im Neuenheimer Feld 581 (TP4), Office 3.301, D-69120 Heidelberg, Germany e-mail: v.grinevich@ dkfz-heidelberg.de; Valery.Grinevich@ mpimf-heidelberg.mpg.de; Françoise Muscatelli, Institut de Neurobiologie de la Méditerranée, INMED UMR U901, INSERM, Parc Scientifique de Luminy, Aix-Marseille Université, INMED UMR 901, 13273 Marseille, France e-mail: francoise.muscatelli@ inserm.fr*

Oxytocin (OT), the main neuropeptide of sociality, is expressed in neurons exclusively localized in the hypothalamus. During the last decade, a plethora of neuroendocrine, metabolic, autonomic and behavioral effects of OT has been reported. In the urgency to find treatments to syndromes as invalidating as autism, many clinical trials have been launched in which OT is administered to patients, including adolescents and children. However, the impact of OT on the developing brain and in particular on the embryonic and early postnatal maturation of OT neurons, has been only poorly investigated. In the present review we summarize available (although limited) literature on general features of ontogenetic transformation of the OT system, including determination, migration and differentiation of OT neurons. Next, we discuss trajectories of OT receptors (OTR) in the perinatal period. Furthermore, we provide evidence that early alterations, from birth, in the central OT system lead to severe neurodevelopmental diseases such as feeding deficit in infancy and severe defects in social behavior in adulthood, as described in Prader-Willi syndrome (PWS). Our review intends to propose a hypothesis about developmental dynamics of central OT pathways, which are essential for survival right after birth and for the acquisition of social skills later on. A better understanding of the embryonic and early postnatal maturation of the OT system may lead to better OT-based treatments in PWS or autism.

**Keywords: oxytocin, oxytocin receptor, ontogenesis, somatodendritic release, axonal release, autisn, Prader-Willi syndrome**

*†These authors have contributed equally to this work.*

#### **INTRODUCTION**

Oxytocin (OT) is a neuropeptide which is synthesized in defined nuclei of the hypothalamus, paraventricular (PVN), supraoptic (SON), and accessory (AN) nuclei and secreted both in the blood circulation as a hormone and in the brain as a neuromodulator. During the last decade, hundreds of original reports and reviews have been published, clearly showing that the OT system is a key regulator of all the aspects of social behavior, including those involved in reproduction and care of the offspring (Lee et al., 2009). Notably, in humans, OT facilitates the processing of social information and improves cognitive emphatic abilities, representing a potential new approach for the treatment of autism spectrum disorders, as extensively reviewed elsewhere (Meyer-Lindenberg et al., 2011). Even if OT is currently regarded as a "prosocial" drug, this does not implicate *per se* a "positive" connotation. OT has been found to increase trust and generosity (Kosfeld et al., 2005), but also gloating and envy (Shamay-Tsoory et al., 2009), which are also manifestations of complex "social" behaviors. Similarly, OT increases in-group but not out-group cooperation, an ethnocentric behavior that can promote prejudice, xenophobia, and intergroup violence (De Dreu et al., 2011). Furthermore, OT has been found to induce adverse behavior in borderline-personality disorders (Bartz et al., 2011). These results suggest that the selection of patients will play a crucial role in determining the outcome of the symptomatic treatment with this neuropeptide, the use of which should be restricted to those individuals who could benefit from it. An understanding of the OT-mediated cellular and molecular mechanisms on neuronal networks underlying social and feeding behavior is needed for a successful employment of this peptide in neuropsychiatric and genetic disorders. However, despite the large body of knowledge about physiology and central pathways of OT (see reviews from Landgraf and Neumann, 2004; Ross and Young, 2009; Lee et al., 2009; Knobloch and Grinevich, 2014), the developmental aspect of the OT system remains the *"locus minoris"* of neuroscience and neuroendocrinology. Here, we collected virtually all of the available information, including our personal ongoing research, and provide some general conclusions regarding the dynamics of central OT pathways in prenatal and early postnatal ontogenesis. Furthermore, we clearly demonstrate, by investigating a mouse model for the Prader-Willi syndrome (PWS), that changes in the developmental maturation of the OT system cause metabolic and social alterations. These can be significantly improved or even fully compensated by OT treatment in the first days after birth, opening the door for a powerful pharmacological therapy for PWS in early infancy.

### **THE BIRTH OF OT-ERGIC NUCLEI**

In many species (rodent, human, zebrafish, chicken), all oxytocin (OT) neurons are generated from the proliferative ("convoluted") neuroepithelium of the diamond shaped third ventricle (**Table 1** and **Figure 1**). Birth-dating studies revealed that these hypothalamic neurons are generated in the second half of the gestational period in rodents, within the first quarter of the gestational period (E30-43; length of pregnancy ∼165 days) in macaques (Markakis, 2002) and at the middle of pregnancy in humans (Swaab, 1995).

In rodents, the SON and PVN appear very early. At embryonic day (E) 12.5 dpc (days post-coîtum), two groups of cells are identified in the mouse: one near the third ventricle and the other moved lateral to the surface pial to give rise to the SON (Dongen and Nieuwenhuys, 1989).  At this stage, an antibody recognizing the Neurophysin-I (the carrier protein for OT) reveals a positive immunosignal (**Figure 1B**), consistent with the expression of the OT-prohormone, not yet detected at E12.5 dpc. Furthermore, an antibody recognizing the intermediate forms of OT (VA10) reveals a positive immunosignal of the OT-intermediate forms at E16.5 dpc (**Figure 1C**), but not at E14.5 dpc. Importantly, the mature, amidated form of OT is detected at birth (see **Table 1**) and co-exists with immature forms of OT during the entire postnatal life (**Figure 1D**). The role of an earlier production of the non-mature forms of OT has not been studied, although a functional role of these forms during the embryonic development has been suggested (Tribollet et al., 1988).

In humans, the SON and PVN are completely formed at 25 weeks of gestation (Dörner and Staudt, 1972) and OTimmunoreactivity is first detected at the age of 26 weeks (Wierda et al., 1991). At that age, the number of stained OT neurons is relatively similar in the fetal and adult hypothalamus (Van der Woude et al., 1995), while the morphological analysis of individual magnocellular neurons suggests that these cells are still immature, as can be seen by the gradual increase of their nuclear volume (Rinne et al., 1962; was also observed in the rat by Crespo et al., 1988).

#### **GENETIC HIERARCHY OF OT NEURON FORMATION**

The signaling molecules and transcription factors that are involved in the determination and differentiation of OT neurons are not well known, only a few mouse studies reported factors

involved in the early stages of development of the hypothalamicneurohypophyseal system (Carrel and Allen, 2000; Caqueret et al., 2006; Szarek et al., 2010).

The early patterning of hypothalamus has been shown to depend on a cascade of transcription factors (**Figure 1A**): firstly

the sonic hedgehog (SHH) is necessary to establish the hypothalamus primordium (E9.5 dpc in mouse) (Mathieu et al., 2002; see also Blaess et al. (2014) in this Research Topic of Frontiers in Neuroanatomy); the local production of the bone morphogenetic proteins (BMPs) is then required to down-regulate SHH and establish region specific transcription profiles (Ohyama et al., 2008). The bHLH-PAS (basic helix-loop-helix PER-ART-SIM) transcription factor SIM1 is expressed in the incipient PVN, SON and AN from E10.5 dpc (Caqueret et al., 2006) where it dimerises with ARNT2 (Michaud et al., 2000; Hosoya et al., 2001). A key downstream target of SIM1/ARNT2 is Brn2, a POU domain transcription factor required for OT (as well as for two other types of neuroendocrine neurons, expressing arginine-vasopressin (AVP) and corticotropin releasing hormone neurons of the PVN/SON/AN and expressed from E11.5 dpc, Nakai et al., 1995; Schoneman et al., 1995). In a parallel or convergent SIM1/ARNT2 pathway, the homeobox orthopedia (OTP) factor is also necessary for Brn2 expression (Caqueret et al., 2006), which is still expressed at E15.5 dpc, with Nkx2.2. All of these factors are required to define at E12.5 dpc the prospective PVN domain, however, from this stage on, the factors that will specify the parvocellular1 and magnocellular OT neurons have not been identified. The ablation of Brn2 results in a loss of all neurons of the PVN, SON and presumably of the AN (Nakai et al., 1995; Schoneman et al., 1995). Importantly the lack of axonal projections of magnocellular OT and AVP neurons in the Brn2 knock-out mice to the pituitary (Schoneman et al., 1995), as also observed in the Arnt2-knockout mice (Hosoya et al., 2001), leads to progressive loss of pituicytes (pituitary astrocyte-like glial cells). All together, these results suggest a role of OT and/or AVP in the formation of the neurohypophysis. Indeed, in the absence of OT, the neurovascular interface in the neurohypophysis does not form in zebra fish (Gutnick et al., 2011).

One approach to identify such factors involved in the determination and differentiation of OT cells is the analysis of the OT promoter and its upstream and downstream sequences. Interestingly, genomic DNA constructs, including more than 500 base pairs (bp) upstream and 3.6 kb downstream of the OT gene, enabled an expression of OT or EGFP-reporter in OT-neurons only (Young et al., 1990; Belenky et al., 1992) in adult transgenic rodents. Later, a genomic analysis of the OT-promoter (500 bp) defined a minimal promoter sufficient for expression in OT-neurons (magnocellular neurons) of adult rodents (Fields et al., 2012; Gainer, 2012). Several binding sites for transcription factors (ERE, COUP-TF, SF1) have been identified in this sequence, but not the ones for the transcription factors described above (**Figure 1A**), suggesting that the DNA sequence required for the development of OT cells is not present in these 500 bp.

In conclusion, from the E12.5 dpc stage, the transcriptional factors that will specify the parvocellular and magnocellular OT neurons have not yet been characterized.

Several other genes such as *fibroblast growth factor 8* (Brooks et al., 2010), the *Mage-D1*, *Necdin* and *Magel2* genes, *CD38*, *Peg3* (**Figure 1** and **Table 3**), are known to be expressed in mouse hypothalamus during development. Their knock-out (KO) alters the number and/or function of OT-neurons, but their role in OT-neurons is not clarified yet.

#### **THE BIOSYNTHESIS OF OT IN ONTOGENESIS**

In the rat and mouse, the OT can be detected by several techniques (such as radioimmunoassay, enzyme immunoassay, immunohistochemistry) from the beginning of the second gestational week (**Table 1**). However, all of these techniques are based on the use of OT antibodies. The specificity of these antibodies, in particular against the different forms of OT, is not always established. The most reliable and relevant studies have been performed with the use of antibodies characterized by Harold Gainer's laboratory or using Mass Spectrometry Ànalysis, which, unfortunately, has not been used in developmental studies. Based on the tools used for OT detection, the OT prohormone is found in embryos just after the appearance of the OT mRNA, while the mature OT peptide is only released from birth on (**Table 1** and **Figure 1**). The OT gene encodes for the Pre-Pro-OT-Neurophysin I (pre-pro-hormone), which is cleaved by different enzymes to give rise to different OT intermediate forms and to the Neurophysin I, and finally to the mature amidated form that is released (**Figure 2**). It has been shown that the steady state of the mature OT form can be controlled by an oxytocinase (P-LAP) that is produced in periphery and centrally by the OTmagnocellular neurons. Noticeably, P-LAP is also expressed in parvocellular OT neurons and in other brain structures (Tobin et al., 2014).

Before birth, there is a delay in OT prohormone processing with an accumulation of the OT intermediate forms in OT neurons until birth. This delay was not observed for AVP processing as the amidated AVP form is detected as early as E16.5 dpc (Whitnall et al., 1985; Altstein and Gainer, 1988). Furthermore, morphological and electrophysiological properties of the magnocellular OT neurons are not mature at birth; their morphological and electrophysiological properties develop progressively during the first two postnatal weeks (see below, as well as Swaab, 1995, 1997a,b). Thus, although functional at birth, the OT-ergic system undergoes a progressive maturation during early postnatal life and is not mature before weaning in rodents and, most likely, in human infants as well. However, a comparison of immature and mature forms of OT in human development has never been studied.

#### **ELECTRICAL PROPERTIES OF OT NEURONS AFTER BIRTH**

To our knowledge, no electrophysiological studies have been performed on embryonic OT neurons. However, the groups of Francoise Moos and Michel G. Desarmenien systematically analyzed electrical activity of magnocellular neurons (although without distinction between OT and AVP) in the SON of rats during early postnatal life. Three main features arise from these studies: the importance of the second postnatal week (PW2), the role of autocontrol by OT, presumably released from soma and dendrites, and the determinant role of the development of electrophysiological features on the morphological maturation of the neurons. At birth and during PW1, the membrane potential is unstable and cells fire small and large erratic action potentials. During PW2, the membrane potential stabilizes to a more

1Parvocellular OT neurons belong to a relatively small population of PVN cells, which project to the brainstem and spinal cord (Sawchenko and Swanson, 1982) and control appetite, nociception and autonomic functions. The development of these particular neurons has not been studied.

hyperpolarized value, the spontaneous activity becomes organized and the action potential progressively increases in size and decreases in duration, leading to a decrease in action potentialevoked calcium entry (Widmer et al., 1997; Chevaleyre et al., 2000). At the end of PW2 and during PW3, a switch in regulation of intracellular free calcium from extrusion to sequestration into the reticulum also occurs (Lee et al., 2007a). These two modifications of calcium entry and regulation may have important consequences on peptide secretion. The PW2 is also the period during which the chloride equilibrium potential becomes hyperpolarized, GABA becomes inhibitory and glutamatergic activity appears (Chevaleyre et al., 2001), together with an increase in NMDA receptor expression (Hussy et al., 1997). Most interestingly, this period of action potential and synaptic activity maturation is concomitant with the appearance of a major feature of SON neurons: autocontrol (**Figure 3**).

OTR has been shown to play animportant rolein autoregulation of magnocellular neuronal activity (Richard et al., 1997) and it is well established that OTRs are expressed in adult PVN (van Leeuwen et al., 1985; Freund-Mercier et al., 1987; Tribollet et al., 1988;Yoshimuraetal.,1993;Adanetal.,1995).Duringontogenesis, cells expressing OTR mRNA have been observed in SON and PVN starting from P1 (Yoshimura et al., 1996), a finding consistent with the faint and diffuse expression of OTR present throughout the hypothalamus at this stage (Tribollet et al., 1989). An increase in OTR mRNA expression is detected from P7 and is then maintained fairly stably to adulthood (Yoshimura et al., 1996).

Consistently with changes in OTR expression during PW2, OT and its related analog are most efficient in increasing electrical activity on one-third of SON neurons (supposed to be OT-ergic neurons since they were insensitive to AVP) and somatodendritic release of native OT (Chevaleyre et al., 2000).

The somatodendritic release of OT not only activates action potential firing, it is also determinant for the maturation of glutamatergic synaptic activity and of the neuronal morphology. Indeed at birth, supraoptic neurons display an oblong soma from which 2-3 dendrites with few proximal branches arise (Chevaleyre et al., 2001). However, during PW2, the interplay between incoming glutamatergic inputs and autocontrol induces an intense sprouting of dendritic branches (Chevaleyre et al., 2002), as if neurons were exploring the environment to establish new connections. This sprouting is transient and the neurons acquire their mature morphology (Randle et al., 1986) by the end of PW2. Although partial and concerning only SON OT neurons, these data point to a determinant role of autocontrol of OT neurons during PW2 in rats. This information should be taken into account in our understanding of how OT treatments during infancy can have lifelong consequences on OT-related social diseases (see the chapter below).

### **ESTABLISHMENT OF OT CIRCUITRY**

Since neuroendocrinology was established as a new discipline, it has been clearly demonstrated that magnocellular (both OT and AVP) neurons of adult vertebrate species, including mammals, are primarily projecting to the posterior pituitary lobe to release these hormones into the systemic blood stream (Knobloch and Grinevich, 2014 and references therein). However, embryogenesis of pituitary OT projections remains unexplored. In fact, only two studies demonstrate such projection in rats without discrimination between OT and AVP components. The first study by the Ann-Judith Silverman group (Silverman et al., 1980) showed the existence of neurophysin-positive (i.e. without

In the rat SON during the second PN week, locally released OT promotes calcium mobilization and OT release, and favors the maturation of

activity and mobilization of calcium from intracellular stores and promotes growth of new dendritic branches.

discrimination between OT and AVP) fibers in the posterior pituitary. Another study by the Michael Ugrumov and André Calas groups (Makarenko et al., 2000, 2002), employing DiIbased retrograde tracing in fixed brains, showed certain dynamics of these projections: first connections between the main part of the SON and pituitary are established earlier (detected at E15—earliest time point of the experiment), while the PVN and retrochiasmatic parts of the SON project to the pituitary later at E17 (Makarenko et al., 2000). Intriguingly, the AN, composed mostly of OT neurons, projects to the pituitary only after birth (Makarenko et al., 2002).

Remarkable work on zebra fish larvae showed that OT is essential for the formation of an effective neurovascular interface (i.e., contacts of axonal OT terminals with fenestrated capillaries in the posterior pituitary; Gutnick et al., 2011). Unfortunately, such observation has not been extended to mice lacking OT or OTR, in which the analysis of the pituitary structure has not been reported.

Although central projections of OT neurons in adult rodents (mice, rats, and voles) have been significantly explored during last years (Ross et al., 2009; Knobloch et al., 2012; Dölen et al., 2013), the literature lacks reports on embryogenesis and early postnatal development of OT projections. This concerns both ascending projections of magnocellular OT neurons to the forebrain and descending projections of parvocellular OT neurons terminating in the brain stem and spinal cord.

The major inputs carrying visceral sensory information to the SON and PVN are relayed by catecholaminergic and noncatecholaminergic (glucagon-like peptide 1) neurons whose soma are located in the nucleus of the solitary tract and ventrolateral medulla. Both direct and indirect arguments indicate that these afferents are not functional at birth and develop during the first postnatal week (see review by Rinaman, 2007). Similarly, although no anatomical studies on the dynamics of innervation of OT neurons by glutamate and GABA have been performed, electrophysiological data summarized above suggest long-term postnatal maturation of synaptic inputs.

#### **THE DEVELOPMENTAL TRAJECTORIES OF OT RECEPTOR EXPRESSION**

The developmental trajectories of OT receptor (OTR) expression in the nervous system have been investigated in mice, rats, voles and humans by different experimental approaches such as autoradiography, *in situ* hybridization and transcriptomic analysis, which provide complementary information. While autoradiography maps high affinity OTR at subcellular sites, which grossly correspond to receptor site(s) of activity on neuronal cell bodies and processes, *in situ* hybridization maps the neuronal bodies in which OTR mRNA accumulates. Transcriptomic analysis provides clues on brain regions actively synthesizing OTR mRNA without giving details on the cell types and subpopulations involved (see **Table 2** for the available literature).

Among mammalian species, the rat has been by far the most extensively investigated. In this animal, the three aforementioned techniques resulted in highly comparable and consistent results, which made it possible to trace a developmental trajectory of OTR ontogenesis. This has been schematically summarized in **Table 3** and **Figure 4** (Shapiro and Insel, 1989; Snijdewint et al., 1989;

#### **Table 2 | Studies of OTR trajectories in development.**

**Table 3 | Brain structures expressing OTR in different periods of prenatal and early postnatal life in rats.**

◦*In the adult, binding in the accumbens has been reported to almost completely disappear (Tribollet et al., 1989) or to be greatly reduced as compared to its pick at PN20 (Shapiro and Insel, 1989).*

*\*The appearance of OTR in the hypothalamic ventromedial nucleus has been reported to appear at PN1 (Tribollet et al., 1989) or to emerge only in the adult brain (Shapiro and Insel, 1989).*

Tribollet et al., 1989; Yoshimura et al., 1996; Lukas et al., 2010; Workman et al., 2013). Throughout the embryonic development and the first post-natal days, OTR progressively appears in several brain regions, reaching a well-defined "infant" pattern of distribution around PN10. After PN13, an abrupt decline of OTR density is observed in several areas, accompanied by expression in novel brain regions; this phase has been referred to as the first transition to the adult pattern and is basically completed at PN18. Around and after weaning, a second transition occurs, characterized by a novel reshaping of OTR expression, which slowly disappears from some areas and increases in others. Finally, the adult pattern of OTR expression is achieved at P60-90. Brain structures expressing OTR in different periods of prenatal and early postnatal life in rats (according to Shapiro and Insel, 1989; Tribollet et al., 1989; Lukas et al., 2010) are reported in **Table 3**.

OTR appears as early as E14 pcd is the posterior portion of the neuronal tube that will become the vagal motor nucleus. Even though the mature OT is not detected until much later in development (see above), immature C-terminal extended forms of OT have been visualized much earlier (E16 pcd, see above). It is thus tempting to speculate that some immature OT forms may have an unrecognized role during early development in the vagal subregion. Mechanistically, the delivery of immature OT to the vagal nucleus can be achieved either via the neuropeptide diffusion through the brain tissue after somatodendritic release or via the transventricular pathway (Knobloch and Grinevich, 2014).

Strongly labeled areas of the "infant pattern" around PN10, schematically reported in **Figure 4B**, include the anterior olfactory nucleus, caudate putamen, accumbens, cingulate cortex, bed nucleus of the stria terminalis, some septal, thalamic and amygdaloid nuclei and the dorsal peduncular cortex. A faint but specific signal is observed at this stage in the hypothalamic ventral medial nucleus, a region that will become strongly labeled in the adult brain, while binding in the lateral septum and bed nucleus of the stria terminalis will undergo an intra-regional reshaping during post-natal development. Of relevance, between PN10 and PN25, is the disappearance of binding in the dorsal subiculum, accompanied by the concomitant appearance of increasingly strong binding in the ventral subiculum. During the same period, binding drops in the thalamus, cingulate cortex and CA1 but increases sharply in the accumbens, where it peaks at PN20 to subsequently decline during the second phase of transition to the adult brain. Changes in selected brain regions between week 5 and week 8 have been reported, such as an increase in OTR binding in the ventromedial hypothalamus and a decrease in the lateral septum (Lukas et al., 2010). However, a detailed, comprehensive study of OTR expression trajectories around weaning in males and females is, at present, missing.

Unfortunately, the studies available in literature for mice, voles and humans do not allow for similar comprehensive compilation of OTR expression trajectories during development. Nevertheless, a recent autoradiography study in mice revealed that regionspecific trajectories of OTR expression are present in this species as well (Hammock and Levitt, 2013). A qualitative inspection of OTR binding from P0 to PN60 outlined a progressive strong increase of OTR from P0 to PN14, followed by region-specific up and down regulation of receptor expression. Different temporal expression profiles were reported in the three areas of the brain in which the OTR binding profile was quantified (hippocampus, lateral septum, and neocortex) with no significant differences between sexes. A very high degree of individual variability in OTR expression during ontogenesis was also reported in prairie voles, hampering the detailed delineation of developmental trajectories in this species; however, as observed in mice and rats, a higher density of OTR was observed in some forebrain regions (the septo-hippocampal nucleus and the hippocampus) between PN6 and PN21 as compared to PN1 and PN60 (Prounis and Ophir, 2013). Similarly, in the lateral septum, OTR density increased during the post-natal age, reaching the adult level at weaning; in particular, the binding increased more rapidly in mountain than in prairie voles, resulting in species differences at weaning and adulthood (Wang and Young, 1997). Finally, it is worth noting that in the different species, the overall pattern of the OTR expression during development does not match completely. The

strong transient expression of OTR in the neocortex reported in mice was suggested to account for a species-specific role played by OTR during brain development (Hammock and Levitt, 2013). However, it should be mentioned that a transient OTR expression in the parietal cortex has also been reported in the rat brain (Snijdewint et al., 1989).

As in humans, the transcriptomic analysis of OTR reported a progressive increase in OTR mRNA during embryonic life in five out of six brain areas analyzed (Kang et al., 2011 and http:// hbatlas*.*org/). Remarkably, the receptor level appeared to reach a maximum already before birth and to remain quite stable thereafter, at least in the first 5 years of life, although with some wide individual variations.

An important consideration when comparing developmental trajectories in translational medicine approaches is that the neurodevelopmental stage of the human brain at birth corresponds to rat and mouse brain at PN10 (Workman et al., 2013 and related web tool at http://www*.*translatingtime*.*net/home). Consistently, the human infant pattern of OTR expression is achieved before parturition while, in the rat, the infant pattern of OTR expression is achieved around PN10, and, in mice, between PN7 and PN14. To extrapolate the effects of pharmacological and environmental manipulation of the OT/OTR system in the human newborn brain from experiments performed in mice and rats, treatments should thus be performed in rodents around PN10, when a comparable maturation of the brain and of the OTR system has been reached in these species.

Several environmental factors have been reported to affect OTR expression in embryogenesis and early postnatal life, among which a predominant role is played by social and sensorial experiences, as more extensively discussed in the next paragraph. Furthermore, exposure to drugs and toxic agents can also modulate the OT/OTR system, as outlined by the interesting finding that nicotine and ethanol administration to pregnant rats increases OTR binding in the nucleus accumbens and in the CA3 region of the hippocampus of male offspring (Williams et al., 2009). Recently, an up-regulation of OTR in the nucleus accumbens, medial anterior olfactory nucleus and central and medial nuclei of amygdala has been reported in µ-opioid receptor knock out mice (Gigliucii et al., 2014), which suggests a close link between OTR expression and the opioid reward system.

#### **OT AND SOCIAL EXPERIENCE IN DEVELOPMENT**

Effects of OT during embryogenesis and early postnatal ontogenesis on social life are extensively summarized in recent reviews (Carter, 2014; Hammock, 2014), but the reverse—i.e., the effects of social stimuli on maturation of OT system—are less explored. It has been reported that early social experience tremendously affects physiology of the OT system (Bales and Perkeybile, 2012; Hammock, 2014). Recently it has been demonstrated that early sensory experience (in the newborn) regulates development of sensory cortices via OT-signaling (Zheng et al., 2014).

This oxytocin early regulation may have long-term consequences in adults. It has been known for many years that exogenous OT in neonates can revert the long-term behavioral effects of prenatal stress (Lee et al., 2007b) and has consequences on other endocrine systems (i.e., the estrogen receptor, Pournajafi-Nazarloo et al., 2007) as well as on blood pressure (Holst et al., 2002) in adults. The effects of OT, or of the environmental and familial circumstances resulting in increased production of mature neonatal OT, on adult social behavior mainly remain to be investigated. With respect to OTR expression, increased levels have been observed in offspring after communal rearing (Curley et al., 2012), increased maternal licking/grooming (Champagne et al., 2001) and social enrichment (Champagne and Meaney, 2007). On the contrary, late weaning has been reported to reduce OTR density in selected, socially relevant, brain regions (Curley et al., 2009). Furthermore, maternal separation has been found to induce a complex modulation of OTR expression with regionspecific up and down regulation of OTR (Lukas et al., 2010). It is important to note, at the end of this section, that exploration of early life experience on OT/OTR system in animals provides a scientific basis for child care and new therapeutical approaches to ameliorating social alterations occurring in adult patients afflicted with Autism Spectrum Disorders or the PWS (see below).

#### **OT AND DEVELOPMENTAL NEUROLOGICAL DISORDERS**

Many reviews are exhaustive on the involvement of OT in shaping and regulating the social brain (Meyer-Lindenberg et al., 2011; Striepens et al., 2011; McCall and Singer, 2012) or in learning and memory (Chini et al., 2014). The strongest line of demonstration of the OT system in social behavior, feeding behavior and maternal care is via the study of knockout mice in which either the *OT* or the *OTR* genes are inactivated (**Table 4**). Mice constitutively lacking OT (*Oxt*−*/*−) are unable to release milk and have impaired social memory (Ferguson et al., 2000). Mice

#### **Table 4 | OT and neurodevelopmental disorders.**