PhD Vanessa Frangville

RACE AND NATIONALITIES: THE ETHNIC CLASSIFICATION PROJECT WITHIN IDEOLOGICAL AND POLITICAL STRATEGIES by

From the first full-scale census in the early 1950s to the present day, the question of ethnic minorities has featured high on the PRC political agenda. The official discourse on minorities and the representation of minorities are particularly interesting to understand how the classification of the Chinese population into a majority Han and numerous minorities has been undertaken to fit to the dominant ideology.

I will attempt to explain, through representation of minorities, the dominant ideology and the political strategies on a nation-level implied in the ethnic classification. I will argue that the representation of minorities and the treatment of ethnicity question in this period hint a wider strategy that attempts to create a Han national identity and to define a new model of a universal China. Therefore, the study of minorities' representation at the first stage of the classification may highlight the origin of political pattern of "diversity in unity" described in the 1990s by Fei Xiaotong.

PAPER

From the first full-scale census in the early 1950s to the present day, the question of ethnic minorities has featured high on the People's Republic of China (PRC) political agenda. 1 Ethnic groups come into play as new material for conveying the political ideology of the Party, and setting up a new subject in its propaganda. Behind very simple images of ethnic communities through minorities in posters or magazines a complex discourse on the search for Chinese national identity and on the need for political legitimization take shape in the young PRC. I distinguish three periods in the question of ethnicity in China since the foundation of the PRC: a first period from 1949 to the end of the 1970s, while ethnic identification is still in process and Han majority sovereignty is being established; from the early 1980s to the middle of the 1990s, in the heart of the economic reform era, when "ethnic" conflicts start to appear repeatedly in the Chinese newspapers while each is encouraged to grow rich; from the end of the 1990s on, the minorities have managed to impose themselves as part of the (local) economic life, even though still ruled by Han supremacy. The period this essay will deal with is the one I identified as the first period. I argue that any classification of population into ethnicities is an answer to a political and ideological purpose to justify inequalities and the hegemony of a dominant group. As Nicholas B. Dirks shows brilliantly in his work on Indian castes, systematic classification of population into distinct groups is " […] not some unchanged survival of ancient India, not some single system that reflects a core civilizational value, not a basic expression of India tradition." 2 In short, divisions are the facts of construction. They are neither fundamental nor specific, they are only valorised (or despised in some case) for one peculiar reason or another. Indeed, the Ethnic Classification Project teams consisted of some scientists and mainly of official local and national leaders. Ethnologists and anthropologists were trained in Western schools or with Western methods: we know that those disciplines in 1 See Fei Xiaotong's works on ethnicity, and especially the concept of « diversity in unity » developed in Fei Xiaotong, Zhonghua minzu duo yuan yi ti geju (Pattern of diversity in unity of the Chinese nation), Beijing, Zhongyang minzu daxue chubanshe, 1999. Western were largely influenced by colonialism and colonial thoughts (and vice versa).

National and local leaders were also involved in this project, sometimes even more than scientists, to make sure that the project would be accomplished according to the national policies. The purpose of this short presentation would be to analyse the official discourse on ethnicity in early PRC. We will argue that representations of ethnic minorities imply a wider ideological discourse in keeping with the general pattern of the governement's policy.

Chinese propaganda posters are an inexhaustible mine of information to tackle the historic events in China over the last 50 years. 3 They also provide a body of material that tells us more about ideals and perspectives ("what this or this should be") than about concrete facts ("what this or this is"). Art in all its forms, under the influence of Mao's famous Yan'an speech on literature and art in 1942, is clearly expected to support and illustrate the official ideology of the Chinese Communist Party (CCP or gongchandang 共产党). 4 In the event, this approach was particularly used-and effective because widely spread-in propaganda posters, till they lost their appeal and credibility in the era of reforms. Visibly, propaganda posters accompanied every historic and political change in the first period of the PRC, and the Ethnic Classification

我们今天开会,就是要使文艺很好地成为整个革命机器的一个组成部分,作为团结 人民、教育人民、打击敌人、消灭敌人的有力的武器,帮助人民同心同德地和敌人 作斗争.
The purpose of our meeting today is to ensure that literature and art fit well into the whole revolutionary machine as a component part that they operate as powerful weapons for uniting and educating the people and for attacking and destroying the enemy, and that they help the people fight the enemy with one heart and mind.

These words clearly made literature and art part of the CCP's propaganda organ to establish its legitimacy and authority.

Project is one of the most important breaks with the Republican period that preceded the Communist era.

Representations of ethnic minorities on propaganda posters enable us to witness the political strategies implied in the construction of a new Chinese nation-state. The division of the population and the identification of several minorities was not intended merely to recognize a multiethnic reality nor to reward non-Han participation in the struggle against the Guomindang, but mainly to pave the way for a new Chinese national identity that needed to be legitimated and redefined by the whole population.

The Ethnic Classification Project was 30-year long project undertaken at the same time as the first representations of minorities appeared in propaganda media.

Hence, I will attempt in this essay to analyse and deconstruct the discourse of the CCP on ethnicity through the representation of playful and colourful minorities. The ethnic division/ di-vision, I argue, seek a justification for Han supremacy; it attempts to dictate a new social order through a strict classification; it legitimates the new Chinese national identity propounded by the PRC government.5

Ethnic minorities on posters: objectification and ideology

I argue that representations of minorities imply a justification of the communist state among the China's peoples. Posters were mostly made by Han artists for a Han public, as a product of the CCP ideology. The minorities on posters are never represented for themselves: they are never the main theme of the discourse; they only support the discourse, like scenery.

On this poster (see below), the main characters are Mongol children, sitting on the grass and concentrating on a radio. The most obvious remark to be made is that Mongol children are not the main theme of the poster. The composition of the poster clearly aims to focus the spectator's attention on the radio set. The central position of a white radio, the children's happy facial expressions with eyes and ears turned toward the radio (even the small white lamb on the right seems to look at the radio), the way the child holds the radio at arm's length, every detail tell us about the outstanding importance of the radio as object.

Plate 1: 草原联北京

The Grassland is Connected to Beijing, author unknown, 1977 Besides, the minority children are passively listening to the radio: they are receiving in a captive posture. At the same time, the radio is broadcasting, as we surmise from the inclined head of the little girl on the left: the radio, despite an inanimate object, is expansive, actively transmitting something to lively but inactive minorities. Ethnic minorities are then objectified as lifeless items, while the inanimate item is personified.

From then on, minorities are not represented for themselves but to represent (and therefore to identify and to acknowledge) the Chinese state. The radio set represents modernity and technology, which the Chinese state tries to promote. This poster shows that anyone, even minorities living far away from the capital Beijing may have access to modernity and high technology.

Posters are almost systematically coupled with texts that point to the real subject of the picture. Verbal texts are of some importance too, since they give a second reading of the picture. For this poster in particular, the caption confirms our analysis: the radio is broadcasting from Beijing, that is to say an official programme from the government. The written texts also assert that posters need a supplementary reading: more than showing a simple image, there is something said that is meant to be understood by every one. Moreover, we can even argue for a third reading, one hidden behind image and text: the sub-text, the ideological message. Connected to Beijing, minorities are therefore under Beijing's control and are destined to follow the government's politic at line. In short, minorities hold the official ideology.

Peculiarly absent for all their presence, the image of minorities on posters is adapted to fit the official ideology. While constructing an inanimate minority and implying an active presence of the state (sometimes through modernity, sometimes through the Han majority as we will see below), these posters are made by Han artists for a Han public: they are the product of CCP ideology.

Divide et Impera: Han's Supremacy

The Ethnic Classification Project directed by scientists (sociologists, ethnologists… mostly trained in Western schools) and mainly CCP local and national leaders, constructed homogeneous and mutually exclusive "ethnic" communities. The communist government applied the "divide et impera" (divide and rule) strategy: in creating one majority and a myriad of small minorities groups, the CCP asserted the supremacy of the majority Han. 6 In some ways, colonialist theory may help us to understand the relationship that binds minorities and Han to each other: the minority was an effect of the majority and also the means for its consolidation as a majority. 7 It is significant that disciplines like sociology, anthropology or ethnology which tend to identify and classify the population into separate groups, were born in Europe when colonialism reached its high point.

The PRC state largely employed discourse of protection of minorities to justify the constant need of minorities to be ruled by the majority: posters illustrate this discourse dramatically.

Plate 2 : 阿姨又送糌粑来了 Auntie ("Ayi") Has Even Come to Bring Tsampa, author unknown, 1970.

In propaganda posters, the Han is constantly represented as the unmarked category. Whereas each minority is recognizable from its colourful and typical clothing or attire, the Han is identifiable through its non-marked characteristics. In this picture, we can easily distinguish the Tibetan woman on the right side from her multi-coloured apron and her hairdo. The child, as we learn from the word "tsampa" in the title, is a Tibetan child cared for by a Han nurse (right in the centre of the poster and therefore the most salient element in the composition). We can surmise from the red cross on the bed top sheet that this Tibetan child is sick.

Creating its own weak, backward, even sick, minority Other, the official discourse imposes the Han model as the dominant one. 8 The Han nurse here brings values like science (here, medicine and knowledge), modernity (western attire) and benevolence (the nurse brings both care and food-here a "tsampa", a traditional Tibetan drink), while the minority is desperately incompetent or in need. The central position of the Han nurse in the picture, between the child and his mother, marks the essential role the Han can play inside the minority. Moreover, the Han nurse has both the ability to care for and to feed the child like his own mother, who seems here incapable of so doing.9

Benevolence is another characteristic of the Han: the nurse here cares very much about the Tibetan customs, offering the little child tsampa, a Tibetan milk-based drink, while we know from the text on the nurse attire that the scene is located in Shanghai:

What do Tibetans do in Shanghai, so far away from Tibet? Where does this tsampa come from then? In addition, we can say from the umbrella the Han nurse brings, that she came even though the rain or the wind: Han are not afraid of braving the danger to protect the minorities. Meanwhile, the child is excessively grateful to the Han nurse, as we can see from his smiling face and his raised arm.

This picture gives us a lot of information about the Han, though unmarked. As a matter of fact, we can understand from the analysis of propaganda period, that the ethnic "di-vision" is a process to establish and impose the young PRC rules through the Han majority.

New Social Order: Civilizing Project

Han ethnicity is also representative of the government policy. The Han hegemony, as government policy representative, is justified by two main arguments: civilization and modernisation. They are dramatically visible on this poster below. Representing the minorities as incapable and backward implies a stable and sovereign Han. As a result, the Han make it a rule to "civilize" minorities. This poster features a Han "big brother" transferring his technological knowledge (technology here represented by tractors) to the Yi "sisters" and "brothers". Again in the central position of the picture, the Han is explaining something to Yi women and men dressed in ethnic clothes. The Yi are literally learning from the Han experience, like this woman holding a notebook, ready to take down the Han youth's every word. We can note as well that the minority Yi is represented in traditional clothing and attire: they represent the past, the old China, what China is not supposed to be anymore. The Han is the only one here that wears modern clothing. Modernisation of China is in Han hands, that is to say in the CCP hands. Modernisation is often presented as the condition for an economic development. Civilizing mission and economic modernisation are two arguments used in colonial times to justify colonization. This is disturbing that they are used to justify the Han hegemony as well…

The function of each member of the PRC is clearly defined through ethnic classification: the social organisation becomes undeniable and assigns people specific set-role. Here colonialist theory seems to be an useful framework thinking about minority representation in China. In what Partha Chatterjee called the "colonial rule of difference", the paradox is: the Han entice minorities with promises of modernity (or freedom in some cases), yet the promise can not be kept since minorities are represented essentially as limited by their own "inaptitude".10 Moreover, it is in Han interest to perpetuate this representation of the minorities' inaptitude, so as to maintain their own leading role.

Once the supremacy of the Han and the CCP rule has been established and legitimated by the inferiority of minorities, the new social order it imposes has to be justified and promoted as well.

Plate 4:

Chairman Mao Trusted Chairman Hua Completely; The People and Army

Warmly Endorse Him Too, Liu Renqing, 1976, Beijing It is necessary for the CCP to assert that a multiethnic state is some unchanged surviving reality from a multi-millennial China. Thus, "ethnicity" (minzu) becomes the reference capable of articulating and organizing China's diverse form of social identity, as well as social class.

Here, posters of Presidents Mao and Hua are held aloft together by a peasant, a worker, a soldier and an intellectual, all representatives of social classes, plus several ethnic representatives: ethnicity is used to mark people. This division of the population seeks to be historically natural, as Chinese society had always been divided into a leading majority Han and a multitude of to-be-guided minorities. The minorities here are expressly represented as "back-ward" and in the background.

The support of the whole population (every class and every ethnicity) is a sine qua non in the justification of the leaders' sovereignty. The two minorities represented just behind the class representatives and in front of the other minorities are a Tibetan woman and a Uygur woman: this has indeed to be connected with the fact that the Tibetans and the Uygurs are precisely the two minorities whose inclusions into Chinese modern nation-state have posed (and still pose) vast problems.

Under these circumstances, the ethnic division system that now defines Chinese social order has to seem "natural" and to be an integral part of Chinese history.

Multiethnic thus becomes an essential feature to define and describe China.

New National Identity: Territorial and Temporal Identity

It is remarkable that representations of minorities often attempt to emphasize China's territorial identity. The most represented minority is most certainly the Tibetan minority for the reasons we mentioned above. about the precise time (it could be four hundreds year ago as well as yesterday).

However, as being a part of China, representing minorities in such decors is also a way to represent China as an unchanging entity. However, it gives China an opportunity to set a new universality: calling itself a multiethnic state, representing its diversity through multiple minorities' cultures, languages, and clothes, China claims to be a melting pot, concentrating various aspects of human cultural practices. 14 From then on, China accedes to a new universal status, the one it lost while constructing a modern nation-state. The world is not concentrated around a Chinese unlimited empire, and China is not a magnetic centre to the barbarians anymore; but still, the modern Chinese nation-state remains a universalistic 14 A French scholar, Anne-Marie Thiesse, assumes that the Third French Republic emphasized the local to assert the national : picturing a large range of landscapes and ethnicities, France tried to find the universalizing and gathering power it has lost in the defeat against the Prussians in the end of the 19 th century. See Anne-Marie Thiesse, Ils apprenaient la France : l'exaltation des régions dans le discours patriotique, Paris, Editions de la Maison des sciences de l'homme, 1997. model: minorities represent its diversity, and the Han majority is defined as the core of this universe.

Conclusion

The representation of minorities as a collective group is clearly a political move toward a definition of a new Chinese national identity. Creating its Other, the Han identity (and the national identity) is established and justified at the same time.

Classification into ethnicities involves the securing a dominant majority. The new social order thus establishes who the dominant are and who the dominated are, and this organization, claiming to be universal, can never be questioned.

Of course these comments on representation of minorities are not limited to propaganda posters: we could draw the same conclusion from an analysis of other visual forms such as films, video clips, performances... Posters are a part of a much larger visual culture.

Similarly, the instrumentalisation of (ethnic) minorities for political ends is not confined to China: France, UK, America and other countries have all used this kind of process to assert their imperial authority (and in some ways still do so in a contemporary postcolonial context).

2

  See Nicholas B. Dirks, Castes of Mind, Colonialism and the Making of Modern India, Princeton University Press, 2001.

Plate 3 :

 3 学习好经验, 建设新山区Learn From Good Experience and Develop the New Mountain Area, Shan Xihe, 1974.

Plate 5 :

 5 Chairman Hua Comes to Our Tibetan Family, Liu Zhisen, 1978 On this poster, Chairman Hua sits with Tibetans who are listening carefully to Hua's every word. Beside them is a peach tree full of peaches: as Li Yu noticed rightly in her work on minorities' representation, you cannot find peach trees on the Tibetan Plate 6: 农奴女儿上大学, Serf's Daugther goes to University, Pan Xinshun, 1973 Plate 7: Title and author unknown, 1978 To be analysed with the first poster (children in the grass with a radio), these two pictures-among many others-illustrate what we have suggested before. In each of these posters, different symbols of innocence can be noted: figures are mostly children; little white lambs appear too. The presence of children has obviously something to do with an infantilization (and feminization) of the minority, and the lambs may emphasize their naivety. 13 But it refers an original "purity" as well. If we consider the décor in which the figures are represented, we can note: desert landscapes with mountains and infinite greenery; non spoiled landscapes with no machines, no plants, and no marks of alteration by men. Everything is represented as if minorities live in a different China, from different times. Even their clothes are out of time: traditional clothes cannot tell us 13 An abundant literature on feminization of the Chinese minorities can be found. See for instance Louisa Schein's articles on feminization of ethnicity such as "Gender and Internal Orientalism in China."in Modern China 23(1), 1997; and her book: Minority Rules: The Miao and the Feminine in China's Cultural Politics.Durham, NC: Duke University Press, 2000. See also Dru Gladney, "Representing Nationality in China: Refiguring Majority/Minority Identities" in The Journal of Asian Studies Vol. 53, No. 1: 92-123, 1994. Less has been written on infantilization of ethnicity.

  Despite modernization and industrialisation, China keeps being "authentic" and become rooted as a-temporal. It would be impossible to represent only Han people in such landscape without questioning their superiority in technology for instance. The Han is a definitively supposed to be a city dweller. By representing minorities in countryside, China's temporal boundaries can be pushed back; its history is rooted deeper and deeper. New China: Harmony and Unity Plate 8 : 团结起来, 争取更大的胜利 Stand United to Gain a Larger Victory, author unknown, 1975. This poster celebrates "tuanjie" (unity and unification) among the China's peoples. Minorities stand behind a worker, a peasant and a soldier. This concept of tuanjie involves the idea that all the differences, which are indeed foregrounded and promoted, have to be complementary but never conflicting. The title of the poster (minzu tuanjie, zhengqu gengda de shengli) translated into various minority languages (Tibetan, Mongolian, Manchu or Korean) is a slogan promoting national consensus, inclusive of all nationalities. The slogan translated into several languages and pictorially represented within the poster, seeks to indicate a simultaneous respect for linguistic differences-different yet united. But the book everyone holds here is the same one: visibly it is a selection of Mao Zedong thoughts. And it looks written in Chinese characters… We can find here the limits the CCP intentionally sets in the question of multiculturalism and alterity. Plate 9: 祖国歌 Song for our Motherland, author unknown, 1982 This poster particularly interesting because it is emphasizes the diversity of peoples in China, living under the CCP rules. Minorities are here represented in a harmonious concert, playing music and dancing. Although their differences (symbolised by different clothing and musical instruments), they all live in harmony, in a great symphony.

Pictures often occur with comments by the authors, historians or sinologists. Stefan Landsberger's collection is one of the most complete reference. See his book: Chinese Propaganda Posters: From Revolution to Modernization. Amsterdam; Singapore: The Pepin Press, 1995; and his website: "Stefan Landsberger's Chinese

  Mao's Talks of the Yan'an forum on Literature and Arts in May 1942 begins with the following sentence:

	Propaganda Poster Pages." http://www.iisg.nl/~landsberger/ (checked in April 2006).
	See also: Harriet Evans and Stephanie Donald, Picturing Power in the People's Republic of China:
	Posters of the Cultural Revolution, Lanham, Md.: Rowman & Littlefield, 1999; Claude Gorsky, La Chine à
	l'affiche, Paris, Ramsay, 1997 ; Victoria and James Edison, Cultural Revolution : Posters & Memorabilia,
	Atglen, Shiffer Pub, 2005; Catalogue of Vintage Chinese Posters c. 1939-1990 : Sale 583, London, Bloomsbery
	Auctions, 2006.

3 Several books on Chinese propaganda posters and « New Year » posters (nianhua) have been published.

4

8

  See an interesting and relevant paper on representations of Mexicans in Los Angeles by public health officials: Natalia Molina, Constructing Race Through Visual Culture: Medicalized Representations of Mexican Communities in Early Twentieth Century Los Angeles, 2003. http://latino.si.edu/researchandmuseums/presentations/molina_paper.html (checked on October 2006)

« Di-vision » is to describe here the ambivalence of the ethnic identification and classification: this is both about dividing population into groups, and producing a new way of visualizing and imagining Chinese nation.

The adage of "divide and impera" (divide and rule) in colonialist context has been developed by Indian scholars among others. Partha Chatterjee talks about "colonial difference" rule, the fact that colonialism is fundamentally based upon stressing absolute difference between colonizers and colonized, often in terms of "race." See Partha Chatterjee, The Nation and Its Fragments, Princeton University Press, 1993. Refering to Chatterjee's work, Nicholas B. Dirks argues that by highliting divisions of the Indian population itself into casts ,the British imposed their superiority representing themselves as "united ".

On the relationship between colonizer and colonized , see Albert Memmi, The Colonizer and theColonized, Beacon Press, 1971 ; or see Frantz Fanon, Black Skin, White Masks, Grove Press, 1971.

Note that the Chinese word "Ayi" can be used to call a woman around the same age or older than your own mother ( "Auntie" but not necessarly from your family), and to call a woman who looks after children ("nanny").

See Partha Chattejee, The Nation and Its Fragments : Colonial and Postcolonial Histories.

 Plateau. 11How did this tree arrive in Tibet? Would Hua have offered them peach trees? Does the scene take place in Tibet? If not, where does the scene take place and what would Tibetans do out of Tibet? 12 If we bring up the poster number two again (sick Tibetan child cared by a Han nurse), we can notice the same process. If we look very carefully in minute detail, a disconcerting element is the words sewed on the white sheet: "上海市儿童医院": Shanghai shi ertong yiyuan: Shanghai Children's Hospital. This suggests that the scene takes place in Shanghai: what would a Tibetan child and mother do in Shanghai, so far away for their hometown? One possibility is that they travelled to Shanghai; or that they are actually in Tibet but the sheet comes from Shanghai hospital.

According to me, whatever the answer is, this confusion is not insignificant: it attempts to justify the China government's hegemony on Tibet. This lack of realism or clear and concrete information is really significant: it attempts to muddle the spectator and to blur the inner frontiers of China by "deterritorializing" the characters. It seeks visibly to strengthen China's territoriality identity.

On the other hand, it appears very clearly that representations of minorities attempt to widen the temporal boundaries of China. We can state this from the recurrent symbols of "purity" and "authenticity" that appear in many posters representing Chinese minorities.

11 Li Yu, Representations of Ethnic Minorities in Chinese Propaganda Posters, 1957-1983. Send by the author, and available now at: http://mclc.osu.edu/rc/pubs/minzu/ (posted in April 2006) 12 Someone in Singapore suggested this may not be peach but apples, which can be found profusely on the Tibetan Plateau. I am grateful for his remark, but I maintain that those fruits look like peaches: peaches are very often represented on Chinese illustrations such as nianhua (New Year Pictures), paintings or posters. They represent prosperity, affluence and longevity. Even if they really are apples, they look strangely like peaches, which cannot be a coincidence: these posters were designed by Han for Han spectators.


Arnauld Belmer

Luc

Regulation of raphe serotonin neurons by serotonin 1A

and 2B receptors

Regulation of raphe serotonin neurons by serotonin 1A and 2B receptors

Arnauld Belmer* and Luc Maroteaux

Several lines of evidence implicate serotonin (5-hydroxytryptamine, 5-HT) in the etiology of mood disorders including major depressive disorders. Serotonergic neurons have long been recognized as key contributors to the regulation of mood and anxiety as the main target of serotonin selective reuptake inhibitor (SSRI) antidepressants. The therapeutic effects of SSRIs are initially triggered by blockade of the serotonin transporter SERT increasing local extracellular serotonin. Serotonin neurotransmission is tightly regulated by autoreceptors (serotonin receptors expressed by serotonin neurons) known to act through negative feedback inhibition at the cell bodies (5-HT 1A receptors) of the raphe nuclei or at the axon terminals (5-HT 1B receptors). Beneficial SSRI effects rely on long-term adaptations that are, at least partially, ascribed to a selective desensitization of somatodendritic 5-HT 1A autoreceptors [START_REF] Richardson-Jones | 5-HT1A autoreceptor levels determine vulnerability to stress and response to antidepressants[END_REF].

A positive regulation of serotonergic neurons by 5-HT 2B receptors has been detected in mice. Local agonist-stimulation of 5-HT 2B receptors in dorsal raphe nuclei increased extracellular serotonin suggesting a functional role of this receptor within serotonergic neurons [START_REF] Doly | Serotonin 5-HT2B receptors are required for 3,4-methylenedioxymethamphetamine-induced hyperlocomotion and 5-HT release in vivo and in vitro[END_REF]. Expression of 5-HT 2B receptors has been detected in subset of serotonergic neurons albeit at low levels [START_REF] Diaz | 5-HT2B receptors are required for serotonin-selective antidepressant actions[END_REF]. Both acute and long-term behavioral and neurogenic effects of SSRIs are abolished in mice knockout for 5-HT 2B receptor gene, (Htr2b -/-) or after exposure to selective 5-HT 2Breceptor antagonists. Conversely, chronic stimulation of 5-HT 2B receptors by selective agonists mimicked chronic SSRI actions on behavior and hippocampal neurogenesis, which were abolished in Htr2b -/-mice [START_REF] Diaz | 5-HT2B receptors are required for serotonin-selective antidepressant actions[END_REF]. Comparable lack of SSRI effects was recently reported in mice knockout for 5-HT 2B receptors only in serotonergic neurons (Htr2b 5-HTKO mice), in which dorsal raphe serotonin neurons displayed a reduced firing frequency, and a stronger hypothermic effect following 5-HT 1A -autoreceptor stimulation [START_REF] Belmer | Positive regulation of raphe serotonin neurons by serotonin 2B receptors[END_REF]. Cell autonomous effects were confirmed by the increased excitability of serotonergic neurons observed upon rapheselective 5-HT 2B -receptor overexpression. Correlative findings have been described in Humans, in which expression of 5-HT 2B receptors can be found in brain stem and a loss-of-function polymorphism of 5-HT 2B receptors has been associated with serotonindependent phenotypes including increased impulsivity and suicidality [START_REF] Bevilacqua | A population-specific HTR2B stop codon predisposes to severe impulsivity[END_REF].

Serotonin released within raphe nuclei is known to induce feedback inhibition of serotonergic neuron firing activity by stimulating dendritic 5-HT 1A negative autoreceptors. Unlike soma and terminals, the dendritic serotonin release is independent of action potentials, relies on L-type Ca2+ channels, can be induced by NMDA, and displays distinct sensitivity to the SSRI antidepressants [START_REF] Colgan | Action potential-independent and pharmacologically unique vesicular serotonin release from dendrites[END_REF]. Dendritic serotonin release, and hence 5-HT 1A receptor-mediated autoinhibition, is thus engaged by excitatory glutamatergic inputs to the dorsal raphe, via locally triggered calcium influx, rather than by neuronal firing. The unique control of dendritic serotonin release has important implications for the antidepressant action of SSRIs. The lack of 5-HT 2B receptor in serotonergic neurons is associated with a higher 5-HT 1A -autoreceptor reactivity and thus a lower activity of these neurons [START_REF] Belmer | Positive regulation of raphe serotonin neurons by serotonin 2B receptors[END_REF]. The excess of inhibitory control exerted by 5-HT 1A receptors in Htr2b 5-HTKO mice may thus explain the lack of response to chronic SSRI in these mice.

The serotonergic tone of raphe neurons and thus the SSRI therapeutic effects likely results from the opposite control exerted by 5-HT 1A and 5-HT 2B receptors via a mechanism that remains to be described.

Affiliations

:

  INSERM U 839, Paris, 75005, France; UMR-S839 Sorbonne Université, Paris, 75005, France; Institut du Fer à Moulin, Paris, 75005, France. *Present address: Translational Research Institute, Queensland University of Technology, Brisbane, QLD 4059, Australia Correspondence should be addressed to: Luc Maroteaux INSERM UMR-S839, 17 rue du Fer a Moulin Paris, 75005, France; E-mail : luc.maroteaux@upmc.fr

Funding and Disclosure


Imane Moutkine

Emily Lauren Collins

Catherine Béchade

Luc Maroteaux
email: luc.maroteaux@upmc.fr

Evolutionary considerations on 5-HT 2 receptors

Keywords: serotonin, receptor, evolution, vertebrates, proteasome

Serotonin is a neurotransmitter widely conserved from ancient organisms lacking nervous systems through man, and its presence precedes the appearance of nervous systems on both developmental and evolutionary time scales. Serotonin receptor subtypes diversified approximately at the time period during which vertebrates diverged from invertebrates. The biological and clinical importance of serotonin receptors, may benefit from studies on their evolution. Although potentially informative about their pathophysiological functions, reviews on this topic are sparse. Several observations support basic functions mediated by serotonin, both in periphery and central nervous system. In particular, 5-HT 2B receptors have been implicated in embryonic development, including cell proliferation, survival, and/or differentiation, in either neural crest cell derivatives, myeloid cell lineage, or heart embryogenesis. In this review, we collected existing data about the genomic association between the RPN2 proteasome subunit gene Psmd1 and the 5-HT 2B receptor gene Htr2b. We discuss about the possibility that, during genome duplications, a single copy of this pair of genes has been conserved, suggesting a strong selective pressure. Many basic physiological functions in which serotonin system is involved could be linked to the early association of these two genes in pre-vertebrates. Their evolutionary association highlights the possibility that the 5-HT 2B receptor gene, Htr2b, is the common ancestor of 5-HT 2A/2B/2C -receptor subfamily. Disentangling these possibilities could bring new understanding of the respective importance of these receptors in pathophysiology of serotonin.

Introduction.

Biogenic amines including serotonin can be detected in virtually all metazoans and exert many behavioral and physiological functions. In vertebrates, most receptors, which mediate biogenic amine physiological activities, belong to the broad family of G protein-coupled receptors (GPCRs), one of the largest eukaryotic receptor families. Indeed, GPCRs are mediating such a diversity of biological functions that they evolutionarily expanded in such a way that they are considered as one of the most successful gene families [START_REF] Fredriksson | The G-protein-coupled receptors in the human genome form five main families. Phylogenetic analysis, paralogon groups, and fingerprints[END_REF]. Within the Rhodopsin-like GPCR family, biogenic amine receptors likely emerged together with the Fungi and Metazoan lineage. In Metazoan, the diversification of biogenic amine GPCRs has largely contributed to the physiological expansion of peripheral and of central nervous system complexity [START_REF] Spielman | Comprehensive, structurally-informed alignment and phylogeny of vertebrate biogenic amine receptors[END_REF].

Serotonin receptors have the highly-conserved structure of seven transmembrane domains common to all GPCRS, including three extracellular loops involved in ligand recognition and three intracellular loops involved in intracellular signaling mainly through Gi-, Gs-, or Gq-proteinmediated pathways. Many pharmaceuticals used to treat diseases such as cardiac diseases, hypertension, migraine, schizophrenia, or depression are directly acting at serotonin receptors. Although biologically and clinically important, studies on the evolution of sub families of serotonin receptors are limited.

Comparisons among orthologous proteins in various species is a way to estimate the gene evolutionary relationships. The percentage of amino acid homology between species allows to estimate the rate of gene evolution. For GPCRs, this rate has been estimated at approximately 1% per 10 million years [START_REF] Peroutka | The molecular evolution of G protein-coupled receptors: focus on 5hydroxytryptamine receptors[END_REF]. From this calculation, it can be hypothesized that primitive serotonin receptors have diverged more than 800 million years ago since the 3 major classes of serotonin receptors (i.e. 5-HT 1 , 5-HT 2 and 5-HT 4-6-7 receptors) show less than 25% homology and orthologous receptor are present in many invertebrates. Other serotonin receptor subtypes differentiated approximatively at the time period during which vertebrates diverged from invertebrates [START_REF] Peroutka | The molecular evolution of G protein-coupled receptors: focus on 5hydroxytryptamine receptors[END_REF]. The serotonin receptor subtypes have further differentiated over the vertebrate evolution although their respective relationships have not been characterized.

In this review, we started from the observation that the Htr2b gene, coding for the 5-HT 2B receptor is encoded within an intron of a large subunit of the proteasome Psmd1, in humans, mice and rats. Existing data from genome sequencing support the hypothesis that association between these two genes appeared in pre-vertebrates. Previous published work indicated that serotonin and 5-HT 2B receptors regulate neural crest cells, heart, and bone marrow stem cells differentiation and proliferation. In addition, these data revealed that during genome duplications, a strong selective pressure seems to keep a single copy of this genetic association to the proteasome Psmd1 gene. Together with its early appearance in pre-vertebrates and its persistence throughout evolution, this association may have functional consequences. These data raised the possibility that the 5-HT 2B receptor is the common ancestor of the 5-HT 2A/2B/2C -receptor subfamily.

Genomic organization of the 5-HT 2B receptor gene.

In humans, the HTR2B gene is located on the reverse strand at the tip of the second chromosome at 2q37.1. The 5-HT 2B receptor's cDNA, which is 2246 base pairs (bp) long, is encoded by four exons, three of which are coding, and produce a pre-mRNA transcript of approximately 51 Kbp before splicing [START_REF] Schmuck | Cloning and functionnal characterisation of the human 5-HT2B serotonin receptor[END_REF][START_REF] Kursar | Molecular cloning, functional expression, and mRNA tissue distribution of the human 5-hydroxytryptamine2B receptor[END_REF][START_REF] Choi | The human serotonin 5-HT2B receptor: pharmacological link between 5-HT2 and 5-HT1D receptors[END_REF][START_REF] Wainscott | Pharmacologic characterization of the human 5-hydroxytryptamine2B receptor: evidence for species differences[END_REF]. Interestingly, the PSMD1 gene, which encodes the non-ATP regulatory subunit 1 (RPN2) of the 26S proteasome, is located at the same locus on the forward strand and overlaps HTR2B, which is entirely encoded within intron 16 as revealed in the Ensembl database [START_REF] Zerbino | Ensembl[END_REF] (Fig. 1). Similar organization with the 5-HT 2B receptor's cDNA encoded in the reverse strand of a large intron of the proteasome RPN2 can be found not only in humans, rats, and mice but also in all mammals. In all other vertebrates from Teleost fishes, such as zebrafish Danio rerio, to jawless fishes, such as lamprey Petromyzon marinus, similar association can be observed (Fig. 1). Interestingly, in ascidian pre-vertebrates such as Ciona savigny (ascidian, tunicates, chordates), a simple survey of the largest intron of the Psmd1-like gene (ENSCAVG00000000318) revealed that it also includes a coding region that encodes for a protein very close to 5-HT 2 receptors (Fig. 1). None of the other serotonin receptor genes is embedded in other genes.

Evolutionary relationship within Htr2 genes.

Focusing on 5-HT 2 receptor genes in all tetrapods including mammals, three Htr2 genes can be identified, when, in teleost fishes such as zebrafish, five Htr2 genes are present. Closer relationships can be found between Htr2a, and to Htr2c compared to Htr2b genes as shown by the Ensembl gene tree comparison [START_REF] Zerbino | Ensembl[END_REF] (Fig. 2A-B). In more primitive species, two Htr2 genes have been identified in the jawless fish agnatha, Lamprey. Only one Htr2-like gene can be found encoded in the reverse strand of Psmd1-like gene (ENSCAVG00000000318) in Ciona savigny. Concerning Psmd1 evolutionary relationships, an almost identical evolutionary tree to that of Htr2b gene is found in the Ensembl gene tree comparison [START_REF] Zerbino | Ensembl[END_REF] (Fig. 2C). One of the two lamprey Htr2 genes is associated to Psmd1 as well as the uniquely identified Ciona Htr2-like gene, which is embedded in Psmd1 gene. Furthermore, only one of the five Htr2 genes found in teleost such as zebrafish is associated to Psmd1 gene and identified as Htr2b. Importantly, none of the invertebrate Htr2 genes is associated with Psmd1/RPN2 coding gene in fruit fly or nematode.

Evolutionary considerations in the context of Htr2 genes.

A whole genome duplication event has been proposed to arise at the non-vertebrate chordate divergence that gave rise to the jawless vertebrates (Agnatha-today lampreys and hagfish) [START_REF] Mulley | Comparative genomics: small genome, big insights[END_REF]. A gene present in the ancestral prochordate lineage would give rise to two copies of that gene in agnathans. Function of the ancestral gene can be retained by duplicated genes (sub functionalization), could acquire new function (neo functionalization), or could lose its original function and become a pseudogene. At the divergence of the agnathans toward the jawed vertebrates (Gnathostomata), a second whole genome duplication has been proposed to occur [START_REF] Mulley | Comparative genomics: small genome, big insights[END_REF]. From these two genome duplication events, a potential for four copies of the original ancestral prochordate gene could be present in gnathostomes. Whole genome duplication events were followed by loss of some redundant genes. Interestingly, in the non-vertebrate chordate Ciona, Psmd1 gene is found associated with a Htr2-like gene. Later, in jawless fishes, the Psmd1 gene is found associated with only one of the two Htr2 gene. Then, the Htr2b gene of cartilaginous fishes, of all tetrapods, and of zebrafish is associated with the Psmd1 gene (Fig. 3). Tetrapods have one Htr2a and one Htr2c gene, whereas teleosts such as zebrafish display two Htr2a-and two Htr2clike genes. One likely explanation would be that an association between Htr2 and Psmd1 gene appeared in pre-vertebrates like Ciona. Following the scheme proposed in reference [START_REF] Mulley | Comparative genomics: small genome, big insights[END_REF], this ancestral gene could have been duplicated in jawless fishes and only one of the two resulting Htr2 gene remained associated with Psmd1, giving rise to one Htr2a-and one Htr2b-like gene in lamprey. Following the subsequent whole genome duplication that gave rise to tetrapods, the Htr2a-like gene could have been duplicated, giving rise to the Htr2a and Htr2c of vertebrates; the Htr2b associated to Psmd1 having lost its duplicated copy (Fig. 3). Finally, the most recent whole genome duplication that appeared at the origin of teleost fishes [START_REF] Mulley | Comparative genomics: small genome, big insights[END_REF] may have led to two Htr2a and two Htr2c genes, the Htr2b associated to Psmd1 having likely again lost its duplicated copy. Thus, this interpretation would support that a strong selective pressure exists in order to keep only one copy of Psmd1/Htr2. This would make Htr2b as the common ancestor of the other Htr2 genes in vertebrates; existing data could support that Htr2b is associated with basic functions of serotonin in embryonic differentiation and development.

Evolutionary consideration about early vertebrates and serotonin implication

At the transition from invertebrates to vertebrates, the shift from a passive to an active mode of predation led to the appearance of many features specific to vertebrates:

1-Neural crest-The acquisition of the neural crest cells and its derivatives in vertebrate lineage was of high evolutionary importance [START_REF] Gans | Neural crest and the origin of vertebrates: a new head[END_REF][START_REF] Le Douarin | The Neural Crest[END_REF]. The neural crest cells are a population of multipotent stem-cells originating from the neuroectodermal cells located at the junction between neural plate and future epidermis. Concomitant to the neural tube closure, neural crest cells delamination generates migratory cells that will populate various regions of the embryo. An epithelio-mesenchymal transition makes cells ready to migrate within embryonic tissues. Neural crest cells differentiate into melanocytes, craniofacial bones, or enteric neurons, glia, and smooth muscle [START_REF] Yu | The evolutionary origin of the vertebrate neural crest and its developmental gene regulatory network--insights from amphioxus[END_REF]. Neural crest cells generate many features specific to vertebrates like jaw-based predation [START_REF] Gans | Neural crest and the origin of vertebrates: a new head[END_REF]. It was recently shown that, in the tunicate Ciona intestinalis, a cephalic melanocyte lineage appears to be similar to neural crest since expression of the transcription factor Twist was sufficient to reprogram this lineage into migrating 'ectomesenchyme'. This work supported that the neural crest melanocyte lineage appeared before the divergence of tunicates and vertebrates [START_REF] Abitua | Identification of a rudimentary neural crest in a non-vertebrate chordate[END_REF]. Thus, features considered as specific to vertebrates can be identified in the common ancestor of agnathans and gnathostomes [START_REF] Ota | Hagfish embryology with reference to the evolution of the neural crest[END_REF].

The question is to know if serotonin could be implicated in such evolutionary processes. Interestingly, Jean Lauder, in late 1970 th , described the participation of serotonin to embryogenesis of many species [START_REF] Lauder | Roles for serotonin in neuroembryogenesis[END_REF]. In vertebrates, she described that serotonin participates in neural crest cell migration [START_REF] Lauder | Serotonin and morphogenesis: I. Sites of serotonin uptake and -binding protein immunoreactivity in the midgestation mouse embryo[END_REF]. Our group showed about 20 years ago that Htr2b was expressed by migratory neural crest cells in mice (Fig. 4A-B) and that its blockade induced defects in embryonic development affecting neural crest cells survival [START_REF] Choi | 5-HT2B receptor-mediated serotonin morphogenetic functions in mouse cranial neural crest and myocardiac cells[END_REF]. Subsequently, the group of Michaela Ori, working with Xenopus embryos, showed that the 5-HT 2B -receptor signaling had a crucial role in neural crest cell derivatives and craniofacial morphogenesis, being involved in shaping the first branchial arch and the jaw joint [START_REF] Ori | Unraveling new roles for serotonin receptor 2B in development: key findings from Xenopus[END_REF]. More recently the group of Michel Bader confirmed in Zebrafish an organizing role of serotonin in pharyngeal arch morphogenesis since the 5-HT 2B receptor was found to be expressed in the pharyngeal arches and its pharmacological inhibition also induced defects in pharyngeal arch morphogenesis [START_REF] Bashammakh | Serotonin is required for pharyngeal arch morphogenesis in zebrafish[END_REF]. These results have been completed by the group of Michael Gershon who showed that the 5-HT 2B receptor is expressed by enteric neurons, another neural crest derivative, and suggested that by stimulating 5-HT 2B receptors, serotonin affects the fate of the large subset of enteric neurons [START_REF] Fiorica-Howells | Serotonin and the 5-HT2B receptor in the development of enteric neurons[END_REF]. Furthermore, serotonin can stimulate proliferation of melanocytes, also neural crest derivatives, via 5-HT 2B receptors [START_REF] Slominski | Functional activity of serotoninergic and melatoninergic systems expressed in the skin[END_REF]. These studies indicated that serotonin via 5-HT 2B receptors regulates proliferation, differentiation, survival, and/or apoptosis of neural crest cells, which were already acquired by pre-vertebrates.

2-Heart structure-

The transition from invertebrates to vertebrates was associated with an increased sophistication of the heart. From a simple single-monolayered heart, the ascidian heart started being multilayered before the apparition of multichamber heart in vertebrates. The embryonic development of vertebrate heart requires a complex coordination between genetic programs and cellular and morphogenetic events. The genetic coordination of the heart embryonic development and anatomy in pre-vertebrates may be informative of important basic biological mechanisms. In the tunicate Ciona, the heart displays the basic cardiac genetic program and developmental cell specification events of the chordate heart [START_REF] Anderson | Ciona as a Simple Chordate Model for Heart Development and Regeneration[END_REF]. Pre-vertebrate tunicate heart is composed of a single-chamber peristaltic pump formed by a single layer of myocardium surrounded by a pericardium. In vertebrates, the heart is composed of at least a two-chambered myocardium with cardiac myocytes, an endocardium, valves that separate inflow and outflow chambers and a surrounding pericardium; the embryonic heart derives from different embryonic territories, including primary and secondary heart fields. Embryonic heart development starts with the formation of a heart tube; looping of the tube initiates the formation of heart chambers through a conserved process among vertebrates [START_REF] Jensen | Evolution and development of the building plan of the vertebrate heart[END_REF]; then, the secondary heart field gives rise to the right ventricle and outflow tract. The organization of Ciona heart represents a simplified version of the cardio-pharyngeal program in which progenitor cells produce the first and second heart field including pharyngeal muscles precursors and associated heart stem cells [START_REF] Anderson | Ciona as a Simple Chordate Model for Heart Development and Regeneration[END_REF]. Heart progenitor cells of simple chordates also generate precursors of the atrial siphon muscles. It appears thus that the last common ancestor between tunicates and vertebrates already possessed multipotent cardiopharyngeal precursors that contributed to the emergence of the secondary heart field [START_REF] Stolfi | Early chordate origins of the vertebrate second heart field[END_REF].

We showed that the 5-HT 2B receptor, which is expressed by embryonic cardiomyocytes, was involved in heart development (Fig. 4C-D) [START_REF] Nebigil | Serotonin 2B receptor is required for heart development[END_REF]. The Htr2b inactivation leads to the appearance of dilated cardiomyopathy in surviving mice. Serotonin protects newborn cardiomyocytes against apoptosis via cross-talks between phosphatidylinositol-3 kinase (PI3K)/Akt and extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathways that regulate mitochondrial functions [START_REF] Nebigil | Functional consequences of serotonin 5-HT2B receptor signaling in heart: A role of mitochondria for the transition between hypertrophy and heart failure?[END_REF]. These findings support a role for serotonin via 5-HT 2B receptor regulatory events during heart development, which also started appearing at the prevertebrate stage.

3-Blood system-

The transition from invertebrates to vertebrates was associated with improved blood system formation. In reaction to the environment, immune system evolved to protect individuals from microbial infection and to recycle their damaged tissues. The two components of the immune system consist in innate and adaptive immunity. Innate immunity is common to all species while adaptive immune system appeared with vertebrates. Adaptive immune system relies on near limitless genetic diversity with long-term memory of lymphocytes. In Ciona, chitin-binding proteins containing variable regions, display high allelic complexity that may reflect functional specificity [START_REF] Dishaw | The amphioxus genome provides unique insight into the evolution of immunity[END_REF]. Agnathans have completed innate responses with adaptive elements; in lampreys, leucine rich repeat-containing proteins appear as variable lymphocyte receptors. In higher vertebrates, the major histocompatibility complex, T-cell and B-cell receptors (immunoglobulins) also assists innate responses as adaptive elements [START_REF] Buchmann | Evolution of Innate Immunity: Clues from Invertebrates via Fish to Mammals[END_REF].

Hematopoietic stem cells initially derive from blood islands on the yolk sac at E7.5 in mice from endothelial progenitor cells (hemangioblasts). Hematopoiesis then occurs later in the aortagonad-mesonephros (AGM) region at E10.5 in mice and moves to fetal liver at E12.5 before migrating to bone marrow around birth. Hematopoietic stem cells give rise to blood cells, including platelets, erythrocytes, myeloid and lymphoid cells. In mice, when looking at the expression of 5-HT 2B receptors in embryos, there were mRNA and protein located at the distal end of the embryo and along the neural tube similar to AGM (Fig. 4AB). In Htr2b -/-mice, the E12.5 embryos are smaller and paler than controls embryos with light pink liver (Fig. 4E). AGM and liver are sites of embryonic hematopoiesis. Furthermore, the absence of 5-HT 2B receptors generates permanent changes in the composition of the blood and bone-marrow lineage, particularly in myeloid lineage and in endothelial cell progenitors in adult mice [START_REF] Launay | Serotonin 5-HT2B receptors are required for bone-marrow contribution to pulmonary arterial hypertension[END_REF] (Fig. 4F). Serotonin participates in pulmonary hypertension [START_REF] Launay | Function of the serotonin 5-hydroxytryptamine 2B receptor in pulmonary hypertension[END_REF] and valvulopathies [START_REF] Hutcheson | Serotonin receptors and heart valve disease--it was meant 2B[END_REF]; we and other have shown that the initial functions of 5-HT 2B receptors in these diseases are restricted to bone-marrow precursor cells [START_REF] Launay | Serotonin 5-HT2B receptors are required for bone-marrow contribution to pulmonary arterial hypertension[END_REF][START_REF] Ayme-Dietrich | Serotonergic 5-HT2B receptors in mitral valvulopathy: bone marrow mobilization of endothelial progenitors[END_REF].

Together these data pointed Htr2b as a regulator of neural crest cell, heart development and bone marrow stem cells differentiation and proliferation. These features appeared in pre-vertebrates and remained throughout evolution, supporting a putative early appearance of the functions mediated by this receptor subtype in evolution. It has to be noticed that no developmental defect has been described in either Htr2a -/-or Htr2c -/-mice.

Putative link between Htr2b and Psmd1

The 26S proteasome is a highly conserved multicatalytic protease from yeast to mammals, which functions to degrade proteins following ubiquitination via the ubiquitin-proteasome system. The proteasome is required for critical cellular functions including cell cycle control and protein homeostasis. The 26S proteasome contains many distinct subunits and is predominantly composed of two large structures; the 19S complex (regulatory particle), within which RPN2 is found, functions to recognize ubiquitinated proteins; it then processes and transports them to the 20S complex (core particle), where proteolysis occurs. RPN2 functions as a molecular scaffold and mediates the binding of a variety of factors including additional proteasome subunits. RPN1 and RPN2 emerge as the primary coordinators of the ubiquitin processing machinery at the base of the 26S proteasome. RPN1 and RPN2 are characterized by eleven tandem copies of a 35-40 amino acid repeat motif termed the proteasome/cyclosome repeat, and RPN13, the ubiquitin receptor, binds to the C-terminal 20 residues of RPN2 [START_REF] He | The structure of the 26S proteasome subunit Rpn2 reveals its PC repeat domain as a closed toroid of two concentric α-helical rings[END_REF]. As a result, the RPN2 protein plays a crucial role as it is a major site of proteasomal regulation.

The genomic arrangement between Htr2b and Psmd1, which has been evolutionarily conserved, could indicate a functional interaction between these two genes. As overlapping genes are relatively common in DNA and RNA viruses, they are quite rare in non-viral organisms, with only few examples described in mammalian genomes. The most common form of overlap seen in eukaryotes between protein coding genes is a nested structure whereby, the internal gene, like Htr2b, lies entirely within an intron of the external gene, here Psmd1. It is thought that this genetic arrangement may result in a co-regulatory relationship [START_REF] Assis | Nested genes and increasing organizational complexity of metazoan genomes[END_REF], leading to a negative correlation due to transcriptional collusion or having a neutral effect; previous studies have shown significant negative correlation between nested genes with little positive correlation [START_REF] Yu | Nested genes in the human genome[END_REF]. Several potential mechanisms could be suggested including direct competition for the transcriptional apparatus or complementary binding between the pre-mRNA of Htr2b and Psmd1 intron. Therefore, it remains to be determined if the two genes are expressed coordinately.

Conclusion.

Several observations indicate that some embryonic functions attributed to serotonin are performed by 5-HT 2B receptors. Existing data support that the association of Htr2b with Psmd1 appeared early in evolution. Many developmental physiological functions, in which serotonin system is involved, could be linked to the early association of these two genes in pre-vertebrates. More work is needed to clearly establish the importance of this association and the possibility that Htr2b is the common ancestor of 5-HT 2 -receptor genes. Determining the molecular basis if this gene association may provide new insight into the embryonic, differentiation, and/or physiological functions of serotonin both in the brain and periphery and bring new understanding of their respective importance in pathophysiology of serotonin. [START_REF] Mulley | Comparative genomics: small genome, big insights[END_REF]. The whole genome duplication that arrived at the divergence between chordates and jawless fishes may have generated the two Htr2 genes in Jawless fishes. The second whole genome duplication that aroused between Jawless fish and cartilaginous fish and all tetrapods may have generate the three Htr2 genes in tetrapods. And the later whole genome duplication that took place at the divergence of teleost fish may have generated the five Htr2 genes in teleost fishes. Gene exons from Htr2a are in blue, from Htr2c in orange, and fom Htr2b in pink with exons surrounding the largest intron of Psmd1 in grey; lost copy X. [START_REF] Nebigil | Serotonin 2B receptor is required for heart development[END_REF]. (D) Histological analysis of newborn mutant hearts shows an obvious loss of ventricular (v) mass (hypoplasia), adapted from [START_REF] Nebigil | Serotonin 2B receptor is required for heart development[END_REF]. (E) The Htr2b -/-(-/-) embryos at E12.5 are smaller and paler with light pink liver, a place for embryonic hematopoiesis, adapted from [START_REF] Nebigil | Serotonin 2B receptor is required for heart development[END_REF], "Copyright (2000) National Academy of Sciences.". (F) The absence of 5-HT 2B receptors generates permanent changes in the composition of the blood and bone-marrow myeloid lineage, particularly in endothelial cell progenitors, adapted from [START_REF] Launay | Serotonin 5-HT2B receptors are required for bone-marrow contribution to pulmonary arterial hypertension[END_REF] with permission.

Figure 1 :

 1 Figure 1: Conserved gene structure of the HTR2B gene.The HTR2B gene is encoded on the reverse strand of the PSMD1 gene located at the same locus, which encodes the non-ATP regulatory subunit RPN2 of the proteasome 19S in human. In other vertebrates from the jawless fish lamprey to Teleost fishes such as zebrafish, similar organization can also be observed. Analysis of ascidian pre-vertebrates such as Ciona savigny locus revealed identical organization with a large intron of Psmd1-like gene encoding a Htr2-like gene. All data are extracted from the Ensembl database[START_REF] Zerbino | Ensembl[END_REF].

Figure 2 :

 2 Figure 2: Evolutionary relationships between Htr2 genes; comparison with Psmd1 genes. Close but clearly distinct relationships can be found between Htr2a, Htr2b, Htr2c genes. (A-B) Htr2 gene tree. (C) The Psmd1 gene tree is identical to Htr2b subtree (B). All data are extracted from the Ensembl database [8].

Figure 3 :

 3 Figure3: Evolutionary consideration about Htr2 genes. Species evolution has been favored by whole genome duplications (WGD)[START_REF] Mulley | Comparative genomics: small genome, big insights[END_REF]. The whole genome duplication that arrived at the divergence between chordates and jawless fishes may have generated the two Htr2 genes in Jawless fishes. The second whole genome duplication that aroused between Jawless fish and cartilaginous fish and all tetrapods may have generate the three Htr2 genes in tetrapods. And the later whole genome duplication that took place at the divergence of teleost fish may have generated the five Htr2 genes in teleost fishes. Gene exons from Htr2a are in blue, from Htr2c in orange, and fom Htr2b in pink with exons surrounding the largest intron of Psmd1 in grey; lost copy X.

Figure 4 :

 4 Figure 4: The 5-HT 2B receptor is associated with development of neural crest cells derivatives, heart, and hematopoietic system. (A-B) On E10.5 (embryonic day 10.5) embryos, the 5-HT 2Breceptor expression as seen by mRNA (A) and protein (B) is located in the cephalic region (b), neural crest cells (nc) derivatives, heart (h), and at the distal end of the embryo (*), adapted from[START_REF] Choi | 5-HT2B receptor-mediated serotonin morphogenetic functions in mouse cranial neural crest and myocardiac cells[END_REF] with permission. (C) Inactivation of 5-HT 2B receptors (Htr2b -/-) (-/-) causes cardiac development defects leading to partial embryonic and perinatal lethality, adapted from[START_REF] Nebigil | Serotonin 2B receptor is required for heart development[END_REF]. Semithin sagittal sections of E9.5 embryos demonstrated a severe reduction in the thickness of the ventricle (v) including both the compact zone and the trabeculae in Htr2b -/-(-/-) embryos. a, Atrium; en, endocardium (e). Higher magnification shows a reduction of trabecular cells (tr) in the mutant heart, whereas elongated cells (white arrowhead) are visible in the compact subepicardial layer (sep). Epicardial cells (ep) are normally developed, adapted from[START_REF] Nebigil | Serotonin 2B receptor is required for heart development[END_REF]. (D) Histological analysis of newborn mutant hearts shows an obvious loss of ventricular (v) mass (hypoplasia), adapted from[START_REF] Nebigil | Serotonin 2B receptor is required for heart development[END_REF]. (E) The Htr2b -/-(-/-) embryos at E12.5 are smaller and paler with light pink liver, a place for embryonic hematopoiesis, adapted from[START_REF] Nebigil | Serotonin 2B receptor is required for heart development[END_REF], "Copyright (2000) National Academy of Sciences.". (F) The absence of 5-HT 2B receptors generates permanent changes in the composition of the blood and bone-marrow myeloid lineage, particularly in endothelial cell progenitors, adapted from[START_REF] Launay | Serotonin 5-HT2B receptors are required for bone-marrow contribution to pulmonary arterial hypertension[END_REF] with permission.

Funding Sources

This work has been supported by funds from the Centre National de la Recherche Scientifique, the Institut National de la Santé et de la Recherche Médicale, the Sorbonne Universités, and by grants from the Fondation pour la Recherche sur le Cerveau, the Fondation de France, the Fondation pour la Recherche Médicale "Equipe FRM DEQ2014039529", the French Ministry of Research (Agence Nationale pour la Recherche ANR-17-CE16-0008 and the Investissements d'Avenir programme ANR-11-IDEX-0004-02). LM's team is part of the École des Neurosciences de Paris Ile-de-France network and of the Bio-Psy Labex and as such this work was supported by French state funds managed by the ANR within the Investissements d'Avenir programme under reference ANR-11-IDEX-0004-02.

Conflict of interest

The authors declare no competing financial interests.