Julia Astegiano

Jimena Andrieu

Matías Wajner

Victoria Marquez

Valentina Saur Palmieri

Julieta Karina

Torrico Chalabe

François Massol

Ana Calviño

Fernando Zamudio

; Andrieu

Matías ; Wajner

; Marquez

Saur Palmieri

François ; Massol

; Calviño

& Zamudio

Commoning social-ecological networks through the lens of relational ontologies and other economies: How ecologists can diversify their notions of human-non-human relationships

Keywords: Ethnobiology, multiplex ERGMs, relational values, social, solidarity, popular and feminist economies, the common

Introduction

The way we conceive our relationships with nature clearly influences how we act as scholars, stakeholders and members of a community in the face of the current biodiversity crisis [START_REF] Balvanera | The role of the values of nature and valuation for addressing the biodiversity crisis and navigating towards more just and sustainable futures[END_REF][START_REF] Leff | Saber ambiental: sustentabilidad, racionalidad, complejidad, poder[END_REF][START_REF] Pascual | Valuing nature's contributions to people: the IPBES approach[END_REF]. An increasing number of ecologists now emphasize the need to shift our perspective from ecosystems to social-ecological systems (SESs), which emerge as a result of human-nature relationships [START_REF] Balvanera | The role of the values of nature and valuation for addressing the biodiversity crisis and navigating towards more just and sustainable futures[END_REF][START_REF] Berkes | Navigating social-ecological systems: building resilience for complexity and change[END_REF][START_REF] Díaz | Assessing nature's contributions to people: Recognizing culture, and diverse sources of knowledge, can improve assessments[END_REF]. This is in line with the impressive rise of studies on social-ecological [START_REF] Kluger | Studying humannature relationships through a network lens: A systematic review[END_REF][START_REF] Sayles | Social-ecological network analysis for sustainability sciences: a systematic review and innovative research agenda for the future[END_REF]. However, these studies mostly focused on the structure of either social or ecological interactions, and few ones have really challenged the full characterization of the social, ecological and social-ecological interactions that produce SESs ("fully articulated SENs" in [START_REF] Sayles | Social-ecological network analysis for sustainability sciences: a systematic review and innovative research agenda for the future[END_REF]"type III networks" in Kluger et al., 2020; see also [START_REF] Barnes | The social structural foundations of adaptation and transformation in social-ecological systems[END_REF], and the third section of [START_REF] Labeyrie | Networking agrobiodiversity management to foster biodiversity-based agriculture. A review[END_REF].

Even though SEN studies have advanced our understanding of human dependence on nature, they are still dominated by the modern conception of nature (e.g. Felipe-Lucía, 2022; [START_REF] Kluger | Studying humannature relationships through a network lens: A systematic review[END_REF]The QUINTESSENCE Consortium, 2016; but see [START_REF] Sayles | Social-ecological network analysis for sustainability sciences: a systematic review and innovative research agenda for the future[END_REF]. Such a conception makes nature both external to society -i.e., nature is conceived as a matrix over which human societies develop-and universal -i.e., can be governed by universal laws established by modern sciences [START_REF] Robertson | Measurement and alienation: making a world of ecosystem services[END_REF][START_REF] Smith | Desarrollo desigual. Naturaleza, capital y producción de espacio[END_REF]. This implies that, even when the existence of several cultures is recognized, there is just one nature [START_REF] Escobar | Territorios de diferencia: Lugar, movimientos, vida, redes. Envión editores[END_REF], that in turn can be dominated and manipulated, and its reproduction (sensu [START_REF] Moore | El capitalismo en la trama de la vida[END_REF]) can be separated from that of human societies [START_REF] Moore | El capitalismo en la trama de la vida[END_REF][START_REF] Smith | Desarrollo desigual. Naturaleza, capital y producción de espacio[END_REF]. That is, a nature that neglects interdependencies between culture and nature. Thus, most current SEN studies may implicitly reproduce a narrative of human-nature relationships functional to capitalism, where nature(s) is(are) something that can be exploited, appropriated or enclosed by humans for value production, which ultimately is one of the main causes of the current biodiversity crisis [START_REF] Escobar | Thinking-feeling with the Earth: territorial struggles and the ontological dimension of the epistemologies of the South[END_REF][START_REF] Moore | El capitalismo en la trama de la vida[END_REF][START_REF] Navarro-Trujillo | Capitaloceno, luchas por lo común y disputas por otros términos de interdependencia en el tejido de la vida. Reflexiones desde América Latina[END_REF]Navarro-Trujillo and Machado Aráoz, 2020). If the ultimate aim of SENs studies is to understand the reproduction of SESs in the face of such crisis, we urgently need to diversify our narratives about nature, i.e. to incorporate other conceptions of human-nonhuman interdependencies that may produce diverse and integrated worlds instead of obliterating them [START_REF] Escobar | Thinking-feeling with the Earth: territorial struggles and the ontological dimension of the epistemologies of the South[END_REF][START_REF] Haraway | Staying with the trouble: Making kin in the Chthulucene[END_REF][START_REF] Moore | El capitalismo en la trama de la vida[END_REF][START_REF] Navarro-Trujillo | Capitaloceno, luchas por lo común y disputas por otros términos de interdependencia en el tejido de la vida. Reflexiones desde América Latina[END_REF].

In this vein, conceptualizing SESs, and therefore SENs, as the common that needs to be produced (Gutiérrez Aguilar and Rátiva Gaona, 2020;[START_REF] Aguilar | Producing the common and reproducing life: Keys towards rethinking the political[END_REF] Trujillo and Linsalata, 2021; Roca-Servat, 2020) may be key to move forward. SENs studies have traditionally conceived commons as natural resources, goods or services, the maintenance of which depends on collective access and the use of which is regulated by collectively constructed norms [START_REF] Barnes | Social-ecological alignment and ecological conditions in coral reefs[END_REF][START_REF] Biggs | The Routledge handbook of research methods for social-ecological systems[END_REF]Felipe-Lucía et al., 2022;[START_REF] Ostrom | A general framework for analyzing sustainability of social ecological systems[END_REF]; but see [START_REF] Mazé | Commoning the seeds: Alternative models of collective action and open innovation within French peasant seed groups for recreating local knowledge commons[END_REF]. In contrast, the framework based on producing the common, developed by sociologists and communitarian feminist scholars in the last two decades (Gutiérrez Aguilar and Rátiva Gaona, 2020;[START_REF] Aguilar | Producing the common and reproducing life: Keys towards rethinking the political[END_REF][START_REF] Navarro-Trujillo | Capitaloceno, luchas por lo común y disputas por otros términos de interdependencia en el tejido de la vida. Reflexiones desde América Latina[END_REF], considers social-ecological goods and processes as parts of the common that needs a communal production. Such a framework builds on the idea of the common as being co-produced by humans and non-human entities, recognizing the dialectic nature of the web of life [START_REF] Moore | El capitalismo en la trama de la vida[END_REF]. This need of a communal production of the common is about establishing diverse, creative and situated spatiotemporal political practices of interdependence to produce human and non-human life [START_REF] Navarro-Trujillo | Capitaloceno, luchas por lo común y disputas por otros términos de interdependencia en el tejido de la vida. Reflexiones desde América Latina[END_REF]. Thus, building SENs through the lens of frameworks that conceive SESs as the common may allow ecologists to diversify their ideas about interdependencies between human and non-human entities.

In this roadmap, we briefly present two frameworks (Figure 1), relational ontologies and other economies that, approached with the lens of ethnobiology and put into a dialogue with the production of the common framework may, from our own experience, diversify ecologists' ideas about interdependencies among human and non-human entities -i.e. about nature. We have built this proposal on a series of interdisciplinary seminars during which we have discussed the literature that exists across Ethnobiology, Political Ecology and Economy with a complex network approach. In this vein, we believe that by investigating how SESs components emerge in a relational way, i.e. they "are" because of co-constructed relationships between human and non-human entities, and how the cooperative and reciprocal human-human relationships that underpin other economies produce other human-non-human links, we may improve our understanding on the reproduction of diverse SESs. multilayered SEN built under a modern conception of nature and a "total market" economy (see section 2.2. Other economies) is displayed on the right side. Asymmetric relationships showing how the hegemonic SEN exploits, appropriates and encloses what is commonly produced (the common) is illustrated by overlapped color composition in the middle of the two SENs. From bottom to top, the layers represent: land, wildlife, goods and humans. Ontologies underpinning such layers and their related links are shown at the top, depicted by violet and yellow circular arrows. The complex social-ecological system (SES) that emerges from the integration of the two SENs is at the top. Icons are from the Noun Project (https://thenounproject.com). Details on icon credits are in Supplementary material_S1.

Diversifying ideas about human-non-human interdependencies:

frameworks that built social-ecological systems as the common

Relational ontologies and values

Relational ontologies

In the last two decades, ecologists have produced diverse SENs to represent complex SESs and facilitate our understanding of relationships among humans and nature, in order to propose management strategies to preserve such systems [START_REF] Kluger | Studying humannature relationships through a network lens: A systematic review[END_REF][START_REF] Sayles | Social-ecological network analysis for sustainability sciences: a systematic review and innovative research agenda for the future[END_REF]. The adoption of a social-ecological perspective of networks, formerly characterized as "ecological", made explicit the human impacts on nature by highlighting interactions among the "social" and the "biological" components of a given landscape [START_REF] Biggs | The Routledge handbook of research methods for social-ecological systems[END_REF]. That academic change certainly helped integrate such components and thus build the idea that SESs emerged from social-ecological interactions (Felipe-Lucía et al., 2022;[START_REF] Kluger | Studying humannature relationships through a network lens: A systematic review[END_REF]. However, the naturalistic modern conception of the world based on the culture-nature dichotomy dominates the SEN literature and, therefore, our understanding of SESs, which fails to represent the complexity of biocultural landscapes [START_REF] Toledo | La memoria biocultural: la importancia ecológica de las sabidurías tradicionales[END_REF]. This is an interpretative bias that prevents us from noticing the coexistence of multiple worlds and identifying the socialecological relationships that may produce them, obliterating the conception of each SES as the common that we need to co-produce with non-human entities.

Relational ontologies encompass ways of being and living that are quite different from the individualistic narrative imposed by modernity [START_REF] Escobar | Thinking-feeling with the Earth: territorial struggles and the ontological dimension of the epistemologies of the South[END_REF][START_REF] Hernando | La fantasía de la individualidad. Sobre la construcción sociohistórica del sujeto moderno[END_REF]. Relational ontologies assume that things and beings do not preexist the relations that constitute them, in other words, they are their relations [START_REF] Escobar | Thinking-feeling with the Earth: territorial struggles and the ontological dimension of the epistemologies of the South[END_REF]. With the lens of relational ontologies, new worlds emerge beyond the one conceived under the modern culture-nature dichotomy, dissolving the limits that such dichotomy imposes on our understanding of SESs. Worlds are constituted by particular entities that acquire a sense of existence based on the rules and codes of each world [START_REF] Blaser | Political ontology: cultural studies without 'cultures'?[END_REF][START_REF] Descola | Más allá de la naturaleza y la cultura[END_REF][START_REF] Viveiros De Castro | La mirada del jaguar. Tinta Limón[END_REF]. Within the relational ontology approach, humans coexist and co-construct worlds with other entities, i.e., worlds are enacted by relations [START_REF] Escobar | Thinking-feeling with the Earth: territorial struggles and the ontological dimension of the epistemologies of the South[END_REF][START_REF] Varela | Ethical know-how: Action, wisdom, and cognition[END_REF]. Thus, it can be easily recognized how the adoption of the relational ontology approach by ecologists developing SENs may encourage them to diversify ideas regarding the co-production of SESs (the common) by human and non-human entities.

In the SEN representation of SES, relationality is easy to conceptualize because networks focus on interactions between the system's parts. Each entity is related to the position it occupies in the network of interrelations with other entities. Following the relational ontology approach, entities are conceived in a particular and unique way in each world. In a SEN, nodes cannot be understood in isolation from the links they weave with other nodes, a conception that is key to understanding how relationships between entities enact each world (sensu [START_REF] Varela | Ethical know-how: Action, wisdom, and cognition[END_REF]; see a recent use in [START_REF] Louafi | Communities of practice in crop diversity management: from data to collaborative governance[END_REF]. In this vein, ethnographic studies unraveling the conflicts underpinning wildlife conservation may provide insights on the relationship between the emergence of conflicts and the different ontological backgrounds of local and foreign actors of the territory, and on the importance of non-human entities in "mediating" conflicts by being part of a more-than-human sociability (e.g. [START_REF] Blaser | Political ontology: cultural studies without 'cultures'?[END_REF][START_REF] Furlan | Ethnobiological equivocation' and other misunderstandings in the interpretation of natures[END_REF][START_REF] Manzano-García | El Cóndor Andino (Vultur gryphus): ¿predador o carroñero?: Pluralidad de percepciones entre los saberes locales y el discurso académico en las sierras centrales de Argentina[END_REF][START_REF] Marquez | El cabrero' guardián de las cabras en el Chaco árido[END_REF][START_REF] Martínez-Dueñas | Pensando la conservación desde el multi-naturalismo en una localidad indígena de los andes colombianos[END_REF][START_REF] Martínez-Medina | Osos vaqueros en el páramo incomún: Hacia una conservación cosmopolítica del oso andino en el páramo de Chingaza, Colombia[END_REF]Tsing, 2013 ;Box 1).

Relational values

A relational ontology approach can also help ecologists think critically about the diversity of ways in which the values of social-ecological goods and processes are created, and to visualize the importance of considering such values within the common that needs to be co-produced.

Social-ecological goods and processes have been defined by classic ecology as either "ecosystem services" [START_REF] Mulder | 10 Years Later: Revisiting Priorities for Science and Society a Decade After the Millennium Ecosystem Assessment[END_REF] or, more recently, as "nature contributions to people" or "naturebased solutions" [START_REF] Díaz | Assessing nature's contributions to people: Recognizing culture, and diverse sources of knowledge, can improve assessments[END_REF][START_REF] O'hogain | A technology portfolio of nature based solutions[END_REF][START_REF] Pascual | Valuing nature's contributions to people: the IPBES approach[END_REF]. In this vein, the Intergovernmental Science-Policy Platform on Biodiversity and Ecosystem Services (IPBES) has mainly considered values as intrinsic -value as an end-in-itself or not linked to human purposes-or instrumental -as a means to an end- [START_REF] Balvanera | The role of the values of nature and valuation for addressing the biodiversity crisis and navigating towards more just and sustainable futures[END_REF], and only recently started to include the idea of relational values as another category for specific values (e.g. [START_REF] Unks | People should also look after the people: relational values of wildlife and collectively titled land in Ilkisongo Maasai group ranches in Southern Kenya[END_REF]. Relational values, by contrast, emerge, when importance shifts from values attached to "things" to values derived from "networks of relationships and responsibilities" [START_REF] Chan | Editorial overview: relational values: what are they, and what's the fuss about?[END_REF]. If entities do not preexist the relations that constitute them [START_REF] Escobar | Thinking-feeling with the Earth: territorial struggles and the ontological dimension of the epistemologies of the South[END_REF], such a thing as intrinsic value does not exist. Only relational values remain. Entities do not just have instrumental value, they may have value through the relations they are part of [START_REF] Tsing | The mushroom at the end of the world: On the possibility of life in capitalist ruins[END_REF]. This contrasts with the logic of capitalism where entities can be torn from their worlds to become objects of exchange. Although tightly related with understanding the value of nodes (entities) and links in SEN (e.g. [START_REF] Felipe-Lucia | Conceptualizing ecosystem services using social-ecological networks[END_REF], such a different conception of specific values has been poorly explored in SES studies using the network approach.

BOX 1. Constructing ethnobiological social-ecological networks (ESEN) to integrate relational ontologies and values into SENs and conceive SESs as the common

Defining SES components (nodes) and relationships (links) and, thus, what "is" and what "is not" a SES, has so far been limited to scientists trying to do a big effort to address the incommensurability of socioecosystems (see Ludwing and El-Hani, 2020). This is, in part, why an integrated analysis of "social" and "ecological" elements remains challenging [START_REF] Bodin | Theorizing benefits and constraints in collaborative environmental governance: a transdisciplinary social-ecological network approach for empirical investigations[END_REF][START_REF] Cumming | Network analysis in conservation biogeography: challenges and opportunities[END_REF]. Nodes and links may be differently conceptualized by ecologists, social scientists and local people, who use different systems of classification, epistemologies and have different scopes or worlds [START_REF] Bodin | Improving network approaches to the study of complex social-ecological interdependencies[END_REF]Felipe-Lucía et al., 2022;[START_REF] Kluger | Studying humannature relationships through a network lens: A systematic review[END_REF][START_REF] Sayles | Social-ecological network analysis for sustainability sciences: a systematic review and innovative research agenda for the future[END_REF]. In this regard, ethnobiology investigates relationships between societies and "nature(s)" from multiple perspectives; utilitarian, cognitive, and more recently relational [START_REF] Hunn | Ethnobiology in four phases[END_REF][START_REF] Mcalvay | Ethnobiology phase VI: Decolonizing institutions, projects, and scholarship[END_REF][START_REF] Wolverton | Ethnobiology 5: interdisciplinarity in an era of rapid environmental change[END_REF]. It is a transdisciplinary science that integrates ecological and evolutionary theory and social sciences methodology [START_REF] Casas | Evolutionary ecology and ethnobiology[END_REF][START_REF] Albuquerque | Methods and techniques in ethnobiology and ethnoecology[END_REF] and can be conceived as another form of "scientific knowledge" [START_REF] Furlan | Ethnobiological equivocation' and other misunderstandings in the interpretation of natures[END_REF]. Therefore, ethnobiology may improve our ability to diversify SES representation by broadening the notions about nodes, links and the emergence of values as part of the common that needs to be co-produced by the SEN, since this discipline can deeply explore both academic and local knowledge systems. Considering the relational value framework [START_REF] Chan | Editorial overview: relational values: what are they, and what's the fuss about?[END_REF], ethnobiology may also create space for systematizing expressions and practices about what matters to people on their own terms.

Until now social network analysis has focused on environmental management and local ecological knowledge and has used the interpretative power of SENs mainly to study information transmission, resource governance or seed circulation [START_REF] Salpeteur | Networking the environment: social network analysis in environmental management and local ecological knowledge studies[END_REF]. SENs have mainly analyzed relationships between different social actors around "resources" or "common goods" (reviewed in [START_REF] Salpeteur | Networking the environment: social network analysis in environmental management and local ecological knowledge studies[END_REF] or those between plant species and plant ecosystem services, represented by people's knowledge about plants and their uses (e.g., [START_REF] Cámara-Leret | Indigenous knowledge networks in the face of global change[END_REF]. However, such studies have poorly explored how people or communities selfperceived the links between them and the "used", "exploited" or "managed" nature. That is, how relations with non-humans were conceived, and therefore, how they were represented by ideas, knowledge, practices and cosmologies of local people. In this vein, relational states and relational events of dyadic (pairwise) links have been defined by questions formulated by scientists (etic SEN) and not by the local population (emic SEN; see [START_REF] Salpeteur | Networking the environment: social network analysis in environmental management and local ecological knowledge studies[END_REF]. Changing "who" defines the nodes and the links and, therefore SENs, could certainly shed light on ways of diversifying our understanding of SES through the lens of relational ontologies and other economies (see section 2.2.). Such networks can be referred to as ethnobiological social-ecological networks (ESENs). ESENs may differ from other already formulated SENs in at least two main points. First, as previously mentioned, the way and the type of information researchers use to construct nodes and links. For example, considering local taxonomies for node definition, as local and scientific classification does not always coincide [START_REF] Martínez-Medina | Osos vaqueros en el páramo incomún: Hacia una conservación cosmopolítica del oso andino en el páramo de Chingaza, Colombia[END_REF], and the type of relationship (and relational value assigned) obtained in narratives to define links. Second, the direction and intention of the question(s) through which the SENs are built. For instance, investigating the origin and destination of seeds will be different from inquiring about the entities that are linked to seeds. The first question will inevitably refer to a person (markets, gardens, etc.) while the second one is likely to lead us to a diversity of human and non-human entities and relationships, like non-animals included in myths, animals as birds and mammals included in the extended socio-ecological web in which seeds may be involved (e.g. [START_REF] Kujawska | The relationship between a western Amazonian society and domesticated sedges (Cyperus spp.)[END_REF]. (usually a dog) that takes care of goats when they are feeding in the "monte" far from the pen. The "cabrero" acts as a livestock guardian dog and becomes part of the more than human sociability of the arid Chaco. . This dog is important in peasant social life because of its role as "mediator" of conflicts between humans and non-humans (e.g. goats, Puma concolor) and, ultimately, among humans (e.g. peasants, conservationists). . The "cabrero" has their own agency; hence, the "cabrero" along with humans co-produce the SES that they inhabit (and that produce them; Tsing, 2013). Icons are from the Noun Project (https://thenounproject.com).

Details on icon credits are given in Supplementary material_S1).

We think that ESENs can contribute to broad our notions of SESs by both allowing access to the diversity of ways of understanding (socio)ecological complexity and to the emic ways in which relationships (reciprocity, commensalism, or predation) between entities are conceived, and thus creating tangible and empirically accountable visual representations of local conceptualizations of SESs (the common that needs to be produced; see [START_REF] Atran | Folkecology, Cultural Epidemiology, and the Spirit of the Commons: A Garden Experiment in the Maya Lowlands, 1991-2001[END_REF][START_REF] Levine | Theories of the deep: combining salience and network analyses to produce mental model visualizations of a coastal British Columbia food web[END_REF]. Following the proposal of [START_REF] Levine | Theories of the deep: combining salience and network analyses to produce mental model visualizations of a coastal British Columbia food web[END_REF], the use of ESENs can particularly help analyze environmental conflicts derived from the clash between the multiple ideas that compose different worlds. For example, "conflicts" between rural producers and the puma (Puma concolor) in the arid Chaco of central Argentina are generally conceived by scientists as a typical human-wildlife fauna conflict arising from puma predation on goat herds [START_REF] Marquez | El cabrero' guardián de las cabras en el Chaco árido[END_REF].

However, such a conflict can be conceptualized as a conflict between rural producers and conservationists that want to produce a different SES, i.e. a human-human conflict [START_REF] Blaser | Political ontology: cultural studies without 'cultures'?[END_REF] that affects the production of the common, or even as a condition in which the "cabrero" (i.e., the livestock guardian dog), by acting as an "intermediary" of the conflict between humans and non-human animals (Fig 2; [START_REF] Marquez | El cabrero' guardián de las cabras en el Chaco árido[END_REF], allows the production of a SES in which peasants can coexist with the puma. In this vein, following [START_REF] Goldman | Beyond ritual and economics: Maasai lion hunting and conservation politics[END_REF] and their study on lion hunting in Africa, we can propose that the conflict around the pumas (Figure 2) is about "the overlap of motivations that are simultaneously social, emotional and political" (Goldman et al., 2013: p. 490). That is, to a series of relational values that are supported by a particular relational ontology that produces a particular SES.

Other economies

Inquiring about the production of the common implies recognizing how social-ecological interactions become functional to capital, to the construction of other economies or both (Gutiérrez Aguilar and Rátiva Gaona, 2020). In the words of Gutiérrez Aguilar and Rátiva Gaona (2020: p. 44; personal translation by J. Astegiano), the production of the common "(...) far from showing an essentially harmonic or transhistorical view of communitarian relationships or of human relationships in nature, this framework enlightens a deep sense of interdependence that emerges from the concrete action of work -collective and individual-and from the imprints of non-human entities on human social live".

Thus, (re)producing the common is about "the multiform productive activity that generates and re-actualizes ties and shared meanings beyond, against and beyond the separations imposed by capital accumulation" (Gutiérrez Aguilar and Rátiva Gaona, 2020: p. 50; personal translation by J. Astegiano).

Understanding each SESs as the common through the lens of complex networks will imply that SENs are constructed based on those economic perspectives that incorporate the life-capital conflict into their formulation. We are interested in introducing the other economies' framework as the value systems of such economies may allow ecologists to focus on the reproduction of the web of life (and not profit). By characterizing SENs using such value systems we want, first, to establish a distance from those frameworks that link economic relationships with the idea of a "total market", defined by Coraggio (2009: p. 2): "the dominant paradigm (...) proposes a universal answer to the question of which is the best system to optimize the use of the scarce resources (...) That solution is the market, which, when considered as an exclusive rational institution, results in the "total market"". Second, we want to highlight the need to make visible the invisible, which means to explicitly recognize interdependencies between humans and non-human entities and its dialectic relationship (produce / be produced; [START_REF] Moore | El capitalismo en la trama de la vida[END_REF][START_REF] Navarro-Trujillo | Capitaloceno, luchas por lo común y disputas por otros términos de interdependencia en el tejido de la vida. Reflexiones desde América Latina[END_REF]. Third, we want to go beyond the split imposed by the "total market" on labor and use and exchange values [START_REF] Aguilar | Reproducción comunitaria de la vida. Pensando la trans-formación social en el presente[END_REF]. Last, to recognize that such other economies can be functional to the reproduction of capital but, we insist, at the same time can show ways that disconnect them from the logics of capital and create new logics beyond those of capital, enabling the emergence of other human-human and human-non-human relationships that may produce the common.

Social, solidarity, popular and feminist economies

The economic perspectives embraced under the term other economies are those that problematize the neo-classical economic logic (i.e., an individualistic and universal logic centered in the homo oeconomicus) and discuss the role of capital and the "total market" [START_REF] Coraggio | Territory and alternative economies[END_REF].

Social Economy denotes the idea that every economic practice needs to be interpreted linked to its social aspect and the other multiple dimensions which make up the context of that practice [START_REF] Coraggio | Territory and alternative economies[END_REF]. Social Economy can also be defined by the actions of entities that assume different legal forms to organize economic activities (e.g. cooperatives, mutual aid organizations or associations; Chaves-Ávila and Monzón-Campos, 2019). Solidarity Economy brings shared values (e.g., cooperation, reciprocity, communalism,) as central to conceive economic rationality, decentering such rationality from both individuals and monetary profit [START_REF] Miller | Solidarity economy: Key concepts and issues[END_REF][START_REF] Razeto | La economía de solidaridad: concepto, realidad y proyecto[END_REF]. A different value system is also proposed as a project in which the boundaries between each perspective (Solidarity and Social Economies) are not drawn rigidly [START_REF] Razeto | ¿Qué es la economía solidaria?[END_REF][START_REF] Razeto | La economía de solidaridad: concepto, realidad y proyecto[END_REF].

Popular Economy, in turn, broadens the definition of work beyond the crisis of formal employment in the context of social exclusion systematically imposed by the logic of capital accumulation [START_REF] Weeks | The Problems with Work[END_REF]. The emergence of this economic perspective is usually attached to the context of social emergency and increased labor informality that characterize the recurrent "socialeconomical" crises in Latin America [START_REF] Chena | La economía popular y sus relaciones determinantes[END_REF][START_REF] Fernández-Álvarez | Más allá de la precariedad: prácticas colectivas y subjetividades políticas desde la economía popular argentina[END_REF][START_REF] Roitman | ¿Exclusión o reconocimiento? La economía popular argentina en la Revolución 4[END_REF]. However, we are interested in highlighting the possibility of experimentation with both theory and praxis to "empower and expand the scope of (...) people's lives" that such practice brings [START_REF] Gago | What Are Popular Economies? Some Reflections from Argentina[END_REF] p. 1).

In the context of our proposal, it is also important to consider a central discussion developed by feminist economies (Rodríguez-Enríquez, 2012): which work is recognized as productive (and which not), and which is paid (and unpaid). In this line of thought, highlighting the inter-relationship between care work (invisible) and productive work (visible) will be key to go beyond the discussion of gender economic inequalities expressed just as a lower participation of women in productive work and a higher participation in care work [START_REF] Rodríguez-Enríquez | Economía feminista y economía del cuidado. Aportes conceptuales para el estudio de la desigualdad[END_REF]. We consider that these discussions that take place in the theoretical and practical framework that defines feminist economies will help ecologists make visible the invisible that builds SENs and, therefore, reproduces SESs in two important ways. First, by transforming unpaid care work into paid work as a mechanism to make it visible, the discussion about the role of salary as a way to recognize interdependencies between productive and reproductive work (i.e., care work) will be introduced [START_REF] Denning | Wageless Life[END_REF][START_REF] Federici | El patriarcado del salario. Críticas feministas al marxismo[END_REF]Rodríguez-Enriquez, 2016;[START_REF] Weeks | The Problems with Work[END_REF].

Second, by solving the unrecognition of care work in a different way from that proposed by the "total market" (i.e., individually and paying for care services), feminist economies will introduce a discussion on the centrality of communal care to produce the common and, therefore, to reproduce human and non-human life [START_REF] Navarro-Trujillo | Capitaloceno, luchas por lo común y disputas por otros términos de interdependencia en el tejido de la vida. Reflexiones desde América Latina[END_REF][START_REF] Perez-Orozco | El conflicto capital-vida[END_REF][START_REF] Rodríguez-Enríquez | Care: the missing link in economic analysis?[END_REF].

Sociologists and communitarian feminists scholars provide key elements for recognizing other forms of organization within, against and beyond capitalism by placing communitarian work on the roots of producing the common (Gutiérrez Aguilar and Rátiva Gaona, 2020). There is "(…) a necessary work for the reproduction of the common as a social relation with power to create material wellness and political strength" (Gutiérrez Aguilar and Rátiva Gaona, 2020: p. 56; personal translation by J. Andrieu). Such community-based work can assume multiple forms and can present its own spatio-temporal specificities (Gutiérrez [START_REF] Aguilar | Reproducción comunitaria de la vida. Pensando la trans-formación social en el presente[END_REF]. These forms of relationship potentially go beyond the logic of the "total market" and the conflict capital-life, although sometimes they are not exempt from contradictions [START_REF] Andrieu | Crisis multisistémicas y resistencias en los territorios latinoamericanos. Diálogo con Maria Mies y Vandana Shiva desde el ecofeminismo[END_REF]. Thus, the crossovers with other economies are opportune; even if the coincidences we find are involuntary and can show contradictions [START_REF] Villalba-Eguiluz | Social and Solidarity Economy and Circular Economy. Necessary complementarities for a socio-ecological transition[END_REF].

BOX 2. Human and non-human co-production of SESs: commoning honeybees, honey and beekeepers' economies

Argentina is one of the main honey producers worldwide (Andrieu et al., 2021a). 63% of beehives are concentrated in the central pampas, where most produced honeys are "monofloral" and light yellow, and an exportation-oriented economy based on the supply-dependent paradigm of "the green revolution" dominates honey production [START_REF] Andrieu | De tranqueras y candados. Las mujeres, su acceso a la tierra y bienes comunes[END_REF]. Overall, honey in Argentina is mostly conceived and produced as a commodity [START_REF] Folguera | Un problema no es sólo un problema. Posiciones, hermenéuticas y políticas públicas relativas a la salud de las abejas[END_REF][START_REF] Nimmo | Apiculture in the Anthropocene: Between Posthumanism and Critical Animal Studies[END_REF].

Moreover, honeybees are mostly treated as conventionally managed cattle, i.e. beehives are supplied with different synthetic substances that either feed ("sugar syrup" and "protein cakes") or treat them against different diseases [START_REF] Nimmo | Apiculture in the Anthropocene: Between Posthumanism and Critical Animal Studies[END_REF][START_REF] Salizzi | Reestructuración económica y transformaciones en el agro pampeano: la expansión del cultivo de la soja y sus efectos sobre la apicultura bonaerense en los inicios del siglo XXI. Estudios Socioterritoriales[END_REF][START_REF] Shanahan | Honey bees and industrial agriculture: what researchers are missing, and why it's a problem[END_REF]. Honey is also produced in other SESs, such as those found in the Cuyo region in western Argentina.

These SESs, and the honeys they produce, are conceived as marginal because of their small contribution to the exportation market, which mostly commercializes undifferentiated honey (Andrieu et al., 2021a). Interestingly, such marginalized SESs produce a high diversity of honeys in terms of colors -brown or dark yellow-, flavors, consistency (Andrieu et al., 2021b), and pollen and nectar-supplying species that feed honeybees (and other bees) and make honeys "multifloral" [START_REF] Andrieu | Aportes para la economía social: valorización de mieles de San Juan a partir de su caracterización botánica desde el enfoque agroecológico[END_REF]. Beekeepers' management practices are also diverse, ranging from agroecological practices (i.e. with almost no supply of provision to beehives and a strong focus on the analyses of honeybee food and water sources to decide beehives location) to those highly dependent on supplies [START_REF] Andrieu | Aportes para la economía social: valorización de mieles de San Juan a partir de su caracterización botánica desde el enfoque agroecológico[END_REF]. Such a diversity in the way honey production is conceived is necessarily produced by and will produce different relationships among beekeepers, and between beekeepers and other people producing "food" for honeybees (e.g. agroecological farmers) as well as beekeepers and honeybees (Cely-Santos, 2020; Gutiérrez Aguilar and Rátiva Gaona 2020;[START_REF] Navarro-Trujillo | Capitaloceno, luchas por lo común y disputas por otros términos de interdependencia en el tejido de la vida. Reflexiones desde América Latina[END_REF]Nimo, 2015;[START_REF] Shanahan | Honey bees and industrial agriculture: what researchers are missing, and why it's a problem[END_REF]. Therefore, in these SESs, honey is more than a commodity; it is also about nourishment, commensality, encounters between humans and non-human entities, and the production of flavors and colors that co-produce the web of life.

The work of beekeepers is also organized in a diversity of collective forms in the Cuyo region, including traditional forms of the Social Economy such as cooperatives, but also others more related to the Popular Economy [START_REF] Andrieu | Reflexiones desde la economía social y solidaria sobre la apicultura sanjuanina[END_REF]. Thus, collaborative practices and forms of reciprocity that are rarely considered as "labor" by the "total market" economy may be key for honey production [START_REF] Denning | Wageless Life[END_REF]. Moreover, as most beekeepers need to negotiate the land to their beehives [START_REF] Andrieu | De tranqueras y candados. Las mujeres, su acceso a la tierra y bienes comunes[END_REF], collaborative practices among beekeepers may influence the way they relate with landowners. Land negotiations, in turn, may imply paying a rent but usually may be associated with other relationships like gifting some produced honey, offering honeybees to pollinate crops, buying farmers' production or just starting a friendship. Thus, beekeeping in the Cuyo region can be understood in terms of a collective (economic or socialecological) practice that (re)produces the common (the SES that produces honey, honeybees and beekeeping itself), by diversifying both human-human and human-non-human relationships that reproduce live [START_REF] Andrieu | De tranqueras y candados. Las mujeres, su acceso a la tierra y bienes comunes[END_REF].

Characterizing SESs as the common: How the network approach can help us

The study of networks resulting from SES studies and the building blocks that produce and are produced by the common (the SES itself) can make use of a variety of existing tools, from a variety of disciplines including social sciences and statistical ecology. Social network analysis has made heavy use of models to explain the occurrence of links (e.g. reciprocity relationships among people) in networks based on the rest of the other links and external variables (e.g. people belonging to different social groups), culminating with exponential random graph models (ERGMs; [START_REF] Handcock | statnet: Software Tools for the Representation, Visualization, Analysis and Simulation of Network Data[END_REF][START_REF] Jasny | Shifting echo chambers in US climate policy networks[END_REF][START_REF] Robins | An introduction to exponential random graph (p*) models for social networks[END_REF][START_REF] Snijders | New specifications for exponential random graph models[END_REF] as one of their most theoretically advanced statistical methods [START_REF] Jasny | Working together: the roles of geographic proximity, homophilic organizational characteristics, and neighborhood context in civic stewardship collaboration networks in Philadelphia and New York City[END_REF][START_REF] Labeyrie | Seed exchange networks, ethnicity, and sorghum diversity[END_REF]. Although initially conceived to deal with networks with only one type of links (e.g. social networks or bipartite ecological networks), recent developments have led to the use of multiplex ERGMs [START_REF] Barnes | Social-ecological alignment and ecological conditions in coral reefs[END_REF][START_REF] Ferrare | Measuring Issue Preferences, Idea Brokerage, and Research-Use in Policy Networks: A Case Study of the Policy Innovators in Education Network[END_REF][START_REF] Guerrero | Achieving social-ecological fit through bottom-up collaborative governance: an empirical investigation[END_REF][START_REF] Wang | Exponential random graph models for multilevel networks[END_REF], i.e. models which could explain the occurrence of a social link (e.g. cooperation between a beekeeper and a farmer) based on other social links (e.g. cooperative relationships among beekeepers) and associated social-ecological links (e.g. beekeepers locating a beehive in a farm that produces a crop that feeds honeybees and whose reproduction is facilitated by them). Thus, which cooperative or reciprocal links among humans and non-humans co-produce the common (e.g. the SES that produces and is produced by honey production), can be evaluated with multiplex ERGMs.

Another important tool for such studies is the family of stochastic block models (SBM; Govaert andNadif, 2010, 2008), which aim at defining groups of nodes (blocks) that interact in a similar fashion, i.e. such that the patterns of interactions within and among blocks can be the most efficiently modeled with as few parameters as possible. In this vein, using ethnobiological and social methods (e.g. structured, semi-structured and in-depth interviews or participant observation research; [START_REF] Albuquerque | Methods and techniques in ethnobiology and ethnoecology[END_REF] to define which set of social practices are produced as and can be characterized as communal forms of politics oriented to produce the common will be crucial (e.g. Box 1 and 2). As SBM approaches have drawn the interest of ecologists for some time [START_REF] Baskerville | Spatial guilds in the Serengeti food web revealed by a Bayesian Group Model[END_REF][START_REF] Leger | Clustering methods differ in their ability to detect patterns in ecological networks[END_REF][START_REF] Miele | Revealing the hidden structure of dynamic ecological networks[END_REF], they have been progressively adapted to the study of SENs. SBMs have been extended to multipartite (i.e. networks in which nodes belong to different levels and never interact with a single level) and multilevel/multiplex networks, which make them a tool of choice for the analysis of multiplex networks such as SEN [START_REF] Bar-Hen | Block models for generalized multipartite networks: Applications in ecology and ethnobiology[END_REF][START_REF] Chabert-Liddell | A stochastic block model approach for the analysis of multilevel networks: An application to the sociology of organizations[END_REF].

While networks provide an important tool for the study of SES as the common, it is worth keeping in mind that networks only represent pairwise interactions between entities (nodes), and thus fail to accurately represent non-pairwise interactions, i.e. interactions that involve three or more entities [START_REF] Felipe-Lucia | Conceptualizing ecosystem services using social-ecological networks[END_REF]. Non-pairwise interactions can most often be "rearranged" into multipartite networks -e.g. communal actions such as communal flailing of harvested millet [START_REF] Garine | An ethnographic account to the many roles of millet beer in the culture of the Duupa agriculturalists[END_REF] could possibly be represented using a bipartite network with farmers on one level and failing groups on the other -but this is not necessarily the best representation to evidence social-ecological relationships, especially when the nodes involved in non-pairwise interactions are of different types (e.g. when interactions are management discussions involving individuals, institutions, and several wildlife species). Another choice of representation is the hypergraph, which works on all types of interactions (pairwise or otherwise), and has begun to garner some interest from ecologists [START_REF] Golubski | Ecological networks over the edge: hypergraph trait-mediated indirect interaction (TMII) structure[END_REF]. However, the above-mentioned methods (ERGM and SBM) are not yet fully functional for the study of large hypergraphs (but see [START_REF] Brusa | Model-based clustering in simple hypergraphs through a stochastic blockmodel[END_REF] for small hypergraph SBMs).

Final remarks

We wrote this roadmap because we believe that it is time for ecologists to question the hegemony of the culture-nature dichotomy imposed by modernity on the construction of SESs as SENs. We urgently need to find frameworks that help us generate other understandings around collaborative interdependencies between humans and non-humans that reproduce the web of life (SESs, the common). Many scholars have previously highlighted such a need and proposed other starting points [START_REF] Despret | Habitar como un pájaro[END_REF][START_REF] Federici | Reencantar el mundo. El feminismo y la política de los comunes[END_REF][START_REF] Haraway | Staying with the trouble: Making kin in the Chthulucene[END_REF][START_REF] Thomsen | Posthumanist Pluralities: Advocating for nonhuman species' rights, agency, and welfare in ecosystem governance[END_REF]. In this vein, disturbance-based ecologies, in which many species sometimes live together without either harmony or conquest, has been recently proposed by [START_REF] Tsing | The mushroom at the end of the world: On the possibility of life in capitalist ruins[END_REF]. As she states "We are contaminated by our encounters; they change who we are as we make way for others" (Tsing, 2015: p. 26).

Because of these transformative encounters, the idea of conceiving a SES as the common, which produces and needs to be produced by communal political practices promoting both human and non-human lives, is promising. We advocate for the potential of relational ontologies and other economies to build more diverse ideas of social relationships co-producing (and being produced by) the common. These frameworks in dialogue with current ethnobiological approaches may certainly help ecologists think about "a world where many worlds fit" (Zapatista dictum in [START_REF] Escobar | Thinking-feeling with the Earth: territorial struggles and the ontological dimension of the epistemologies of the South[END_REF].

Figure 3 (

 3 Figure 2 (within Box 1)

Figure 1

 1 Figure1

Figure 1 .

 1 Figure 1. A simplified social-ecological system (SES) produced by two different social-

Figure 2 .

 2 Figure 2. Relational ontologies in the arid Chaco, central Argentina (Marquez et al.,

Figure 3 .

 3 Figure 3. Economies and the production of SESs: : beekeeping practices that co-produce

Acknowledgments

To all communities that allowed us to know and to enact other worlds.

Businessman icon created by alrigel from the Noun Project Bumblebee icon created by Nicky Spencer from the Noun Project Shipment icon created by Mas Dhimas from the Noun project (here modified) Barrel icon created by Trevor Mowry from the Noun Project (here modified) Honey icon created by Patrick Straub from the Noun Project (here modified) Trees icon created by Artem Yurov from the Noun Project Soy plant icon created by Oleksandr Lavreniuk from the Noun Project.


Timothée Bigot

Elisa Gabinaud

Laurent Hannouche

Véronique Sbarra

Elisa Andersen

Delphine Bastelica

Céline Falaise

Manal Denis Bernot

Pierre-Emmanuel Morange

Manal Ibrahim-Kosta

Morange Pierre-Emmanuel

Marie Loosveld

Paul Saultier

Dominique Payet-Bornet

Marie-Christine Alessi

Delphine Potier

C2VN Marjorie Poggi
email: marjorie.poggi@univ-amu.fr

Single-cell analysis of megakaryopoiesis in peripheral CD34+ cells: insights into ETV6-related thrombocytopenia

Keywords: 7, Tables: 0, Supplementary data: 10 Figures and 8 Tables. Essentials: ETV6, Megakaryopoiesis, Ribosomal Protein S6, Single-cell RNA sequencing, Thrombocytopenia

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

ETV6-related thrombocytopenia (ETV6-RT) is a highly penetrant form of inherited thrombocytopenia with autosomal dominant inheritance [START_REF] Paola | ETV6-related thrombocytopenia and leukemia predisposition[END_REF]. The common phenotype observed in ETV6-RT includes moderate thrombocytopenia sometimes associated with bleeding and predisposition to acute T-or B-cell lymphoblastic leukemia. The precise role that ETV6 plays in megakaryocyte (MK) differentiation and pro-platelet formation currently remains poorly understood. Studying Etv6 function in murine models is challenging because complete loss of the gene is lethal [START_REF] Wang | Yolk sac angiogenic defect and intra-embryonic apoptosis in mice lacking the Ets-related factor TEL[END_REF] and heterozygous Etv6 mice have unperturbed hematopoiesis [START_REF] Zhou | Germline ETV6 mutation promotes inflammation and disrupts lymphoid development of early hematopoietic progenitors[END_REF]. Human induced pluripotent stem cells (iPSCs) harboring a pathogenic heterozygous ETV6 mutation do not give rise to an increase in hematopoietic progenitor cells and MKs. However, iPSCs carrying the homozygous ETV6 mutation give rise to an increase in hematopoietic progenitor cells and immature MKs, as observed in heterozygous patients using an in vitro model of CD34 + -derived MKs [START_REF] Borst | Study of inherited thrombocytopenia resulting from mutations in ETV6 or RUNX1 using a human pluripotent stem cell model[END_REF][START_REF] Poggi | Germline variants in ETV6 underlie reduced platelet formation, platelet dysfunction and increased levels of circulating CD34+ progenitors[END_REF]. Therefore, CD34 + -derived MK differentiation appears to be a valuable model to understand defective megakaryopoiesis associated with aberrant ETV6. This model has been successfully used to characterize acquired and inherited thrombocytopenia [START_REF] Grodzielski | Multiple concomitant mechanisms contribute to low platelet count in patients with immune thrombocytopenia[END_REF], investigate novel mechanisms in MK differentiation and platelet function [START_REF] Kollmann | A novel signalling screen demonstrates that CALR mutations activate essential MAPK signalling and facilitate megakaryocyte differentiation[END_REF][START_REF] Zeddies | MEIS1 regulates early erythroid and megakaryocytic cell fate[END_REF][START_REF] Basak | miR-15a-5p regulates expression of multiple proteins in the megakaryocyte GPVI signaling pathway[END_REF][START_REF] Song | Inhibition of ferroptosis promotes megakaryocyte differentiation and platelet production[END_REF], and evaluate the effect of infectious diseases or therapeutic agents on megakaryoblast differentiation [START_REF] Lin | Suppressive effect of dengue virus envelope protein domain III on megakaryopoiesis[END_REF][START_REF] Hu | Radiation-induced bystander effects impair transplanted human hematopoietic stem cells via oxidative DNA damage[END_REF]. Using CD34 + -derived cells from controls and patients harboring the ETV6 variants, we examined the transcriptome of every cell type in the differentiation pathway from hematopoietic stem/progenitor cells (HSPCs) to MKs. We provide detailed insight into peripheral CD34 + megakaryopoiesis. Furthermore, to fully characterize the megakaryocytic differentiation stages, regulon signatures and the corresponding regulatory networks were assessed. We found that ETV6 deficiency leads to the development of aberrant megakaryocyte-erythroid progenitor (MEP) and MK populations with some hyperactive regulons and a highly deregulated core "ribosome biogenesis" pathway involving ribosomal protein S6 (RPS6). Our findings provide insight into the pathology of ETV6 variants and potential targets for both diagnostic purposes and therapeutic approaches to restore proplatelet formation.

Methods

Patients and DNA sequencing

Patients were recruited at the Center for the Investigation of Hemorrhagic and Thrombotic Pathologies (University Hospital of Marseille) after informing written consent was obtained, in accordance with protocols approved by the national review process (authorization number 20200T2-02). Two high-throughput sequencing methods were used to identify ETV6 variants as previously described [START_REF] Poggi | Germline variants in ETV6 underlie reduced platelet formation, platelet dysfunction and increased levels of circulating CD34+ progenitors[END_REF][START_REF] Saultier | Macrothrombocytopenia and dense granule deficiency associated with FLI1 variants: ultrastructural and pathogenic features[END_REF]. Two ETV6 variants were evaluated in this study, the new variant pF417LTer4 (NM_001987, c.1251del) and a previously described variant p.P214L (NM_001987, c.C641T). Blood samples were collected from five different patients, on several occasions (at least twice for each, with an interval of six months minimum).

In vitro Megakaryocyte Differentiation

After density gradient separation (Eurobio), circulating CD34 + cells were purified using positive selection with magnetic beads (Miltenyi-Biotec) and then cultured in StemSpan Serum-Free Expansion Medium II supplemented with Megakaryocyte Expansion Supplement (Stemcell Technologies) as previously described [START_REF] Saultier | Macrothrombocytopenia and dense granule deficiency associated with FLI1 variants: ultrastructural and pathogenic features[END_REF]. In some experiments, at day 11 or day 13, cells were pre-incubated for 48 hours with CX-5461 (Sigma Aldrich) or cycloheximide (Merck) as previously described [START_REF] Schmidt | SUnSET, a nonradioactive method to monitor protein synthesis[END_REF][START_REF] Adamik | Distinct metabolic states guide maturation of inflammatory and tolerogenic dendritic cells[END_REF] Cell Preparation and Single-Cell RNA Sequencing CD34 + -differentiated cells from two healthy controls (Control 1: male, born in 1996 and control 2: female, born in 1980) and two patients harboring ETV6 variants (a male; born in 1985 and female, born in 1986) were harvested from culture at days 6 and 11 (Figure 1A).

Cell concentrations and viability were assessed after trypan blue staining with an automated cell counter (EveTM NanoEntek). The cell samples from each volunteer were labeled with a distinct hashtag oligo (TotalSeq, Biolegend) and pooled. Single-cell isolation was then carried out with the 10x Genomics Technology using the Chromium Next GEM Single Cell 5' Kit v2 (ref 1000263) according to the manufacturer's protocol. Single-cell cDNA synthesis and sequencing libraries (days 6 and 11) were prepared with a single cell 5' Library and Gel Bead kit. Libraries were sequenced applying a 75-bp paired end reads format with a Next-seq500 (GBiM platform) (parameters, read 1: 26 cycles, i7: 8 cycles, read 2: 57 cycles).

Statistical Analyses

Quantitative variables are expressed as the mean±SEM. Experimental data analyses were performed using GraphPad Prism software. Statistical differences were determined using oneway ANOVA with Kruskal-Wallis post-hoc test, Mann Whitney test or one-sample t-test when appropriate. P-value <0.05 was considered statistically significant.

Results

Characterization of differentiation in control CD34 + -cells

We performed UMAP (Uniform Manifold Approximation and Projection) dimensional reduction to analyze cell transcriptome heterogeneity. The variations between the transcriptomic profiles of cells after 6 and 11 days in culture indicated that distinct gene sets were involved in each stage of differentiation (Figure 1B).

Using unsupervised clustering, we identified a total of 15 clusters (Figure 1C), which were present in both controls (Supplementary figure 1A-C). We performed a detailed characterization of each cell cluster based on known gene sets (signatures) [START_REF] Schmidt | SUnSET, a nonradioactive method to monitor protein synthesis[END_REF][START_REF] Adamik | Distinct metabolic states guide maturation of inflammatory and tolerogenic dendritic cells[END_REF][START_REF] Psaila | Single-Cell Analyses Reveal Megakaryocyte-Biased Hematopoiesis in Myelofibrosis and Identify Mutant Clone-Specific Targets[END_REF][START_REF] Chen | Transcriptional diversity during lineage commitment of human blood progenitors[END_REF][START_REF] Estevez | RUNX1 haploinsufficiency causes a marked deficiency of megakaryocyte-biased hematopoietic progenitor cells[END_REF] (Figure 1D and supplementary Figure 1F) and the top differentially expressed genes (DEGs) (Figure 1E, Supplementary Figure 2, Supplementary table 1).

Cluster 10 was designated HSPC based on the expression profiles of PROM1, CRHBP, FLT3, HOPX and AVP. Similarly, clusters 11 and 7 were designated CMP based on the expression profiles of CPA3, PRG2, CLC and TPSAB1. Cluster 11 clearly differed from cluster 7 as it shared marker genes with the MKP/MK clusters (VWA5A, LMNA, CAVIN2, LAT, CD9 and ITGA2B) and expressed the genes KIT, KRT1, HPGDS and TPSB2. We thus defined this cluster 11 as MK-primed CMP. Cluster 13 was designated GMP because it expressed specific markers such as MPO, ELANE and PRTN3. Clusters 9, 5, 4, 0 and 2 were designated MEP.

Although these clusters shared a strong common MEP signature (CD38, TFRC, DEPTOR, KLF1, TFR2), we deepened the characterization of these five subpopulations. Cluster 9 was the most immature population and was designated "early MEP" based on the remaining expression levels of HSPC and CMP genes. Subsetting of cluster 9 confirms the presence of transitional steps with different levels of engagement towards MEP (HSPC-MEP and CMP-MEP; data not shown). Cluster 5 was designated a MEP-ERP subpopulation based on the genes associated with erythroid progenitors (ERPs) such as HBB, TFR2 and ANK1. Clusters 4, 0 and 2 were designated a MEP-MK subpopulation based on the expression levels of MK genes, such as GP9, GP6, GP1BA and MPIG6B, and the gradual increase in MYH9 expression levels between clusters 4, 0 and 2 (Supplementary figure 1D). Clusters 1, 6, 3, 8 and 12 were primarily observed at day 11 and were designated MK based on the expression of genes associated with classical MK. Specific MK markers (GP6, GP9, PF4, P2RY1) gradually increased from clusters 1 to 12 (Figure 1D, Supplementary figures 1D). Of these five clusters, cluster 1 was the most immature stage (MKP) and cluster 12 was the most mature. Cluster 14 was mainly observed at day 11 and was designated platelet-like particles (PLPs) based on the low number of genes expressed (mean ± SD: 1,418±347 vs. 4,208 ±1,399), the low overall RNA count (mean ± SD: 2,243±751 vs. 23,855±14,826) as compared with the MKP/MK populations (Figures 1F-G, Supplementary figure 1D). Also, their proportion increases from day 6 to day 11 (0.04 % to 1.55 % of total cells). Furthermore, cluster 14 expressed the same genes as cluster 12, with higher expression levels of STXBP5 and RAB27b involved in platelet granule metabolism and RGS18, GP6 and ITGA2B (Figures 1D-E, Supplementary figures 1D-F). For each cell type, we proposed new enriched gene signatures that included known cell-type specific genes and top-ranked DEGs (Supplementary table 2). Lineage signature scores were then computed in each cell and cell types were defined accordingly (Figure 1H-I).

Inference of Megakaryopoiesis Regulon Activity in Control Cells

The cell state transitions in megakaryopoiesis are tightly controlled by transcription factors (TFs). Regulons are inferred groups of genes controlled as a unit by the same repressor or activator TF [START_REF] Pellin | A comprehensive single cell transcriptional landscape of human hematopoietic progenitors[END_REF]. Cell type-specific regulons provide a great opportunity to identify key regulators of cell fate decisions and to improve signatures. Analysis of our control data set using the single-cell regulatory network inference and clustering (SCENIC) workflow provided new insight into cell type-specific regulons that drive cellular heterogeneity and megakaryocytic differentiation. For each regulon, the activity score was calculated in each cell based on the number of transcripts for all genes included in the regulon (TFs and targets).

Comparing each cell type, we analyzed the five most active regulons to isolate key regulons at each stage of differentiation and identify a regulon signature (Figure 2 A-B, Supplementary table 2). Using Cytoscape (v.3.9.1), we then constructed the TF gene regulatory network (TF-GRN) based on the top five regulons of each cell type and their direct TF targets (Figure 2C).

Next, we established a list of specific and common active TFs for each cell type (Supplementary table 3). Among the hyperactive TFs that characterize the MK-primed CMPbiased population, we unexpectedly observed a TF that has never been associated with MK differentiation (ZMAT4; zinc finger matrin, type 4) and two highly specific TFs, AhR (aryl hydrocarbon receptor) and BATF (basic leucine zipper ATF-like transcription factor).

Characterization of the ETV6 Variants

We performed a functional study of the novel ETV6 variant pF417LTer4 compared with the previously described p.P214L [START_REF] Poggi | Germline variants in ETV6 underlie reduced platelet formation, platelet dysfunction and increased levels of circulating CD34+ progenitors[END_REF] (Figure 3A). The nonsense mutation p.F417Lter4 is located in the ETS domain, while the missense mutation p.P214L is localized in the linker domain (Figure 3B, lower panel). The clinical and laboratory characteristics of the patients are shown in Supplementary table 4. We analyzed the repressive activity of the two variants using a dual-luciferase reporter assay. Co-transfection of the reporter plasmid containing the ETSbinding site along with the expression of a plasmid encoding wild type (WT) ETV6 resulted in almost 90% inhibition of luciferase activity. Substitution of WT ETV6 with any of the ETV6 variants led to a significant reduction in repressive activity (85% to 100%) (Figure 3B, left panel). Western-blot analysis showed that mutant ETV6 protein was expressed in the GripTite293 macrophage scavenger receptor (MSR) cell line (figure 3B). Subcellular fractionation of GripTite293 MSR cells showed increased ETV6 protein levels in the cytoplasmic fraction and decreased levels or absence of ETV6 in the nuclear fraction in cells expressing the p.P1214L or p.F417Lter4 variants compared with cells expressing the WT protein (Supplementary figure 3A). Microscopy confirmed that WT ETV6 concentrated primarily in cell nuclei, whereas both ETV6 variants were predominantly located in the cytoplasm (Supplementary figure 3B). 5).

Single-cell transcriptional profiling of ETV6-variant CD34 + cells during megakaryopoiesis

The cell types found in controls were also observed in patients. Clusters 3 and 7 corresponded to HSPC; cluster 4 corresponded to CMP; cluster 10 corresponded to MK-primed CMP; cluster 9 corresponded to GMP; clusters 5, 2, 6, 8 and 0 corresponded to MEP; and cluster 1 corresponded to MKP/MK. No PLP cluster was observed (figure 3D-E, Supplementary figure 4). The proposed lineage genetic signatures (Supplementary table 2, left panel) were computed in each cell and cell types were assigned (Figures 3F-G).

Developmental Trajectory of Megakaryocyte Differentiation

We then assessed the single-cell transcriptome for pseudotemporal ordering of differentiation stages during megakaryopoiesis in controls and patients. Using Slingshot, we inferred differentiation trajectories in cells from controls and ETV6-variant carriers independently. For each condition, we determined the overall trajectory structure of each lineage (rooted tree) by generating a transcriptomic distance matrix using the manually set root cluster (HSPCs; cluster 10 for controls and cluster 3 for patients). We observed an unexpected lineage of MK differentiation that bypassed the CMP cell type and differentiated directly from HSPCs into MEPs (Figures 4A).

Analyzing the differentiation trajectory in control cells, stem cell markers such as CD34, CD38 and HLA-DRA displayed decreased expression, while the MK markers GP9, PF4 and ITGA2B were upregulated (Figure 4B). The GATA1 and FLI1 TFs displayed increased expression, while GATA2 displayed high expression levels in immature cells and reduced expression in later stages. During differentiation, ETV6 and RUNX1 expression levels were low and stable (Figure 4B). By contrast, ETV6-deficient cells displayed stable expression of CD34, HLA-DRA and GATA2 (Figure 4B) during MK differentiation; these genes remained highly expressed at the MKP/MK stage (MKP/MK: fold change patients vs. controls = 2.5 (CD34), 1.5 (HLA-DRA), 1.6 (GATA2), adjusted p-value <0.05). The expression levels of GATA1 and FLI1 were lower than that observed in controls. Analyzing GP9 and PF4, we observed lower expression levels and a delayed response time (Figure 4B). These results indicate that ETV6-variant carriers exhibit a delay in differentiation.

Aberrant populations in ETV6-variant cells identified via single-cell RNA sequencing

The full data set (including both control and patients) was analyzed to compare controls and patients. The cell types identified in control and patient data sets were transferred to the full data set using the cell identity barcode (Figure 5A). In the early cell stages (HSPCs, CMPs, MK-primed CMPs and GMPs), the transcriptome profiles of controls and patients were markedly similar. By contrast, distinct gene expression profiles were observed as early as the MEP stage, which intensified at the MKP/MK stage.

Cell type distribution differed between controls and patients (Figures 5B). Patients harboring

ETV6 variants displayed an increased proportion of HSPC (D6: 17.6±6.1 vs 5.2±1.1%; D11: 16±8 vs 0.6±0.04%) and a decline in MKP/MK (D11: 37±1 vs 78±0.6%) compared with controls (Figure 5B).

We applied the SCENIC workflow to further characterize the aberrant MEP and MKP/MK populations. The UMAP reduction using the 312 combined regulon activity scores in each cell is shown in Figure 5C. Compared to the early stages, we observed marked differences between patients and controls at the MEP and MKP/MK stages. The top 20 differentially active regulons are shown in Figure 5D. Some of the top 20 regulons were common for the two cell stages and demonstrated hyperactivity in patient cells (Supplementary Table 3). After analyzing the regulons using tree hierarchical algorithms with Cytoscape software, we found that SPI1 and GATA2 hyperactivity represented initial events in the modification of regulon activity observed in patients (Supplementary figure 5). Overall, these results indicate that ETV6 mutations alter megakaryopoiesis, resulting in the development of aberrant MEP and MK populations as well as the disruption of key TF activity, with GATA2 and SPI1 hyperactivity representing initial events.

Deregulated "DNA repair" and "translation" pathways in ETV6-variant cells

When comparing controls and patients, the number of DEGs (fold change > 1.3, adjusted pvalue < 0.05) increased over the course of cell differentiation, especially at the MEP and MK stages (HSPC, DEGs=38, 23 up-regulated genes and 15 down-regulated genes; CMP, DEGs=56, 29 up-regulated genes and 27 down-regulated genes; MK-primed CMP, DEGs=30, 15 up-regulated genes and 15 down-regulated genes; GMP, DEGs=32, 21 up-regulated genes and 11 down-regulated genes; MEP, DEGs=239, 100 up-regulated genes and 139 downregulated genes; and MKP/MK, DEGs=942, 339 up-regulated genes and 603 down-regulated genes) (Figure 6A). Focusing on MEP and MKP/MK populations, DEGs were enriched in various biological pathways in multiple gene set databases (GO Biological process (Figures 6B), KEGG (Supplementary figure 6) and Reactome (Supplementary figure 7). Top 10 GO deregulated pathways are available in the supplementary tables 6 and 7.

Several pathways associated with DNA repair and cellular response to DNA damage were downregulated in patient MEPs and MKs (Supplementary table 8). Some genes involved in major DNA repair pathways displayed reduced expression levels: FEN1 (base excision repair), MGMT (direct reversal of DNA damage), RAD23A and RAD23B (nucleotide excision repair), and XRCC6 and PRKDC (non-homologous end joining) [START_REF] Roy | Transitions in lineage specification and gene regulatory networks in hematopoietic stem/progenitor cells over human development[END_REF]. Caretaker genes indirectly involved in maintaining genomic stability (e.g., TTK, NUDT1, DUT and UBE2V2) were also downregulated in MKs harboring ETV6 variants (Figure 6C). The top 10 upregulated pathways in ETV6-variant MKs were associated with "translation genes" (Supplementary table 7). The most upregulated genes, RPS and RPL, code for ribosomal protein small subunits and ribosomal protein large subunits, respectively (Supplementary figure 8).

Increased Translation and RPS6 in Patients Harboring an ETV6 Variant

Translation was the strongly dominant upregulated pathway. Translation levels were evaluated in HEL cells transduced with P214L or WT ETV6 before and after PMA-induced differentiation. As expected, PMA stimulation led to reduced translation in all samples (Figure 7A). Translation levels were significantly higher in ETV6-deficient HEL cells at the non-stimulated basal state and after PMA stimulation (Figure 7A). Compared with control cells, higher translation levels were also observed in CD34 + -derived MK from ETV6-variant carriers (Figure 7B). Among all upregulated genes in ETV6-variant MKPs/MKs, 1,488 genes contained 2,189 sequences that correspond to the canonical ETV6-binding site (i-cisTarget: normalized enrichment score 4.85, rank 40e/512, Supplementary table 9), including RPS6, which is a cornerstone of translation [START_REF] Aibar | SCENIC: single-cell regulatory network inference and clustering[END_REF] (Figure 7C). Furthermore, the ETV6-binding site in RPS6 is highly conserved across seven species (2 nd /2,189 detected ETV6-binding regions). Analyzing the differentiation trajectory in control cells, we found that RPS6 mRNA levels gradually decreased, with the lowest levels observed in the most mature MKs (cluster 12). By contrast, ETV6-deficient cells displayed stable RPS6 expression levels throughout the differentiation pathway (Figure 7D).

Our data suggest that ETV6 regulates RPS6 expression. Therefore, we obtained ETV6 chromatin immunoprecipitation (ChIP)-sequence data on the GM12876 cell line from the Gene Expression Omnibus database (GSM2574795, GSM2534228, GSM2574796). The peak enrichment on chromosome 9, which includes the RPS6 locus, was extracted and displayed in the UCSC genome browser (Figure 7E). This analysis showed one enriched binding region for ETV6. The binding peak is located in the promoter.

The expression of total RPS6 and phosphorylated RPS6 (p-RPS6, Ser235/236) were analyzed by western blot and flow cytometry in washed platelets, CD34 + -derived MKs from ETV6variant carriers, and the respective age-and gender-matched controls. RPS6 and p-RPS6 were overexpressed in native platelets issued from ETV6-variant carriers compared with controls (Figures 7F, left panel and Supplementary figure 9). We confirmed RPS6 and p-RPS6 overexpression in CD34 + -derived MKs issued from three ETV6-variant carriers using flow cytometry (Figures 7F, right panel).

To investigate the consequences of increased translation, we treated control and patient CD34 + -derived cell cultures with CX-5461, a specific RNA Pol I inhibitor. Healthy control CD34 + -derived MKs (day 11) treated with CX-5461 exhibited reduced translation and p-RPS6 levels, whereas the expression of total RPS6 was unaffected (Figure 7G). In CD34 +derived cells issued from three ETV6-variant carriers, CX-5461 treatment increased the production of PLPs by CD42a + MKs in all cases (Figure 7G). In addition, cells exposed to cycloheximide (CHX) to inhibit protein synthesis, exhibited reduced translation, RPS6 and phopspho-RPS6 levels. CHX treatment was able to increase the production of PLPs (Supplementary Figure 10).

Discussion

Using single-cell transcriptome profiling of MK cell cultures derived from peripheral CD34 + cells, we established a specific signature for each cell stage based on both distinct gene expression profiles and regulon activities. Cells harboring ETV6 variants displayed distinct transcriptomic/regulon profiles starting at the MEP stage and intensifying at the MKP/MK stage, with SPI1 and GATA2 regulon hyperactivity representing early events. Translation pathways were upregulated in patient MEPs and MKs. Puromycin incorporation results as well as RPS6 and p-RPS6 immunoblots were consistent with these findings. Overall, ETV6 deficiency affects key cellular processes that may represent promising mechanistic targets.

In CD34 + -derived MK differentiation, we observed a biased megakaryopoiesis pathway that may be due to stress-driven hematopoiesis as a result of the culture conditions [START_REF] Chatterjee | Mechanisms of DNA damage, repair and mutagenesis[END_REF]. HSPCs directly gave rise to MEPs without passing through the CMP stage, as previously suggested [START_REF] Chauvin | Ribosomal protein S6 kinase activity controls the ribosome biogenesis transcriptional program[END_REF]. Furthermore, it has been hypothesized that MK-biased hematopoietic stem cells (HSCs) represent the hierarchical apex with MEPs developing directly from HSCs [START_REF] Psaila | Single-cell approaches reveal novel cellular pathways for megakaryocyte and erythroid differentiation[END_REF], although this theory has been challenged. We did not observe a MK-biased HSPC population [START_REF] Adolfsson | Identification of Flt3+ Lympho-Myeloid Stem Cells Lacking Erythro-Megakaryocytic Potential: A Revised Road Map for Adult Blood Lineage Commitment[END_REF] because either the analysis time points were too late to detect these cell types or mRNA did not enable detection of MK-biased HSPCs, as previously reported [START_REF] Sanjuan-Pla | Platelet-biased stem cells reside at the apex of the haematopoietic stem-cell hierarchy[END_REF]. However, we identified a MKprimed CMP population that co-expressed megakaryocytic genes and displayed high KIT expression levels (27% of the CMP population). High KIT expression levels represented a good marker of MK-biased CMPs. Previous murine in vitro and in vivo functional studies have demonstrated that HSCs with higher levels of c-Kit signaling preferentially differentiate into MKs [START_REF] Noetzli | New Insights into the Differentiation of Megakaryocytes from Hematopoietic Progenitors[END_REF]. In human bone marrow, MK-primed CMPs likely represented the major megakaryopoiesis pathway independent of the canonical MEP lineage [START_REF] Haas | Inflammation-Induced Emergency Megakaryopoiesis Driven by Hematopoietic Stem Cell-like Megakaryocyte Progenitors[END_REF]. This in vitro model is a valuable tool to investigate these biased pathways.

Hematopoiesis is a progressive process controlled by an elaborate network of TFs. We characterized each cell type by analyzing the activity of specific regulons, to better define hematopoietic populations and establish a TF-GRN of megakaryopoiesis. HSPCs were characterized by the expression of the regulons HLF [START_REF] Shin | High c-Kit expression identifies hematopoietic stem cells with impaired self-renewal and megakaryocytic bias[END_REF], HOXA9 [START_REF] Estevez | RUNX1 haploinsufficiency causes a marked deficiency of megakaryocyte-biased hematopoietic progenitor cells[END_REF] and TCF4 [START_REF] Miyawaki | Identification of unipotent megakaryocyte progenitors in human hematopoiesis[END_REF], which are involved in stem cell maintenance and myeloid commitment. C/EBP family members are critical for myelopoiesis [START_REF] Lehnertz | HLF expression defines the human hematopoietic stem cell state[END_REF][START_REF] Hout | TCF4 promotes erythroid development[END_REF]. Accordingly, we observed noticeable C/EBP activity at the CMP and GMP stages. Interestingly, ZMAT4 was newly identified as an active TF in MK-primed CMP populations. ZMAT4 is a TF of the zinc finger protein family and is associated with "programed cell death" [START_REF] Radomska | CCAAT/enhancer binding protein alpha is a regulatory switch sufficient for induction of granulocytic development from bipotential myeloid progenitors[END_REF], innate lymphoid cells and natural killer cells [START_REF] Shyamsunder | Identification of a novel enhancer of CEBPE essential for granulocytic differentiation[END_REF]. Although copy number variants of ZMAT4 are associated with leukemia [START_REF] Apostolidis | Proposed megakaryocytic regulon of p53: the genes engaged to control cell cycle and apoptosis during megakaryocytic differentiation[END_REF], the role that ZMAT4 plays in myeloid differentiation remains unclear. The MK-primed CMP population was also characterized by AhR expression, which is involved in MK maturation [START_REF] Allan | Transcriptome analysis reveals similarities between human blood CD3-CD56bright cells and mouse CD127+ innate lymphoid cells[END_REF][START_REF] Wan | The association between the copy-number variations of ZMAT4 and hematological malignancy[END_REF][START_REF] Lindsey | The aryl hydrocarbon receptor (AHR) transcription factor regulates megakaryocytic polyploidization[END_REF] and BATF hyperactivation. BATF hyperactivation was evidenced as a marker of MK commitment. We also observed KLF1 activity in MEPs, which is involved in MEP lineage decisions and commitment [START_REF] Strassel | Aryl hydrocarbon receptor-dependent enrichment of a megakaryocytic precursor with a high potential to produce proplatelets[END_REF]. Finally, NFE2 and GATA1 were expected markers of MKs [START_REF] Smith | The aryl hydrocarbon receptor directs hematopoietic progenitor cell expansion and differentiation[END_REF][START_REF] Tallack | Megakaryocyte-erythroid lineage promiscuity in EKLF null mouse blood[END_REF]. Overall, these findings demonstrate that characterizing TF activity at the single-cell level is useful to phenotype cell types and decipher unexpected key functions of new TFs in each cell type.

Applying the lineage signature used in control cells, we found that ETV6-deficient cells displayed the same cell types with a higher proportion of HSPCs, a lower proportion of MKs and a lack of PLPs, which correlates with the thrombocytopenia phenotype observed in patients harboring an ETV6 variant. We also detected marked differences in regulon activities.

Several regulons displayed higher activity levels in the MEP and MK stages. This finding may be due to a loss of ETV6 repressor activity as documented in all ETV6-mutant carriers [START_REF] Poggi | Germline variants in ETV6 underlie reduced platelet formation, platelet dysfunction and increased levels of circulating CD34+ progenitors[END_REF]. We observed GATA2 hyperactivity in MKs from ETV6-mutant carriers, directly regulating SPI1 activity. SPI1 has previously been described as a common downstream target gene of GATA2 in human and murine embryonic stem cells 52,53 . High GATA2 expression levels have been described in REH cells, a B-cell acute lymphoblastic leukemia cell line harboring the ETV6/RUNX fusion transcript, which is the most frequent structural cytogenetic abnormality in children with acute lymphoblastic leukemia [START_REF] Tsang | Failure of megakaryopoiesis and arrested erythropoiesis in mice lacking the GATA-1 transcriptional cofactor FOG[END_REF]. Beyond this observation, the connection between ETV6 deficiency and GATA2 hyperactivity remains poorly understood. This relationship merits further investigation as deficient or excess GATA-2 expression are both pathogenetic [START_REF] Levin | Pathophysiology of Thrombocytopenia and Anemia in Mice Lacking Transcription Factor NF-E2[END_REF].

Analysis of several gene enrichment databases displayed significant differences. Assessing the ten highest scores and after grouping the pathways with common genes, we observed deregulation of pathways linked with translation and DNA repair in MEP and MKP/MK patient cells. Translation was the most upregulated pathway. Altered expression levels of numerous ribosomal protein genes were observed, including upregulation of more than 30 ribosomal proteins. Recent data has emphasized the unexpected contribution of ribosomal biogenesis in hematopoiesis and megakaryopoiesis. Additionally, ribosome biogenesis is involved in mediating the transition between proliferation and differentiation of erythroid progenitors [START_REF] Gaine | GATA2 regulates the erythropoietin receptor in t(12;21) ALL[END_REF]. GTPase-Dynamin-2 deletion in platelets and MKs induces severe thrombocytopenia and bleeding diathesis in mice and results in the upregulation of genes involved in ribosome biogenesis in erythroblasts [START_REF] Katsumura | The GATA factor revolution in hematology[END_REF]. Furthermore, increased translation was confirmed at the functional level. Protein synthesis was increased in CD34 + -derived MKs in patients and hematopoietic cell lines transduced with an ETV6 variant. Analysis of the RPS6 gene revealed a putative ETV6-binding site, which was confirmed via ChIP-sequencing [START_REF] Goff | p53 activation during ribosome biogenesis regulates normal erythroid differentiation[END_REF]. As Rps6-deficient mice display features of megakaryocytic dysplasia with thrombocytosis, this gene likely plays a role in thrombopoiesis [START_REF] Eaton | Endocytosis of the thrombopoietin receptor Mpl regulates megakaryocyte and erythroid maturation in mice[END_REF]48]. Moreover, overexpression of RPS6 or p-RPS6 in murine Sertoli cells cultured in vitro has been shown to disrupt cell function through changes in the organization of actin and microtubule-based cytoskeletons via AKT inhibition [START_REF] Mcgowan | Reduced ribosomal protein gene dosage and p53 activation in low-risk myelodysplastic syndrome[END_REF][START_REF] Keel | Establishing Rps6 hemizygous mice as a model for studying how ribosomal protein haploinsufficiency impairs erythropoiesis[END_REF]. In ETV6-variant carriers, overexpression of RPS6 was observed in platelets and may act on pro-platelet formation, which is dependent on massive reorganization of the actin cytoskeleton. Translation inhibition led to reduced RPS6 phosphorylation and increased numbers of PLPs in ETV6-deficient cultures, thereby suggesting that overexpression of p-RPS6 in patient cells causes a decrease in actin polymerization rates and pro-platelet formation.

Overall, our study demonstrates the heterogeneity of human CD34 + cell-induced MK differentiation in vitro and a major differentiation trajectory in which HSPCs develop into MEPs, bypassing the CMP stage. We found that ETV6 variations result in aberrant MEP and MK populations, which are associated with GATA2 hyperactivity and deregulated translation pathways, with RPS6 and p-RPS6 overexpression in CD34 + -derived MKs and platelets. These findings provide novel insight into megakaryopoiesis and ETV6 function that may be applied to develop targeted therapeutic strategies to alleviate platelet defects.

  We performed UMAP dimensional reduction to analyze cell transcriptome heterogeneity and clusters. The day 6 and day 11 transcriptome profiles of patient cells overlapped considerably more than those of control cells, thereby suggesting that differentiation was delayed with accumulation of early-stage cells in ETV6-variant carriers (Figure 3C, left panel). Using unsupervised clustering, a total of 11 clusters were identified (Figure 3C, right panel). As previously, we performed a detailed characterization of each cell cluster based on known gene sets and the top DEGs (Figure 3D-E, Supplementary Figure 4, Supplementary table

Figure 1 .

 1 Figure 1. Single-cell RNA sequencing analysis in megakaryopoiesis cell stages in CD34 + -cells isolated from healthy volunteers.A-Schematic diagram of the experimental design. Fresh CD34 + cells from human

Figure 2 .

 2 Figure 2. Inference of genes regulatory network during megakaryopoiesis: control CD34 + -cell culture.A-Heatmap of top 5 differentially active regulons for each cell type in control CD34 + -cell

Figure 3 .

 3 Figure 3. Single-cell RNA sequencing of CD34 + -cells from patients with an ETV6

Figure 4 .

 4 Figure 4. Megakaryocyte differentiation in healthy controls and ETV6 patients:

Figure 5 .

 5 Figure 5. Single-cell RNA sequencing of cells derived from ETV6-variant carriers and

Acknowledgments

The authors acknowledge the members of the French Reference Center for Inherited Hereditary Platelet Disorders for their contribution regarding clinical analyses, the highperformance computing center of Aix-Marseille for granting access to its high-performance computing resources, the GBiM platform for the sequencing and Dr Argüello for providing reagents to study translation. This work was supported by Aix-Marseille University (AMIDEX "Emergence et innovation" ngSUMMIT), the Agence Nationale de la Recherche (JCJC MOST) and the Institut National de la Santé et de la Recherche Médicale (PIA Biofit).

Competing interests

The authors declare that they have no competing interests.