Astrogenesis in the hypothalamus: A life-long process contributing to the development and plasticity of neuroendocrine networks

Astrocytes are now recognized as integral components of neural circuits, regulating their maturation, activity and plasticity. Neuroendocrinology has provided fertile ground for revealing the diverse strategies used by astrocytes to regulate the physiological and behavioural outcomes of neural circuit activity in response to internal and environmental inputs. However, the development of astrocytes in the hypothalamus has received much less attention than in other brain regions such as the cerebral cortex and spinal cord. In this review, we synthesize our current knowledge of astrogenesis in the hypothalamus across various life stages. A distinctive feature of hypothalamic astrogenesis is that it persists life-long, and involves multiple cellular sources corresponding to radial glial cells during early development, followed by tanycytes, parenchymal progenitors and locally dividing astrocytes. Astrogenesis in the hypothalamus is closely coordinated with the maturation of hypothalamic neurons. This coordination is exemplified by recent findings in neurons producing gonadotropin-releasing hormone, which actively shape their astroglial environment during infancy to integrate functionally into their neural network and facilitate sexual maturation, a process vulnerable to endocrine disruption. While hypothalamic astrogenesis shares common principles with other brain regions, it also exhibits specific features in its dynamics and regulation, both at the inter-and intra-regional levels. These unique properties emphasize the importance of further exploration. Additionally, we discuss the experimental strategies used to assess astrogenesis in the hypothalamus and their potential bias and limitations. Understanding the mechanisms of hypothalamic astrogenesis throughout life will be crucial for comprehending the development and function of the hypothalamus under both physiological and pathological conditions.


Introduction
The hypothalamus is an evolutionarily ancient brain structure containing highly conserved neural circuits that control basic life functions, including feeding and energy metabolism, reproduction, sleep, thermoregulation, and electrolyte balance.These functions are handled by a myriad of different neuronal populations organized in a large number of specific circuits that constantly adapt their output activity to incoming nervous and humoral signals in order to maintain body homeostasis (Fong et al., 2023;Saper and Lowell, 2014).The last decades of research in neuroscience have brought to light the importance of astrocytes in regulating the development and function of neural circuits and their physiological and behavioral outcomes.Astrocytes, with their highly ramified, bushy-like, structure, constitute a heterogeneous glial cell population that both sense and regulate the activity of their neuronal, vascular and glial partners.They manage an impressive number of tasks including ion and water homeostasis, neurotransmitter recycling, metabolic support for neurons, blood brain barrier control, regulation of local blood flow, and control of synapse formation, maintenance and plasticity (Barros et al., 2024;Chung et al., 2024;Mishra et al., 2024;Oliveira and Araque, 2022;Verkhratsky and Nedergaard, 2018).Pioneering research in the field of neuroendocrinology has greatly contributed to reveal the significance of astrocytes for the whole-body physiology (Fig. 1).In the magnocellular neuroendocrine system, astrocytes undergo morphological plasticity in response to physiological challenges such as dehydration, parturition and lactation.By modulating the levels of neurotransmitters, neuromodulators and gliotransmitters present in the extracellular space, this process influences the excitability of magnocellular neurons and the resulting secretion of oxytocin and vasopressin, which consequently regulate body fluid homeostasis and milk ejection (Clasadonte and Prevot, 2018;Oliet et al., 2004;Panatier, 2009).Astrocytes are also intimate partners of kisspeptin and gonadotropin-releasing hormone (GnRH) neurons, the hypothalamic neurons controlling fertility in mammals.Indeed, astrocytes contribute to relay the actions of the neurotransmitter glutamate and the neuropeptides oxytocin and kisspeptin on reproduction by engaging in juxtacrine and paracrine interactions with GnRH neurons to modulate their electrical and secretory activity, and hence coordinate puberty and fertility in response to gonadal steroids and to the metabolic status (Prevot and Sharif, 2022;Torres et al., 2024).Beyond controlling neuroendocrine systems, hypothalamic astrocytes regulate the sleepwake cycle by providing metabolic supply to the wake-promoting orexin neurons in the lateral hypothalamic area (Braga et al., 2024;Clasadonte et al., 2017), and adenosine to modulate the activity of sleeppromoting neurons in the ventrolateral preoptic nucleus (Choi et al., 2022;Kim et al., 2020).Moreover, astrocytes in the suprachiasmatic nucleus were recently shown to actively control circadian timekeeping by regulating the levels of extracellular glutamate and GABA thanks to their cell-autonomous clock (Smyllie et al., 2024).Accumulating studies performed these last years also revealed the importance of hypothalamic astrocytes in modulating the activity of the neuronal circuits controlling energy and hemodynamic homeostasis through multiple strategies including glucose transport from blood into the brain, release of gliotransmitters, or glial coverage plasticity in response to nutrients and metabolic peripheral signals (Bouyakdan et al., 2019;García-Cáceres et al., 2019;Gruber et al., 2021;Herrera Moro Chao et al., 2022;Nampoothiri et al., 2022;Nuzzaci et al., 2020;Varela et al., 2021aVarela et al., , 2021b)).Despite the rising recognition of the significant role of astrocytes in regulating hypothalamic neural circuits and their physiological and behavioral outputs, the principles and molecular machinery underlying the development of these glial cells, specifically in the hypothalamus, remain little studied.Moreover, a distinctive feature of the hypothalamus that has only been recognized recently, is its ability for ongoing neuro-and gliogenesis throughout life (Migaud et al., 2010;Sharif et al., 2021Sharif et al., , 2014;;Yoo and Blackshaw, 2018), providing yet another substrate for neural circuit plasticity in response to an everchanging internal and environmental state.
In this review, we examine our current understanding of hypothalamic astrogenesis across different life stages, from embryogenesis through adulthood.Particularly, the nodal position of the hypothalamus in orchestrating sexual maturation prompted researchers to have a closer look at the postnatal period, which can be further decomposed into different stages, the prepubertal period, puberty, and adulthood, each with distinct features in astrogenesis sources and dynamics.We also discuss technical aspects in evaluating astrogenesis, and how they can impact the detection of this process.

Embryogenesis
The vertebrate hypothalamus develops from the rostral diencephalon of the forebrain.Following induction of the hypothalamic primordium during neural plate formation, the developing hypothalamus is patterned in distinct antero-posterior and dorso-ventral subregions that further subdivide into functional areas and nuclei, in response to the finely-tuned spatio-temporal expression of secreted morphogens and downstream transcription factors (Bedont et al., 2015;Xie and Dorsky, 2017).Production of the different cell types follows the same general principles as in other brain regions, with periventricular radial neural stem cells giving rise first to neurons followed by glial cells (Romanov et al., 2020;Zhang et al., 2021;Zhou et al., 2022).As in the neocortex, hypothalamic neural stem cells correspond to radial glial cells that extend a short apical process to the ventricular surface and a long basal process contacting the pial surface with a cell body located in the ventricular zone that undergoes interkinetic nuclear migration.These Fig. 1.Astrocytes and hypothalamic functions.Astrocytes are endowed with a number of basic key properties that enable them to support and regulate the development and function of neuronal networks (upper left box).Beyond these basic properties, astrocytes exhibit morphological, molecular and functional heterogeneity both between and within different regions of the mammalian central nervous system (Bartels et al., 2024).In the hypothalamus, astrocytes contribute to the regulation of major physiological functions by modulating the activity of specific neuronal populations (indicated in italic) located in different nuclei (shown in circles) (see main text for details).Green stars indicate sites of persistent astrogenesis beyond the embryonic period.These include the subventricular zone lining the lateral ventricles, the dentate gyrus of the hippocampus and some hypothalamic regions.Ctx, cerebral cortex; Hipp, hippocampus; Hyp, hypothalamus; LH, lateral hypothalamic area; LV, lateral ventricle; MBH, mediobasal hypothalamus; POA, preoptic area; PVN, paraventricular nucleus; SCN, suprachiasmatic nucleus; SON, supraoptic nucleus; VLPO, ventrolateral preoptic nucleus.Schematic created with Biorender.(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)ventricular radial glial cells give rise to a second population of radialglia-like cells that maintain their pial contact but lose their apical process with their cell bodies dispersed throughout the mantle zone.They undergo mitotic somal translocation during cell division before giving birth to progenitors and/or neurons that migrate to reach their final location (Zhou et al., 2020) (Fig. 2).
Embryonic hypothalamic neurogenesis occurs between E9 and E18 in mice.Most neurons are formed between E11 and E14, with slight variations in their peak generation depending on the nuclei (Lopez-Rodriguez et al., 2022;Shimada and Nakamura, 1973).However, we lack such detailed information on the timing of hypothalamic astrogenesis during embryogenesis.Alvarez-Bolado and collaborators explored the contribution to hypothalamic development of progenitors expressing Sonic Hedgehog (Shh), a secreted morphogen critical for the induction and patterning of the hypothalamus (Alvarez-Bolado et al., 2012).Lineage-tracing during early embryogenesis (E7.5 to E12.5) showed that Shh-expressing progenitors labelled from E7.5 onwards produced both neurons and astrocytes while after E12.5, they generated almost exclusively astrocytes.Notably, Shh shows a dynamic expression pattern during embryogenesis and Shh-expressing progenitors variably contribute to distinct hypothalamic nuclei.Indeed, the area of Shhprogenitor-derived cells shifts from posterior to more rostral regions between E7.5 and E12.5, with the exception of the anterior hypothalamus where no Shh-derived cells are found within the time window analyzed.While Shh-expressing progenitors are first found in the mammillary and tuberal hypothalamus, where they give rise to neurons and astrocytes, they appear later in the preoptic region, at E10.5, where they mostly produce astrocytes (Alvarez-Bolado et al., 2012).At present, the origin of astrocytes populating the anterior hypothalamus remains to be determined, as well as the extent of the hypothalamic astrocyte population that derives from Shh-expressing progenitors in the preoptic, tuberal and mammillary regions.
Besides neuroanatomical studies based on birth dating strategies and lineage tracing, the recent advancement of high-throughput molecular approaches at a single-cell level has provided a wealth of data on gene expression throughout mouse and human hypothalamic development, with the aim of reconstituting lineage hierarchies and identify the underlying regulatory gene networks (Kim et al., 2020;Romanov et al., 2020;Zhang et al., 2021;Zhou et al., 2022Zhou et al., , 2020)).The main focus of these studies is to understand how the impressive heterogeneity of hypothalamic neurons emerges and unveil its transcriptional code.While a particular interest has also been given to the ontogeny of tanycytes, which are specialized ependymoglial cells lining the floor and ventral walls of the third ventricle (Prevot et al., 2018), the development of other cell populations, in particular astrocytes, remains under-explored.A major limitation in studying astrocyte development is the lack of knowledge of the different stages of astroglial lineage and corresponding markers, in contrast to oligodendrogenesis for which the differentiation path is far better characterized (Elbaz and Popko, 2019;van Bruggen et al., 2017).However, developmental trajectories can be inferred from single-cell RNA sequencing (scRNA-seq) data using specific analyses.Using such approach, Kim and collaborators identified transitional Fig. 2. General principles of astrogenesis in the hypothalamus throughout life.During embryogenesis, the primordial hypothalamic neural stem cells are the radial glial cells (hRG), which are bipolar cells extending a short apical process to the 3 V wall and a long basal process to the pial surface.These cells divide and give rise to mantle zone radial glial cells (hmRG), which keep their pial contact but lose their luminal one.hmRG proliferate and give birth to neurons, which presumably migrate along radial processes to reach their final destination, as in the cerebral cortex, other hmRG, and progenitors that are dispersed in the mantle zone and have lost their pial contact (MZ progenitors).Whether astrocytes originate from the direct transformation of radial glial cells, and/or from progenitors of the mantle zone (hmRG and MZ progenitors) remains to be explored (Zhou et al., 2020).Soon after birth, radial glial cells transform into tanycytes in the ventral part of the 3 V (Coutteau-Robles et al., 2023;Mirzadeh et al., 2017).During the prepubertal postnatal period, the astrocyte population expands thanks to astrocyte proliferation (Rottkamp et al., 2015;Shoneye et al., 2020), and differentiation from tanycytes (Goodman et al., 2020;Yoo et al., 2021) and parenchymal progenitors (Pellegrino et al., 2021).This period is marked by astrocyte maturation, as evidenced by increased expression of the astrocytic markers GFAP, S100, Connexin-43 (Cx43), and Aldh1L1 (Coutteau-Robles et al., 2023;Marsters et al., 2016;Munekawa et al., 2000).In the adult hypothalamus, astrocytes are produced from tanycytes (Chaker et al., 2016;Robins et al., 2013a), parenchymal progenitors expressing Sox2 and/or Olig2 (Li et al., 2012;Tatsumi et al., 2018) and, to a low extent, from the local proliferation of astrocytes (Gouazé et al., 2013;Ohlig et al., 2021).Ongoing astrogenesis occurs concomitantly with neurogenesis throughout the postnatal life.states between gliogenic progenitors and hypothalamic astrocytes, associated to an upregulation of Notch signaling components across mouse astrocyte development (Kim et al., 2020).Along the same line, Romanov and colleagues identified Hes5, Sox9 and the nuclear factor I/A (Nfia) as master transcription factors specifying the astroglial fate in mice.Accordingly, mice deficient in Nfia expression showed impaired embryonic hypothalamic astrogenesis while neurogenesis was not affected (Romanov et al., 2020).Another scRNA-seq study of the progeny of hypothalamic neuroepithelial cells expressing the retina and anterior neural fold homeobox (Rax) transcription factor led Zhang and collaborators to propose a "state-switching" model of cell diversification, in which radial glial cells can produce astroglial lineage cells very early during embryogenesis, concomitantly with neurogenesis, as opposed to the sequential model in which radial glial cells first produce neuronal and then astroglial progeny (Zhang et al., 2021).In the human embryonic hypothalamus, progenitors specified toward the astroglial fate showed enriched expression of the FOXJ1, GLIS3 and AXNA1 transcription factors.Notably, comparison of transcriptional profiles between humans and mice suggests differences in molecular and temporal features of hypothalamic astrocyte development.Indeed, expression of the astrocytic marker glial fibrillary acidic protein (GFAP) starts at gestational week 15 in humans but after birth in mice.Moreover, the expression of certain genes, such as HOPX, is enriched in developing hypothalamic astrocytes in humans but not in mice (Zhou et al., 2022).Therefore, analysis of scRNA-seq datasets offers the opportunity to better characterize the astroglial lineage, identify candidate genes and/ or pathways regulating astrocyte specification and differentiation, and explore evolutionary divergence.

The prepubertal postnatal period
The postnatal period that extends from birth to puberty is marked not only by terminal differentiation, circuit connectivity refinement and maturation of neurons, under the influence of internal and external cues such as hormones and nutritional regulators (Sominsky et al., 2018), but also by astrocyte maturation and ongoing astrogenesis.In rodents, this period has been subdivided into a sequence of different phases: the neonatal period (i.e. the first postnatal week), the infantile period (i.e. the second and third postnatal weeks, ending at weaning) and the juvenile period (from weaning to the pubertal period), each marked by key events in sexual maturation and acquisition of the ability to reproduce at puberty (Ojeda et al., 1980;Prevot, 2015).

Maturation of embryonic astrocytes
While astrogenesis starts during embryogenesis, maturation mostly occurs postnatally, as attested by the expression of the astrocytic markers GFAP, S100, Connexin-43 or Aldehyde dehydrogenase 1 (Aldh1L1) that is low at birth in the parenchyma and significantly increases during the first postnatal weeks (Coutteau-Robles et al., 2023;Marsters et al., 2016;Munekawa et al., 2000).Hypothalamic astrocyte maturation appears to be under the influence of hormonal and neuronal activity-related factors.Estradiol promotes the differentiation of rat hypothalamic astrocytes in vitro while perinatal androgens increase GFAP mRNA and protein levels, and astrocyte morphological complexity in the rat arcuate nucleus without affecting the number of newborn astrocytes (Garcia-Segura et al., 1995;Mong et al., 1999).The resulting sexual dimorphism in GFAP levels that are higher in males than females may contribute to the differential astroglial coverage and synaptic connectivity of arcuate neurons between the two sexes (Garcia-Segura et al., 1995).In the rat suprachiasmatic nucleus, eye-enucleation at birth blunted the postnatal increase in GFAP immunoreactivity and the proper development of the astroglial coverage of neurons (Munekawa et al., 2000).Since this hypothalamic region is the master regulator of circadian physiology, receiving photic information from the retina (Bedont and Blackshaw, 2015), these observations suggest that astrocyte maturation is regulated by the activity of their neuronal partners through signals that remain to be determined.

Timely coordination of prepubertal postnatal astrogenesis and maturation of hypothalamic neurons
As prenatally-born astrocytes mature, new astrocytes continue to be born after birth.Using the thymidine analog bromodeoxyuridine (BrdU), that incorporates into the DNA of cells undergoing the S-phase of the cell cycle and is then transmitted to their progeny, the proliferation dynamics and differentiation trajectories of newborn cells was evaluated in the female rat preoptic region, the hypothalamic region hosting GnRH neurons.Proliferation was high until weaning with most newborn cells differentiating into GFAP-expressing astrocytes, while during the juvenile period, both the proliferative activity and the commitment of newborn cells to the astroglial lineage decreased, as oligodendrogenesis rose (Pellegrino et al., 2021).Remarkably, early postnatal astrogenesis develops in close interaction with GnRH neurons.The GnRH system is unique since GnRH neurons originate in the olfactory placode, migrate to the preoptic region of the hypothalamus during embryogenesis and then undergo a long process of postnatal maturation ending with puberty onset and acquisition of fertility (Prevot, 2015).At the beginning of the infantile period, GnRH neurons actively shape their astroglial entourage by recruiting in their vicinity progenitors, which differentiate into astrocytes that remain associated with GnRH neurons into adulthood (Fig. 3).This glial attraction is mediated by the phospholipid-derived signalling molecule prostaglandin D 2 (PGD 2 ), produced by the PGD 2 synthase whose levels rise into the GnRH neuron population during the infantile period, and that activates the DP1 receptor in neighbouring progenitors.This wave of astrogenesis in the environment of GnRH neurons is critical for sexual maturation.Indeed, blocking cell proliferation in the preoptic region during the infantile period by the local infusion of an antimitotic compound delays puberty onset and perturbs adult estrous cyclicity.By more selectively blunting the recruitment of progenitors by GnRH neurons without affecting their generation using a DP1 antagonist, puberty is not affected but the onset of regular estrous cyclicity is delayed.Notably, this treatment has major detrimental effects on the GnRH system that are already visible during the infantile period, such as altered minipuberty (Pellegrino et al., 2021).Minipuberty, which is marked by a surge of follicle-stimulating hormone at P12 in rodents and 1 month in humans, corresponds to the first centrally driven and gonad-independent activation of the hypothalamic-pituitary-gonadal (HPG) axis.This transient phase of HPG axis stimulation triggers gonadal steroid synthesis and gonadal maturation that is essential for subsequent gametogenesis and fertility at puberty (François et al., 2017;Kuiri-Hänninen et al., 2014;Prevot, 2015;Rohayem et al., 2024).The blunted minipuberty induced by DP1 antagonism is associated to an altered integration of GnRH neurons into their neural circuit, as attested by a decreased number of glutamatergic inputs on GnRH neuronal somata and a reduced firing rate (Pellegrino et al., 2021), further supporting the key role of astrocytes in synapse formation and function (Chung et al., 2024;Pfrieger and Barres, 1997;Ullian et al., 2001).Altogether, these data show that astrogenesis in the preoptic region during the infantile period is critically involved in the correct integration of GnRH neurons into their neuroglial network and hence sexual maturation.
Astrogenesis also occurs in the tuberal region during the first two weeks of postnatal life (Rottkamp et al., 2015).This region hosts key neuronal populations for the control of energy homeostasis, which are born during embryogenesis but develop their projections postnatally, in response to the neurotrophic action of the adipokine leptin (Bouret et al., 2004), whose levels dramatically rise between P4 and P14 in mice (Ahima et al., 1998).Interestingly, treatment with leptin during the second postnatal week stimulates the production of astrocytes, while removing the leptin receptor from GFAP-expressing cells decreases the neonatal proliferation of astrocytes (Rottkamp et al., 2015), suggesting that the early postnatal trophic action of leptin on the hypothalamic feeding circuits extends to their astrocytic component.While leptin exerts a direct stimulatory action on hypothalamic neurite outgrowth (Bouret et al., 2004), these results raise the possibility that leptin also contributes to the maturation of the energy homeostasis circuits by promoting the establishment of a proper astroglial entourage.

Origin of prepubertal postnatal astrocytes?
Different sources have been identified for the new postnatal astrocytes: amplification of the astrocytic pool by the proliferation of alreadyformed astrocytes, or differentiation from tanycytes or parenchymal progenitors (Fig. 2).
In the postnatal cerebral cortex, a major source of glia is the local proliferation of differentiated astrocytes, with nearly half of the astrocytes originating from local division during the first two weeks of postnatal life.This proliferative activity is most prominent during the first postnatal week and then drops sharply (Ge et al., 2012).Immunofluorescent or genetic labelling of astrocytes coupled to thymidine analog-based labelling of proliferating cells showed that mouse hypothalamic astrocytes also robustly proliferate during the first postnatal weeks (Rottkamp et al., 2015;Shoneye et al., 2020).A comparative analysis between different brain regions showed that the density of proliferating astrocytes is higher in the cerebral cortex than in the hypothalamus during the first postnatal week, similar during the second postnatal week while it is significantly higher in the hypothalamus from P15 to P30, with ~ 40 % of astrocytes being proliferating in this region as opposed to ~ 10 % in the cerebral cortex.Cortical and hypothalamic astrocytes also differ in their morphological maturation since most hypothalamic astrocytes do not significantly increase their volume from P7 to P26 while that of cortical astrocytes shows a dramatic growth during the same period.Indeed, most hypothalamic astrocytes at P30 exhibit a volume of ~ 2000 µm 3 , being morphologically less complex and smaller than cortical astrocytes that develop a considerable arborization of branches and reach average volumes of 6000 to 7000 µm 3 (Shoneye et al., 2020).These data show that once the first astrocytes are produced from radial glia during embryogenesis, the astrocyte population massively expands postnatally via the local proliferation of resident astrocytes, and that hypothalamic astrocytes maintain a robust proliferative activity longer than cortical astrocytes during the first month of In early infancy, astrocytes in the vicinity of GnRH neurons secrete factors that stimulate expression of the Ptgds gene, which codes for the prostaglandin D2 (PGD2)-synthesizing enzyme, in these neurons (1).PGD2 released by GnRH neurons (2) binds to its DP1 receptor on glial progenitors, attracting them to the vicinity of neuronal cell bodies (3).This recruitment process is strongly affected by early exposure to bisphenol A (BPA).As recruited progenitors differentiate into astrocytes, they supply more astrocytic factors, helping to amplify the attraction phenomenon.The establishment of astrocytic coverage stimulates the electrical activity of GnRH neurons by promoting the development of glutamatergic synapses on their cell bodies (4), supplying them with more of the excitatory gliotransmitter prostaglandin E2 (PGE2) (5), and increasing the number of their PGE2 EP receptors.All these mechanisms contribute to the maturation of the electrical and secretory activity of GnRH neurons, enabling the first ovulation to occur, marking puberty, and the onset of estrous cycles.Glu: glutamatergic afference; GABA: GABAergic afference.Adapted from (Pellegrino et al., 2021) with permission.(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)postnatal life.
The other source of postnatal astrocytes corresponds to tanycytes.Tanycytes are ependymoglial radial cells lining the ventro-lateral wall of the third ventricle in the tuberal region of the hypothalamus, which act as master regulators of reproduction and energy homeostasis.These cells constitute a heterogeneous cell population in terms of morphological, molecular and functional properties.They originate from embryonic radial glia and keep acting as neural stem cells in the postnatal brain (Fong and Kurrasch, 2022;Prevot et al., 2018).Lineage-tracing of tanycytes using the Rax promoter, which is expressed in all tanycyte populations, coupled to scRNA-seq, showed that neonatal tanycytes generate multiple cell types including astrocytes in the mediobasal hypothalamus.Interestingly, deletion of the NFI family of transcription factors Nfia/b/x in Rax-expressing tanycytes increases tanycyte proliferation and neurogenesis, at the expense of gliogenesis, suggesting a role for NFI family genes in promoting glial specification and differentiation from tanycytes (Yoo et al., 2021), in line with the pro-astrogenic action of Nfia during embryonic development (Romanov et al., 2020).Similarly, lineage-tracing of the subpopulation of tanycytes expressing fibroblast growth factor 10 (Fgf10), which line the floor of the third ventricle, showed that during the first postnatal week, these tanycytes give birth to mainly neurons and a few glial cells including astrocytes that populate the mediobasal hypothalamus (Goodman et al., 2020).Notably, this property seems to be lost thereafter since lineage-tracing of adult Fgf10-expressing tanycytes shows no GFAP-expressing progeny (Haan et al., 2013).
Finally, postnatal astrocytes can be produced from parenchymal progenitors.In the female rat preoptic region at the beginning of the infantile period, Sox2 + /GFAP -progenitors proliferate and give rise to GFAP-expressing astrocytes 7 days later (Pellegrino et al., 2021) (Fig. 3B-D).The origin of these parenchymal progenitors remains, however, to be determined.Since the adjacent organum vasculosum laminae terminalis (OVLT) contains a population of tanycyte-like cells (Langlet et al., 2013), it could be suggested that progenitors are born from these tanycytes.Moreover, whether such parenchymal progenitors are found in other hypothalamic regions remains unknown.
Altogether, the prepubertal postnatal period is marked by intense astrogenesis in different hypothalamic regions that is coordinated with the maturation of neuronal circuits controlling reproduction and metabolism.The multiple sources of postnatal astrogenesis may provide heterogeneous populations of astrocytes able to adapt to the specific requirements of select neuronal circuits.

Puberty
Puberty, defined as the period of postnatal development during which the capacity for sexual reproduction is achieved, is the result of a complex array of events that unfold throughout the postnatal period (Prevot, 2015).While the timely acquisition of reproductive competence involves cell neogenesis in prepubertal postnatal development, as seen with the infantile wave of astrogenesis that controls the functional maturation of the GnRH system (Pellegrino et al., 2021) (Fig. 3), the production of new cells in the hypothalamus during the peripubertal period also appears determinant.Indeed, during the peripubertal period, gonadal hormones contribute to establishing or maintaining sexual dimorphism in rats' brain by regulating in a sex-and region-dependent manner the addition of new cells to sexually-dimorphic nuclei such as the anteroventral periventricular nucleus of the hypothalamus (AVPV) and the sexually dimorphic nucleus of the preoptic area (SDN) (Ahmed et al., 2008).The AVPV, which is larger in females than in males, is critically involved in the female-specific ability to generate a preovulatory surge of luteinizing hormone (LH) in response to estradiol, leading to ovulation (Wang and Moenter, 2020).During the peripubertal period, more cells are produced in the female than in the male rat AVPV (Ahmed et al., 2008;Mohr et al., 2016).These pubertally newborn cells appear functionally integrated into the AVPV circuit as some of them express the activation marker Fos following a hormone-induced LH surge.Moreover, some of the newborn cells express the estrogen receptor ERα, showing their ability to directly sense estrogen levels.Blocking cell proliferation in the periventricular regions, including the AVPV, by intracerebroventricular infusion of a mitotic inhibitor starting at P30 did not affect the onset of puberty that occurred a few days later.However, it perturbed estrous cyclicity, with less days in proestrus, when the preovulatory surge of gonadotrophins occurs.It also blunted and delayed the surge of LH induced by ovarian steroid treatment of ovariectomized females, suggesting an altered capacity of the neuroendocrine brain to generate a positive feedback in response to elevated levels of gonadal steroids (Mohr et al., 2017).Phenotyping of pubertally newborn cells revealed multiple lineages, including neurons, astrocytes and microglia, associated to non-identified cells, possibly oligodendroglial lineage cells or immature cells (Mohr et al., 2017(Mohr et al., , 2016)).While the phenotype of proliferative cells at the moment of their birth was not analysed, the observation of cell pairs within the AVPV parenchyma 2 h after the BrdU injection suggests that newborn cells arise from the local proliferation of parenchymal progenitors (Mohr et al., 2016).In the AVPV, the master regulators of the estrogen positive feedback are the kisspeptin neurons, which are highly sexually dimorphic, express gonadal steroid receptors, and stimulate the activity of GnRH neurons in response to rising estrogen levels to trigger the GnRH/LH surge (Wang and Moenter, 2020).It remains, however, to be determined whether the pubertally newborn neurons in the AVPV belong to the kisspeptin population.Interestingly, a significant fraction of pubertally newborn cells were astrocytes (Mohr et al., 2017(Mohr et al., , 2016)), thought to be integral components of the estrogen positive feedback.Indeed, in vitro studies have shown that estradiol stimulates the production of progesterone by hypothalamic astrocytes.The proposed underlying mechanism involves the activation by estradiol of membrane ERα complexed with the metabotropic glutamate receptor-1a in astrocytes, leading to the mobilization of intracellular calcium stores that promotes the transport of cholesterol into the inner mitochondrial membrane where it is converted to pregnenolone.Pregnenolone is then converted to progesterone that is secreted from astrocytes and acts on kisspeptin neurons to augment estradiol-induced kisspeptin expression and increase kisspeptin release (reviewed in Sinchak et al., 2020).While it remains to be determined whether this astrocyte-to-neuron interaction exists in vivo and participates to the rise in kisspeptin expression in the AVPV that drives the GnRH/LH surge, these data raise the possibility that the addition of new neurons and astrocytes to the peripubertal female AVPV contributes to the pubertal acquisition of the estrogen positive feedback leading to ovulation.Notably, the intracerebroventricular delivery of the antimitotic drug used by Mohr and colleagues (Mohr et al., 2017) precludes any specific targeting of a particular brain region.As a consequence, one cannot exclude that the deleterious effects of the drug on the magnitude and timing of the LH surge might have been due to cell proliferation inhibition in other brain regions than the AVPV, such as the suprachiasmatic nucleus, which times the preovulatory LH surge (Williams and Kriegsfeld, 2012), the preoptic or the tuberal regions, where glial cells regulate the electrical and secretory activity of GnRH neurons and downstream release of LH (Prevot and Sharif, 2022).
In addition to the acquisition of the reproductive competence, the addition of new cells in the hypothalamus at puberty has been suggested to be involved in sexual behaviour.In male Syrian hamsters, pubertally newborn neurons and astrocytes were detected in the medial preoptic area (MPOA) and in the arcuate nucleus of the hypothalamus, albeit in different proportions.While newborn astrocytes outnumber newborn neurons in the arcuate nucleus, the proportion is similar in the MPOA.Notably, in both regions, the majority of newborn cells were not identified as neurons or astrocytes, raising the possibility that they belong to the oligodendroglial lineage, remain undifferentiated or correspond to microglia.These two hypothalamic regions significantly differed from the dentate gyrus of the hippocampus, where most newborn cells differentiated into neurons.The number of newborn cells was increased by environmental enrichment in the adult MPOA but not in the arcuate nucleus.While the effect of enrichment on the phenotype of newborn cells was not determined, these data show differential, region-specific, regulation of the production and/or survival of newborn cells during puberty.In both regions, a small fraction of pubertally newborn cells was activated after interaction with a receptive female in adulthood, suggesting that this neogenic activity may be involved in sociosexual behaviour in male Syrian hamsters (Mohr and Sisk, 2013).While additional experiments are required to better characterize the phenotype and function of newborn cells, these data suggest that the addition of new cells, including astrocytes, to the hypothalamus at puberty contributes to multiple aspects of the acquisition of the reproductive competence, from physiological to behavioural dimensions.

Adulthood
A hallmark of the hypothalamus is its capacity for ongoing cell renewal beyond the developmental period, a property that was long thought to be restricted to the subventricular zone of the lateral ventricles (SVZ) and the subgranular zone of the hippocampal dentate gyrus (SGZ) (Fig. 1).A number of studies have now reported neurogenesis and gliogenesis in the adult hypothalamus, although the mechanisms regulating this process and its functional significance still remain poorly understood (Migaud et al., 2010;Sharif et al., 2021Sharif et al., , 2014;;Yoo and Blackshaw, 2018).While these studies consistently show significant levels of neurogenesis, astrogenesis is detected at highly variable rates, from few if any newborn astrocytes (Kokoeva et al., 2007(Kokoeva et al., , 2005;;Matsuzaki et al., 2015Matsuzaki et al., , 2009)), to more appreciable levels (Chaker et al., 2016;Li et al., 2012;Migaud et al., 2011;Mohr et al., 2017;Pencea et al., 2001;Robins et al., 2013a).Since the experimental strategies, protocols and analyses greatly differ between studies, it is difficult to draw a comprehensive picture of the distribution and extent of astrogenesis in the adult hypothalamus (Box 1).In most BrdU studies, the majority of newborn cells take on a neuronal fate (Kokoeva et al., 2007(Kokoeva et al., , 2005;;Li et al., 2012;Matsuzaki et al., 2015Matsuzaki et al., , 2009;;Pencea et al., 2001).However, in the adult female rat AVPV, around a third of newborn cells were GFAP-expressing astrocytes while a lower proportion became NeuN + neurons (Mohr et al., 2017).A high rate of gliogenesis was also found in circumventricular organs of adult mice where 30 to 50 % of newborn cells were identified as astrocytes in the subfornical organ, median eminence and OVLT but none adopted a neuronal identity (Bennett et al., 2009;Morita et al., 2013).Interestingly, in the arcuate nucleus of adult mice, which is adjacent to the median eminence, newborn cells mainly become neurons while less than 10 % differentiate into astrocytes (Li et al., 2012), suggesting a strict control of differentiation trajectories within each nucleus.

Origin of adult-born astrocytes?
Lineage tracing and genetic labelling studies have shown that, as during the prepubertal postnatal period, astrocytes can be produced from tanycytes, parenchymal progenitors or from the proliferation of astrocytes (Fig. 2).
Tanycytes show a high proliferative activity until the first postnatal week, which then drops as they mature (Coutteau-Robles et al., 2023;Lopez-Rodriguez et al., 2022;Mirzadeh et al., 2017).While proliferative tanycytes are very rarely seen in adult mice, lineage-tracing studies have shown their ability to give birth to new neurons and glial cells in the long term.Lineage-tracing of adult tanycytes using the nestin promotor, which is expressed in all tanycyte populations, showed the production of newborn astrocytes in the mediobasal hypothalamus, albeit in much lower proportion compared to newborn neurons.Notably, this process appears to follow a very slow kinetics, as the few newborn astrocytes were detected at 6 and 13 months after induction of the nestin-driven reporter expression, but not after 1 month (Chaker et al., 2016).When following the fate of a subpopulation of lateral tanycytes, using a GLAST-driven reporter mouse model, astrocytic progeny was seen to accumulate in the mediobasal hypothalamus over time, being confined to the periventricular region at 6 weeks after induction of the reporter expression but being detected deeper in the parenchyma 9 months later.At this late time point, 44 % of the GLAST + tanycyte progeny corresponded to astrocytes, while less than 2 % differentiated into neurons, Box 1 .Technical considerations in detecting astrogenesis.
Two experimental strategies have been used to evaluate astrogenesis.The first, and most frequent one, is based on the administration of BrdU (intraperitoneally, intracerebroventricularly, intravenously or via drinking water) followed by the co-immunodetection of BrdU with GFAP or S100 as astrocytic markers.The second one evaluates the expression of astrocytic markers by cells expressing a reporter gene driven by a neural stem/progenitor cell-specific promoter to perform lineage tracing experiments.In most rodent BrdU studies, newborn astrocytes were identified based on the co-expression of BrdU and GFAP.With the exception of the female rat AVPV (Mohr et al., 2017), these studies report extremely low levels of astrogenesis (Kokoeva et al., 2007(Kokoeva et al., , 2005;;Matsuzaki et al., 2015Matsuzaki et al., , 2009;;Pencea et al., 2001) while studies using the S100 marker report higher levels of astrogenesis (Bennett et al., 2009;Li et al., 2012;Morita et al., 2013).When comparing the two astrocytic markers, the use of GFAP to identify astrocytes in the hypothalamus presents several limitations (Fig. 4).First, GFAP, as a cytoskeletal protein, is mainly detected in major processes while its expression in the astrocytic soma is sometimes equivocal, hence the unambiguous conclusion that a cell co-expresses BrdU and GFAP can be difficult to reach.In contrast, S100 is detected in the soma and proximal major processes, making the co-detection with BrdU non-ambiguous.Of note, in lineage tracing studies, the reporter gene is usually expressed in the whole cell volume, enabling easy superimposition of the GFAP immunolabeling with the reporter expression (Fig. 4A-C).Accordingly, fate mapping of nestin-or GLAST-expressing tanycytes revealed non-ambiguous GFAP-expressing progeny (Chaker et al., 2016;Robins et al., 2013a).Second, the levels of GFAP expression in at least some regions of the hypothalamic parenchyma appear lower compared to other brain regions such as the hippocampus.It is particularly true in the mediobasal hypothalamus, where GFAP is mostly detected in some tanycyte subpopulations, and in periventricular, pial and perivascular astrocytes (Langlet et al., 2013;Moore et al., 2022;Sharif et al., 2014), while the remaining parenchyma contains many S100positive astrocytes that lack GFAP immunoreactivity (Fig. 4D-H).As a further support and as discussed in this review, lineage tracing of Olig2and GFAP-expressing cells in the adult mouse brain showed that the hypothalamus contains two subpopulations of astrocytes, the Olig2-lineage astrocytes, which express high levels of mature astrocyte markers such as S100β or Sox9 but low to undetectable levels of GFAP, and the GFAPpositive astrocytes, which co-express S100β (Tatsumi et al., 2018).Altogether, using GFAP immunodetection to evidence newborn astrocytes in the hypothalamus likely greatly underestimates the extent of astrogenesis while S100 appears to be a more convenient and broader astrocyte maker.

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A. Sharif and V. Prevot Frontiers in Neuroendocrinology 75 (2024) suggesting that adult GLAST + tanycytes are mainly gliogenic under normal conditions (Robins et al., 2013a).Lineage tracing of parenchymal progenitors also revealed their ability to give birth to astrocytes.Fate mapping of parenchymal cells expressing the sex determining region Y-box 2 (Sox2) transcription factor, a marker of neural stem and progenitor cells (Ellis et al., 2004;Encinas et al., 2006;Suh et al., 2007), revealed their capacity to give birth to astrocytes in the arcuate nucleus 80 days after the reporter induction, albeit in much lower proportion compared to neurons (Li et al., 2012).The transcription factor Olig2 is generally ascribed to the oligodendroglial lineage, being expressed from oligodendrocyte precursor cells (OPCs) to myelinating oligodendrocytes in the adult brain (El Waly et al., 2014).Interestingly, lineage tracing of Olig2-expressing cells in the adult mouse brain showed that they give rise to astrocytes that express several mature astrocyte markers such as Sox9, S100β and glutamine synthetase, but low to undetectable levels of GFAP.Mapping of these Olig2-lineage astrocytes in the whole brain showed their wide distribution and enrichment in some regions such as the hypothalamus, where they co-habit in a mutually exclusive manner with GFAP-positive astrocytes (Tatsumi et al., 2018).Notably, lineage-tracing of the population of OPCs in the mediobasal hypothalamus of adult mice, using the NG2 promotor expression, revealed oligodendrocytic and neuronal progeny but no GFAP-positive descendants (Robins et al., 2013b).Considering that OPCs co-express NG2 and Olig2 (Campbell et al., 2017;Ligon et al., 2006), and that Olig2-lineage astrocytes lack significant expression of GFAP (Tatsumi et al., 2018), one cannot exclude that NG2positive OPCs give rise to astrocytes that express other markers than GFAP.Co-immunolabeling experiments showed that expression of Sox2, Olig2 and NG2 partially overlaps, highlighting the heterogeneity of the progenitor population.Almost all NG2-positive cells were found to express Olig2 in all brain regions of adult mouse and rats, while NG2positive cells represent not more than a third of the Olig2-positive population, with varying levels according to brain regions (Ligon et al., 2006).In the mediobasal hypothalamus specifically, NG2-positive cells represent less than 10 % of the total Sox2-positive population while 37 % of NG2-positive cells co-express high levels of Sox2 (Robins et al., 2013b).Therefore, the contribution of the different subpopulations of progenitors to astrogenesis still remains to be determined.Moreover, as discussed for the prepubertal postnatal period, whether one or several subpopulations of parenchymal progenitors derive from tanycytes remains to be determined.
Finally, new astrocytes can be produced from the local proliferation of astrocytes.However, while this process significantly contributes to amplify the pool of astrocytes during the prepubertal postnatal period, its extent appears very limited in the adult brain.Genetic labelling and clonal analyses showed that adult diencephalic (i.e.thalamic and hypothalamic) astrocytes can proliferate, even in 8-month-old mice, but this event is rare and produces only small clones of 2-3 cells (Ohlig et al., 2021).The scarcity of this process may explain the variable results obtained from immunolabeling studies using Ki67, a marker of active cell cycle (Andrés-Sánchez et al., 2022), with some authors reporting low numbers of proliferative astrocytes in the adult mouse hypothalamus (Gouazé et al., 2013) while others failed to detect such event in peripubertal mice (Shoneye et al., 2020).The transcription factor Smad4, a downstream effector of the Transforming Growth Factor β/Bone Morphogenetic Protein signalling, was shown to promote adult diencephalic astrocyte proliferation.Intriguingly, transcriptomic analyses revealed that proliferation genes are widely expressed in diencephalic astrocytes (Ohlig et al., 2021), suggesting that large populations of astrocytes are slowly progressing through the cell cycle rather than being quiescent.In agreement with a facilitatory state for cell division, hypothalamic astrocytes respond to pathophysiological stimuli with cell proliferation.Indeed, intracerebroventricular administration of insulinlike growth factor I stimulates the proliferation of subependymal astrocytes of the adult rat third ventricle (Pérez-Martín et al., 2010), while high-fat diet feeding rapidly promotes the proliferation of astrocytes in the whole hypothalamus of adult mice (Gouazé et al., 2013).

Functional significance of adult hypothalamic astrogenesis?
The functional significance of cell neogenesis in the adult hypothalamus has been addressed in several studies that inhibited this process and showed consequences on major hypothalamic functions such as energy metabolism, reproduction, thermoregulation and sleep (reviewed in Sharif et al., 2021;Yoo and Blackshaw, 2018).The most widely used strategy to inhibit cell neogenesis is blocking cell proliferation by intracerebroventricular infusion of the antimitotic drug cytosine-β-D-arabinofuranoside (Ara-C) (Batailler et al., 2018;Borg et al., 2014;Djogo et al., 2016;Gouazé et al., 2013;Kokoeva et al., 2005;Kostin et al., 2019;Matsuzaki et al., 2017;Mohr et al., 2017;Pierce and Xu, 2010) or focal X-ray irradiation of the mediobasal hypothalamus (Djogo et al., 2016;Lee et al., 2014Lee et al., , 2012)).It is important to note, however, that none of these strategies enable to target a specific cell type, e.g.tanycytes or a subpopulation of parenchymal progenitors, and potential extra-hypothalamic sites can be affected.A more selective approach was used by Li and colleagues, who combined genetic targeting to viral delivery to specifically ablate Sox2-expressing progenitors in the mediobasal hypothalamus of adult male mice and showed adverse consequences on energy metabolism including increased food intake, bodyweight and glucose intolerance (Li et al., 2014(Li et al., , 2012)).However, as discussed above, both neuro-and gliogenesis occur together in varying proportions depending on the region and the cell of origin, and blocking cell proliferation or ablating hypothalamic progenitors offers no specific targeting of one of the two processes.Therefore, we still need to develop more specific strategies in order to determine the selective contribution of astrogenesis in the regulation of hypothalamic functions.
Why does astrogenesis selectively persist in certain regions in the adult brain?While a definitive answer would require the development of the appropriate experimental tools, it is possible to speculate on the reasons for this phenomenon.Hypothalamic astrocytes fulfil indispensable supportive and regulatory functions for the proper activity of neuronal networks (Clasadonte and Prevot, 2018), as they do in other brain regions (Verkhratsky and Nedergaard, 2018).However, the Fig. 4. Detection of astrocytes in the adult mouse mediobasal hypothalamus.(A-C) Immunofluorescent labelling of GFAP (green) in adult mice expressing the td Tomato red fluorescent protein under the control of the tamoxifen-inducible GLAST creERT2 promoter (as described in (Sharif et al., 2013).The three GLAST-expressing astrocytes pointed by numbered arrows express GFAP at variable levels (1: strong; 2: low; 3: undetectable).(D-H) Co-immunofluorescent labelling of S100 (red) and GFAP (green) in the mediobasal hypothalamus of an adult C57Bl/6J male mouse.Nuclei were counterstained with DAPI (blue) (see (Coutteau-Robles et al., 2023) for the methods).In the low magnification view shown in (D-G), note that GFAP immunoreactivity is mostly detected in dorsal tanycytes (yellow arrows), in the periventricular parenchyma, at the ventral pial surface (pink arrows), and around blood vessels (arrowheads).(H) is a high-magnification view of the boxed area in (G) showing S100 + /GFAP + astrocytes (arrows) and S100 + /GFAP -astrocytes (arrowheads) in the VMH.The blood vessel is indicated by an asterisk.The astrocytes pointed by numbered arrows and arrowheads are shown at higher magnification in the right panels.1: astrocyte with non-equivocal S100 and GFAP immunolabeling; 2, 3: perivascular astrocytes.Note that the astrocyte #2 has strong GFAP labelling in processes but low staining in its soma, making its identification equivocal if GFAP is used as the sole marker; 4: S100 + astrocyte with very low GFAP immunoreactivity in its left process, that would not have been identified as an astrocyte using GFAP labelling only; 5: S100 + astrocyte with undetectable GFAP immunoreactivity.Scale bars = 20 µm in (C); 100 µm in (G); 50 µm in (H, left panel); 20 µm in (H, right panels). 3 V, third ventricle; ARH, arcuate nucleus of the hypothalamus; ME, median eminence; VMH, ventromedial nucleus of the hypothalamus.(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)hypothalamus is distinctive in receiving privileged access to humoral signals thanks to its peculiar vascularization.Indeed, it contains circumventricular organs, i.e. brain structures vascularized by fenestrated endothelial cells, such as the OVLT and the median eminence (Langlet et al., 2013), allowing nutritional and hormonal messengers to reach hypothalamic neurons and glial cells (Prevot et al., 2018;Prevot et al., 2021).Thanks to their repertoire of transporters and receptors, hypothalamic astrocytes are particularly sensitive to the nutritional status and to the ongoing fluctuations of peripheral signals, such as leptin, insulin or glycemia, to which they respond by dramatic morphological plasticity and consequent modulation of neuronal activity (Frago et al., 2024;García-Cáceres et al., 2016;Kim et al., 2014;Nuzzaci et al., 2020;Zhang et al., 2017b).Interestingly, such plastic changes in astrocyte morphology in response to metabolic inputs are less or not observed in other brain regions such as the hippocampus or the cerebral cortex (García-Cáceres et al., 2016;Kim et al., 2014;Zhang et al., 2017b).From these observations, it may be speculated that ongoing cell renewal enables hypothalamic astrocytes to maintain their optimal neuronsupporting and regulatory activity in the face of constant and intense solicitations by peripheral signals.It is noteworthy that astrogenesis also persists in the other adult neurogenic niches, the SVZ (Delgado et al., 2021) and the dentate gyrus of the hippocampus (Schneider et al., 2022).Interestingly, in the adult dentate gyrus, the neuron-to-astrocyte ratio among the population of newborn cells follows that of the total cell population.Moreover, voluntary physical exercise concomitantly stimulates neuro-and astrogenesis, while keeping unchanged the relative percentage of newborn neurons to newborn astrocytes (Schneider et al., 2022), raising the possibility that astrogenesis may simply allow to maintain a stable balance in the relative numbers of neurons and astrocytes.In all cases, the intrinsic proliferative potential of adult hypothalamic astrocytes (Ohlig et al., 2021), allowing them to rapidly enter cell division in response to pathophysiological stimuli (Gouazé et al., 2013;Pérez-Martín et al., 2010), provides another level of plasticity to finely regulate and adapt the activity of neuronal circuits to incoming signals.

Clinical relevance: Endocrine disruption and ageing
Considering the critical role of astrocytes in the development and function of neuronal networks, a defect in astrogenesis at any stage of life is likely to translate into pathological states.Accordingly, alterations in astrocyte production and circuit integration have been reported in response to early-life exposure to detrimental environmental factors.
The development of the hypothalamus is highly sensitive to environmental influence such as nutritional and hormonal factors that, when deregulated, can have long-term adverse consequences on hypothalamic circuit activity, a process referred to as hypothalamic mal-programming (Sominsky et al., 2018).In this context, one of the most studied hypothalamic circuits is the melanocortin system, which plays a central role in the regulation of feeding behavior and energy homeostasis.This system is particularly affected by maternal obesity, which disrupts neurogenesis and circuit formation, leading to the development of metabolic diseases in the offspring (Bouret, 2022).While the consequences of maternal obesity are essentially studied on neuronal elements, astrocytes also appear affected.Indeed, maternal obesity is associated with increased proliferation of hypothalamic astrocytes in the offspring during late pregnancy and at birth, presumably in response to the elevated circulating levels of interleukin 6 (Kim et al., 2016).While the consequences of an adverse maternal environment on hypothalamic astrogenesis awaits further investigations, these results raise the possibility that dysregulated astrogenesis may also participate to the developmental mal-programming of the hypothalamus.
Another adverse environmental condition affecting hypothalamic development is exposure to endocrine-disrupting chemicals (EDCs).Endocrine disruptors are environmental natural or man-made substances capable of interfering with endocrine systems.The reproductive axis is particularly sensitive to EDCs, mostly when exposure occurs during early life.While the gonads have long been considered the main targets of EDCs, data accumulated these last years have shown that the brain, and particularly the GnRH system, is also affected by EDCs (Lopez-Rodriguez et al., 2021).In addition to altering the neuronal network controlling GnRH neurons, a few studies have suggested that EDCs could disrupt the astrocytic regulation of GnRH secretion (Prevot and Sharif, 2022).However, the precise neuroendocrine mechanisms by which EDCs affect puberty and fertility are still largely unknown.Notably, exposure of female rats during their first 15 days of postnatal life to the plasticizer bisphenol A (BPA) at a very low dose (ng per kilogram body weight per day range), which is consistent with current human exposure (Geens et al., 2012), delayed sexual maturation.Indeed, BPA treatment counteracted the rise in the frequency of GnRH secretory pulses that normally precedes puberty, and tended to delay vaginal opening, a marker of puberty onset in rats.This effect involved increased GABAergic tone, and was associated to altered gene expression in GABA neurotransmission in the hypothalamus (Franssen et al., 2016).Intriguingly, the ability of GnRH neurons to sculpt their astrocytic microenvironment during the infantile period also appeared affected by early exposure to a very low dose of BPA.While treatment with BPA during the first 2 weeks of postnatal life did not affect cell birth in the vicinity of GnRH neurons at the beginning of the infantile period, nor the total number of GnRH neurons in the preoptic region, it decreased the proportion of GnRH neurons associated with newborn cells 7 days later (Pellegrino et al., 2021), suggesting that BPA interferes with the ability of GnRH neurons to recruit neighboring newborn glial progenitors and build the astrocytic environment supporting their network integration at minipuberty (Fig. 3).While additional studies will be needed to further shed light on the cellular and molecular mechanisms underlying the deleterious effects of BPA on the GnRH neuron-glial progenitor dialogue, these results show that glial cells are part of the scenario by which early life exposure to EDCs alters the maturation of the GnRH system.
At the other end of lifespan, ageing has been associated with a decreased number and proliferative activity of hypothalamic neural stem cells and progenitors (Chaker et al., 2016;Haan et al., 2013;Matsuzaki et al., 2015;Pérez-Martín et al., 2016;Zhang et al., 2017a), and decreased neurogenesis (Matsuzaki et al., 2015), conditions that are also seen in response to long-term high-fat-diet feeding in male rodents (Yoo and Blackshaw, 2018).Intriguingly, the hypothalamus has been shown to play a programmatic role in ageing progression and lifespan (Zhang et al., 2013(Zhang et al., , 2017a)).This effect was found to involve the integrity of the neural stem cell pool, since experimental depletion or implantation of hypothalamic neural stem cells accelerated or slowed down ageing, respectively, and modulated longevity accordingly.A mechanism proposed to underlie the rapid anti-ageing effect of hypothalamic neural stem cells was their high capacity to secrete exosomal miRNAs (Zhang et al., 2017a).Whether an altered neuro-and/or gliogenic activity is also causally linked to ageing progression remains to be explored.Interestingly, in the aging adult hippocampus, besides a similar decay in neurogenesis and neuroprogenitors proliferation, the niche activity switches from neurogenic to neuro-and astrogenic, as a result of an increased yield of astrocytes from proliferating progenitors (Beccari et al., 2017).While the consequences of this switch on the hippocampal pathophysiology are still to determine, it is likely that any perturbation in the finely regulated balance between neuro-and astrogenesis will affect the activity and functional output of neuronal networks.Future studies aiming at characterizing the activity and dynamics of the hypothalamic niche throughout adulthood may help get insights into the central mechanisms controlling the aging speed and lifespan.

Conclusions and future directions
Our current knowledge about astrogenesis is primarily derived from studies conducted in the developing cerebral cortex and spinal cord, where the spatio-temporal principles of astrocyte network formation, maturation and underlying molecular determinants are increasingly well understood (Akdemir et al., 2020;Bartels et al., 2024;Clavreul et al., 2022;Lattke and Guillemot, 2022;Vivi and Di Benedetto, 2024).However, the general principles derived from these studies do not necessarily reflect the subtle differences that may exist between brain regions.As discussed here, hypothalamic astrocytes show different proliferative and morphological maturation dynamics compared to cortical ones during the first postnatal month (Shoneye et al., 2020).During adulthood, comparing different neogenic niches also reveals distinctive features.Even though adult diencephalic astrocytes retain proliferative capacity (Ohlig et al., 2021), newborn hypothalamic astrocytes appear to be mainly produced from tanycytes and parenchymal progenitors (Chaker et al., 2016;Li et al., 2012;Robins et al., 2013a;Tatsumi et al., 2018), whereas in the adult hippocampus, astrogenesis is predominantly driven by the proliferation of local astrocytes (Schneider et al., 2022).Beyond inter-regional differences, diversity also emerges at the intra-regional level, as illustrated by the astrocytic progeny of Shhexpressing progenitors that populate all hypothalamic regions except the anterior hypothalamus during early embryogenesis (Alvarez-Bolado et al., 2012) or the differential astrogenic potential of distinct subpopulations of adult tanycytes (Haan et al., 2013;Robins et al., 2013a).These examples stress the need to explore the specific dynamics and regulation of hypothalamic astrogenesis throughout life, which may be instrumental in tackling the still poorly understood question of astrocyte heterogeneity (Bartels et al., 2024).Indeed, while astrocytes have long been known to be morphologically heterogeneous, with the fibrous astrocytes of the white matter and the protoplasmic astrocytes of the gray matter, their molecular heterogeneity has been recognized more recently thanks to the advent of high-throughput molecular profiling, and has been shown to translate into functional diversification (Bartels et al., 2024;Batiuk et al., 2020;Endo et al., 2022;Karpf et al., 2022).For instance, rodent and human hypothalamic astrocytes differ from their cortical counterparts by a distinct repertoire of erbB tyrosine kinase receptor expression, activation and biological response to ligands (Sharif et al., 2009;Sharif and Prevot, 2010).Future work will be needed to characterize in-depth hypothalamic astrocyte inter-and intra-regional heterogeneity, when it emerges during development, and how it is regulated.
A comprehensive understanding of astrocyte genesis and physiology necessitates an integrated view of their intimate relationships with neurons.Accumulating studies, mainly focusing on cortical regions, have revealed the importance and molecular underpinnings of the bidirectional communication between astrocytes and neurons during brain development, where paracrine and juxtacrine interactions between the two cell types shape both synaptogenesis and astrocyte morphogenesis (Farizatto and Baldwin, 2023).As discussed here, such a dialogue was recently identified in the postnatal hypothalamus, where GnRH neurons secrete PGD 2 in order to build their astrocytic environment that in turn promotes their functional maturation (Pellegrino et al., 2021).However, a number of questions remain to be investigated.What are the astrocytic factors and underlying mechanisms that regulate the synaptic integration of GnRH neurons?Do gonadal hormones control this neuron-astrocyte dialogue?Such a possibility is supported by the ability of BPA, an endocrine disruptor known to interfere with estrogen signaling (Rubin, 2011) to disrupt this dialogue.Is this process sexually dimorphic, as recently shown for the synaptogenic action of astrocytederived thrombospondin-2 in cortical neurons (Mazur et al., 2021)?Beyond the GnRH neuronal population, what are the mechanisms used by other hypothalamic neurons to interact with their astrocytic partners?Related to the intra-regional heterogeneity of astrocytes, a full understanding of the mechanisms of astrogenesis will involve considering this process on the scale of the neuronal circuit in which newborn astrocytes are integrated.
As a first step to explore further hypothalamic astrogenesis, a precise description of the spatio-temporal dynamics of hypothalamic astrocyte production during the developmental period remains to be done.BrdUbased studies, already applied to explore the ontogenesis of hypothalamic neurons and tanycytes (Lopez-Rodriguez et al., 2022) will enable to birthdate astrocytes and determine whether, as for neurons and tanycytes, spatio-temporal gradients in the production of astrocytes exist and how they relate to neurogenesis.Moreover, while we have gained insights into the histogenesis of the hypothalamus during embryogenesis with the description of hypothalamic stem/progenitor cells and how they produce neurons (Zhou et al., 2020), the principles of astrocyte production from these cells remains to be described.A major gap to be filled in the astrogenesis field is the precise molecular description of the different steps leading from a neural stem cell to a mature astrocyte.The recent emergence of scRNA-seq studies (Kim et al., 2020;Ohlig et al., 2021;Romanov et al., 2020;Zhang et al., 2021;Zhou et al., 2022Zhou et al., , 2020)), associated with new analysis strategies (Gulati et al., 2020), should enable to identify cell differentiation hierarchies, their underlying molecular signature and regulatory gene networks.Identifying a marker of immature astrocytes will be very instrumental in directly probing astrogenesis, in particular in tissues in which lineage tracing is not possible such as in human brain samples.Indeed, while the adult human hypothalamus contains putative neural stem cells (Pellegrino et al., 2018) and newborn neurons (Batailler et al., 2014), as determined from the immunodetection of doublecortin, a microtubuleassociated protein transiently expressed in immature neurons that is used as a surrogate marker of neurogenesis (Couillard-Despres et al., 2005), we currently have no tool to probe whether astrogenesis also occurs in our species during adulthood.Beyond the description of the process, identifying the molecular control of astrocyte fate commitment and differentiation from neural stem cells will allow to tackle the key question of the specific contribution of astrogenesis to hypothalamic development and function, by enabling to selectively blunt it without affecting other cell lineages.Finally, whereas the origins of hypothalamic pathologies are essentially sought in neuronal defects, the few studies that probed the astrocytic compartment and showed that it was affected in response to early-life exposure to detrimental factors call for in-depth characterization of astrogenesis under various pathological conditions and question its contribution to disease initiation and progression.

Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Fig. 2.General principles of astrogenesis in the hypothalamus throughout life.During embryogenesis, the primordial hypothalamic neural stem cells are the radial glial cells (hRG), which are bipolar cells extending a short apical process to the 3 V wall and a long basal process to the pial surface.These cells divide and give rise to mantle zone radial glial cells (hmRG), which keep their pial contact but lose their luminal one.hmRG proliferate and give birth to neurons, which presumably migrate along radial processes to reach their final destination, as in the cerebral cortex, other hmRG, and progenitors that are dispersed in the mantle zone and have lost their pial contact (MZ progenitors).Whether astrocytes originate from the direct transformation of radial glial cells, and/or from progenitors of the mantle zone (hmRG and MZ progenitors) remains to be explored(Zhou et al., 2020).Soon after birth, radial glial cells transform into tanycytes in the ventral part of the 3 V(Coutteau-Robles et al., 2023;Mirzadeh et al., 2017).During the prepubertal postnatal period, the astrocyte population expands thanks to astrocyte proliferation(Rottkamp et al., 2015;Shoneye et al., 2020), and differentiation from tanycytes(Goodman et al., 2020;Yoo et al., 2021) and parenchymal progenitors(Pellegrino et al., 2021).This period is marked by astrocyte maturation, as evidenced by increased expression of the astrocytic markers GFAP, S100, Connexin-43 (Cx43), and Aldh1L1(Coutteau-Robles et al., 2023;Marsters et al., 2016;Munekawa et al., 2000).In the adult hypothalamus, astrocytes are produced from tanycytes(Chaker et al., 2016; Robins et al., 2013a), parenchymal progenitors expressing Sox2 and/or Olig2(Li et al., 2012;Tatsumi et al., 2018) and, to a low extent, from the local proliferation of astrocytes(Gouazé et al., 2013;Ohlig et al., 2021).Ongoing astrogenesis occurs concomitantly with neurogenesis throughout the postnatal life.Arrows indicate lineage relationships between cells; dashed grey arrows indicate hypothetical relationships; circled arrows indicate that cells are able to proliferate.3 V, third ventricle; MZ, mantle zone; VZ, ventricular zone.Schematic created with Biorender.

Fig. 3 .
Fig. 3. Infantile hypothalamic astrogenesis regulates sexual maturation.(A-D) Immunofluorescent detection of GnRH (green), BrdU (magenta) and Sox2 (B, white) or GFAP (C, D, white) in the female rat preoptic region during the infantile period.Animals were injected with BrdU at P8 and analysed 2 h (A, B, C) or 7 days later (D) to evaluate cell proliferation and differentiation, respectively.(A) The arrow and arrowheads point to GnRH neuron cell bodies.The inset shows a high magnification view of the GnRH neuron pointed by the arrow in the main panel, which is morphologically associated to a BrdU + cell (crossed arrow).At the moment of their birth (B, C), the BrdU + cells associated with GnRH neurons (yellow arrows) are Sox2 + /GFAP -progenitors, while 7 days later (D), they have differentiated into GFAP-expressing stellate cells.Nuclei in (A) were counterstained with Hoechst (white).Scale bars: 100 µm (A, main panel); 10 µm (A, inset); 20 µm (B, C, D).OVLT, organum vasculosum laminae terminalis.(E) Proposed model for the regulation of female sexual maturation by astrogenesis.In early infancy, astrocytes in the vicinity of GnRH neurons secrete factors that stimulate expression of the Ptgds gene, which codes for the prostaglandin D2 (PGD2)-synthesizing enzyme, in these neurons (1).PGD2 released by GnRH neurons (2) binds to its DP1 receptor on glial progenitors, attracting them to the vicinity of neuronal cell bodies (3).This recruitment process is strongly affected by early exposure to bisphenol A (BPA).As recruited progenitors differentiate into astrocytes, they supply more astrocytic factors, helping to amplify the attraction phenomenon.The establishment of astrocytic coverage stimulates the electrical activity of GnRH neurons by promoting the development of glutamatergic synapses on their cell bodies (4), supplying them with more of the excitatory gliotransmitter prostaglandin E2 (PGE2) (5), and increasing the number of their PGE2 EP receptors.All these mechanisms contribute to the maturation of the electrical and secretory activity of GnRH neurons, enabling the first ovulation to occur, marking puberty, and the onset of estrous cycles.Glu: glutamatergic afference; GABA: GABAergic afference.Adapted from(Pellegrino et al., 2021) with permission.(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)