Cell proliferation and glial cell marker expression in the wall of the third ventricle in the tuberal region of the male mouse hypothalamus during postnatal development

The third ventricle (3 V) wall of the tuberal hypothalamus is composed of two types of cells; specialized ependymoglial cells called tanycytes located ventrally and ependymocytes dorsally, which control the exchanges between the cerebrospinal fluid and the hypothalamic parenchyma. By regulating the dialogue between the brain and the periphery, tanycytes are now recognized as central players in the control of major hypothalamic functions such as energy metabolism and reproduction. While our knowledge of the biology of adult tanycytes is progressing rapidly, our understanding of their development remains very incomplete. To gain insight into the postnatal maturation of the 3 V ependymal lining, we conducted a comprehensive immunofluorescent study of the mouse tuberal region at four postnatal ages (postnatal day (P) 0, P4, P10, and P20). We analyzed the expression profile of a panel of tanycyte and ependymocyte markers (vimentin, S100, connexin‐43 [Cx43], and glial fibrillary acidic protein [GFAP]) and characterized cell proliferation in the 3 V wall using the thymidine analog bromodeoxyuridine. Our results show that most changes in marker expression occur between P4 and P10, with a switch from a 3 V mostly lined by radial cells to the emergence of a tanycytic domain ventrally and an ependymocytic domain dorsally, a drop in cell proliferation and increased expression of S100, Cx43, and GFAP that acquire a mature profile at P20. Our study thus identifies the transition between the first and the second postnatal week as a critical time window for the postnatal maturation of the 3 V wall ependymal lining.

the brain and the periphery, tanycytes are now recognized as central players in the control of major hypothalamic functions such as energy metabolism and reproduction. While our knowledge of the biology of adult tanycytes is progressing rapidly, our understanding of their development remains very incomplete. To gain insight into the postnatal maturation of the 3 V ependymal lining, we conducted a comprehensive immunofluorescent study of the mouse tuberal region at four postnatal ages (postnatal day (P) 0, P4, P10, and P20). We analyzed the expression profile of a panel of tanycyte and ependymocyte markers (vimentin, S100, connexin-43 [Cx43], and glial fibrillary acidic protein [GFAP]) and characterized cell proliferation in the 3 V wall using the thymidine analog bromodeoxyuridine. Our results show that most changes in marker expression occur between P4 and P10, with a switch from a 3 V mostly lined by radial cells to the emergence of a tanycytic domain ventrally and an ependymocytic domain dorsally, a drop in cell proliferation and increased expression of S100, Cx43, and GFAP that acquire a mature profile at P20. Our study thus identifies the transition between the first and the second postnatal week as a critical time window for the postnatal maturation of the 3 V wall ependymal lining. and specialized cells called tanycytes in its ventral part. 1 Tanycytes are described as ependymoglial cells given their dual phenotype. On the one hand, they are organized as a simple epithelium lining the ventricular surface, similar to ependymocytes, and share with them the expression of vimentin. 1,2 On the other hand, they express molecular markers of astrocytes such as glial fibrillary acidic protein (GFAP), the calcium-binding protein S100, [3][4][5] the gap junction protein connexin-43 (Cx43), [6][7][8] the glutamate/aspartate transporter GLAST, 2,9 glutamine synthetase 10 and the water channel aquaporin-9. 11 Tanycytes are characterized by a radial morphology, with an elongated cell body sending a long and slender basal process running into the parenchyma to eventually contact vascular and neuronal elements. 12 These morphological characteristics, which place tanycytes at the interface between the blood, the CSF and the hypothalamic parenchyma, allow them to master the bidirectional dialogue between the brain and the periphery, and hence regulate major hypothalamic functions, in particular reproduction and energy homeostasis (reviewed in 13 ). By dynamically controlling the secretion of the neurohormones gonadotropin-releasing hormone 14 and thyrotropinreleasing hormone 15 into the portal vasculature, they determine the output of the hypothalamic-pituitary-gonadal and hypothalamicpituitary-thyroid axes, respectively. Furthermore, they control energy homeostasis by relaying peripheral metabolic signals to hypothalamic neurons through diverse strategies. They modulate the access of bloodborne molecules to arcuate nucleus neurons by dynamically reorganizing the blood-CSF barrier according to the energy status 16 and by shuttling circulating metabolic hormones, in particular leptin, into the CSF through transcytotic transport. 17,18 They also relay information about peripheral nutrient levels to hypothalamic neurons controlling food intake and energy homeostasis. 6,13,19,20 Finally, they may affect energy metabolism by remodeling hypothalamic neuronal networks thanks to their neural stem cell properties and neurogenic competence. 4,[21][22][23][24][25] The broad spectrum of tanycyte functions is underpinned not only by the great versatility of these cells but also by their heterogeneity, leading to the recognition of different tanycyte subpopulations. 13 While we are making rapid progress in understanding the molecular and functional properties of adult tanycytes, our knowledge of their ontogenesis remains very incomplete. Early studies performed in rats showed that tanycyte cytogenesis starts during the last 2 days of pregnancy, following that of ependymal cells, and continues during the early postnatal period while their differentiation is thought to be complete by the end of the first postnatal month. 26 More recent studies in mice confirmed and expanded these observations by providing insights into their origin and molecular control. Tanycytes are derived from sonic hedgehog-expressing progenitors. 5,27 Expression of Lhx2 and its downstream target Rax have been shown to be essential for tanycyte specification and differentiation. 28,29 Molecular marker analyses showed that radial glial cells convert into tanycytes by P10 while maturation of their apical specializations is complete by P35. 5 To increase our knowledge of the postnatal maturation of the 3 V ependymal lining, we performed a comprehensive ventrodorsal and anteroposterior characterization of the proliferation and expression profile of a series of tanycyte and ependymocyte markers (vimentin, S100, Cx43, and GFAP) along the 3 V wall in the mouse tuberal region of the hypothalamus.

| BrdU administration
Mouse pups aged postnatal day (P) 0, that is, the day of birth, P4, P10, or P20 received a single intraperitoneal (i.p.) injection of BrdU (300 mg/kg bodyweight; Sigma) diluted in 0.01 M phosphate buffer saline (PBS) (pH 7.4) at 10 a.m. and were sacrificed 2 h later. This dose of BrdU has previously been shown to label all S-phase cells in the adult rat brain without toxicity. 30 The 2-h chase time corresponds to the BrdU clearance time, as previously determined in the adult rat hippocampus 30 and postnatal rat hypothalamus. 31

| Tissue preparation
P0 and P4 mouse pups were decapitated, and their heads were immersed in a solution of 4% paraformaldehyde (PFA) in PBS for 48 h at 4 C. P10 and P20 mice were anesthetized by i.p. injection of xylazine 17 mg/kg (Paxman, Virbac, France) and ketamine 170 mg/kg (Ketamidor, Axience, France), perfused transcardially with a rinse of saline solution (0.9% NaCl), followed by 20 ml of 4% PFA in PBS (pH 7.4). The brains were removed and immersed in the same fixative for 2 h at 4 C.
The tissues were then transferred to PBS containing 20% sucrose until they had sunk, embedded in Tissue Tek (Sakura Finetek, Villeneuve d'Ascq, France), and frozen in liquid nitrogen. Frozen 14-μm coronal sections were prepared using a cryostat (CM3050S, Leica, Nussloch, Germany) and mounted on chrome-alum-gelatin coated slides, air-dried, and subjected to immunofluorescent stainings.

| Immunofluorescence labeling
After rehydration in PBS, the sections were systematically subjected to microwave pretreatment in a solution of sodium citrate 0.01 M (pH 6) (citrate buffer) for 4 min at 800 W followed by two cycles of 5 min each at 400 W for antigen retrieval. After cooling at room temperature (RT) and washing in PBS, sections were incubated with primary antibodies diluted in PBS containing 0.3% Triton X-100 and 10% normal donkey serum (PBSTS) for one to two nights at 4 C in a humid chamber. The characteristics of the primary antibodies used are shown in Table 1. The specificity of primary antibodies has been described in detail in previous publications listed in Table 1   Anatomical landmarks were determined using the mouse brain atlas from Paxinos and Franklin. 32 Analyses were done in the tuberal region, where tanycytes are found in adult mice. The region was subdivided into four zones along the anteroposterior axis, according to the shape of the 3 V and the neuroanatomical characteristics of adjacent hypothalamic nuclei ( Figure 1) as previously done. 12 In adult male mice, zone 1 extends from Bregma À1.3 to À1.6 mm, zone two from Bregma À1.6 to À1.8 mm, zone 3 from Bregma À1.8 to À2.1 mm, and zone 4 from Bregma À2.1 to À2.5 mm. The morphometric criteria defining these zones in adult male mice were applied to postnatal animals. In addition, the 3 V wall was divided into five areas along the ventrodorsal axis, according to the adjacent parenchymal nucleus it faces 13 : median eminence (ME, i.e., the floor of the 3 V), arcuate nucleus (ARH), ventromedial nucleus (VMH), dorsomedial nucleus (DMH) and posterior area (PH). The ARH was further subdivided into a ventromedial (vmARH) and a dorsomedial part (dmARH).

Quantification of BrdU labeling and analysis of vimentin expression
was done on 6 to 8 mice per age group. Analysis of S100, Cx43, and GFAP expression was done on three mice per age group. An average of 11.8 ± 0.5 sections were analyzed per animal.

| Statistical analysis
All analyses were performed using the GraphPad Prism 9. F I G U R E 3 Expression of S100 in the postnatal tuberal region. Images are lowmagnification views of the 3 V at P0 (A-D), P4 (E-H), P10 (I-L), and P20 (M-P) at different rostrocaudal levels (zone 1-4). White arrows point to S100-immunoreactive (white) cells in the 3 V wall and white arrowheads show S100-immunoreactive cells in the parenchyma. Empty arrows point to 3 V wall cells lacking S100 immunoreactivity. Empty arrowheads in A-D point to autofluorescent red blood cells. Inset in (J) is a high-magnification view of the boxed area in the main panel. Scale bars = 20 μm in the inset; 100 μm in all other panels.
analyzed using the two-way ANOVA with Tukey's multiple comparisons test. p < .05 was considered statistically significant.

| RESULTS
We conducted our neuroanatomical study of the postnatal 3 V wall at postnatal day 0 (P0), P4, P10, and P20. We divided the tuberal region of the hypothalamus into four zones along the anteroposterior axis as previously done 12 and into five areas along the ventrodorsal axis, according to the adjacent parenchymal nucleus 13 (Figure 1). At P10 and P20, ependymocytes had short to absent vimentinimmunoreactive processes (inset in Figure 2N). The relative distribution of radial cells and ependymocytes is summarized in Table 3A.

| Distribution of radial glia/tanycytes and ependymocytes in the 3 V wall
These data show that the distribution pattern of radial cells and ependymocytes along the 3 V wall, as assessed by their vimentin immunoreactivity, mostly changes between P4 and P10 with the emergence of the tanycytic domain ventrally, the ependymocytic domain dorsally and the overlapping region in-between.
3.2 | Expression of the glial markers S100, connexin-43 and GFAP 3.2.1 | S100 At P0, 3 V wall cells showed low to undetectable levels of S100 ( Figure 3A-D). At P4, an immunoreactive signal appeared along the 3 V wall except in the ME and vmARH ( Figure 3E-H). At P10 and P20, the expression pattern was similar to that observed at P4 but the signal intensity increased and was highest in the dorsal part of the 3 V ( Figure 3I-P), following a profile previously described in postnatal and adult mice. [3][4][5] The distribution and intensity of S100 immunoreactivity in the 3 V wall are summarized in Table 3B. Notably, the rise in S100 expression in the 3 V wall between the first and the second postnatal week was associated with the appearance of S100-immunoreactive cells in the parenchyma, corresponding to astrocytes ( Figure 3I-P, inset in Figure 3J).
At P0 and P4, the immunolabeling was mostly confined to the apical

| GFAP
GFAP expression in the 3 V wall was restricted to the area extending from the dmARH to the DMH and was highest in the anterior tuberal region ( Figure 5 and Table 3D). At P0 and P4, GFAP-immunoreactive 3 V wall cells were mostly seen at the level of the dmARH and DMH in zones 1 and 2, except the dorsal tip of the 3 V that lacked GFAP immunoreactivity. A low signal was seen in the DMH in zone 3 and the 3 V wall was devoid of GFAP immunoreactivity in zone 4 ( Figure 5A-H). At P10 and P20, the labeling intensity and the number of immunoreactive cells increased. Immunoreactive cells were visible in zones 3 and 4 with decreasing density. The dorsal tip of the 3 V was always devoid of GFAP immunoreactivity ( Figure 5I-P). GFAPimmunoreactive cells were also detected in the parenchyma from P4 and with increasing density at P10 and P20 ( Figure 5E-P and inset in Figure 5I, yellow arrows). The expression profile observed at P20 was similar to that previously described in adult mice. 5,24,33,34

| Proliferation
To evaluate the proliferative activity of cells bordering the 3 V wall, we used the thymidine analog bromodeoxyuridine (BrdU). Animals Tanycytes and ependymocytes derive from embryonic radial glia. 5,36 Given their morphological, molecular, and functional resemblance, tanycytes are considered persistent radial glia in the adult brain, even though they also display distinct characteristics. 26 Accordingly, our vimentin expression analysis showed that the 3 V wall is mostly lined by radial cells at P0 and P4 while distinct domains hosting radial cells only, ependymocytes only, or a mix of both cell types, appear at P10.
The transition between the first and the second postnatal week is also characterized by a marked increase in the expression of the glial markers S100 and GFAP in dorsal tanycytes facing the dmARH, VMH, and DMH (also known as α tanycytes 13 ) associated to a wider distribution of Cx43 immunoreactivity in tanycyte cell bodies. This temporal window of maturation had previously been identified by others.  Indeed, cells lining the mouse 3 V wall lose the expression of the radial glia marker RC2 over the first 10 postnatal days, while GFAP expression progressively appears in α tanycytes, 5 in agreement with our present analysis. Interestingly, fully mature apical specializations are only seen after 1 month, 5 in line with previous cytological, histochemical, and ultrastructural studies in rats, 26 indicating that tanycyte maturation is a protracted process.
Our study also provides insights into the maturation of 3 V ependy- at P10, their basal process regressed. In addition to its expression in dorsal tanycytes, S100 is highly expressed in ependymocytes. 3,5,37 Our analysis showing low to undetectable levels of S100 at P0, progressive increase at P4, and acquisition of high levels at P10 suggests that S100 expression is a rather late event during ependymocyte maturation.
Analysis of BrdU labeling showed that cell proliferation in the 3 V wall is high during the first postnatal week and then drops sharply to reach almost 0 at P20, in line with a previous Ki67 labeling study. 5  and not significantly different between P0, P4, and P10 in the ME, while they were high during the first postnatal week in the middle part of the 3 V wall. These results suggest that the production of ME tanycytes is mostly complete before birth while that of more dorsal tanycytes continues postnatally. Since tanycytes retain neural stem/progenitor properties during early postnatal life and give birth to neurons and glial cells, 4,22,23,25 it will be interesting to evaluate how the different subtypes of tanycytes contribute to hypothalamic circuit development. Notably, numerous BrdU + cells were seen scattered throughout the hypothalamic parenchyma. Whether these cells are progenitors originating from tanycytes or whether they belong to another lineage remains to be explored.
An early 3 H-thymidine labeling study in the rat showed that the birth of ependymocytes precedes that of tanycytes, with most of them born on E17 and E18. 38 Accordingly, we did not identify any BrdU + ependymocytes at P0 and P4. However, we saw a few BrdU + ependymocytes at P10 and one was detected at P20. These observations raise the question of a short period of ependymocyte production around P10. Alternatively, since tanycytes have been suggested to give birth to ependymocytes during the first postnatal week, 25 it is possible that some of the BrdU + cells that we identified as tanycytes at P0 and P4 were rather differentiating ependymocytes that had not already acquired the typical vimentin immunoreactive profile.
Previous studies have suggested a relationship between the expression of the glial markers GFAP, Cx43, and S100 and the cell proliferative activity or potential. Fibroblast growth factor 2 (FGF2) was shown to stimulate the proliferation of tanycytes in vitro by a sequence of events involving increased Cx43 expression and hemichannel activity. 39 Moreover, in vivo and in vitro experiments have suggested that the subpopulation of dorsal tanycytes expressing GFAP (i.e., dmARH or dorsal α2) selectively exhibits neural stem cell properties in adulthood. 24 Furthermore, the acquisition of S100 expression in forebrain astrocytes has been correlated with their maturation and loss of neural stem cell potential. 40 Our coimmunolabeling experiments showed that BrdU + cells were heterogeneous in terms of S100, Cx43, and GFAP expression profiles. During the first postnatal week, when proliferation was high, 3 V wall cells exhibited low levels of S100 and GFAP, associated with apical localization of Cx43. In particular, the highest proliferative activity seen in zone 4 at P0 was associated with an absence of S100 and GFAP expression, and low to moderate expression of Cx43. Notably, ME tanycytes, which showed consistently low proliferative activity all along postnatal development, exhibited this same pattern of marker expression. Moreover, BrdU + cells expressing S100, GFAP, or high Cx43 were also detected, preventing correlations between the F I G U R E 8 Analysis of S100, Cx43, and GFAP expression in BrdU + cells of the postnatal 3 V wall. (A-E) Coimmunolabeling of BrdU (green), S100 (red), and GFAP (white) in P4 (A, B) and P10 mice (C-E). Arrows and arrowheads point to BrdU + cells in the 3 V wall. (A 1 -A 4 ) shows a BrdU + /S100 + /GFAP À cell. (B 1 -B 4 ) shows a BrdU + cell that expresses low levels of S100 (arrow) and BrdU + cells that do not express S100 Whether a switch in the activity of these factors and associated pathways occurs between the first and the second postnatal week to induce the cell cycle exit of tanycytes remains to be explored.
A hallmark of tanycytes is their morphological and functional connection with capillaries and neurons. 12,13 Interestingly, the P5-P15 period has been identified as a critical time window for the maturation of the gliovascular unit in the mouse brain, with the establishment of the perivascular astrocyte coverage. 41,42 Whether the P4-P10 period shown here to be important for the maturation of tanycytes also correlates with the maturation of the interface between tanycyte endfeet and capillaries remains to be explored. Similarly, the first postnatal weeks are known to be critical for the terminal differentiation, circuit connectivity refinement, and maturation of neural circuits, in particular those controlling energy metabolism in the tuberal region. 43,44 While astrocytes play a key role in the postnatal maturation of neuronal circuits, 45,46 including in the hypothalamus, 31 whether tanycytes, which morphologically and functionally interact with hypothalamic neurons, 6,12 also control their postnatal development awaits future investigations.
Altogether, our neuroanatomical study identifies the transition between the first and the second postnatal week as a key temporal window for the maturation of tanycytes and ependymocytes with the progressive acquisition of their adult morphological and molecular features. Recent high-throughput molecular profiling of the developing mouse hypothalamus using single-cell RNA-seq technology 47 has provided a database that will enable to explore further the molecular determinants of 3 V ependymal cell development, and how it relates to that of capillaries and neurons with which tanycytes morphologically and functionally interact.