# 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.

Adrien Coutteau-Robles, Vincent Prevot, Ariane Sharif

<figure><img src="image_1.png" /></figure>

## To cite this version:

Adrien Coutteau-Robles, Vincent Prevot, Ariane Sharif. 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. Journal of Neuroendocrinology, 2023, Journal of Neuroendocrinology, 35 (3), pp.e13239. 10.1111/jne.13239. hal-04613818

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ORIGINAL ARTICLE

Journal of Neuroendocrinology

WILEY

# 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

Adrian Coutteau-Robles

Vincent Prevot

Ariane Sharif

CHU Lille, Laboratory of Development and Plasticity of the Neuroendocrine Brain, Lille Neuroscience & Cognition, University of Lille, Inserm, Lille, France

## Correspondence

Ariane Sharif, University of Lille, Inserm, CHU Lille, Laboratory of Development and Plasticity of the Neuroendocrine Brain, Lille Neuroscience & Cognition, UMR-S 1172, Bâtiment Biserte, Place de Verdun, 59045, Lille, France.

Email: ariane.sharif@inserm.fr

## Funding information

Association pour l'Etude des Anomalies Congénitales; European Research Council ERC; Institut National de la Santé et de la Recherche Médicale; University of Lille

## Abstract

The third ventricle (3 V) wall of the tuberal hypothalamus is composed of two types of cells; specialized ependymoglia 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 tanicytic 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.

## KEYWORDS

hypothalamus, postnatal, proliferation, tanycytes, third ventricle

# 1 | INTRODUCTION

The third ventricle (3 V) presents a complex interface between the cerebrospinal fluid (CSF) and the surrounding hypothalamic parenchyma. In the tuberal region, its wall is made of one cell layer

composed of two types of cells: cuboidal ciliated ependymocytes, as found elsewhere along cerebral ventricular borders, in its dorsal part, and specialized cells called tanycytes in its ventral part.¹

Tanycytes are described as ependymoglia cells given their dual phenotype. On the one hand, they are organized as a simple

This is an open access article under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

© 2023 The Authors. *Journal of Neuroendocrinology* published by John Wiley & Sons Ltd on behalf of British Society for Neuroendocrinology.
ORIGINAL ARTICLE

Journal of Neuroendocrinology

WILEY

# 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

Adrian Coutteau-Robles

Vincent Prevot

Ariane Sharif

CHU Lille, Laboratory of Development and Plasticity of the Neuroendocrine Brain, Lille Neuroscience & Cognition, University of Lille, Inserm, Lille, France

## Correspondence

Ariane Sharif, University of Lille, Inserm, CHU Lille, Laboratory of Development and Plasticity of the Neuroendocrine Brain, Lille Neuroscience & Cognition, UMR-S 1172, Bâtiment Biserte, Place de Verdun, 59045, Lille, France.

Email: ariane.sharif@inserm.fr

## Funding information

Association pour l'Etude des Anomalies Congénitales; European Research Council ERC; Institut National de la Santé et de la Recherche Médicale; University of Lille

## Abstract

The third ventricle (3 V) wall of the tuberal hypothalamus is composed of two types of cells; specialized ependymoglia 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 tanicytic 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.

## KEYWORDS

hypothalamus, postnatal, proliferation, tanycytes, third ventricle

# 1 | INTRODUCTION

The third ventricle (3 V) presents a complex interface between the cerebrospinal fluid (CSF) and the surrounding hypothalamic parenchyma. In the tuberal region, its wall is made of one cell layer

composed of two types of cells: cuboidal ciliated ependymocytes, as found elsewhere along cerebral ventricular borders, in its dorsal part, and specialized cells called tanycytes in its ventral part.¹

Tanycytes are described as ependymoglia cells given their dual phenotype. On the one hand, they are organized as a simple

This is an open access article under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

© 2023 The Authors. *Journal of Neuroendocrinology* published by John Wiley & Sons Ltd on behalf of British Society for Neuroendocrinology.
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 S100,³⁻⁵ the gap junction protein connexin-43 (Cx43),⁶⁻⁸ the glutamate/aspartate transporter GLAST,²,⁹ glutamine synthetase¹⁰ and the water channel aquaporin-9.¹¹ Tanocytes 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 tanocytes 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 in¹³). By dynamically controlling the secretion of the neurohormones gonadotropin-releasing hormone¹⁴ and thyrotropin-releasing hormone¹⁵ into the portal vasculature, they determine the output of the hypothalamic-pituitary-gonadal and hypothalamic-pituitary-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 status¹⁶ 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.⁴,²¹⁻²⁵ The broad spectrum of tanocyte functions is underpinned not only by the great versatility of these cells but also by their heterogeneity, leading to the recognition of different tanocyte subpopulations.¹³

While we are making rapid progress in understanding the molecular and functional properties of adult tanocytes, our knowledge of their ontogenesis remains very incomplete. Early studies performed in rats showed that tanocyte 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. Tanocytes are derived from sonic hedgehog-expressing progenitors.⁵,²⁷ Expression of *Lhx2* and its downstream target *Rax* have been shown to be essential for tanocyte specification and differentiation.²⁸,²⁹ Molecular marker analyses showed that radial glial cells convert into tanocytes 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 tanocyte and ependymocyte markers (vimentin, S100, Cx43, and GFAP) along the 3 V wall in the mouse tuberal region of the hypothalamus.

## 2 | MATERIALS AND METHODS

### 2.1 | Animals

Experiments were performed on postnatal C57BI/6J male mice (Janvier Labs, Le Genest-Saint-Isle, France; RRID:IMSR_JAX:000664). The mothers and their pups were maintained at 23-25°C on a 12-h light-dark cycle (lights on at 7:00 a.m.), with chow diet (SAFE A03, Scientific Diets) and water available ad libitum. Animal studies were approved by the Institutional Ethics Committee for the Care and Use of Experimental Animals of the University of Lille (APAFIS# 29172-2020212811279767 v5). All experiments were performed in accordance with the guidelines for animal use specified by the European Union Council Directive of September 22, 2010 (2010/63/EU).

### 2.2 | 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 hippocampus³⁰ and postnatal rat hypothalamus.³¹

### 2.3 | 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.

### 2.4 | 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
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 S100,³⁻⁵ the gap junction protein connexin-43 (Cx43),⁶⁻⁸ the glutamate/aspartate transporter GLAST,²,⁹ glutamine synthetase¹⁰ and the water channel aquaporin-9.¹¹ Tanocytes 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 tanocytes 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 in¹³). By dynamically controlling the secretion of the neurohormones gonadotropin-releasing hormone¹⁴ and thyrotropin-releasing hormone¹⁵ into the portal vasculature, they determine the output of the hypothalamic-pituitary-gonadal and hypothalamic-pituitary-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 status¹⁶ 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.⁴,²¹⁻²⁵ The broad spectrum of tanocyte functions is underpinned not only by the great versatility of these cells but also by their heterogeneity, leading to the recognition of different tanocyte subpopulations.¹³

While we are making rapid progress in understanding the molecular and functional properties of adult tanocytes, our knowledge of their ontogenesis remains very incomplete. Early studies performed in rats showed that tanocyte 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. Tanocytes are derived from sonic hedgehog-expressing progenitors.⁵,²⁷ Expression of *Lhx2* and its downstream target *Rax* have been shown to be essential for tanocyte specification and differentiation.²⁸,²⁹ Molecular marker analyses showed that radial glial cells convert into tanocytes 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 tanocyte and ependymocyte markers (vimentin, S100, Cx43, and GFAP) along the 3 V wall in the mouse tuberal region of the hypothalamus.

## 2 | MATERIALS AND METHODS

### 2.1 | Animals

Experiments were performed on postnatal C57Bl/6J male mice (Janvier Labs, Le Genest-Saint-Isle, France; RRID:IMSR_JAX:000664). The mothers and their pups were maintained at 23-25°C on a 12-h light-dark cycle (lights on at 7:00 a.m.), with chow diet (SAFE A03, Scientific Diets) and water available ad libitum. Animal studies were approved by the Institutional Ethics Committee for the Care and Use of Experimental Animals of the University of Lille (APAFIS# 29172-2020212811279767 v5). All experiments were performed in accordance with the guidelines for animal use specified by the European Union Council Directive of September 22, 2010 (2010/63/EU).

### 2.2 | 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 hippocampus³⁰ and postnatal rat hypothalamus.³¹

### 2.3 | 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.

### 2.4 | 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
<table><caption>TABLE 1 Primary antibodies used in the study</caption><thead><tr><th>Antigen</th><th>Host & Clonality</th><th>Dilution</th><th>Reference (company) RRID number</th><th>Specificity</th></tr></thead><tbody><tr><td>BrdU</td><td>Rat monoclonal</td><td>1/500</td><td>ab6326 (Abcam)<br>RRID:AB_305426</td><td>31</td></tr><tr><td>Ki67</td><td>Rabbit polyclonal</td><td>1/500</td><td>ab15580 (Abcam)<br>RRID:AB_443209</td><td>2</td></tr><tr><td>Vimentin</td><td>Chicken polyclonal</td><td>1/2000</td><td>AB5733 (Millipore)<br>RRID:AB_11212377</td><td>2</td></tr><tr><td>Vimentin</td><td>Chicken polyclonal</td><td>1/2000</td><td>919,101, clone Poly29191 (BioLegends)<br>RRID:AB_2565208</td><td>BioLegends</td></tr><tr><td>GFAP</td><td>Rabbit polyclonal</td><td>1/500</td><td>Z0334 (Agilent)<br>RRID:AB_10013382</td><td>2</td></tr><tr><td>GFAP</td><td>Mouse monoclonal</td><td>1/200</td><td>14-9892-82,<br>Clone GA5, (Thermo Fisher Scientific)<br>RRID:AB_10598206</td><td>Thermo Fisher Scientific</td></tr><tr><td>S100</td><td>Rabbit polyclonal</td><td>Ready-to-use</td><td>IR504 (Dako/Agilent)</td><td>31</td></tr><tr><td>Connexin-43</td><td>Rabbit polyclonal</td><td>1/200</td><td>AB1727 (Millipore)<br>RRID:AB_2294609</td><td>6</td></tr></tbody></table>

<table><caption>TABLE 2 Secondary antibodies used in the study</caption><thead><tr><th>Species</th><th>Conjugated</th><th>Dilution</th><th>Reference (company) RRID number</th></tr></thead><tbody><tr><td>Donkey anti-rat IgG</td><td>Alexa 488</td><td>1/500</td><td>A-21208 (Molecular Probes) RRID:AB_141709</td></tr><tr><td>Donkey anti-mouse IgG</td><td>Alexa 647</td><td>1/500</td><td>A-31571 (Molecular Probes) RRID:AB_162542</td></tr><tr><td>Donkey anti-rabbit IgG</td><td>Alexa 568</td><td>1/500</td><td>A10042 (Thermo Fisher Scientific) RRID:AB_2534017</td></tr><tr><td>Donkey anti-rabbit IgG</td><td>Alexa 647</td><td>1/500</td><td>A-31573 (Molecular Probes) RRID:AB_2536183</td></tr><tr><td>Donkey anti-chicken IgG</td><td>Alexa 594</td><td>1/500</td><td>703-585-155 (Jackson ImmunoResearch Labs)<br>RRID:AB_2340377</td></tr><tr><td>Donkey anti-chicken IgG</td><td>Alexa 647</td><td>1/500</td><td>703-605-155 (Jackson ImmunoResearch Labs)<br>RRID:AB_2340379</td></tr></tbody></table>

<figure><img src="image_1.png" /><figcaption>FIGURE 1 Schematic representation of the third ventricle and adjacent nuclei at different rostrocaudal levels. Zone 1 (A, B) corresponds to the most anterior region, before the median eminence; in zone 2 (C), the ventricle is lined by well-developed ARH, VMH, and DMH with the ME at its base; in zone 3 (D, E), the VMH progressively disappears while the ventricle dives ventrally as the pituitary stalk develops; zone 4 (F, G) corresponds to the ending of the ME and progressive reduction in the size of the 3 V that adopts a diamond shape. 3 V, third ventricle; ARH, arcuate nucleus of the hypothalamus; dm, dorsomedial part of the arcuate nucleus of the hypothalamus; DMH, dorsomedial nucleus of the hypothalamus; DTM, dorsal tuberomammillary nucleus; ME, median eminence; PH, posterior hypothalamic area; vm, ventromedial part of the arcuate nucleus of the hypothalamus; VMH, ventromedial nucleus of the hypothalamus.</figcaption></figure>

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. The two anti-vimentin antibodies yielded a similar expression profile, as the
<table><caption>TABLE 1 Primary antibodies used in the study</caption><thead><tr><th>Antigen</th><th>Host & Clonality</th><th>Dilution</th><th>Reference (company) RRID number</th><th>Specificity</th></tr></thead><tbody><tr><td>BrdU</td><td>Rat monoclonal</td><td>1/500</td><td>ab6326 (Abcam)<br>RRID:AB_305426</td><td>31</td></tr><tr><td>Ki67</td><td>Rabbit polyclonal</td><td>1/500</td><td>ab15580 (Abcam)<br>RRID:AB_443209</td><td>2</td></tr><tr><td>Vimentin</td><td>Chicken polyclonal</td><td>1/2000</td><td>AB5733 (Millipore)<br>RRID:AB_11212377</td><td>2</td></tr><tr><td>Vimentin</td><td>Chicken polyclonal</td><td>1/2000</td><td>919,101, clone Poly29191 (BioLegends)<br>RRID:AB_2565208</td><td>BioLegends</td></tr><tr><td>GFAP</td><td>Rabbit polyclonal</td><td>1/500</td><td>Z0334 (Agilent)<br>RRID:AB_10013382</td><td>2</td></tr><tr><td>GFAP</td><td>Mouse monoclonal</td><td>1/200</td><td>14-9892-82,<br>Clone GA5, (Thermo Fisher Scientific)<br>RRID:AB_10598206</td><td>Thermo Fisher Scientific</td></tr><tr><td>S100</td><td>Rabbit polyclonal</td><td>Ready-to-use</td><td>IR504 (Dako/Agilent)</td><td>31</td></tr><tr><td>Connexin-43</td><td>Rabbit polyclonal</td><td>1/200</td><td>AB1727 (Millipore)<br>RRID:AB_2294609</td><td>6</td></tr></tbody></table>

<table><caption>TABLE 2 Secondary antibodies used in the study</caption><thead><tr><th>Species</th><th>Conjugated</th><th>Dilution</th><th>Reference (company) RRID number</th></tr></thead><tbody><tr><td>Donkey anti-rat IgG</td><td>Alexa 488</td><td>1/500</td><td>A-21208 (Molecular Probes) RRID:AB_141709</td></tr><tr><td>Donkey anti-mouse IgG</td><td>Alexa 647</td><td>1/500</td><td>A-31571 (Molecular Probes) RRID:AB_162542</td></tr><tr><td>Donkey anti-rabbit IgG</td><td>Alexa 568</td><td>1/500</td><td>A10042 (Thermo Fisher Scientific) RRID:AB_2534017</td></tr><tr><td>Donkey anti-rabbit IgG</td><td>Alexa 647</td><td>1/500</td><td>A-31573 (Molecular Probes) RRID:AB_2536183</td></tr><tr><td>Donkey anti-chicken IgG</td><td>Alexa 594</td><td>1/500</td><td>703-585-155 (Jackson ImmunoResearch Labs)<br>RRID:AB_2340377</td></tr><tr><td>Donkey anti-chicken IgG</td><td>Alexa 647</td><td>1/500</td><td>703-605-155 (Jackson ImmunoResearch Labs)<br>RRID:AB_2340379</td></tr></tbody></table>

<figure><img src="image_3.png" /><figcaption>FIGURE 1 Schematic representation of the third ventricle and adjacent nuclei at different rostrocaudal levels. Zone 1 (A, B) corresponds to the most anterior region, before the median eminence; in zone 2 (C), the ventricle is lined by well-developed ARH, VMH, and DMH with the ME at its base; in zone 3 (D, E), the VMH progressively disappears while the ventricle dives ventrally as the pituitary stalk develops; zone 4 (F, G) corresponds to the ending of the ME and progressive reduction in the size of the 3 V that adopts a diamond shape. 3 V, third ventricle; ARH, arcuate nucleus of the hypothalamus; dm, dorsomedial part of the arcuate nucleus of the hypothalamus; DMH, dorsomedial nucleus of the hypothalamus; DTM, dorsal tuberomammillary nucleus; ME, median eminence; PH, posterior hypothalamic area; vm, ventromedial part of the arcuate nucleus of the hypothalamus; VMH, ventromedial nucleus of the hypothalamus.</figcaption></figure>

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. The two anti-vimentin antibodies yielded a similar expression profile, as the
**FIGURE 2** Expression of vimentin in the postnatal tuberal region. Images are low-magnification 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 vimentin-immunoreactive (white) radial processes corresponding to radial glia/tanycytes, and white arrowheads show ependymocytes. Empty arrows indicate the part of the 3 V wall where vimentin immunoreactivity is low. Insets in (B), (C), and (N) are high-magnification views of the boxed areas. Scale bars = 20 µm in insets; 100 µm in all other panels.

<figure><img src="image_2.png" /></figure>
**FIGURE 2** Expression of vimentin in the postnatal tuberal region. Images are low-magnification 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 vimentin-immunoreactive (white) radial processes corresponding to radial glia/tanycytes, and white arrowheads show ependymocytes. Empty arrows indicate the part of the 3 V wall where vimentin immunoreactivity is low. Insets in (B), (C), and (N) are high-magnification views of the boxed areas. Scale bars = 20 µm in insets; 100 µm in all other panels.

<figure><img src="image_4.png" /></figure>
<figure><img src="image_3.png" /><figcaption>**TABLE 3** Summary of the expression profile of vimentin, S100, connexin-43, and GFAP in the postnatal 3 V wall at different rostrocaudal levels</figcaption></figure>

Note: (A) Distribution of radial cells and ependymocytes along the 3 V wall as determined by the pattern of vimentin immunoreactivity. Radial cells correspond to radial glia or tanycytes. The color code indicates absence/presence but does not refer to the relative abundance of cells. (B-D) Expression pattern of S100 (B), connexin-43 (C), and GFAP (D) in the 3 V wall. The color code represents a semi-quantitative scoring of the expression level based on the intensity of the immunoreactive signal (B, C) and the number of immunoreactive cells (D). Boxes marked with a cross mean that the nucleus/area does not exist at this level. Note that in zone 4b, the roof of the 3 V wall is lined by the PH and/or dorsal tuberomammillary nucleus (DTM) (Figure 1) but since no staining showed a distinctive expression profile between these two nuclei, we collectively refer to the roof of the 3 V as the PH for simplification reasons.

Abbreviations: dmARH, dorsomedial arcuate nucleus of the hypothalamus; DMH, dorsomedial nucleus of the hypothalamus; ME, median eminence; PH, posterior hypothalamic area; vmARH, ventromedial arcuate nucleus of the hypothalamus; VMH, ventromedial nucleus of the hypothalamus.

two anti-GFAP antibodies. After rinsing in PBS, secondary antibodies diluted in PBSTS were deposited on sections and incubated for 1.5 h at RT. The characteristics of the secondary antibodies used are shown in Table 2. After washing in PBS, cell nuclei were stained with DAPI (D9542, Sigma) diluted at 1/5000 in PBS for 4 min, washed, and covered slipped in Mowiol mounting medium (20% Mowiol, 2.5% DABCO in Tris 0.2 M pH 8.5). Control sections were incubated in the absence of primary antibodies. The omission of the primary antibody resulted in no staining.

## 2.5 | Microscopy and analyses

Analysis of sections and acquisition of images was done using an Axio Imager Z2 Apo-Tome microscope equipped with a motorized stage (Zeiss, Germany) and an ORCA-Flash 4.0 V2 camera (Hamamatsu, Japan) driven by the Zen 2.3 (blue edition) imaging software (Zeiss; RRID:SCR_013672). Photomicrographs represent maximal intensity projections derived from ApoTome images collected using the z-stack module of the Zen imaging software. Images to be used for figures were pseudocolored, adjusted for brightness and contrast, and merged using Adobe Photoshop 22.1.0 (Adobe Systems, San Jose, CA; RRID:SCR_014199).

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 –1.3 to –1.6 mm, zone two from Bregma –1.6 to –1.8 mm, zone 3 from Bregma –1.8 to –2.1 mm, and zone 4 from Bregma –2.1 to –2.5 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 faces¹³: 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.

## 2.6 | Statistical analysis

All analyses were performed using the GraphPad Prism 9.2.0 software (GraphPad; RRID:SCR_002798). Quantification of the total number of BrdU⁺ cells in the 3 V wall at different postnatal ages was analyzed using the Kruskal-Wallis one-way analysis of variance (ANOVA) on ranks test followed by the Dunn's multiple comparison test because of non-normally distributed data according to a Shapiro-Wilk normality test. The distribution of BrdU⁺ cells in the 3 V wall along the anteroposterior or ventrodorsal axis at different postnatal ages was
<figure><img src="image_5.png" /><figcaption>**TABLE 3** Summary of the expression profile of vimentin, S100, connexin-43, and GFAP in the postnatal 3 V wall at different rostrocaudal levels</figcaption></figure>

Note: (A) Distribution of radial cells and ependymocytes along the 3 V wall as determined by the pattern of vimentin immunoreactivity. Radial cells correspond to radial glia or tanycytes. The color code indicates absence/presence but does not refer to the relative abundance of cells. (B-D) Expression pattern of S100 (B), connexin-43 (C), and GFAP (D) in the 3 V wall. The color code represents a semi-quantitative scoring of the expression level based on the intensity of the immunoreactive signal (B, C) and the number of immunoreactive cells (D). Boxes marked with a cross mean that the nucleus/area does not exist at this level. Note that in zone 4b, the roof of the 3 V wall is lined by the PH and/or dorsal tuberomammillary nucleus (DTM) (Figure 1) but since no staining showed a distinctive expression profile between these two nuclei, we collectively refer to the roof of the 3 V as the PH for simplification reasons.

Abbreviations: dmARH, dorsomedial arcuate nucleus of the hypothalamus; DMH, dorsomedial nucleus of the hypothalamus; ME, median eminence; PH, posterior hypothalamic area; vmARH, ventromedial arcuate nucleus of the hypothalamus; VMH, ventromedial nucleus of the hypothalamus.

two anti-GFAP antibodies. After rinsing in PBS, secondary antibodies diluted in PBSTS were deposited on sections and incubated for 1.5 h at RT. The characteristics of the secondary antibodies used are shown in Table 2. After washing in PBS, cell nuclei were stained with DAPI (D9542, Sigma) diluted at 1/5000 in PBS for 4 min, washed, and covered slipped in Mowiol mounting medium (20% Mowiol, 2.5% DABCO in Tris 0.2 M pH 8.5). Control sections were incubated in the absence of primary antibodies. The omission of the primary antibody resulted in no staining.

## 2.5 | Microscopy and analyses

Analysis of sections and acquisition of images was done using an Axio Imager Z2 Apo-Tome microscope equipped with a motorized stage (Zeiss, Germany) and an ORCA-Flash 4.0 V2 camera (Hamamatsu, Japan) driven by the Zen 2.3 (blue edition) imaging software (Zeiss; RRID:SCR_013672). Photomicrographs represent maximal intensity projections derived from ApoTome images collected using the z-stack module of the Zen imaging software. Images to be used for figures were pseudocolored, adjusted for brightness and contrast, and merged using Adobe Photoshop 22.1.0 (Adobe Systems, San Jose, CA; RRID:SCR_014199).

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 -1.3 to -1.6 mm, zone two from Bregma -1.6 to -1.8 mm, zone 3 from Bregma -1.8 to -2.1 mm, and zone 4 from Bregma -2.1 to -2.5 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 faces¹³: 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.

## 2.6 | Statistical analysis

All analyses were performed using the GraphPad Prism 9.2.0 software (GraphPad; RRID:SCR_002798). Quantification of the total number of BrdU⁺ cells in the 3 V wall at different postnatal ages was analyzed using the Kruskal-Wallis one-way analysis of variance (ANOVA) on ranks test followed by the Dunn's multiple comparison test because of non-normally distributed data according to a Shapiro-Wilk normality test. The distribution of BrdU⁺ cells in the 3 V wall along the anteroposterior or ventrodorsal axis at different postnatal ages was
<figure><img src="image_4.png" /><figcaption>**FIGURE 3** Expression of S100 in the postnatal tuberal region. Images are low-magnification 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.</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>**FIGURE 3** Expression of S100 in the postnatal tuberal region. Images are low-magnification 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.</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>FIGURE 4 Legend on next page.</figcaption></figure>
<figure><img src="image_7.png" /><figcaption>FIGURE 4 Legend on next page.</figcaption></figure>
analyzed using the two-way ANOVA with Tukey's multiple comparisons test. *p* < .05 was considered statistically significant.

## 3 | 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 done¹² and into five areas along the ventrodorsal axis, according to the adjacent parenchymal nucleus¹³ (Figure 1).

### 3.1 | Distribution of radial glia/tanycytes and ependymocytes in the 3 V wall

We used vimentin, an intermediate filament expressed in all 3 V wall cells¹ to distinguish radial glia/tanycytes from ependymocytes. Radial glia and tanycytes were identified by their morphology, characterized by an elongated cell body with a long basal process running into the parenchyma while ependymocytes had a cuboidal cell body and a stronger vimentin immunoreactivity circling their cell body (Figure 2). At P0, most of the 3 V wall was lined by vimentin-expressing radial cells (Figure 2A-D). The dorsal part of the 3 V was characterized by lower vimentin immunoreactivity. Ependymocytes were visible in the upper third of the 3 V wall with an increasing number from the anterior-to-posterior regions. These cells still carried a process running into the parenchyma (inset in Figure 2B,C). In the most posterior region, when the 3 V was divided into different segments in the ventrodorsal axis, the ventral, diamond-shaped, part was mostly lined by radial cells while more dorsal parts were only lined by ependymocytes (Figure 2D). At P4, a similar profile was seen except that in zones 2 and 3, the entire dorsal part of the 3 V showed intense vimentin immunoreactivity with process-bearing ependymocytes (Figure 2E-H). At P10, the ventral portion of the 3 V was mostly lined by radial cells while its dorsal part was mostly lined by cuboidal cells with strong vimentin immunoreactivity (Figure 2I-L). These two cell populations overlapped to an extent that varied according to the anteroposterior axis. Cuboidal cells were seen from the roof of the 3 V until the dmARH in zone 1, while their ventral limit progressed dorsally towards more posterior regions. Conversely, radial cells were seen from the floor of the 3 V until the DMH with a decreasing density along the ventral-to-dorsal axis. In zones 3 and 4, most of the 3 V wall was lined by radial cells while ependymocytes were confined to the roof of the ventricle (Figure 2K,L). At

P20, the vimentin immunoreactivity pattern was similar to that observed at P10 (Figure 2M-P) and to that described in adult mice.¹ At P10 and P20, ependymocytes had short to absent vimentin-immunoreactive processes (inset in Figure 2N). The relative distribution of radial cells and ependymocytes is summarized in Table 3A. 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.³⁻⁵ 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).

#### 3.2.2 | Connexin-43

Expression of Cx43 was detected all along the 3 V wall at all ages examined (Figure 4). At P0, the immunoreactive signal was strongest in the middle part of the 3 V wall in anterior regions, i.e. zone 1 to 3 (Figure 4A-C) while the signal was low in zone 4 (Figure 4D). At P4 (Figure 4E-H) and P10 (Figure 4I-L), the territory of strongly immunoreactive cells extended along the ventrodorsal and anteroposterior axis. At P20, the expression profile was similar to P10 (Figure 4M-P) and comparable to that previously described in adult mice.⁶⁻⁸ Lower expression levels were consistently observed in cells bordering the ME compared to the rest of the 3 V at all ages examined. The distribution and intensity of Cx43 immunoreactivity in the 3 V wall are summarized in Table 3C. At P0 and P4, the immunolabeling was mostly confined to the apical

**FIGURE 4** Expression of connexin-43 in the postnatal tuberal region. (A-P) Images are low-magnification 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 to 4). White arrows point to Cx43-strongly immunoreactive (white) cells in the 3 V wall and white arrowheads show 3 V wall cells with less intense Cx43 immunoreactivity. Scale bar = 100 µm in all panels. (B₁, B₂, F₁, F₂, J₁, O₁) are high-magnification views of the boxed area indicated in the corresponding low-magnification views. The right panels show an overlay between Cx43 immunoreactivity (white) and nuclei counterstaining (DAPI, blue). White arrows point to Cx43-immunoreactive cells in the 3 V wall, white arrowheads show Cx43 immunosignal in the parenchyma, and yellow asterisks indicate capillaries. Scale bar = 100 µm in all panels. ARH, arcuate nucleus of the hypothalamus; ME, median eminence; VMH, ventromedial nucleus of the hypothalamus.
analyzed using the two-way ANOVA with Tukey's multiple comparisons test. *p* < .05 was considered statistically significant.

## 3 | 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 done¹² and into five areas along the ventrodorsal axis, according to the adjacent parenchymal nucleus¹³ (Figure 1).

### 3.1 | Distribution of radial glia/tanycytes and ependymocytes in the 3 V wall

We used vimentin, an intermediate filament expressed in all 3 V wall cells¹ to distinguish radial glia/tanycytes from ependymocytes. Radial glia and tanycytes were identified by their morphology, characterized by an elongated cell body with a long basal process running into the parenchyma while ependymocytes had a cuboidal cell body and a stronger vimentin immunoreactivity circling their cell body (Figure 2). At P0, most of the 3 V wall was lined by vimentin-expressing radial cells (Figure 2A-D). The dorsal part of the 3 V was characterized by lower vimentin immunoreactivity. Ependymocytes were visible in the upper third of the 3 V wall with an increasing number from the anterior-to-posterior regions. These cells still carried a process running into the parenchyma (inset in Figure 2B,C). In the most posterior region, when the 3 V was divided into different segments in the ventrodorsal axis, the ventral, diamond-shaped, part was mostly lined by radial cells while more dorsal parts were only lined by ependymocytes (Figure 2D). At P4, a similar profile was seen except that in zones 2 and 3, the entire dorsal part of the 3 V showed intense vimentin immunoreactivity with process-bearing ependymocytes (Figure 2E-H). At P10, the ventral portion of the 3 V was mostly lined by radial cells while its dorsal part was mostly lined by cuboidal cells with strong vimentin immunoreactivity (Figure 2I-L). These two cell populations overlapped to an extent that varied according to the anteroposterior axis. Cuboidal cells were seen from the roof of the 3 V until the dmARH in zone 1, while their ventral limit pro-gressed dorsally towards more posterior regions. Conversely, radial cells were seen from the floor of the 3 V until the DMH with a decreasing density along the ventral-to-dorsal axis. In zones 3 and 4, most of the 3 V wall was lined by radial cells while ependymo-cytes were confined to the roof of the ventricle (Figure 2K,L). At

P20, the vimentin immunoreactivity pattern was similar to that observed at P10 (Figure 2M-P) and to that described in adult mice.¹ At P10 and P20, ependymocytes had short to absent vimentin-immunoreactive processes (inset in Figure 2N). The relative distribution of radial cells and ependymocytes is summarized in Table 3A. 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.³⁻⁵ 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).

#### 3.2.2 | Connexin-43

Expression of Cx43 was detected all along the 3 V wall at all ages examined (Figure 4). At P0, the immunoreactive signal was strongest in the middle part of the 3 V wall in anterior regions, i.e. zone 1 to 3 (Figure 4A-C) while the signal was low in zone 4 (Figure 4D). At P4 (Figure 4E-H) and P10 (Figure 4I-L), the territory of strongly immunoreactive cells extended along the ventrodorsal and anteroposterior axis. At P20, the expression profile was similar to P10 (Figure 4M-P) and comparable to that previously described in adult mice.⁶⁻⁸ Lower expression levels were consistently observed in cells bordering the ME compared to the rest of the 3 V at all ages examined. The distribution and intensity of Cx43 immunoreactivity in the 3 V wall are summarized in Table 3C. At P0 and P4, the immunolabeling was mostly confined to the apical

**FIGURE 4** Expression of connexin-43 in the postnatal tuberal region. (A-P) Images are low-magnification 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 to 4). White arrows point to Cx43-strongly immunoreactive (white) cells in the 3 V wall and white arrowheads show 3 V wall cells with less intense Cx43 immunoreactivity. Scale bar = 100 µm in all panels. (B₁, B₂, F₁, F₂, J₁, O₁) are high-magnification views of the boxed area indicated in the corresponding low-magnification views. The right panels show an overlay between Cx43 immunoreactivity (white) and nuclei counterstaining (DAPI, blue). White arrows point to Cx43-immunoreactive cells in the 3 V wall, white arrowheads show Cx43 immunosignal in the parenchyma, and yellow asterisks indicate capillaries. Scale bar = 100 µm in all panels. ARH, arcuate nucleus of the hypothalamus; ME, median eminence; VMH, ventromedial nucleus of the hypothalamus.
**FIGURE 5** Expression of GFAP in the postnatal tuberal region. (A–P) Images are low-magnification 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) showing GFAP immunoreactivity (white) and nuclei counterstaining (DAPI, blue). White arrows point to GFAP-strongly immunoreactive cells in the 3 V wall, white arrowheads show 3 V wall areas with less intense GFAP immunoreactivity and empty arrowheads point to 3 V wall areas devoid of GFAP immunolabeling. Yellow arrows indicate GFAP-immunoreactive cells in the parenchyma. Inset in (I) is a high-magnification view of the astrocyte pointed by the yellow arrow in the main panel. Scale bars = 20 µm in the inset; 100 µm in all other panels.

<figure><img src="image_6.png" /></figure>
**FIGURE 5** Expression of GFAP in the postnatal tuberal region. (A–P) Images are low-magnification 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) showing GFAP immunoreactivity (white) and nuclei counterstaining (DAPI, blue). White arrows point to GFAP-strongly immunoreactive cells in the 3 V wall, white arrowheads show 3 V wall areas with less intense GFAP immunoreactivity and empty arrowheads point to 3 V wall areas devoid of GFAP immunolabeling. Yellow arrows indicate GFAP-immunoreactive cells in the parenchyma. Inset in (I) is a high-magnification view of the astrocyte pointed by the yellow arrow in the main panel. Scale bars = 20 µm in the inset; 100 µm in all other panels.

<figure><img src="image_8.png" /></figure>
<figure><img src="image_7.png" /><figcaption>**FIGURE 6** Evaluation of cell proliferation in the 3 V wall during the postnatal period. (A) Schematic of the experimental protocol. (B-D) Representative coronal section of the tuberal region (zone 2) of a P4 animal immunolabeled for BrdU (green) and Ki67 (magenta). Nuclei were counterstained with DAPI (blue). White arrows point to BrdU/Ki67 colabeled cells in the 3 V wall. Magenta arrows indicate Ki67⁺ cells devoid of BrdU labeling, corresponding to cells that have entered the cell cycle but have not yet reached the S-phase. Note that the thickness of the 3 V wall varies from one (asterisk in panel D₁) to several layers (double asterisk in panel C₁). Scale bar = 100 µm in (B); 50 µm in (C, D). ARH, arcuate nucleus of the hypothalamus; DMH, dorsomedial nucleus of the hypothalamus; i.p., intraperitoneal; ME, median eminence; VMH, ventromedial nucleus of the hypothalamus.</figcaption></figure>

pole of 3 V wall cell bodies. A punctuate immunosignal was also seen in the parenchyma following a pattern consistent with tanycyte processes and endfeet (Figure 4B₁, B₂, F₁, F₂). This parenchymal staining was highest at the level of the arcuate nucleus (Figure 4B₂, F₂). At P10 and P20, the extent of the immunosignal in 3 V wall cell bodies increased and was no more strictly confined to the apical pole. The punctuate parenchymal signal was most intense at the level of parenchymal capillaries and in the external zone of the ME, suggesting high Cx43 expression in tanycyte endfeet (Figure 4J₁, O₁).

### 3.2.3 | 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). GFAP-immunoreactive 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

### 3.3 | Proliferation

To evaluate the proliferative activity of cells bordering the 3 V wall, we used the thymidine analog bromodeoxyuridine (BrdU). Animals
<figure><img src="image_9.png" /><figcaption>**FIGURE 6** Evaluation of cell proliferation in the 3 V wall during the postnatal period. (A) Schematic of the experimental protocol. (B-D) Representative coronal section of the tuberal region (zone 2) of a P4 animal immunolabeled for BrdU (green) and Ki67 (magenta). Nuclei were counterstained with DAPI (blue). White arrows point to BrdU/Ki67 colabeled cells in the 3 V wall. Magenta arrows indicate Ki67⁺ cells devoid of BrdU labeling, corresponding to cells that have entered the cell cycle but have not yet reached the S-phase. Note that the thickness of the 3 V wall varies from one (asterisk in panel D₁) to several layers (double asterisk in panel C₁). Scale bar = 100 µm in (B); 50 µm in (C, D). ARH, arcuate nucleus of the hypothalamus; DMH, dorsomedial nucleus of the hypothalamus; i.p., intraperitoneal; ME, median eminence; VMH, ventromedial nucleus of the hypothalamus.</figcaption></figure>

pole of 3 V wall cell bodies. A punctuate immunosignal was also seen in the parenchyma following a pattern consistent with tanycyte processes and endfeet (Figure 4B₁, B₂, F₁, F₂). This parenchymal staining was highest at the level of the arcuate nucleus (Figure 4B₂, F₂). At P10 and P20, the extent of the immunosignal in 3 V wall cell bodies increased and was no more strictly confined to the apical pole. The punctuate parenchymal signal was most intense at the level of parenchymal capillaries and in the external zone of the ME, suggesting high Cx43 expression in tanycyte endfeet (Figure 4J₁, O₁).

### 3.2.3 | 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). GFAP-immunoreactive 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

### 3.3 | Proliferation

To evaluate the proliferative activity of cells bordering the 3 V wall, we used the thymidine analog bromodeoxyuridine (BrdU). Animals
<figure><img src="image_8.png" /><figcaption>**FIGURE 7** Legend on next page.</figcaption></figure>
<figure><img src="image_10.png" /><figcaption>**FIGURE 7** Legend on next page.</figcaption></figure>
received a single i.p. injection of BrdU at P0, P4, P10, or P20, and were euthanized 2 h later, in order to label cells that had entered the S-phase of the cell cycle during this time frame, without considering their subsequent amplification and/or survival (Figure 6A). A coimmunolabeling with Ki67, a nuclear protein expressed throughout all active phases of the cell cycle,³⁵ showed that most BrdU-expressing (BrdU⁺) cells expressed detectable levels of Ki67 (91.1 ± 4.5% of BrdU⁺ cells colabeled for Ki67; Figure 6B-D), confirming that BrdU⁺ cells were proliferating. The proliferative activity was highest during the first postnatal week and dropped at P10 to reach almost 0 at P20 (Figure 7A-Q). When looking at the anteroposterior distribution of proliferative cells, the highest proliferative activity was seen in the posterior region of the 3 V at P0 (Figure 7A-D,R), while no such difference was observed at P4 or P10 (Figure 7G-J,R). When looking at the ventrodorsal distribution of proliferative cells, the highest number of BrdU⁺ ventricular cells was seen in the 3 V wall portion facing the ARH and the DMH at P0, and in the 3 V wall portion facing the ARH, VMH, and DMH at P4. At P10, no significant difference was observed in the ventrodorsal distribution of BrdU⁺ cells along the 3 V wall (Figure 7A-P,S).

We determined whether BrdU⁺ cells were radial glia/tanycytes or ependymocytes by analyzing their vimentin immunoreactivity. At P0 and P4, all BrdU⁺ cells had a radial glia/tanocyte morphology (Figure 7E,F,K,L) while at P10, 13.6 ± 4% (n = 7 mice) of BrdU⁺ cells had an ependymocyte aspect (Figure 7M₂,O), the remaining corresponding to tanycytes (Figure 7M₁,N,P). Notably, at P20, only one BrdU⁺ cell was detected in all six animals analyzed, and it had an ependymocyte aspect (not shown).

Finally, we evaluated whether BrdU⁺ cells coexpressed the glial markers S100, Cx43, or GFAP (Figure 8). No strict correlation was found between the level of expression of any of these glial markers

and the absence or presence of BrdU⁺ cells. While the density of BrdU⁺ cells was highest at P0 in zone 4, in regions with low to undetectable levels of S100, Cx43, and GFAP (Figure 8B,G), BrdU⁺ cells coexpressing S100 (Figure 8A,C-E), Cx43 (Figure 8F,H-J) or GFAP (Figure 8C-E) were also detected.

## 4 | DISCUSSION

Using immunofluorescence, we provide here a detailed characterization of the proliferation and expression profile of a series of ependymal cell markers in the 3 V wall lining across the mouse postnatal development. Our results show that most major changes occur between the first and the second postnatal week with a drop in cell proliferation between P4 and P10, associated with marked modifications in the expression level and profile of vimentin, S100, Cx43, and GFAP that acquire mature features at P20.

The development of cells lining the 3 V is still poorly understood. 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 tanycytes¹³) associated to a wider distribution of Cx43 immunoreactivity in tanocyte cell bodies. This temporal window of maturation had previously been identified by others.

**FIGURE 7** Quantification and distribution of proliferative cells in the 3 V wall during the postnatal period. (A-D, G-J) Low-magnification views of the 3 V at P0 (A-D) and P4 (G-J) at different rostrocaudal levels (zone 1 to 4) showing BrdU immunoreactivity (green). White arrows point to BrdU⁺ cells in the 3 V wall. (E, F, K, L) show higher magnification views of BrdU (green)/vimentin (red) coimmunolabeling in zone 4 (E, F) and zone 2 (K, L). Note that BrdU⁺ cells (white arrows) have a vimentin expression pattern of radial glia/tanycytes but none exhibit the strong vimentin immunoreactivity pattern typical of ependymocytes (white arrowheads). (M-P) show examples of BrdU (green)/vimentin (red) coimmunolabeling at P10. (M₁ and M₂) are high-magnification views of the BrdU⁺ cells pointed by the arrows in panel (M) showing a labeled tanocyte (M₁) and a labeled ependymocyte with apical vimentin (M₂) in zone 2. (N) shows two BrdU⁺ tanycytes in the ME in zone 3, (O) shows a BrdU⁺ ependymocyte in the DMH in zone 2, and (P) shows a BrdU⁺ tanocyte in the DMH in zone 3. Nuclei were counterstained with DAPI (blue). Scale bars = 100 µm in (A-D, G-J, M), 50 µm in (E, F, K, L), and 20 µm in (M₁, M₂, O, P). (Q) Quantification of the total number of BrdU⁺ cells in the 3 V wall (Kruskal-Wallis one-way ANOVA on ranks: *H*(3) = 22.63, *p* < .0001, *n* = 6 to seven mice per group; Dunn's multiple-comparison test: P0 vs. P10, *p* = .0198; P0 vs. P20, *p* = .0001; P4 vs. P20, *p* = .0037). (R) Distribution of BrdU⁺ cells in the 3 V wall along the anteroposterior axis at P0, P4, and P10 (*n* = 7 to 8 mice per group; two-way ANOVA: age, *F*(2, 75) = 46.45, *p* < .0001; zone, *F*(3, 75) = 14.22, *p* < .0001; interaction, *F*(6, 75) = 11.02, *p* < .0001; Tukey's multiple-comparison test, P4 and P10: no significant differences between zones; zone 1: no significant differences between ages, zone 2: P4 vs. P10, *p* = .006, zone 3: P0 vs. P10, *p* = .0002, P4 vs. P10, *p* = .0468, zone 4: P0 vs. P4, *p* < .0001, P0 vs. P10, *p* < .0001, P4 vs. P10, *p* = .0032). (S) Distribution of BrdU⁺ cells in the 3 V wall along the ventrodorsal axis at P0, P4 and P10 (*n* = 7 mice per group; two-way ANOVA: age, *F*(2, 90) = 49.50, *p* < .0001; region, *F*(4, 90) = 27.68, *p* < .0001; interaction, *F*(8, 90) = 13.52, *p* < .0001; Tukey's multiple-comparison test, P0: ME vs. ARH, *p* < .0001, ME vs. DMH, *p* < .0001, ARH vs. VMH, *p* < .0001, ARH vs. PH, *p* < .0001, VMH vs. DMH, *p* < .0001, DMH vs. PH, *p* = .0004; P4: ME vs. ARH, *p* = .0332, ME vs. VMH, *p* = .0001, ME vs. DMH, *p* < .0001, ARH vs. PH, *p* = .0494, VMH vs. PH, *p* = .0002, DMH vs. PH, *p* < .0001; P10: no significant differences between regions; ME: no significant differences between ages; ARH: P0 vs. P4, *p* < .0001, P4 vs. P10, *p* = .0426; VMH: P4 vs. P10, *p* = .0006; DMH: P0 vs. P10 and P4 vs. P10, *p* < .0001; PH: no significant differences). The values are means ± s.e.m. \* *p* < .05; \*\* *p* < .01; \*\*\* *p* < .001. 3 V, third ventricle; ARH, arcuate nucleus of the hypothalamus; DMH, dorsomedial nucleus of the hypothalamus; ME, median eminence; PH, posterior hypothalamic area; VMH, ventromedial nucleus of the hypothalamus.
received a single i.p. injection of BrdU at P0, P4, P10, or P20, and were euthanized 2 h later, in order to label cells that had entered the S-phase of the cell cycle during this time frame, without considering their subsequent amplification and/or survival (Figure 6A). A coimmunolabeling with Ki67, a nuclear protein expressed throughout all active phases of the cell cycle,³⁵ showed that most BrdU-expressing (BrdU⁺) cells expressed detectable levels of Ki67 (91.1 ± 4.5% of BrdU⁺ cells colabeled for Ki67; Figure 6B-D), confirming that BrdU⁺ cells were proliferating. The proliferative activity was highest during the first postnatal week and dropped at P10 to reach almost 0 at P20 (Figure 7A-Q). When looking at the anteroposterior distribution of proliferative cells, the highest proliferative activity was seen in the posterior region of the 3 V at P0 (Figure 7A-D,R), while no such difference was observed at P4 or P10 (Figure 7G-J,R). When looking at the ventrodorsal distribution of proliferative cells, the highest number of BrdU⁺ ventricular cells was seen in the 3 V wall portion facing the ARH and the DMH at P0, and in the 3 V wall portion facing the ARH, VMH, and DMH at P4. At P10, no significant difference was observed in the ventrodorsal distribution of BrdU⁺ cells along the 3 V wall (Figure 7A-P,S).

We determined whether BrdU⁺ cells were radial glia/tanycytes or ependymocytes by analyzing their vimentin immunoreactivity. At P0 and P4, all BrdU⁺ cells had a radial glia/tanocyte morphology (Figure 7E,F,K,L) while at P10, 13.6 ± 4% (n = 7 mice) of BrdU⁺ cells had an ependymocyte aspect (Figure 7M₂,O), the remaining corresponding to tanycytes (Figure 7M₁,N,P). Notably, at P20, only one BrdU⁺ cell was detected in all six animals analyzed, and it had an ependymocyte aspect (not shown).

Finally, we evaluated whether BrdU⁺ cells coexpressed the glial markers S100, Cx43, or GFAP (Figure 8). No strict correlation was found between the level of expression of any of these glial markers

and the absence or presence of BrdU⁺ cells. While the density of BrdU⁺ cells was highest at P0 in zone 4, in regions with low to undetectable levels of S100, Cx43, and GFAP (Figure 8B,G), BrdU⁺ cells coexpressing S100 (Figure 8A,C-E), Cx43 (Figure 8F,H-J) or GFAP (Figure 8C-E) were also detected.

## 4 | DISCUSSION

Using immunofluorescence, we provide here a detailed characterization of the proliferation and expression profile of a series of ependymal cell markers in the 3 V wall lining across the mouse postnatal development. Our results show that most major changes occur between the first and the second postnatal week with a drop in cell proliferation between P4 and P10, associated with marked modifications in the expression level and profile of vimentin, S100, Cx43, and GFAP that acquire mature features at P20.

The development of cells lining the 3 V is still poorly understood. 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 tanycytes¹³) associated to a wider distribution of Cx43 immunoreactivity in tanocyte cell bodies. This temporal window of maturation had previously been identified by others.

**FIGURE 7** Quantification and distribution of proliferative cells in the 3 V wall during the postnatal period. (A–D, G–J) Low-magnification views of the 3 V at P0 (A–D) and P4 (G–J) at different rostrocaudal levels (zone 1 to 4) showing BrdU immunoreactivity (green). White arrows point to BrdU⁺ cells in the 3 V wall. (E, F, K, L) show higher magnification views of BrdU (green)/vimentin (red) coimmunolabeling in zone 4 (E, F) and zone 2 (K, L). Note that BrdU⁺ cells (white arrows) have a vimentin expression pattern of radial glia/tanycytes but none exhibit the strong vimentin immunoreactivity pattern typical of ependymocytes (white arrowheads). (M–P) show examples of BrdU (green)/vimentin (red) coimmunolabeling at P10. (M₁ and M₂) are high-magnification views of the BrdU⁺ cells pointed by the arrows in panel (M) showing a labeled tanocyte (M₁) and a labeled ependymocyte with apical vimentin (M₂) in zone 2. (N) shows two BrdU⁺ tanycytes in the ME in zone 3, (O) shows a BrdU⁺ ependymocyte in the DMH in zone 2, and (P) shows a BrdU⁺ tanocyte in the DMH in zone 3. Nuclei were counterstained with DAPI (blue). Scale bars = 100 µm in (A–D, G–J, M), 50 µm in (E, F, K, L), and 20 µm in (M₁, M₂, O, P). (Q) Quantification of the total number of BrdU⁺ cells in the 3 V wall (Kruskal–Wallis one-way ANOVA on ranks: *H*(3) = 22.63, *p* < .0001, *n* = 6 to seven mice per group; Dunn's multiple-comparison test: P0 vs. P10, *p* = .0198; P0 vs. P20, *p* = .0001; P4 vs. P20, *p* = .0037). (R) Distribution of BrdU⁺ cells in the 3 V wall along the anteroposterior axis at P0, P4, and P10 (*n* = 7 to 8 mice per group; two-way ANOVA: age, *F*(2, 75) = 46.45, *p* < .0001; zone, *F*(3, 75) = 14.22, *p* < .0001; interaction, *F*(6, 75) = 11.02, *p* < .0001; Tukey's multiple-comparison test, P4 and P10: no significant differences between zones; zone 1: no significant differences between ages, zone 2: P4 vs. P10, *p* = .006, zone 3: P0 vs. P10, *p* = .0002, P4 vs. P10, *p* = .0468, zone 4: P0 vs. P4, *p* < .0001, P0 vs. P10, *p* < .0001, P4 vs. P10, *p* = .0032). (S) Distribution of BrdU⁺ cells in the 3 V wall along the ventrodorsal axis at P0, P4 and P10 (*n* = 7 mice per group; two-way ANOVA: age, *F*(2, 90) = 49.50, *p* < .0001; region, *F*(4, 90) = 27.68, *p* < .0001; interaction, *F*(8, 90) = 13.52, *p* < .0001; Tukey's multiple-comparison test, P0: ME vs. ARH, *p* < .0001, ME vs. DMH, *p* < .0001, ARH vs. VMH, *p* < .0001, ARH vs. PH, *p* < .0001, VMH vs. DMH, *p* < .0001, DMH vs. PH, *p* = .0004; P4: ME vs. ARH, *p* = .0332, ME vs. VMH, *p* = .0001, ME vs. DMH, *p* < .0001, ARH vs. PH, *p* = .0494, VMH vs. PH, *p* = .0002, DMH vs. PH, *p* < .0001; P10: no significant differences between regions; ME: no significant differences between ages; ARH: P0 vs. P4, *p* < .0001, P4 vs. P10, *p* = .0426; VMH: P4 vs. P10, *p* = .0006; DMH: P0 vs. P10 and P4 vs. P10, *p* < .0001; PH: no significant differences). The values are means ± s.e.m. \* *p* < .05; \*\* *p* < .01; \*\*\* *p* < .001. 3 V, third ventricle; ARH, arcuate nucleus of the hypothalamus; DMH, dorsomedial nucleus of the hypothalamus; ME, median eminence; PH, posterior hypothalamic area; VMH, ventromedial nucleus of the hypothalamus.
<figure><img src="image_9.png" /><figcaption>FIGURE 8 Legend on next page.</figcaption></figure>
<figure><img src="image_11.png" /><figcaption>FIGURE 8 Legend on next page.</figcaption></figure>
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 ependymocytes. Cells with a characteristic high vimentin labeling circling their cell body were already visible at P0 in the upper third of the 3 V with a caudal-to-rostral gradient. Notably, the dorsal tip of the 3 V in zones 2 and 3 was characterized by low vimentin expression at P0, while this region acquired strong vimentin immunoreactivity in ependymocyte-like cells at P4, indicating ventral-to-dorsal maturation in the upper third of the 3 V wall. At P10, the ventricular domain containing ependymocytes expanded ventrally and rostrally. These cells still had long basal processes at P4, in line with their progressive transformation from radial glia, while 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 Moreover, cell proliferation dynamics differed according to the rostrocaudal and ventrodorsal axis. Indeed, proliferation levels were highest at P0 selectively in zone 4. Moreover, while proliferation in zone 2 to 4 was higher at P4 compared to P10, levels were not significantly different in zone 1. These observations suggest a rostral-to-caudal gradient in the generation of 3 V wall cells, with most cells in zone 1 being born before P0 while most cells in zone 4 are produced around P0. Interestingly, a rostral-to-caudal gradient is also seen in the expression of Cx43 and GFAP between P0 and P20, with expression levels that are lowest in posterior regions at P0 and that progressively rise to reach similar levels as anterior regions. Such a developmental time course is further supported by the anterior-to-posterior gradient of RC2 downregulation between P0 and P10.⁵ Analysis of cell proliferation along the ventrodorsal axis also revealed differences in the timing of the production of distinct tanycyte subpopulations. Proliferation levels were consistently low 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 ³H-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

**FIGURE 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. (A₁–A₄) shows a BrdU⁺/S100⁺/GFAP⁻ cell. (B₁–B₄) shows a BrdU⁺ cell that expresses low levels of S100 (arrow) and BrdU⁺ cells that do not express S100 (arrowheads). Both cells are devoid of GFAP immunosignal. (C–E) show examples of BrdU⁺/S100⁺/GFAP⁺ cells (C₁–C₄ cell #1, D₁–D₄ and E cell #2) and BrdU⁺/S100⁺/GFAP⁻ cells (C₁–C₄ cell #2 and E cell #1). (F–J) Coimmunolabeling of BrdU (green), Cx43 (red), and GFAP (white) in P0 (F, G), P4 (H), and P10 mice (I, J). Arrows point to BrdU⁺ cells in the 3 V wall. (F, H, I, J) show BrdU⁺/Cx43⁺ cells. Note that Cx43 immunoreactivity is mostly seen at the apical pole of tanycytes at P0 and P4 while it is more widely distributed in cell bodies at P10. The BrdU⁺ cell in (J) is located in the ME and expresses Cx43 at lower levels than more dorsally along the 3 V wall. (G) shows BrdU⁺ cells with low to undetectable levels of Cx43 in zone 4. None of the BrdU⁺ cells shown in (F–J) express GFAP. Nuclei were counterstained with DAPI (blue). Scale bars = 50 µm in (G), 20 µm in all other panels. 3 V, third ventricle; ME, median eminence.
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 ependymocytes. Cells with a characteristic high vimentin labeling circling their cell body were already visible at P0 in the upper third of the 3 V with a caudal-to-rostral gradient. Notably, the dorsal tip of the 3 V in zones 2 and 3 was characterized by low vimentin expression at P0, while this region acquired strong vimentin immunoreactivity in ependymocyte-like cells at P4, indicating ventral-to-dorsal maturation in the upper third of the 3 V wall. At P10, the ventricular domain containing ependymocytes expanded ventrally and rostrally. These cells still had long basal processes at P4, in line with their progressive transformation from radial glia, while 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 Moreover, cell proliferation dynamics differed according to the rostrocaudal and ventrodorsal axis. Indeed, proliferation levels were highest at P0 selectively in zone 4. Moreover, while proliferation in zone 2 to 4 was higher at P4 compared to P10, levels were not significantly different in zone 1. These observations suggest a rostral-to-caudal gradient in the generation of 3 V wall cells, with most cells in zone 1 being born before P0 while most cells in zone 4 are produced around P0. Interestingly, a rostral-to-caudal gradient is also seen in the expression of Cx43 and GFAP between P0 and P20, with expression levels that are lowest in posterior regions at P0 and that progressively rise to reach similar levels as anterior regions. Such a developmental time course is further supported by the anterior-to-posterior gradient of RC2 downregulation between P0 and P10.⁵ Analysis of cell proliferation along the ventrodorsal axis also revealed differences in the timing of the production of distinct tanycyte subpopulations. Proliferation levels were consistently low 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 ³H-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

**FIGURE 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. (A₁–A₄) shows a BrdU⁺/S100⁺/GFAP⁻ cell. (B₁–B₄) shows a BrdU⁺ cell that expresses low levels of S100 (arrow) and BrdU⁺ cells that do not express S100 (arrowheads). Both cells are devoid of GFAP immunosignal. (C–E) show examples of BrdU⁺/S100⁺/GFAP⁺ cells (C₁–C₄ cell #1, D₁–D₄ and E cell #2) and BrdU⁺/S100⁺/GFAP⁻ cells (C₁–C₄ cell #2 and E cell #1). (F–J) Coimmunolabeling of BrdU (green), Cx43 (red), and GFAP (white) in P0 (F, G), P4 (H), and P10 mice (I, J). Arrows point to BrdU⁺ cells in the 3 V wall. (F, H, I, J) show BrdU⁺/Cx43⁺ cells. Note that Cx43 immunoreactivity is mostly seen at the apical pole of tanycytes at P0 and P4 while it is more widely distributed in cell bodies at P10. The BrdU⁺ cell in (J) is located in the ME and expresses Cx43 at lower levels than more dorsally along the 3 V wall. (G) shows BrdU⁺ cells with low to undetectable levels of Cx43 in zone 4. None of the BrdU⁺ cells shown in (F–J) express GFAP. Nuclei were counterstained with DAPI (blue). Scale bars = 50 µm in (G), 20 µm in all other panels. 3 V, third ventricle; ME, median eminence.
expression of these markers and the proliferative activity. The molecular mechanisms regulating postnatal tanocyte proliferation have just started to be explored. Two recent genetic invalidation studies identified fibroblast growth factor 10 (Fgf10)⁴ and the nuclear factor I (NFI) family of transcription factors²⁵ as critical regulators of tanocyte proliferation and neurogenic competence during the postnatal period. 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 tanocytes remains to be explored.

A hallmark of tanocytes 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 tanocytes also correlates with the maturation of the interface between tanocyte 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 tanocytes, 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 tanocytes 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 technology⁴⁷ 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 tanocytes morphologically and functionally interact.

## AUTHOR CONTRIBUTIONS

**Adrian Coutteau-Robles:** Conceptualization; formal analysis; investigation; methodology; writing – review and editing. **Vincent Prevot:** Conceptualization; formal analysis; funding acquisition; investigation; methodology; writing – review and editing. **Ariane Sharif:** Conceptualization; formal analysis; funding acquisition; investigation; methodology; supervision; writing – original draft.

## ACKNOWLEDGMENTS

This work was supported by a doctoral fellowship from the University of Lille to A. C.-R., Inserm grant U1172, the Association pour l'Etude des Anomalies Congénitales (to A.S.), the European Research Council ERC-Synergy-Grant-2019-WATCH no. 810331 (to V.P.). We thank the Animal Core Facility of the University of Lille (PLBS UAR 2014-US41) for expert technical support.

## CONFLICT OF INTEREST

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

## PEER REVIEW

The peer review history for this article is available at https://publons.com/publon/10.1111/jne.13239.

## DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding authors upon request.

## ORCID

Vincent Prevot https://orcid.org/0000-0001-7185-3615

Ariane Sharif https://orcid.org/0000-0003-0810-400X

## REFERENCES

1. Mullier A, Bouret SG, Prevot V, Dehouck B. Differential distribution of tight junction proteins suggests a role for tanocytes in blood-hypothalamus barrier regulation in the adult mouse brain. *J Comp Neurol.* 2010;518(7):943-962. doi:10.1002/cne.22273

2. Pellegrino G, Trubert C, Terrien J, et al. A comparative study of the neural stem cell niche in the adult hypothalamus of human, mouse, rat and gray mouse lemur (Microcebus murinus). *J Comp Neurol.* 2018; 526(9):1419-1443. doi:10.1002/cne.24376

3. Goodman T, Hajihosseini MK. Hypothalamic tanocytes-masters and servants of metabolic, neuroendocrine, and neurogenic functions. *Front Neurosci.* 2015;9:387. doi:10.3389/fnins.2015.00387

4. Goodman T, Nayar SG, Clare S, et al. Fibroblast growth factor 10 is a negative regulator of postnatal neurogenesis in the mouse hypothalamus. *Development.* 2020;147(13):dev180950. doi:10.1242/dev.180950

5. Mirzadeh Z, Kusne Y, Duran-Moreno M, et al. Bi- and unencilitated ependymal cells define continuous floor-plate-derived tancytic territories. *Nat Commun.* 2017;8:13759. doi:10.1038/ncomms13759

6. Lhomme T, Clasadonte J, Imbernon M, et al. Tancytic networks mediate energy balance by feeding lactate to glucose-insensitive POMC neurons. *J Clin Invest.* 2021;131(18):140521. doi:10.1172/JCI140521

7. Recabal A, Elizondo-Vega R, Philippot C, et al. Connexin-43 gap junctions are responsible for the hypothalamic Tanocyte-coupled network. *Front Cell Neurosci.* 2018;12:406. doi:10.3389/fncnl.2018.00406

8. Szilvásy-Szabó A, Varga E, Beliczai Z, Lechan RM, Fekete C. Localization of connexin 43 gap junctions and hemichannels in tanocytes of adult mice. *Brain Res.* 2017;1673:64-71. doi:10.1016/j.brainres.2017.08.010

9. Berger UV, Hediger MA. Differential distribution of the glutamate transporters GLT-1 and GLAST in tanocytes of the third ventricle. *J Comp Neurol.* 2001;433(1):101-114. doi:10.1002/cne.1128

10. Nilaweera K, Herwig A, Bolborea M, et al. Photoperiodic regulation of glycogen metabolism, glycolysis, and glutamine synthesis in tanocytes of the Siberian hamster suggests novel roles of tanocytes in hypothalamic function. *Glia.* 2011;59(11):1695-1705. doi:10.1002/glia.21216

11. Elkjaer M, Vajda Z, Nejsum LN, et al. Immunolocalization of AQP9 in liver, epididymis, testis, spleen, and brain. *Biochem Biophys Res Commun.* 2000;276(3):1118-1128. doi:10.1006/bbrc.2000.3505

12. Pasquettaz R, Kolotuev I, Rohrbach A, Gouelle C, Pellerin L, Langlet F. Peculiar protrusions along tanocyte processes face diverse neural and nonneural cell types in the hypothalamic parenchyma. *J Comp Neurol.* 2021;529(3):553-575. doi:10.1002/cne.24965

13. Prevot V, Dehouck B, Sharif A, Biofi P, Giacobini P, Clasadonte J. The versatile Tanocyte: a hypothalamic integrator of reproduction and energy metabolism. *Endocr Rev.* 2018;39(3):333-368. doi:10.1210/er.2017-00235

14. Prevot V, Sharif A. The polygamous GnRH neuron: astrocytic and tancytic communication with a neuroendocrine neuronal population. *J Neuroendocrinol.* 2022;e13104:e13104. doi:10.1111/jne.13104
expression of these markers and the proliferative activity. The molecular mechanisms regulating postnatal tanocyte proliferation have just started to be explored. Two recent genetic invalidation studies identified fibroblast growth factor 10 (Fgf10)⁴ and the nuclear factor I (NFI) family of transcription factors²⁵ as critical regulators of tanocyte proliferation and neurogenic competence during the postnatal period. 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 tanocytes remains to be explored.

A hallmark of tanocytes 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 tanocytes also correlates with the maturation of the interface between tanocyte 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 tanocytes, 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 tanocytes 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 technology⁴⁷ 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 tanocytes morphologically and functionally interact.

## AUTHOR CONTRIBUTIONS

**Adrian Coutteau-Robles:** Conceptualization; formal analysis; investigation; methodology; writing – review and editing. **Vincent Prevot:** Conceptualization; formal analysis; funding acquisition; investigation; methodology; writing – review and editing. **Ariane Sharif:** Conceptualization; formal analysis; funding acquisition; investigation; methodology; supervision; writing – original draft.

## ACKNOWLEDGMENTS

This work was supported by a doctoral fellowship from the University of Lille to A. C.-R., Inserm grant U1172, the Association pour l'Etude des Anomalies Congénitales (to A.S.), the European Research Council ERC-Synergy-Grant-2019-WATCH no. 810331 (to V.P.). We thank the Animal Core Facility of the University of Lille (PLBS UAR 2014-US41) for expert technical support.

## CONFLICT OF INTEREST

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

## PEER REVIEW

The peer review history for this article is available at https://publons.com/publon/10.1111/jne.13239.

## DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding authors upon request.

## ORCID

Vincent Prevot https://orcid.org/0000-0001-7185-3615

Ariane Sharif https://orcid.org/0000-0003-0810-400X

## REFERENCES

1. Mullier A, Bouret SG, Prevot V, Dehouck B. Differential distribution of tight junction proteins suggests a role for tanocytes in blood-hypothalamus barrier regulation in the adult mouse brain. *J Comp Neurol.* 2010;518(7):943-962. doi:10.1002/cne.22273

2. Pellegrino G, Trubert C, Terrien J, et al. A comparative study of the neural stem cell niche in the adult hypothalamus of human, mouse, rat and gray mouse lemur (Microcebus murinus). *J Comp Neurol.* 2018; 526(9):1419-1443. doi:10.1002/cne.24376

3. Goodman T, Hajihosseini MK. Hypothalamic tanocytes-masters and servants of metabolic, neuroendocrine, and neurogenic functions. *Front Neurosci.* 2015;9:387. doi:10.3389/fnins.2015.00387

4. Goodman T, Nayar SG, Clare S, et al. Fibroblast growth factor 10 is a negative regulator of postnatal neurogenesis in the mouse hypothalamus. *Development.* 2020;147(13):dev180950. doi:10.1242/dev.180950

5. Mirzadeh Z, Kusne Y, Duran-Moreno M, et al. Bi- and unencilitated ependymal cells define continuous floor-plate-derived tancytic territories. *Nat Commun.* 2017;8:13759. doi:10.1038/ncomms13759

6. Lhomme T, Clasadonte J, Imbernon M, et al. Tancytic networks mediate energy balance by feeding lactate to glucose-insensitive POMC neurons. *J Clin Invest.* 2021;131(18):140521. doi:10.1172/JCI140521

7. Recabal A, Elizondo-Vega R, Philippot C, et al. Connexin-43 gap junctions are responsible for the hypothalamic Tanocyte-coupled network. *Front Cell Neurosci.* 2018;12:406. doi:10.3389/fncnl.2018.00406

8. Szilvásy-Szabó A, Varga E, Beliczai Z, Lechan RM, Fekete C. Localization of connexin 43 gap junctions and hemichannels in tanocytes of adult mice. *Brain Res.* 2017;1673:64-71. doi:10.1016/j.brainres.2017.08.010

9. Berger UV, Hediger MA. Differential distribution of the glutamate transporters GLT-1 and GLAST in tanocytes of the third ventricle. *J Comp Neurol.* 2001;433(1):101-114. doi:10.1002/cne.1128

10. Nilaweera K, Herwig A, Bolborea M, et al. Photoperiodic regulation of glycogen metabolism, glycolysis, and glutamine synthesis in tanocytes of the Siberian hamster suggests novel roles of tanocytes in hypothalamic function. *Glia.* 2011;59(11):1695-1705. doi:10.1002/glia.21216

11. Elkjaer M, Vajda Z, Nejsum LN, et al. Immunolocalization of AQP9 in liver, epididymis, testis, spleen, and brain. *Biochem Biophys Res Commun.* 2000;276(3):1118-1128. doi:10.1006/bbrc.2000.3505

12. Pasquettaz R, Kolotuev I, Rohrbach A, Gouelle C, Pellerin L, Langlet F. Peculiar protrusions along tanocyte processes face diverse neural and nonneural cell types in the hypothalamic parenchyma. *J Comp Neurol.* 2021;529(3):553-575. doi:10.1002/cne.24965

13. Prevot V, Dehouck B, Sharif A, Biofi P, Giacobini P, Clasadonte J. The versatile Tanocyte: a hypothalamic integrator of reproduction and energy metabolism. *Endocr Rev.* 2018;39(3):333-368. doi:10.1210/er.2017-00235

14. Prevot V, Sharif A. The polygamous GnRH neuron: astrocytic and tancytic communication with a neuroendocrine neuronal population. *J Neuroendocrinol.* 2022;e13104:e13104. doi:10.1111/jne.13104
15. Müller-Fielitz H, Stahr M, Bernau M, et al. Tanycytes control the hor-
monal output of the hypothalamic-pituitary-thyroid axis. *Nat Com-
mun.* 2017;8(1):484. doi:10.1038/s41467-017-00604-6

16. Langlet F, Levin BE, Luquet S, et al. Tanycytic VEGF-A boosts blood-
hypothalamus barrier plasticity and access of metabolic signals to the
arcuate nucleus in response to fasting. *Cell Metab.* 2013;17(4):607-
617. doi:10.1016/j.cmet.2013.03.004

17. Balland E, Dam J, Langlet F, et al. Hypothalamic tanycytes are an ERK-gated conduit for leptin into the brain. *Cell Metab.* 2014;19(2): 293-301. doi:10.1016/j.cmet.2013.12.015

18. Duquenne M, Folgueira C, Bourouh C, et al. Leptin brain entry via a tanycytic LepR-EGFR shuttle controls lipid metabolism and pancreas function. *Nat Metab.* 2021;3(8):1071-1090. doi:10.1038/s42255-021-00432-5

19. Bolborea M, Pollatzek E, Benford H, Sotelo-Hitschfeld T, Dale N. Hypothalamic tanycytes generate acute hyperphagia through activation of the arcuate neuronal network. *Proc Natl Acad Sci U S A.* 2020; 117(25):14473-14481. doi:10.1073/pnas.1919887117

20. Geller S, Arribat Y, Netzahualcoyotzi C, et al. Tanycytes regulate lipid homeostasis by sensing free fatty acids and signaling to key hypothalamic neuronal populations via FGF21 secretion. *Cell Metab.* 2019; 30(4):833-844.e7. doi:10.1016/j.cmet.2019.08.004

21. Chaker Z, George C, Petrovska M, et al. Hypothalamic neurogenesis persists in the aging brain and is controlled by energy-sensing IGF-I pathway. *Neurobiol Aging.* 2016;41:64-72. doi:10.1016/j.neurobiolaging.2016.02.008

22. Haan N, Goodman T, Najdi-Samiei A, et al. Fgf10-expressing tanycytes add new neurons to the appetite/energy-balance regulating centers of the postnatal and adult hypothalamus. *J Neurosci.* 2013; 33(14):6170-6180. doi:10.1523/JNEUROSCI.2437-12.2013

23. Lee DA, Bedont JL, Pak T, et al. Tanycytes of the hypothalamic median eminence form a diet-responsive neurogenic niche. *Nat Neurosci.* 2012;15(5):700-702. doi:10.1038/nn.3079

24. Robins SC, Stewart I, McNay DE, et al. α-Tanycytes of the adult hypothalamic third ventricle include distinct populations of FGF-responsive neural progenitors. *Nat Commun.* 2013;4:2049. doi:10.1038/ncomms3049

25. Yoo S, Kim J, Lyu P, et al. Control of neurogenic competence in mammalian hypothalamic tanycytes. *Sci Adv.* 2021;7(22):eabg3777. doi: 10.1126/sciadv.abg3777

26. Rodríguez EM, Blázquez JL, Pastor FE, et al. Hypothalamic tanycytes: a key component of brain-endocrine interaction. *Int Rev Cytol.* 2005; 247:89-164. doi:10.1016/S0074-7696(05)47003-5

27. Alvarez-Bolado G, Paul FA, Blaess S. Sonic hedgehog lineage in the mouse hypothalamus: from progenitor domains to hypothalamic regions. *Neural Dev.* 2012;7:4. doi:10.1186/1749-8104-7-4

28. Miranda-Angulo AL, Byerly MS, Mesa J, Wang H, Blackshaw S. Rax regulates hypothalamic tanocyte differentiation and barrier function in mice. *J Comp Neurol.* 2014;522(4):876-899. doi:10.1002/cne.23451

29. Salvatierra J, Lee DA, Zibetti C, et al. The LIM homeodomain factor Lhx2 is required for hypothalamic tanocyte specification and differentiation. *J Neurosci.* 2014;34(50):16809-16820. doi:10.1523/JNEUROSCI.1711-14.2014

30. Cameron HA, McKay RD. Adult neurogenesis produces a large pool of new granule cells in the dentate gyrus. *J Comp Neurol.* 2001; 435(4):406-417. doi:10.1002/cne.1040

31. Pellegrino G, Martin M, Allet C, et al. GnRH neurons recruit astrocytes in infancy to facilitate network integration and sexual maturation. *Nat Neurosci.* 2021;24(12):1660-1672. doi:10.1038/s41593-021-00960-z

32. Paxinos G, Franklin KBJ. *The Mouse Brain in Stereotaxic Coordinates.* Second ed. Elsevier, Academic Press; 2001.

33. Barahona MJ, Langlet F, Labouèbe G, et al. GLUT2 expression by glial fibrillary acidic protein-positive tanycytes is required for promoting feeding-response to fasting. *Sci Rep.* 2022;12(1):17717. doi:10.1038/s41598-022-22489-2

34. Moore A, Chinnaiya K, Kim DW, et al. Loss of function of the neural cell adhesion molecule NrCAM regulates differentiation, proliferation and neurogenesis in early postnatal hypothalamic Tanycytes. *Front Neurosci.* 2022;16:832961. doi:10.3389/fnins.2022.832961

35. Andrés-Sánchez N, Fisher D, Krasinska L. Physiological functions and roles in cancer of the proliferation marker Ki-67. *J Cell Sci.* 2022; 135(11):jcs258932. doi:10.1242/jcs.258932

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How to cite this article: Coutteau-Robles A, Prevot V, Sharif A. 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. *J Neuroendocrinol.* 2023;35(3):e13239. doi:10.1111/jne.13239
15. Müller-Fielitz H, Stahr M, Bernau M, et al. Tanycytes control the hor-
monal output of the hypothalamic-pituitary-thyroid axis. *Nat Com-
mun.* 2017;8(1):484. doi:10.1038/s41467-017-00604-6

16. Langlet F, Levin BE, Luquet S, et al. Tanycytic VEGF-A boosts blood-
hypothalamus barrier plasticity and access of metabolic signals to the
arcuate nucleus in response to fasting. *Cell Metab.* 2013;17(4):607-
617. doi:10.1016/j.cmet.2013.03.004

17. Balland E, Dam J, Langlet F, et al. Hypothalamic tanycytes are an ERK-gated conduit for leptin into the brain. *Cell Metab.* 2014;19(2): 293-301. doi:10.1016/j.cmet.2013.12.015

18. Duquenne M, Folgueira C, Bourouh C, et al. Leptin brain entry via a tanycytic LepR-EGFR shuttle controls lipid metabolism and pancreas function. *Nat Metab.* 2021;3(8):1071-1090. doi:10.1038/s42255-021-00432-5

19. Bolborea M, Pollatzek E, Benford H, Sotelo-Hitschfeld T, Dale N. Hypothalamic tanycytes generate acute hyperphagia through activation of the arcuate neuronal network. *Proc Natl Acad Sci U S A.* 2020; 117(25):14473-14481. doi:10.1073/pnas.1919887117

20. Geller S, Arribat Y, Netzahualcoyotzi C, et al. Tanycytes regulate lipid homeostasis by sensing free fatty acids and signaling to key hypothalamic neuronal populations via FGF21 secretion. *Cell Metab.* 2019; 30(4):833-844.e7. doi:10.1016/j.cmet.2019.08.004

21. Chaker Z, George C, Petrovska M, et al. Hypothalamic neurogenesis persists in the aging brain and is controlled by energy-sensing IGF-I pathway. *Neurobiol Aging.* 2016;41:64-72. doi:10.1016/j.neurobiolaging.2016.02.008

22. Haan N, Goodman T, Najdi-Samiei A, et al. Fgf10-expressing tanycytes add new neurons to the appetite/energy-balance regulating centers of the postnatal and adult hypothalamus. *J Neurosci.* 2013; 33(14):6170-6180. doi:10.1523/JNEUROSCI.2437-12.2013

23. Lee DA, Bedont JL, Pak T, et al. Tanycytes of the hypothalamic median eminence form a diet-responsive neurogenic niche. *Nat Neurosci.* 2012;15(5):700-702. doi:10.1038/nn.3079

24. Robins SC, Stewart I, McNay DE, et al. α-Tanycytes of the adult hypothalamic third ventricle include distinct populations of FGF-responsive neural progenitors. *Nat Commun.* 2013;4:2049. doi:10.1038/ncomms3049

25. Yoo S, Kim J, Lyu P, et al. Control of neurogenic competence in mammalian hypothalamic tanycytes. *Sci Adv.* 2021;7(22):eabg3777. doi: 10.1126/sciadv.abg3777

26. Rodríguez EM, Blázquez JL, Pastor FE, et al. Hypothalamic tanycytes: a key component of brain-endocrine interaction. *Int Rev Cytol.* 2005; 247:89-164. doi:10.1016/S0074-7696(05)47003-5

27. Alvarez-Bolado G, Paul FA, Blaess S. Sonic hedgehog lineage in the mouse hypothalamus: from progenitor domains to hypothalamic regions. *Neural Dev.* 2012;7:4. doi:10.1186/1749-8104-7-4

28. Miranda-Angulo AL, Byerly MS, Mesa J, Wang H, Blackshaw S. Rax regulates hypothalamic tanocyte differentiation and barrier function in mice. *J Comp Neurol.* 2014;522(4):876-899. doi:10.1002/cne.23451

29. Salvatierra J, Lee DA, Zibetti C, et al. The LIM homeodomain factor Lhx2 is required for hypothalamic tanocyte specification and differentiation. *J Neurosci.* 2014;34(50):16809-16820. doi:10.1523/JNEUROSCI.1711-14.2014

30. Cameron HA, McKay RD. Adult neurogenesis produces a large pool of new granule cells in the dentate gyrus. *J Comp Neurol.* 2001; 435(4):406-417. doi:10.1002/cne.1040

31. Pellegrino G, Martin M, Allet C, et al. GnRH neurons recruit astrocytes in infancy to facilitate network integration and sexual maturation. *Nat Neurosci.* 2021;24(12):1660-1672. doi:10.1038/s41593-021-00960-z

32. Paxinos G, Franklin KBJ. *The Mouse Brain in Stereotaxic Coordinates.* Second ed. Elsevier, Academic Press; 2001.

33. Barahona MJ, Langlet F, Labouèbe G, et al. GLUT2 expression by glial fibrillary acidic protein-positive tanycytes is required for promoting feeding-response to fasting. *Sci Rep.* 2022;12(1):17717. doi:10.1038/s41598-022-22489-2

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How to cite this article: Coutteau-Robles A, Prevot V, Sharif A. 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.
*J Neuroendocrinol.* 2023;35(3):e13239. doi:10.1111/jne.13239